Attorney Docket No. 01318-0014-00PCT OR-043WO SYNTHETIC INTERNAL RIBOSOME ENTRY SITES SEQUENCE LISTING [1] The present application contains a Sequence Listing which has been submitted electronically in XML format. Said XML copy, created on June 24, 2025, is named “01318- 0014-60PCT-SL.xml” and is 31,301,409 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. [2] Conventional gene therapy involves the use of DNA for insertion of desired genetic information into host cells. The DNA introduced into the cell is usually integrated to a certain extent into the genome of one or more transfected cells, allowing for long-lasting action of the introduced genetic material in the host. While there may be substantial benefits to such sustained action, integration of exogenous DNA into a host genome may also have many deleterious effects. For example, it is possible that the introduced DNA will be inserted into an intact gene, resulting in a mutation which impedes or even totally eliminates the function of the endogenous gene. Thus, gene therapy with DNA may result in the impairment of a vital genetic function in the treated host, such as e.g., elimination or deleteriously reduced production of an essential enzyme or interruption of a gene critical for the regulation of cell growth, resulting in unregulated or cancerous cell proliferation. In addition, with conventional DNA based gene therapy it is necessary for effective expression of the desired gene product to include a strong promoter sequence, which again may lead to undesirable changes in the regulation of normal gene expression in the cell. It is also possible that the DNA based genetic material will result in the induction of undesired anti-DNA antibodies, which in turn, may trigger a possibly fatal immune response. Gene therapy approaches using viral vectors can also result in an adverse immune response. In some circumstances, the viral vector may even integrate into the host genome. In addition, production of clinical grade viral vectors is also expensive and time consuming. Targeting delivery of the introduced genetic material using viral vectors can also be difficult to control. Thus, while DNA based gene therapy has been evaluated for delivery of secreted proteins using viral vectors (U.S. Patent No.6,066,626; U.S. Publication No. US2004/0110709), these approaches may be limited for these various reasons. [3] In contrast to DNA, the use of RNA as a gene therapy agent is substantially safer because RNA does not involve the risk of being stably integrated into the genome of the transfected cell, thus eliminating the concern that the introduced genetic material will disrupt the normal functioning of an essential gene, or cause a mutation that results in deleterious or 1
Attorney Docket No. 01318-0014-00PCT OR-043WO oncogenic effects, and extraneous promoter sequences are not required for effective translation of the encoded protein, again avoiding possible deleterious side effects. In addition, it is not necessary for RNA to enter the nucleus to perform its function, while DNA must overcome this major barrier. [4] Circular RNA (circRNA or oRNA®) is a stable form of RNA. Circular RNA can also be particularly interesting and useful for in vivo applications, especially in the research area of RNA-based control of gene expression and therapeutics, including, for example, protein replacement therapy and vaccination. [5] Many eukaryotic mRNAs are translated in the canonical cap-dependent manner (Koch, Nat Struct Mol Biol. 2020 Dec; 27(12): 1095–1104, incorporated by reference herein), in which the 5’ end of the linear precursor RNA polynucleotide contains a 5’ cap that recruits binding of the ribosome 40S subunit to initiate robust translation. (See Renaud-Gabardos, World J Exp Med. 2015 Feb 20; 5(1): 11–20 at Figure 1, incorporated by reference herein in its entirety). This general mechanism depends on the recognition of the 5’ cap by the translation eukaryotic initiation factor (eIF)-4F, which is composed of three polypeptides (eIF4A, eIF4E, and eIF4G). (Salas-Martinez, Front. Microbiol., 03 January 2018 Sec. Virology, Volume 8 Article 2629.) Circular RNA has no 5’ end and therefore no 5’ cap by which robust ribosome binding and translation can be initiated. Some eukaryotic linear mRNAs and viral RNAs contain an internal ribosome entry site (IRES) in the 5’ untranslated region, which can recruit ribosomes in a cap-independent manner. (See Koch 2020; Renaud- Gabardos 2015). “Cellular IRESs are very diverse, which makes them difficult to classify. In other words, there appear to be many different, as of yet uncharacterized, mechanisms that control cap-independent translation using endogenous IRESs.” (See Deviatkin, Vaccines. 2023; 11(2):238, incorporated by reference herein.) However, IRES-driven expression is generally lower than that of cap-dependent translation, for example initial studies showed lower efficiency of IRES-associated translation initiation compared to cap-dependent initiation. (Deviatkin 2023.) [6] Five different types of IRESs (Types I, II, III, IV, V) have been classified based on evolutionary conserved sequences and structural organization. Each type harbors a common RNA structure core maintained by evolutionary conserved covariant substitutions. (See Salas- Martinez 2018, incorporated by reference herein.) The IRESs are made of domains (e.g., Domains I, II, III, IV, V, VI, and VII). Within those domains are motifs or regions (e.g., GNRA, or C-rich, or EIF4G) that have different functions. While the different types of IRESs 2
Attorney Docket No. 01318-0014-00PCT OR-043WO can vary structurally, certain domains and motifs are conserved across the different types of IRESs. (Id.) [7] As set forth herein, in some embodiments, the circular RNA comprises a TIE comprising a synthetic internal ribosome entry site (IRES) or a fragment or variant thereof and a coding element comprising an expression sequence encoding at least one therapeutic protein. In some embodiments, the synthetic IRES is capable of increasing expression of operably linked expression sequences as compared to naturally occurring IRESs. In some embodiments, the synthetic IRESs comprise nucleotide additions, nucleotide deletions, and nucleotide substitutions as compared to the naturally occurring counterpart of the synthetic IRES (i.e. a naturally occurring IRES without the changes, or the starting IRES before the changes are made). In some embodiments, the synthetic IRESs comprise at least one addition, deletion, or substitution of a nucleotide, domain, or motif, as compared to a naturally occurring IRES. In some embodiments, the synthetic IRES comprise at least one addition, deletion, or substitution of a nucleotide or motif in a domain as compared to a corresponding naturally occurring domain, or comprises a deletion or substitution of the domain in whole or in part. In some embodiments, the synthetic IRES comprises at least one naturally occurring domain. As set forth in further detail herein, in some embodiments, the synthetic IRES has improved function and/or expression and/or stability as compared to a naturally occurring IRES. BRIEF DESCRIPTION OF THE DRAWINGS [8] FIG. 1 depicts a graphical representation of exemplary high expressing internal ribosome entry sites (IRES) displayed in clusters for Type I, Type II, and Type V IRES. Expression was measured in primary human hepatocytes (PHH), T cell lymphocytes (TCL) and myotubes (MYO). [9] FIGs.2A-C depict coxsackievirus B3 (CVB3) IRES secondary structure alignment and folding based on homology of its domains. DETAILED DESCRIPTION [10] The present disclosure provides, among other things, precursor RNAs for producing circular RNAs and the produced circular RNAs. In some embodiments, such produced circular RNAs have improved properties, such as improved circularization efficiency. In some embodiments, the precursor RNAs comprise Group I or Group II exon and/or intron segments. In certain embodiments, the precursor RNAs and/or circular RNAs comprise one or more 3
Attorney Docket No. 01318-0014-00PCT OR-043WO modified nucleotides or nucleosides. Also provided herein are related compositions (e.g., template DNAs or lipid nanoparticles). Also provided herein are methods for the selection, design, preparation, manufacture, formulation, and/or use of RNA preparations, such as precursor RNAs or circular RNAs. [11] Reference will now be made in detail to certain embodiments, examples of which are illustrated in the accompanying drawings. While the disclosure is described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the disclosure to those embodiments. On the contrary, the disclosure is intended to cover all alternatives, modifications, and equivalents, which may be included within the disclosure as defined by the appended claims and included embodiments. [12] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. [13] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings. [14] Unless specifically noted in the specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of” or “consisting essentially of” the recited components; embodiments in the specification that recite “consisting of” various components are also contemplated as “comprising” or “consisting essentially of” the recited components; and embodiments in the specification that recite “consisting essentially of” various components are also contemplated as “consisting of” or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims). The term “or” is used in an inclusive sense, i.e., equivalent to “and/or,” unless the context clearly indicates otherwise. [15] The section headings used herein are for organizational purposes only and are not to be construed as limiting the desired subject matter in any way. In the event that any material incorporated by reference contradicts any term defined in this specification or any other express 4
Attorney Docket No. 01318-0014-00PCT OR-043WO content of this specification, this specification controls. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art. 1. DEFINITIONS [16] Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings: [17] As used herein, linear nucleic acid molecules are said to have a “5’-terminus” (or “5’ end”) and a “3’-terminus” (or “3’ end”) because nucleic acid phosphodiester linkages occur at the 5’ carbon and 3’ carbon of the sugar moieties of the substituent mononucleotides. The end nucleotide of a polynucleotide at which a new linkage would be to a 5’ carbon is its 5’ terminal nucleotide. The end nucleotide of a polynucleotide at which a new linkage would be to a 3’ carbon is its 3’ terminal nucleotide. A “terminal nucleotide,” as used herein, is the nucleotide at the end position of the 3’- or 5’-terminus. [18] As used herein, the term “3’ intron segment” (or “3’ intron fragment”) refers to a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% similarity to the 3’-proximal end of a natural intron (e.g., a group I or group II intron). In certain embodiments, the 3’ intron segment includes the 5’ nucleotide of the splice site dinucleotide. “3’ exon segment” (or “3’ exon fragment”) refers to a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% similarity to the 5’-proximal end of an exon adjacent to a “3’ intron segment” as described herein. In certain embodiments, the 3’ exon segment includes the 3’ nucleotide of the splice site dinucleotide. [19] The term “5’ intron segment” (or “5’ intron fragment”) refers to a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher 100% similarity to the 5’-proximal end of a natural intron (e.g., a group I or group II intron). In certain embodiments, the 5’ intron segment includes the 3’ nucleotide of the splice site dinucleotide. “5’ exon segment” (or “5’ exon fragment”) refers to a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher 100% 5
Attorney Docket No. 01318-0014-00PCT OR-043WO similarity to the 3’-proximal end of an exon adjacent to a “5’ intron segment” as described herein. In certain embodiments, the 5’ exon segment includes the 5’ nucleotide of the splice site dinucleotide. [20] In some embodiments, the 3’ intron segment and the 3’ exon segment together form a first portion of an autocatalytic or self-splicing intron-exon sequence. In some embodiments, the 5’ intron segment and the 5’ exon segment together form the remainder (i.e., second portion) of the autocatalytic or self-splicing intron-exon sequence. In these embodiments, a linear nucleic acid molecule, e.g., RNA, comprising the 3’ intron segment and the 3’ exon segment at the 5’ end of the linear nucleic acid molecule and further the 5’ intron segment and the 5’ exon segment at the 3’ end the linear nucleic acid molecule, is capable of autocatalytically self-splicing and thereby capable of forming a circular nucleic acid molecule, e.g., circular RNA. In these embodiments, the 3’ intron segment and the 5’ intron segments are excised from the circular nucleic acid molecule, e.g., circular RNA, and the 3’ exon segment and the 5’ exon segment are retained in the circular nucleic acid molecule, e.g., circular RNA. Each retained post-splicing exon segment may be referred to as a self-splicing or self-spliced exon segment, e.g., a 3’ self-splicing or self-spliced exon segment and a 5’ self-splicing or self- spliced exon segment. [21] In some embodiments, the intron segment is a “Group I intron” and the corresponding exon segment may be referred to as a “Group I exon” or “Group 1 self-splicing exon” or “Group I self-spliced exon segment” or the like. In some embodiments, the intron segment is a “Group II intron” and the corresponding exon segment may be referred to as a “Group II exon” or “Group II self-splicing exon” or “Group II self-spliced exon segment” or the like. [22] In some embodiments, the retained, post-splicing, self-splicing 3’ or 5’ exon segment is a non-coding sequence in the circular nucleic acid molecule, e.g., circular RNA. In some embodiments, the circular nucleic acid molecule, e.g., circular RNA, further comprises a desired coding sequence, and the retained, post-splicing, self-splicing 3’ or 5’ exon segment is (e.g., designed) to be a portion of the desired expression sequence, contiguous with the desired coding sequence, and/or in frame with the desired coding sequence. [23] Within a circular nucleic acid molecule, e.g., derived from a linear nucleic acid precursor, and comprising a coding sequence, the 5’ to 3’ orientation of the coding sequence may be used to inform whether other sequences within the circular nucleic acid are 5’ and/or 3’, e.g., for example, 5’ is nearer to the 5’ of the coding sequence, and the 3’ end is downstream 6
Attorney Docket No. 01318-0014-00PCT OR-043WO of the coding sequence. As used herein, within a circular nucleic acid molecule, e.g., derived from a linear nucleic acid precursor, reference to a “5’” or “3’” portion of the molecule may correspond to the orientation of the sequence within the linear nucleic acid precursor. [24] As used herein, “splice site” refers to the junction consisting of a dinucleotide between an exon and an intron in an unspliced RNA. As used herein, the term “splice site” refers to a dinucleotide that is partially or fully included in a group I or group II intron and/or exon and between which a phosphodiester bond is cleaved during RNA circularization. A “splice site dinucleotide” refers two nucleotides: a 5’ splice site nucleotide and the 3’ splice site nucleotide. A “5’ splice site” refers to the natural 5’ dinucleotide of the intron and/or exon e.g., group I or group II intron and/or exon, while a “3’ splice site” refers to the natural 3’ dinucleotide of the intron and/or exon. Exemplary splice site dinucleotides are shown in the table below. Table: Exemplary Splice Site Dinucleotides
7
Attorney Docket No. 01318-0014-00PCT OR-043WO
[25] As used herein, the term “permutation site” refers to a site in an intron and/or exon 8
Attorney Docket No. 01318-0014-00PCT OR-043WO (e.g., a group I or II intron and/or exon) where a cut is made prior to permutation of the intron/or exon. For example, such a cut generates an intron sequence comprising a 3’ intron segment and a sequence comprising a 5’ intron segment (e.g., group I or group II intron fragments) that are permuted to be on either side of a stretch of precursor RNA to be circularized. The permuted intron segments are thereby called “3’ permuted intron segments” or “3’ permuted elements” and “5’ permuted intron segments” or “5’ permuted elements” in the context of said precursor RNA. As used herein, “permuted intron segment” and “permuted intron element” are used interchangeably. In some embodiments, the permutation site consists of a dinucleotide. [26] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes combinations of two or more cells, or entire cultures of cells; reference to “a polynucleotide” includes, as a practical matter, many copies of that polynucleotide. [27] A used herein, the terms “about,” or “approximately” are understood as within a range of normal tolerance in the art, for example within 2 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 clear from the context, all numerical values provided herein are modified by the term “about.” [28] As used herein, “accessory element” or “accessory sequences” refers to internal spacer(s), external spacer(s), and/or homology arm(s). As used herein, a “combined accessory element” or “combined accessory sequences” comprises the accessory element and further comprises an intron and/or exon segment. In some embodiments, the accessory element increases circularization efficiency and/or translation efficiency in a circular RNA as compared to a control circular RNA without the accessory sequences. [29] As used herein, an “affinity sequence” or “affinity tag” is a region of a polynucleotide sequence ranging from one (1) nucleotide to hundreds or thousands of nucleotides containing a repeated set of nucleotides for the purposes of aiding purification of a polynucleotide sequence. For example, an affinity sequence may comprise, but is not limited to, a polyA or polyAC sequence. In some embodiments, affinity tags are used in purification methods, referred to herein as “affinity-purification,” in which selective binding of a binding agent to molecules comprising an affinity tag facilitates separation from molecules that do not comprise an affinity tag. In some embodiments, an affinity-purification method is a “negative selection” purification method, in which unwanted species, such as linear RNA, are selectively 9
Attorney Docket No. 01318-0014-00PCT OR-043WO bound and removed and wanted species, such as circular RNA, are eluted and separated from unwanted species. [30] An “antigen” refers to any molecule that provokes an immune response or is capable of being bound by an antibody or an antigen binding molecule. The immune response may involve either antibody production, or the activation of specific immunologically - competent cells, or both. A person of skill in the art would readily understand that any macromolecule, including virtually all proteins or peptides, may serve as an antigen. An antigen may be endogenously expressed, i.e. expressed by genomic DNA, or may be recombinantly expressed. An antigen may be specific to a certain tissue, such as a cancer cell, or it may be broadly expressed. In addition, fragments of larger molecules may act as antigens. In some embodiments, antigens are tumor antigens. [31] An “antigen binding molecule,” “antigen binding portion,” or “antibody fragment” refers to any molecule that specifically binds to a desired antigen. In some embodiments, an antigen binding molecule comprises the antigen binding parts (e.g., CDRs) of an antibody or antibody-like molecule. An antigen binding molecule may include the antigenic complementarity determining regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen binding molecules. Peptibodies (i.e., Fc fusion molecules comprising peptide binding domains) are another example of suitable antigen binding molecules. In some embodiments, the antigen binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen binding molecule binds to an antigen on a cell involved in a hyperproliferative disease or to a viral or bacterial antigen. In further embodiments, the antigen binding molecule is an antibody fragment, including one or more of the complementarity determining regions (CDRs) thereof, that specifically binds to the antigen. In further embodiments, the antigen binding molecule is a single chain variable fragment (scFv). In some embodiments, the antigen binding molecule comprises or consists of avimers. [32] The term “antibody” (Ab) includes, without limitation, a glycoprotein immunoglobulin which binds specifically to an antigen. In general, an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding molecule thereof. Each H chain may comprise a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region can comprise three constant domains, CH1, CH2 and CH3. Each light chain can comprise a light chain variable region (abbreviated herein as VL) and a light chain constant region. The 10
Attorney Docket No. 01318-0014-00PCT OR-043WO light chain constant region can comprise one constant domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). CDRs may be described by numbering known in the art, for example, Kabat numbering, Chothia numbering, AbM numbering, or contact numbering. Each VH and VL may comprise three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the Abs may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system. Antibodies may include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain- antibody heavy chain pair, intrabodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single- chain variable fragments (scFv), camelized antibodies, affybodies, Fab fragments, F(ab’)2 fragments, disulfide-linked variable fragments (sdFv), anti-idiotypic (anti-id) antibodies (including, e.g., anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), and antigen-binding fragments of any of the above. In some embodiments, antibodies described herein refer to polyclonal antibody populations. [33] An immunoglobulin may derive from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgG1, IgG2, IgG3 and IgG4. “Isotype” refers to the Ab class or subclass (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes. The term “antibody” includes, by way of example, both naturally occurring and non-naturally occurring Abs; monoclonal and polyclonal Abs; chimeric and humanized Abs; human or nonhuman Abs; wholly synthetic Abs; and single chain Abs. A nonhuman Ab may be humanized by recombinant methods to reduce its immunogenicity in 11
Attorney Docket No. 01318-0014-00PCT OR-043WO humans. Where not expressly stated, and unless the context indicates otherwise, the term “antibody” also includes an antigen-binding fragment or an antigen-binding portion of any of the aforementioned immunoglobulins, and includes a monovalent and a divalent fragment or portion, and a single chain Ab. [34] As used herein, the terms “variable region” or “variable domain” are used interchangeably and are common in the art. The variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. The variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with antigen. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In particular embodiments, the variable region is a primate (e.g., non-human primate) variable region. In some embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs). The terms “VL” and “VL domain” are used interchangeably to refer to the light chain variable region of an antibody or an antigen-binding molecule thereof. The terms “VH” and “VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody or an antigen-binding molecule thereof. [35] As used herein, the terms “constant region” and “constant domain” are interchangeable and have a meaning common in the art. The constant region is an antibody portion, e.g., a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen but which may exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain. [36] As used herein, “aptamer” refers in general to either an oligonucleotide of a single defined sequence or a mixture of said nucleotides, wherein the mixture retains the properties of binding specifically to the target molecule (e.g., eukaryotic initiation factor, 40S ribosome, 12
Attorney Docket No. 01318-0014-00PCT OR-043WO polyC binding protein, polyA binding protein, polypyrimidine tract-binding protein, argonaute protein family, Heterogeneous nuclear ribonucleoprotein K and La and related RNA-binding protein). Thus, as used herein “aptamer” denotes both singular and plural sequences of nucleotides, as defined hereinabove. The term “aptamer” is meant to refer to a single- or double-stranded nucleic acid which is capable of binding to a protein or other molecule. In general, aptamers preferably comprise about 10 to about 100 nucleotides, preferably about 15 to about 40 nucleotides, more preferably about 20 to about 40 nucleotides, in that oligonucleotides of a length that falls within these ranges are readily prepared by conventional techniques. Optionally, aptamers can further comprise a minimum of approximately 6 nucleotides, preferably 10, and more preferably 14 or 15 nucleotides, that are necessary to effect specific binding. [37] As used herein, “autoimmunity” is defined as persistent and progressive immune reactions to non-infectious self-antigens, as distinct from infectious non self-antigens from bacterial, viral, fungal, or parasitic organisms which invade and persist within mammals and humans. Autoimmune conditions include scleroderma, Grave's disease, Crohn's disease, Sjorgen's disease, multiple sclerosis, Hashimoto's disease, psoriasis, myasthenia gravis, autoimmune polyendocrinopathy syndromes, Type I diabetes mellitus (TIDM), autoimmune gastritis, autoimmune uveoretinitis, polymyositis, colitis, and thyroiditis, as well as in the generalized autoimmune diseases typified by human Lupus. [38] “Autoantigen” or “self-antigen” as used herein refers to an antigen or epitope which is native to the mammal and which is immunogenic in said mammal. [39] The term “autologous” refers to any material derived from the same individual to which it is later to be re-introduced. For example, the engineered autologous cell therapy (eACT™) method described herein involves collection of lymphocytes from a patient, which are then engineered to express, e.g., a CAR construct, and then administered back to the same patient. [40] “Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y may generally be represented by the dissociation constant (KD or Kd). Affinity may be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium 13
Attorney Docket No. 01318-0014-00PCT OR-043WO dissociation constant (KD), and equilibrium association constant (KA or Ka). The KD is calculated from the quotient of koff/kon, whereas KA is calculated from the quotient of kon/koff. kon refers to the association rate constant of, e.g., an antibody to an antigen, and koff refers to the dissociation of, e.g., an antibody to an antigen. The kon and koff may be determined by techniques known to one of ordinary skill in the art, such as BIACORE® or KinExA. [41] As used herein, the term, “specifically binds,” refers to molecules that bind to an antigen (e.g., epitope or immune complex) as such binding is understood by one skilled in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, generally with lower affinity as determined by, e.g., immunoassays, BIACORE®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In a specific embodiment, molecules that specifically bind to an antigen bind to the antigen with a KA that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater than the KA when the molecules bind to another antigen. [42] As used herein, “bicistronic RNA” refers to a polynucleotide that includes two expression sequences coding for two distinct proteins. These expression sequences can be separated by a nucleotide sequence encoding a cleavable peptide such as a protease cleavage site. They can also be separated by a ribosomal skipping element. [43] A “cancer” refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. A “cancer” or “cancer tissue” may include a tumor. Examples of cancers that may be treated by the methods disclosed herein include, but are not limited to, cancers of the immune system including lymphoma, leukemia, myeloma, and other leukocyte malignancies. In some embodiments, the methods disclosed herein may be used to reduce the tumor size of a tumor derived from, for example, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, multiple myeloma, Hodgkin's Disease, non- Hodgkin's lymphoma (NHL), primary mediastinal large B cell lymphoma (PMBC), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, cancer of the urethra, cancer of the penis, chronic or acute 14
Attorney Docket No. 01318-0014-00PCT OR-043WO leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non T cell ALL), chronic lymphocytic leukemia (CLL), solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, epidermoid cancer, squamous cell cancer, T cell lymphoma, environmentally induced cancers including those induced by asbestos, other B cell malignancies, and combinations of said cancers. In some embodiments, the methods disclosed herein may be used to reduce the tumor size of a tumor derived from, for example, sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, Kaposi's sarcoma, sarcoma of soft tissue, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, hepatocellular carcinomna, lung cancer, colorectal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (for example adenocarcinoma of the pancreas, colon, ovary, lung, breast, stomach, prostate, cervix, or esophagus), sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, bladder carcinoma, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, carcinoma of the renal pelvis, CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma). The particular cancer may be responsive to chemo- or radiation therapy or the cancer may be refractory. A refractory cancer refers to a cancer that is not amenable to surgical intervention and the cancer is either initially unresponsive to chemo- or radiation therapy or the cancer becomes unresponsive over time. [44] As used herein, the terms “circRNA,” “circular polyribonucleotide,” “circular RNA,” “circularized RNA,” “circular RNA polynucleotide” and “oRNA” are used interchangeably and refer to a single-stranded polyribonucleotide wherein the 3’ and 5’ ends that are normally present in a linear RNA polynucleotide have been joined together, e.g., by covalent bonds. As used herein, such terms also include preparations comprising circRNAs. [45] As used herein, the term “circularization efficiency” refers to a measurement of the rate of formation of amount of resultant circular polyribonucleotide as compared to its 15
Attorney Docket No. 01318-0014-00PCT OR-043WO linear starting material. [46] The expression sequences in the polynucleotide construct may be separated by a “cleavage site” sequence which enables polypeptides encoded by the expression sequences, once translated, to be expressed separately by the cell, e.g., eukaryotic cell. A “self-cleaving peptide” refers to a peptide which is translated without a peptide bond between two adjacent amino acids, or functions such that when the polypeptide comprising the proteins and the self- cleaving peptide is produced, it is immediately cleaved or separated into distinct and discrete first and second polypeptides without the need for any external cleavage activity. [47] As used herein, “co-administering” refers to administering a therapeutic agent provided herein in conjunction with one or more additional therapeutic agents sufficiently close in time such that the therapeutic agent provided herein can enhance the effect of the one or more additional therapeutic agents, or vice versa. [48] As used herein, “coding element,” “coding sequence,” “coding nucleic acid,” or “coding region” is region located within the expression sequence and encodings for one or more proteins or polypeptides (e.g., therapeutic protein). [49] As used herein, a “noncoding element,” “noncoding sequence,” “non-coding nucleic acid,” or “noncoding nucleic acid” is a region located within the expression sequence. This sequence by itself does not encode for a protein or polypeptide, but may have other regulatory functions, including but not limited, allow the overall polynucleotide to act as a biomarker or adjuvant to a specific cell. [50] A “costimulatory ligand,” as used herein, includes a molecule on an antigen presenting cell that specifically binds a cognate co-stimulatory molecule on a T cell. Binding of the costimulatory ligand provides a signal that mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A costimulatory ligand induces a signal that is in addition to the primary signal provided by a stimulatory molecule, for instance, by binding of a T cell receptor (TCR)/CD3 complex with a major histocompatibility complex (MHC) molecule loaded with peptide. A co-stimulatory ligand may include, but is not limited to, 3/TR6, 4-IBB ligand, agonist or antibody that binds Toll-like receptor, B7-1 (CD80), B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT) 3, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), ligand that specifically binds with B7-H3, lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2, or programmed 16
Attorney Docket No. 01318-0014-00PCT OR-043WO death (PD) LI. A co-stimulatory ligand includes, without limitation, an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, ligand that specifically binds with CD83, lymphocyte function- associated antigen-1 (LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT). [51] A “costimulatory molecule” is a cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, 4-1BB/CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD 33, CD 45, CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD 18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (alpha; beta; delta; epsilon; gamma; zeta), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8alpha, CD8beta, CD9, CD96 (Tactile), CD1- la, CDl-lb, CDl-lc, CDl-ld, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, IT GAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CD1 la/CD18), MHC class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG/Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocytic activation molecule, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof. [52] As used herein, an antigen binding molecule, an antibody, or an antigen binding molecule thereof “cross-competes” with a reference antibody or an antigen binding molecule thereof if the interaction between an antigen and the first binding molecule, an antibody, or an antigen binding molecule thereof blocks, limits, inhibits, or otherwise reduces the ability of the reference binding molecule, reference antibody, or an antigen binding molecule thereof to interact with the antigen. Cross competition may be complete, e.g., binding of the binding molecule to the antigen completely blocks the ability of the reference binding molecule to bind the antigen, or it may be partial, e.g., binding of the binding molecule to the antigen reduces 17
Attorney Docket No. 01318-0014-00PCT OR-043WO the ability of the reference binding molecule to bind the antigen. In some embodiments, an antigen binding molecule that cross-competes with a reference antigen binding molecule binds the same or an overlapping epitope as the reference antigen binding molecule. In other embodiments, the antigen binding molecule that cross-competes with a reference antigen binding molecule binds a different epitope as the reference antigen binding molecule. Numerous types of competitive binding assays may be used to determine if one antigen binding molecule competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA); solid phase direct or indirect enzyme immunoassay (EIA); sandwich competition assay (Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (Kirkland et al., 1986, J. Immunol. 137:3614-3619); solid phase direct labeled assay, solid phase direct labeled sandwich assay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (Morel et al., 1988, Molec. Immunol.25:7-15); solid phase direct biotin-avidin EIA (Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). [53] A “cytokine,” as used herein, refers to a non-antibody protein that is released by one cell in response to contact with a specific antigen, wherein the cytokine interacts with a second cell to mediate a response in the second cell. A cytokine may be endogenously expressed by a cell or administered to a subject. Cytokines may be released by immune cells, including macrophages, B cells, T cells, neutrophils, dendritic cells, eosinophils and mast cells to propagate an immune response. Cytokines may induce various responses in the recipient cell. Cytokines may include homeostatic cytokines, chemokines, pro- inflammatory cytokines, effectors, and acute-phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, promote immune cell survival and proliferation, and pro- inflammatory cytokines may promote an inflammatory response. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of pro-inflammatory cytokines include, but are not limited to, IL-la, IL-lb, IL- 6, IL-13, IL-17a, IL-23, IL-27, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF) 2, granulocyte macrophage colony-stimulating factor (GM- CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), TGF-beta, IL-35, and perforin. 18
Attorney Docket No. 01318-0014-00PCT OR-043WO Examples of acute phase-proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA). [54] The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer composed of deoxyribonucleotides. The terms “ribonucleic acid” and “RNA” as used herein mean a polymer composed of ribonucleotides. [55] As used herein, the term “DNA template” refers to a DNA sequence capable of transcribing a linear RNA polynucleotide. For example, but not intending to be limiting, a DNA template may include a DNA vector, PCR product or plasmid. [56] As used herein, the terms “duplexed,” “double-stranded,” and “hybridized” are used interchangeably and refer to double-stranded nucleic acids formed by hybridization of two single strands of nucleic acids containing complementary sequences. Sequences of the two single-stranded nucleic acids can be fully complementary or partially complementary. In some embodiments, a nucleic acid provided herein may be fully double-stranded or partially double- stranded. In most cases, genomic DNA is double-stranded. [57] As used herein, two “duplex sequences,” “duplex forming sequences,” “duplex region,” “duplex forming regions,” “homology arms,” or “homology regions,” complement, or are complementary, fully or partially, to one another when the two regions share a sufficient level of sequence identity to one another’s reverse complement to act as substrates for a hybridization reaction. In some embodiments, two duplex forming sequences are thermodynamically favored to cross-pair in a sequence specific interaction. As used herein, polynucleotide sequences have “homology” when they are either identical or share sequence identity to a reverse complement or “complementary” sequence. The percent sequence identity between a homology region and a counterpart homology region’s reverse complement can be any percent of sequence identity that allows for hybridization to occur. In some embodiments, an internal duplex forming region of a polynucleotide disclosed herein is capable of forming a duplex with another internal duplex forming region and does not form a duplex with an external duplex forming region. [58] As used herein, the term “encode” refers broadly to any process whereby the information in a polymeric macromolecule is used to direct the production of a second molecule that is different from the first. The second molecule may have a chemical structure that is different from the chemical nature of the first molecule. For example, a DNA template (e.g., a DNA vector) may encode a RNA polynucleotide; a precursor RNA polynucleotide (e.g., a linear precursor RNA polynucleotide) may encode a mature RNA polynucleotide (e.g., a 19
Attorney Docket No. 01318-0014-00PCT OR-043WO circular RNA polynucleotide). [59] As used herein, “endogenous” means a substance that is native to, i.e., naturally originated from, a biological system (e.g., an organism, a tissue, or a cell). For example, in some embodiments, a “endogenous polynucleotide” is normally expressed in a cell or tissue. In some embodiments, a polynucleotide is still considered endogenous if the control sequences, such as a promoter or enhancer sequences which activate transcription or translation, have been altered through recombinant techniques. [60] As used herein, the term “heterologous” means from any source other than naturally occurring sequences. [61] As used herein, an “endonuclease site” refers to a stretch of nucleotides within a polynucleotide that is capable of being recognized and cleaved by an endonuclease protein. [62] An “eukaryotic initiation factor” or “eIF” refers to a protein or protein complex used in assembling an initiator tRNA, 40S and 60S ribosomal subunits required for initiating eukaryotic translation. [63] As used herein, an “epitope” is a term in the art and refers to a localized region of an antigen to which an antibody may specifically bind. An epitope may be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can, for example, come together from two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous, or non-contiguous epitope). In some embodiments, the epitope to which an antibody binds may be determined by, e.g., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array -based oligo-peptide scanning assays, and/or mutagenesis mapping (e.g., site- directed mutagenesis mapping). For X-ray crystallography, crystallization may be accomplished using any of the known methods in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1- 23; Chayen NE (1997) Structure 5: 1269- 1274; McPherson A (1976) J Biol Chem 251: 6300- 6303). Antibody: antigen crystals may be studied using well known X-ray diffraction techniques and may be refined using computer software such as X- PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see e.g. Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.; U.S. Patent Publication No. 2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol 20
Attorney Docket No. 01318-0014-00PCT OR-043WO Crystallogr 56(Pt 10): 1316-1323). [64] As used herein, the term “expression sequence” refers to a nucleic acid sequence that encodes a product, e.g., a peptide or polypeptide, regulatory nucleic acid, or non-coding nucleic acid. An exemplary expression sequence that codes for a peptide or polypeptide can comprise a plurality of nucleotide triads, each of which can code for an amino acid and is termed as a “codon.” [65] As used herein, a “fusion protein” is a protein with at least two domains that are encoded by separate genes that have been joined to transcribe for a single peptide. [66] As used herein, the term “genetically engineered” or “engineered” refers to a method of modifying the genome of a cell, including, but not limited to, deleting a coding or non-coding region or a portion thereof or inserting a coding region or a portion thereof. In some embodiments, the cell that is modified is a lymphocyte, e.g., a T cell, which may either be obtained from a patient or a donor. The cell may be modified to express an exogenous construct, such as, e.g., a chimeric antigen receptor (CAR) or a T cell receptor (TCR), which is incorporated into the cell's genome. [67] As used herein, an “immune response” refers to the action of a cell of the immune system (for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including Abs, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from a vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. [68] As used herein, the term “immunogenic” or “immunostimulatory” refers to a potential to induce an immune response to a substance. An immune response may be induced when an immune system of an organism or a certain type of immune cells is exposed to an immunogenic substance. The term “non-immunogenic” refers to a lack of or absence of an immune response above a detectable threshold to a substance. No immune response is detected when an immune system of an organism or a certain type of immune cells is exposed to a non- immunogenic substance. In some embodiments, a non-immunogenic circular polyribonucleotide as provided herein, does not induce an immune response above a pre- determined threshold when measured by an immunogenicity assay. In some embodiments, no innate immune response is detected when an immune system of an organism or a certain type 21
Attorney Docket No. 01318-0014-00PCT OR-043WO of immune cells is exposed to a non-immunogenic circular polyribonucleotide as provided herein. In some embodiments, no adaptive immune response is detected when an immune system of an organism or a certain type of immune cell is exposed to a non-immunogenic circular polyribonucleotide as provided herein. [69] As used herein, an “internal ribosome entry site” or “IRES” refers to an RNA sequence or structural element ranging in size from 10 nt to 1000 nt or more, capable of initiating translation of a polypeptide in the absence of a typical RNA cap structure. An exemplary IRES can be about 500 nt to about 700 nt in length. [70] As used herein, an “intervening region” refers to the portion of an RNA sequence that comprises one or more noncoding or one or more coding elements, or combinations thereof (e.g., translation initiation element, coding element, and/or stop codon) between splice sites. In some embodiments, the intervening regions are between the 5’ combined accessory element and the 3’ combined accessory element or between the 3’ intron fragment and the 5’ intron fragment in a precursor RNA polynucleotide. In some embodiments, the intervening region is between the monotron element and terminal element in other precursor RNA polynucleotides. [71] As used herein, “isolated” or “purified” generally refers to isolation of a substance (for example, in some embodiments, a compound, a polynucleotide, a protein, a polypeptide, a polynucleotide composition, or a polypeptide composition) such that the substance comprises a significant percent (e.g., greater than 1%, greater than 2%, greater than 5%, greater than 10%, greater than 20%, greater than 50%, or more, usually up to about 90%-100%) of the sample in which it resides. In certain embodiments, a substantially purified component comprises at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% of the sample. In additional embodiments, a substantially purified component comprises about, 80%-85%, or 90%-95%, 95-99%, 96-99%, 97-99%, or 95-100% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density. Generally, a substance is purified when it exists in a sample in an amount, relative to other components of the sample, that is more than as it is found naturally. [72] As used herein, a “leading untranslated sequence” is a region of polynucleotide sequences ranging from 1 nucleotide to hundreds of nucleotides located at the upmost 5' end of a polynucleotide sequence. The sequences can be defined or can be random. A leading untranslated sequence is non-coding. [73] As used herein, a “terminal untranslated sequence” is a region of polynucleotide 22
Attorney Docket No. 01318-0014-00PCT OR-043WO sequences ranging from 1 nucleotide to hundreds of nucleotides located at the downmost 3' end of a polynucleotide sequence. The sequences can be defined or can be random. A terminal untranslated sequence is non-coding. [74] As used herein, the terms “terminal sequence” or “terminal element” are used interchangeably to refer to an RNA sequence capable of complexing with a monotron sequence or monotron element. The terminal sequence comprises a splice site nucleotide from the natural group I or group II intron present in the monotron. In some embodiments, the terminal sequence further comprises a natural exon or a fragment thereof and/or a synthetic sequence. [75] The term “lymphocyte” as used herein includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell toxic) lymphocyte that represent a major component of the innate immune system. NK cells reject tumors and cells infected by viruses. It works through the process of apoptosis or programmed cell death. They were termed “natural killers” because they do not require activation in order to kill cells. T cells play a major role in cell-mediated-immunity (no antibody involvement). T cell receptors (TCR) differentiate T cells from other lymphocyte types. The thymus, a specialized organ of the immune system, is the primary site for T cell maturation. There are numerous types of T cells, including: helper T cells (e.g., CD4+ cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T-killer cells, cytolytic T cells, CD8+ T cells or killer T cells), memory T cells ((i) stem memory cells (TSCM), like naive cells, are CD45RO-, CCR7+, CD45RA+, CD62L+ (L- selectin), CD27+, CD28+ and IL-7Ra+, but also express large amounts of CD95, IL-2R, CXCR3, and LFA-1, and show numerous functional attributes distinctive of memory cells); (ii) central memory cells (TCM) express L-selectin and CCR7, they secrete IL-2, but not IFNγ or IL-4, and (iii) effector memory cells (TEM), however, do not express L-selectin or CCR7 but produce effector cytokines like IFNγ and IL-4), regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ or CD4+ FoxP3+ regulatory T cells), natural killer T cells (NKT) and gamma delta T cells. B-cells, on the other hand, play a principal role in humoral immunity (with antibody involvement). B-cells make antibodies, are capable of acting as antigen- presenting cells (APCs) and turn into memory B-cells and plasma cells, both short-lived and long-lived, after activation by antigen interaction. In mammals, immature B-cells are formed in the bone marrow. [76] As used herein, a “miRNA site” refers to a stretch of nucleotides within a polynucleotide that is capable of forming a duplex with at least 8 nucleotides of a natural miRNA sequence. 23
Attorney Docket No. 01318-0014-00PCT OR-043WO [77] As used herein, the terms “monotron,” “monotron sequence,” or “monotron element” are used interchangeably to refer a segment of a precursor RNA polynucleotide that is located at either the 5’ or 3’ end of the polynucleotide, i.e., either 5’ or 3’ from the intervening region. A monotron element refers to a sequence with 70% or higher similarity to a natural group I or group II intron including the splice site dinucleotide. In some embodiments, the monotron is capable of contributing to ribozymatic activity that allows it to enzymatically self- cleave. In some embodiments, the monotron is capable of forming a phosphodiester bond with a terminal sequence, i.e., a sequence containing a splice site dinucleotide and optionally a natural exon sequence or fragment thereof. In some embodiments, the terminal sequence is upstream of the monotron in a linear precursor. In some embodiments, the monotron sequence is upstream of the terminal sequence in a linear precursor. When the terminal sequence is upstream to the monotron in a linear precursor, the monotron can perform two transesterification reactions, e.g., sequentially, self-cleavage and formation of a phosphodiester bond with the terminal sequence. In embodiments in which the terminal sequence is upstream to the monotron in the linear precursor, (a) the monotron is capable of interacting with a nucleophile that is capable of cleaving at the splice site dinucleotide at or near the 5’ end of the monotron, and (b) the cleavage product of (a), i.e., the 5’ splice site nucleotide, e.g., having a 3’ hydroxyl group, engages in a transesterification reaction (cleaves) at the splice site nucleotide of the terminal sequence, yielding a circular RNA or oRNA. In these embodiments, the monotron interacts with the nucleophile (e.g., a guanosine, e.g., a free guanosine that is introduced to the precursor) by forming a binding pocket with the nucleophile, and the linear precursor is capable of adopting a conformation in which the nucleophile is in proximity to and is capable of cleaving at the splice site dinucleotide at or near the 5’ end of the monotron. When the monotron is upstream of the terminal sequence in a linear precursor, the monotron can also perform two transesterification reactions. In embodiments in which the monotron is upstream of the terminal sequence in the linear precursor, (a) the monotron is capable of interacting with a nucleophile that is capable of cleaving at the splice site nucleotide of the terminal element, and (b) the cleavage product of (a), i.e., the 5’ splice site nucleotide, e.g., having a 3’ hydroxyl group, engages in a transesterification reaction (cleaves) at the splice site dinucleotide at or near the 3’ end of the monotron, yielding a circular RNA or oRNA. In these embodiments, the monotron interacts with the nucleophile (e.g., a guanosine, e.g., a free guanosine that is introduced to the precursor) by forming a binding pocket with the nucleophile, and the linear precursor is capable of adopting a conformation in which the nucleophile is in proximity to and 24
Attorney Docket No. 01318-0014-00PCT OR-043WO is capable of cleaving the splice site nucleotide of the terminal element. [78] In some embodiments, the monotron comprises a 5’ proximal end of a natural group I or group II intron including the splice site dinucleotide and optionally a natural exon sequence or fragment thereof. In some embodiments, the 5’ end of the monotron refers to nucleotides within the 5’ half of the monotron. In some embodiments, the 3’ end of the monotron refers to nucleotides within the 3’ half of the monotron. In some embodiments, at or near the 5’ end of the monotron refers to within the 5’ half of the monotron. In some embodiments, at or near the 5’ end of the monotron refers to within the first ten 5’ positions in the monotron. In some embodiments, at the 5’ end of the monotron refers to the first 5’ position(s) in the monotron. In some embodiments, at or near the 3’ end of the monotron refers to within the 3’ half of the monotron. In some embodiments, at or near the 3’ end of the monotron refers to within the last ten 3’ positions in the monotron. In some embodiments, at the 3’ end of the monotron refers to last 3’ position(s) in the monotron. [79] The term “naturally occurring” or “wild-type” (used interchangeably) IRES, or IRES domain or motif as used herein refers to, e.g., an IRES that does not contain a change that is present in a synthetic IRES. A “naturally occurring IRES” or a “wild-type IRES” refers to an IRES that has not been engineered to contain at least one addition, deletion, or substitution of a nucleotide, domain, or motif. Similarly, a “naturally occurring domain,” (e.g., “naturally occurring Domain II”) or “naturally occurring motif,” (e.g., “naturally occurring GNRA motif”) refers to a domain or a motif within a naturally occurring IRES or synthetic IRES where the domain or motif has not been engineered to contain at least one addition, deletion, or substitution of a nucleotide therein, or a deletion or substitution of the domain or motif in whole or in part. A synthetic IRES can comprise naturally occurring domains or motifs but can also comprise synthetic other domains or motifs. For example, a synthetic IRES can contain nucleotide additions, deletions, or substitutions within Domain I, while also containing a wild- type Domain II, Domain III, Domain IV, and Domain V. In such case, there would be no nucleotide additions, deletions, or substitutions within the wild-type Domain II, Domain III, Domain IV, or Domain V and no additions, deletions, or substitutions of Domain II, Domain III, Domain IV, or Domain V in whole or in part. In some embodiments, the “naturally occurring” IRES, domain, or motif refers to the starting IRES, domain, or motif before any changes are induced. As used herein, the term “retaining” or “preserving” or “conserving” a naturally occurring domain or motif refers to preserving the sequence of the naturally occurring domain or motif within an otherwise modified synthetic IRES. For example, if 25
Attorney Docket No. 01318-0014-00PCT OR-043WO Domain II is retained or conserved, the synthetic IRES comprises a naturally occurring Domain II. [80] In certain instances where a synthetic IRES comprises a substitution of an entire domain or motif, the replacement domain or motif may be derived from a different IRES, e.g., from a second higher-expressing, naturally occurring, IRES. Replacing the domain or motif with that of a different IRES renders the initial IRES synthetic, even if the domain or motif does not contain any nucleotide modifications. For example, in some embodiments, the synthetic IRES comprises a substitution of a PPT tract in Domain IV, where the PPT tract sequence is from a second different IRES, e.g., a higher-expressing, naturally occurring, IRES. In that embodiment, the PPT tract in Domain IV of the synthetic IRES is not conserved because it differs from the PPT tract of the starting IRES. In some embodiments, the entirety of Domain IV is substituted with a Domain IV from a second different IRES. In some embodiments, the second different IRES is a higher-expressing, naturally occurring, IRES. In those embodiments, the resultant synthetic IRES neither is naturally occurring nor contains a naturally occurring Domain IV, even if the replacement Domain IV does not contain any individual nucleotide additions or deletions therein as compared to its sequence in the second different naturally occurring IRES. [81] The term “nucleophile” refers to a nucleophilic nucleotide or nucleoside capable of initiating a nucleophilic attack at a splice site and/or transesterification reaction (cleavage) at a splice site. [82] The term “nucleotide” and “nucleoside” refer to a ribonucleotide, a deoxyribonucleotide, or an analog thereof. Nucleotides include species that comprise purines, e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs, as well as pyrimidines, e.g., cytosine, uracil, thymine, and their derivatives and analogs. Nucleosides are similar to nucleotides, e.g., comprising purines and pyrimidines, but without the additional phosphate group. [83] “Modified nucleotide or nucleosides,” or nucleoside or nucleotide “analogs” include nucleotides or nucleoside having modifications in the chemical structure of the base, sugar and/or phosphate, including, but not limited to, 5’-position pyrimidine modifications, 8’- position purine modifications, modifications at cytosine exocyclic amines, and substitution of 5-bromo-uracil; and 2’-position sugar modifications, including but not limited to, sugar- modified ribonucleotides in which the 2’-OH is replaced by a group such as an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety as defined herein. Nucleotide 26
Attorney Docket No. 01318-0014-00PCT OR-043WO or nucleoside modifications are also meant to include nucleotides or nucleoside with bases such as inosine, queuosine, xanthine; sugars such as 2’-methyl ribose; non-natural phosphodiester linkages such as methylphosphonate, phosphorothioate and peptide linkages. Nucleotide or nucleoside modifications include 5-methoxyuridine, 1-methylpseudouridine, and 6- methyladenosine. Exemplary nucleotide or nucleotide modifications are described herein. As exhibited by the exemplary nucleotide or nucleotide modifications described in more detail herein (see section “Modified nucleotides or nucleosides”), such modifications differ from mutations selected from insertions, deletions, addition, or subtraction of nucleotides, for example, the mutations in a permuted Group I and Group II intron segment; or the additions, deletions, or substitutions of a nucleotide, domain, or motif present in a synthetic IRES (as compared to the naturally occurring IRES). [84] As used herein, a nucleotide or nucleoside “comprising no nucleotide or nucleoside modifications” (i.e., comprising 0% modifications) can be interchangeable with “an unmodified nucleotide or nucleoside” in context. A modified nucleotide or nucleoside can have a modification, e.g., between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 100% of the nucleotides or nucleosides are modified. In some embodiments, “% modification” refers to the level of incorporation within a polynucleotide, i.e., the number of modified nucleotides or nucleosides in a polynucleotide sequence divided by the total number of nucleotides or nucleosides (modified or unmodified) in the polynucleotide sequence. In some embodiments, “% modification” refers to the relative quantity of modified nucleotide or nucleoside used to generate the polynucleotide (e.g., 5% modified adenosine refers to feeding 5 mM modified adenosine and 95 mM unmodified adenosine to generate a polynucleotide sequence). [85] All nucleotide sequences disclosed herein can represent an RNA sequence or a corresponding DNA sequence. It is understood that deoxythymidine (dT or T) in a DNA is transcribed into a uridine (U) in an RNA. As such, “T” and “U” may be used interchangeably herein in nucleotide sequences. [86] The terms “nucleic acid”, “polynucleotide”, and “nucleic acid molecule,” are used interchangeably herein to describe a polymer of any length, e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, or up to about 10,000 or more bases, composed of nucleotides, e.g., 27
Attorney Docket No. 01318-0014-00PCT OR-043WO deoxyribonucleotides or ribonucleotides, and may be produced enzymatically or synthetically (e.g., as described in U.S. Pat. No. 5,948,902 and the references cited therein), which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions. A nucleic acid “backbone” can be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (“peptide nucleic acids” or PNA; PCT No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of a nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, e.g., 2’ methoxy or 2’ halide substitutions. Nitrogenous bases can be conventional bases (A, G, C, T, U), analogs thereof (e.g., modified uridines such as 5- methoxyuridine, pseudouridine, or N1-methylpseudouridine, or others); inosine; derivatives of purines or pyrimidines (e.g., N4-methyl deoxyguanosine, deaza- or aza-purines, deaza- or aza- pyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position (e.g., 5- methylcytosine), purine bases with a substituent at the 2, 6, or 8 positions, 2-amino-6- methylaminopurine, O6-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4- dimethylhydrazine-pyrimidines, and O4-alkyl-pyrimidines; US Pat. No. 5,378,825 and PCT No. WO 93/13121). For general discussion see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992). Nucleic acids can include one or more “abasic” residues where the backbone includes no nitrogenous base for position(s) of the polymer (US Pat. No. 5,585,481). A nucleic acid can comprise only conventional RNA or DNA sugars, bases and linkages, or can include both conventional components and substitutions (e.g., conventional bases with 2’ methoxy linkages, or polymers containing both conventional bases and one or more base analogs). Naturally occurring nucleic acids are comprised of nucleotides, including guanine, cytosine, adenine, thymine, and uracil containing nucleotides (G, C, A, T, and U respectively). [87] As used herein, an “oligonucleotide” is a polynucleotide comprising fewer than 1000 nucleotides, such as a polynucleotide comprising fewer than 500 nucleotides or fewer than 100 nucleotides. [88] As used herein, “polyA” means a polynucleotide or a portion of a polynucleotide consisting of nucleotides comprising adenine. As used herein, “polyT” means a polynucleotide or a portion of a polynucleotide consisting of nucleotides comprising thymine. As used herein, “polyAC” means a polynucleotide or a portion of a polynucleotide consisting of nucleotides comprising adenine or cytosine. 28
Attorney Docket No. 01318-0014-00PCT OR-043WO [89] As used herein, the term “ribosomal skipping element” refers to a nucleotide sequence encoding a short peptide sequence capable of causing generation of two peptide chains from translation of one RNA molecule. While not wishing to be bound by theory, it is hypothesized that ribosomal skipping elements function by (1) terminating translation of the first peptide chain and re-initiating translation of the second peptide chain; or (2) cleavage of a peptide bond in the peptide sequence encoded by the ribosomal skipping element by an intrinsic protease activity of the encoded peptide, or by another protease in the environment (e.g., cytosol). [90] The terms “sequence identity,” or “sequence similarity” as used herein, refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid- by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein, typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide. [91] As used herein, a “spacer” refers to a region of a polynucleotide sequence ranging from 1 nucleotide to hundreds or thousands of nucleotides separating two other elements along a polynucleotide sequence. The sequences can be defined or can be random. A spacer is typically non-coding. In some embodiments, spacers include duplex regions. [92] As used here, the term “splicing efficiency” refers to a measurement of the rate of splicing activity (e.g., none, low, or high) in a splicing or self-splicing reaction, for example, in portions of a precursor RNA polynucleotide capable of self-circularization. In some embodiments, the splicing activity of, e.g., a monotron element or intron segment, is affected by the structure and/or sequence of the linear RNA polynucleotide. [93] As used herein, “structured” with regard to RNA refers to an RNA sequence that is predicted by the RNAFold software or similar predictive tools to form a structure (e.g., a 29
Attorney Docket No. 01318-0014-00PCT OR-043WO hairpin loop) with itself or other sequences in the same RNA molecule. As used herein, “unstructured” with regard to RNA refers to an RNA sequence that is not predicted by RNA structure predictive tools to form a structure (e.g., a hairpin loop) with itself or other sequences in the same RNA molecule. In some embodiments, unstructured RNA can be functionally characterized using nuclease protection assays. [94] As used herein, the terms “synthetic IRES” or “synthetic TIE” or “synthetic domain” or “synthetic motif” and the like, refer to IRESs, TIES, and domains or motifs therein that have been engineered to contain structural changes in the IRES, domains, or motifs, or nucleotides therein that are not naturally occurring. As set forth in detail herein, the synthetic IRESs can comprise at least one addition, deletion, or substitution of a nucleotide, domain, or motif such that the sequences in the synthetic IRESs differ from the corresponding a corresponding naturally occurring IRES. Where the synthetic IRES contains a domain or motif has been substituted in whole or in part, the domain or motif itself can be naturally occurring, but the substitution still renders the synthetic IRES “synthetic.” Synthetic IRESs, TIEs, domains, or motifs have been designed to increase expression of operably linked expression sequences as compared to naturally occurring counterparts. These effects differ from the modifications (e.g., modified nucleotides or nucleosides in intronic or exonic sequences) described elsewhere herein that seek to improve circularization efficiency or splicing efficiency. These effects also differ from naturally occurring IRESs derived from naturally occurring untranslated regions (UTR); truncations or deletions of nucleotides/nucleosides of a naturally occurring UTR sequence to select for a sequence that allows for translation initiation comprise derivations from a naturally occurring IRES, but do not comprise engineering of a synthetic IRES. [95] As used herein, the term “therapeutic protein” refers to any protein that, when administered to a subject directly or indirectly in the form of a translated nucleic acid, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect. [96] As used herein, “translation initiation element” or “TIE” refers to a portion of the intervening region comprising a sequence to allow translation efficiency of an encoded protein. In some embodiments, core functional elements comprising one or more coding elements will further comprise one or more TIEs. In some embodiments, where the intervening region comprises one or more noncoding elements, the TIE can be part of the noncoding element. In some embodiments, the TIE comprises an internal ribosome entry site (IRES). In some 30
Attorney Docket No. 01318-0014-00PCT OR-043WO embodiments, the TIE comprises a naturally occurring IRES or synthetic IRES. [97] As used herein, “transcription” refers to the formation or synthesis of an RNA molecule by an RNA polymerase using a DNA molecule as a template. The disclosure is not limited with respect to the RNA polymerase that is used for transcription. For example, in some embodiments, a T7-type RNA polymerase can be used. [98] As used herein, “translation” refers to the formation of a polypeptide molecule by a ribosome based upon an RNA template. [99] As used herein, the term “translation efficiency” refers to a rate or amount of protein or peptide production from a ribonucleotide transcript. In some embodiments, translation efficiency can be expressed as amount of protein or peptide produced per given amount of transcript that codes for the protein or peptide. [100] As used herein, the term “transfect” or “transfection” refers to the intracellular introduction of one or more encapsulated materials (e.g., nucleic acids and/or polynucleotides) into a cell, or preferably into a target cell. The term “transfection efficiency” refers to the relative amount of such encapsulated material (e.g., polynucleotides) up-taken by, introduced into and/or expressed by the target cell which is subject to transfection. In some embodiments, transfection efficiency may be estimated by the amount of a reporter polynucleotide product produced by the target cells following transfection. In some embodiments, a transfer vehicle has high transfection efficiency. In some embodiments, a transfer vehicle has at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% transfection efficiency. [101] As used herein, “transfer vehicle” includes any of the standard pharmaceutical carriers, diluents, excipients, and the like, which are generally intended for use in connection with the administration of biologically active agents, including nucleic acids. In certain embodiments of the present disclosure, the transfer vehicles (e.g., lipid nanoparticles) are prepared to encapsulate one or more materials or therapeutic agents (e.g., circRNA). The process of incorporating a desired therapeutic agent (e.g., circRNA) into a transfer vehicle is referred to herein as or “loading” or “encapsulating” (Lasic, et al., FEBS Lett., 312: 255-258, 1992). The transfer vehicle-loaded or -encapsulated materials (e.g., circRNA) may be completely or partially located in the interior space of the transfer vehicle, within a bilayer membrane of the transfer vehicle, or associated with the exterior surface of the transfer vehicle. [102] The terms “treat,” and “prevent” as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes 31
Attorney Docket No. 01318-0014-00PCT OR-043WO as having a potential benefit or therapeutic effect. The treatment or prevention provided by the method disclosed herein can include treatment or prevention of one or more conditions or symptoms of the disease. Also, for purposes herein, “prevention” can encompass delaying the onset of the disease, or a symptom or condition thereof, e.g., prophylaxis of disease. [103] As used herein, the terms “upstream” and “downstream” refer to relative positions of genetic code, e.g., nucleotides, sequence elements, in polynucleotide sequences. In some embodiments, in an RNA polynucleotide, upstream is toward the 5’ end of the polynucleotide and downstream is toward the 3’ end. In some embodiments, in a DNA polynucleotide, upstream is toward the 5’ end of the coding strand for the gene in question and downstream is toward the 3’ end. [104] As used herein, a “vaccine” refers to a composition for generating immunity for the prophylaxis and/or treatment of diseases. Accordingly, vaccines are medicaments which comprise antigens and are intended to be used in humans or animals for generating specific defense and protective substances upon administration to the human or animal. A. LIPID DEFINITIONS [105] As used herein, the phrase “biodegradable lipid” or “degradable lipid” refers to any of a number of lipid species that are broken down in a host environment on the order of minutes, hours, or days ideally making them less toxic and unlikely to accumulate in a host over time. Common modifications to lipids include ester bonds, and disulfide bonds among others to increase the biodegradability of a lipid. [106] As used herein, the phrase “biodegradable PEG lipid” or “degradable PEG lipid” refers to any of a number of lipid species where the PEG molecules are cleaved from the lipid in a host environment on the order of minutes, hours, or days ideally making them less immunogenic. Common modifications to PEG lipids include ester bonds, and disulfide bonds among others to increase the biodegradability of a lipid. [107] As used herein, the term “cationic lipid” or “ionizable lipid” refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH 4 and a neutral charge at other pHs such as physiological pH 7. [108] As used herein, the term “PEG” means any polyethylene glycol or other polyalkylene ether polymer. [109] As generally defined herein, a “PEG-OH lipid” (also referred to herein as “hydroxy-PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (–OH) groups 32
Attorney Docket No. 01318-0014-00PCT OR-043WO on the lipid. [110] As used herein, a “phospholipid” is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. [111] As used herein, the term “structural lipid” refers to sterols and also to lipids containing sterol moieties. As defined herein, “sterols” are a subgroup of steroids consisting of steroid alcohols. [112] The terms “head-group” and “tail-group,” when used herein to describe the compounds (e.g., lipids) of the present disclosure, and in particular functional groups that are comprised in such compounds, are used for ease of reference to describe the orientation of such compounds or of one or more functional groups relative to other functional groups. For example, in certain embodiments, a hydrophilic head-group (e.g., guanidinium) is bound (e.g., by 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 bound to a hydrophobic tail-group (e.g., cholesterol). In certain embodiments, the compounds disclosed herein comprise, for example, at least one hydrophilic head-group and at least one hydrophobic tail-group, each bound to at least one cleavable group, thereby rendering such compounds amphiphilic. [113] As used herein, the term “amphiphilic” means the ability to dissolve in both polar (e.g., water) and non-polar (e.g., lipid) environments. For example, in certain embodiments, the compounds (e.g., lipids) disclosed herein comprise at least one lipophilic tail-group (e.g., cholesterol or a C6-20 alkyl) and at least one hydrophilic head-group (e.g., imidazole), each bound to a cleavable group (e.g., disulfide). [114] As used herein, the term “hydrophilic” is used to indicate in qualitative terms that a functional group is water-preferring, and typically such groups are water-soluble. For example, disclosed herein are compounds (e.g., ionizable lipids) that comprise a cleavable group (e.g., a disulfide (S—S) group) bound to one or more hydrophilic groups (e.g., a hydrophilic head-group), wherein such hydrophilic groups comprise or are selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, an optionally-substituted alkyl amino (e.g., an alkyl amino such as dimethylamino) and pyridyl. [115] As used herein, the term “hydrophobic” is used to indicate in qualitative terms that a functional group is water-avoiding, and typically such groups are not water soluble. In certain embodiments, at least one of the functional groups of moieties that comprise the compounds disclosed herein is hydrophobic in nature (e.g., a hydrophobic tail-group comprising a naturally 33
Attorney Docket No. 01318-0014-00PCT OR-043WO occurring lipid such as cholesterol). For example, disclosed herein are compounds (e.g., ionizable lipids) that comprise a cleavable functional group (e.g., a disulfide (S—S) group) bound to one or more hydrophobic groups, wherein such hydrophobic groups may comprise, or may be selected from, one or more naturally occurring lipids such as cholesterol, an optionally substituted, variably saturated or unsaturated C6-C20 alkyl, and/or an optionally substituted, variably saturated or unsaturated C6-C20 acyl. [116] As used herein, the term “liposome” generally refers to a vesicle composed of lipids (e.g., amphiphilic lipids) arranged in one or more spherical bilayer or bilayers. Such liposomes may be unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the encapsulated circRNA to be delivered to one or more target cells, tissues and organs. [117] As used herein, the phrase “lipid nanoparticle” or “LNP” refers to a transfer vehicle comprising one or more cationic or ionizable lipids, stabilizing lipids, structural lipids, and helper lipids. [118] In certain embodiments, the compositions described herein comprise one or more liposomes or lipid nanoparticles. Examples of suitable lipids (e.g., ionizable lipids) that may be used to form the liposomes and lipid nanoparticles contemplated include one or more of the compounds disclosed herein (e.g., HGT4001, HGT4002, HGT4003, HGT4004 and/or HGT4005). Such liposomes and lipid nanoparticles may also comprise additional ionizable lipids such as C12-200, dLin-KC2-DMA, and/or HGT5001, helper lipids, structural lipids, PEG-modified lipids, MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE, HGT5000, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA, DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, kLin-K-DMA, dLin-K-XTC2-DMA, HGT4003, and combinations thereof. [119] In some embodiments, a lipid, e.g., an ionizable lipid, disclosed herein comprises one or more cleavable groups. The terms “cleave” and “cleavable” are used in this regard to mean that one or more chemical bonds (e.g., one or more of covalent bonds, hydrogen-bonds, van der Waals' forces and/or ionic interactions) between atoms in or adjacent to the subject functional group are broken (e.g., hydrolyzed) or are capable of being broken upon exposure to selected conditions (e.g., upon exposure to enzymatic conditions). In certain embodiments, the cleavable group is a disulfide functional group, and in particular embodiments is a disulfide group that is capable of being cleaved upon exposure to selected biological conditions (e.g., intracellular conditions). In certain embodiments, the cleavable group is an ester functional 34
Attorney Docket No. 01318-0014-00PCT OR-043WO group that is capable of being cleaved upon exposure to selected biological conditions. For example, the disulfide groups may be cleaved enzymatically or by a hydrolysis, oxidation or reduction reaction. Upon cleavage of such disulfide functional group, the one or more functional moieties or groups (e.g., one or more of a head-group and/or a tail-group) that are bound thereto may be liberated. Exemplary cleavable groups may include, but are not limited to, disulfide groups, ester groups, ether groups, and any derivatives thereof (e.g., alkyl and aryl esters). In certain embodiments, the cleavable group is not an ester group or an ether group. In some embodiments, a cleavable group is bound (e.g., bound by one or more of hydrogen-bonds, van der Waals’ forces, ionic interactions and covalent bonds) to one or more functional moieties or groups (e.g., at least one head-group and at least one tail-group). In certain embodiments, at least one of the functional moieties or groups is hydrophilic (e.g., a hydrophilic head-group comprising one or more of imidazole, guanidinium, amino, imine, enamine, optionally-substituted alkyl amino and pyridyl). B. CHEMICAL DEFINITIONS [120] The disclosure may include compounds and pharmaceutically acceptable salts thereof, pharmaceutical compositions containing such compounds and methods of using such compounds and compositions, and the following terms, if present, have the following meanings unless otherwise indicated. It should also be understood that when described herein any of the moieties defined forth below may be substituted with a variety of substituents, and that the respective definitions are intended to include such substituted moieties within their scope as set out below. Unless otherwise stated, the term “substituted” is to be defined as set out below. It should be further understood that the terms “groups” and “radicals” can be considered interchangeable when used herein. [121] Compounds described herein may also comprise one or more isotopic substitutions. For example, H may be in any isotopic form, including 1H, 2H (D or deuterium), and 3H (T or tritium); C may be in any isotopic form, including 12C, 13C, and 14C; O may be in any isotopic form, including 16O and 18O; F may be in any isotopic form, including 18F and 19F; and the like. [122] When a range of values is listed, it is intended to encompass each value and sub– range within the range. For example, “C1–6 alkyl” is intended to encompass, C1, C2, C3, C4, C5, C6, C1–6, C1–5, C1–4, C1–3, C1–2, C2–6, C2–5, C2–4, C2–3, C3–6, C3–5, C3–4, C4–6, C4–5, and C5–6 alkyl. [123] As used herein, the term “aliphatic” or “aliphatic group,” means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is 35
Attorney Docket No. 01318-0014-00PCT OR-043WO completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocycle,” “cycloaliphatic” or “cycloalkyl”), that has a single point of attachment to the rest of the molecule or multiple points of attachment to the rest of the molecule, as would be readily apparent to a person of ordinary skill in the art based on the context of the described molecule. In some embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refers to a monocyclic, bicyclic, or polycyclic C3-C14 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. Exemplary aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl. Examples of bicyclic and polycyclic cycloalkyls include bridged, fused, and spirocyclic carbocyclyls. [124] As used herein, the term “alkyl” refers to both straight and branched chain C1-40 hydrocarbons (e.g., C6-20 hydrocarbons), and include both saturated and unsaturated hydrocarbons. In certain embodiments, the alkyl may comprise one or more cyclic alkyls and/or one or more heteroatoms such as oxygen, nitrogen, or sulfur and may optionally be substituted with substituents (e.g., one or more of alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester or amide). In certain embodiments, a contemplated alkyl includes (9Z,12Z)-octadeca-9,12- dien. The use of designations such as, for example, “C6-20” is intended to refer to an alkyl (e.g., straight or branched chain and inclusive of alkenes and alkyls) having the recited range carbon atoms. In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C1–10 alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C1–9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C1–8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1–7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C1–6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C1–5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C1–4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1–3 alkyl”). In some embodiments, an 36
Attorney Docket No. 01318-0014-00PCT OR-043WO alkyl group has 1 to 2 carbon atoms (“C1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C1 alkyl”). Examples of C1–6 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, and the like. [125] As used herein, “alkenyl” refers to a radical of a straight–chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon–carbon double bonds (e.g., 1, 2, 3, or 4 carbon–carbon double bonds), and optionally one or more carbon– carbon triple bonds (e.g., 1, 2, 3, or 4 carbon–carbon triple bonds) (“C2–20 alkenyl”). In certain embodiments, alkenyl does not contain any triple bonds. In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C2–10 alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C2–9 alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C2–8 alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C2– 7 alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C2–6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C2–5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C2–4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C2–3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C2 alkenyl”). The one or more carbon– carbon double bonds can be internal (such as in 2–butenyl) or terminal (such as in 1–butenyl). Examples of C2–4 alkenyl groups include ethenyl (C2), 1–propenyl (C3), 2–propenyl (C3), 1– butenyl (C4), 2–butenyl (C4), butadienyl (C4), and the like. Examples of C2–6 alkenyl groups include the aforementioned C2–4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. [126] As used herein, “alkynyl” refers to a radical of a straight–chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon–carbon triple bonds (e.g., 1, 2, 3, or 4 carbon–carbon triple bonds), and optionally one or more carbon–carbon double bonds (e.g., 1, 2, 3, or 4 carbon–carbon double bonds) (“C2–20 alkynyl”). In certain embodiments, alkynyl does not contain any double bonds. In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C2–10 alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C2–9 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C2–8 alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C2– 7 alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C2–6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C2–5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C2–4 alkynyl”). In some 37
Attorney Docket No. 01318-0014-00PCT OR-043WO embodiments, an alkynyl group has 2 to 3 carbon atoms (“C2–3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C2 alkynyl”). The one or more carbon– carbon triple bonds can be internal (such as in 2–butynyl) or terminal (such as in 1–butynyl). Examples of C2–4 alkynyl groups include, without limitation, ethynyl (C2), 1–propynyl (C3), 2– propynyl (C3), 1–butynyl (C4), 2–butynyl (C4), and the like. Examples of C2–6 alkenyl groups include the aforementioned C2–4 alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like. [127] As used herein, “alkylene,” “alkenylene,” and “alkynylene,” refer to a divalent radical of an alkyl, alkenyl, and alkynyl group respectively. When a range or number of carbons is provided for a particular “alkylene,” “alkenylene,” or “alkynylene” group, it is understood that the range or number refers to the range or number of carbons in the linear carbon divalent chain. “Alkylene,” “alkenylene,” and “alkynylene” groups may be substituted or unsubstituted with one or more substituents as described herein. [128] The term “alkoxy,” as used herein, refers to an alkyl group which is attached to another moiety via an oxygen atom (–O(alkyl)). Non-limiting examples include e.g., methoxy, ethoxy, propoxy, and butoxy. [129] As used herein, the term “aryl” refers to aromatic groups (e.g., monocyclic, bicyclic and tricyclic structures) containing six to ten carbons in the ring portion. The aryl groups may be optionally substituted through available carbon atoms and in certain embodiments may include one or more heteroatoms such as oxygen, nitrogen or sulfur. In some embodiments, an aryl group has six ring carbon atoms (“C6 aryl”; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1–naphthyl and 2–naphthyl). [130] As used herein, the term “bicyclic ring” or “bicyclic ring system” refers to any bicyclic ring system, i.e. carbocyclic or heterocyclic, saturated or having one or more units of unsaturation, having one or more atoms in common between the two rings of the ring system. Thus, the term comprises any permissible ring fusion, such as ortho-fused or spirocyclic. As used herein, the term “heterobicyclic” is a subset of “bicyclic” that requires that one or more heteroatoms are present in one or both rings of the bicycle. Such heteroatoms may be present at ring junctions and are optionally substituted, and may be selected from nitrogen (including N-oxides), oxygen, sulfur (including oxidized forms such as sulfones and sulfonates), phosphorus (including oxidized forms such as phosphonates and phosphates), boron, etc. In some embodiments, a bicyclic group has 7-12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. As used herein, the term “bridged 38
Attorney Docket No. 01318-0014-00PCT OR-043WO bicyclic” refers to any bicyclic ring system, i.e. carbocyclic or heterocyclic, saturated or partially unsaturated, having at least one bridge. As defined by IUPAC, a “bridge” is an unbranched chain of atoms or an atom or a valence bond connecting two bridgeheads, where a “bridgehead” is any skeletal atom of the ring system which is bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, a bridged bicyclic group has 7-12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Such bridged bicyclic groups are well known in the art and include those groups set forth below where each group is attached to the rest of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise specified, a bridged bicyclic group is optionally substituted with one or more substituents as set forth for aliphatic groups. Additionally or alternatively, any substitutable nitrogen of a bridged bicyclic group is optionally substituted. Exemplary bicyclic rings include:
Exemplary bridged bicyclics include: 39
Attorney Docket No. 01318-0014-00PCT OR-043WO
[131] The term “cycloalkyl” refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons, referred to herein, e.g., as "C4-8 cycloalkyl," derived from a cycloalkane. Exemplary cycloalkyl groups include, but are not limited to, cyclohexanes, cyclopentanes, cyclobutanes and cyclopropanes. [132] As used herein, “cyano” refers to –CN. [133] As used herein, “heteroaryl” refers to a radical of a 5–10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 electrons shared in a cyclic array) having ring carbon atoms and 1–4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5–10 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is 40
Attorney Docket No. 01318-0014-00PCT OR-043WO either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl/heteroaryl) ring system. Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2–indolyl) or the ring that does not contain a heteroatom (e.g., 5– indolyl). [134] As used herein, “heterocyclyl” or “heterocyclic” refers to a radical of a 3– to 10– membered non–aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3–10 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”), and can be saturated or can be partially unsaturated. Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. The terms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclic group,” “heterocyclic moiety,” and “heterocyclic radical,” may be used interchangeably. [135] The terms “halo” and “halogen” as used herein refer to an atom selected from fluorine (fluoro, F), chlorine (chloro, Cl), bromine (bromo, Br), and iodine (iodo, I). In certain embodiments, the halo group is either fluoro or chloro. [136] As used herein, “oxo” refers to –C=O. [137] In general, the term “substituted”, whether preceded by the term “optionally” or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the 41
Attorney Docket No. 01318-0014-00PCT OR-043WO same or different at each position. Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, for example, 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats. Representative substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic, substituted C3-C20 cyclic, heterocyclic, substituted heterocyclic, aminoacid, peptide, and polypeptide groups. Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term "stable", as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. [138] Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are independently halogen; —(CH2)0-4R∘; —(CH2)0-4OR∘; —O(CH2)0-4R∘, — O—(CH2)0-4C(O)OR∘; —(CH2)0-4CH(OR∘)2; —(CH2)0-4SR∘; —(CH2)0-4Ph, which may be substituted with R∘; —(CH2)0-4O(CH2)0-1Ph which may be substituted with R∘; —CH═CHPh, which may be substituted with R∘; —(CH2)0-4O(CH2)0-1-pyridyl which may be substituted with R∘; —NO2; —CN; —N3; —(CH2)0-4N(R∘)2; —(CH2)0-4N(R∘)C(O)R∘; —N(R∘)C(S)R∘; —(CH2)0- 4N(R∘)C(O)NR∘ 2; —N(R∘)C(S)NR∘ 2; —(CH2)0-4N(R∘)C(O)OR∘; —N(R∘)N(R∘)C(O)R∘; — N(R∘)N(R∘)C(O)NR∘ 2; —N(R∘)N(R∘)C(O)OR∘; —(CH2)0-4C(O)R∘; —C(S)R∘; —(CH2)0- 4C(O)OR∘; —(CH2)0-4C(O)SR∘; —(CH2)0-4C(O)OSiR∘ 3; —(CH2)0-4OC(O)R∘; —OC(O)(CH2)0- 4SR∘, SC(S)SR∘; —(CH2)0-4SC(O)R∘;—(CH2)0-4C(O)NR∘ 2; —C(S)NR∘ 2; —C(S)SR∘; —SC(S)SR∘, —(CH2)0-4OC(O)NR∘ 2; —C(O)N(OR∘)R∘; —C(O)C(O)R∘; —C(O)CH2C(O)R∘; —C(NOR∘)R∘; — (CH2)0-4SSR∘; —(CH2)0-4S(O)2R∘; —(CH2)0-4S(O)2OR∘; —(CH2)0-4OS(O)2R∘; —S(O)2NR∘ 2; — (CH2)0-4S(O)R∘; —N(R∘)S(O)2NR∘2; —N(R∘)S(O)2R∘; —N(OR∘)R∘; —C(NH)NR∘2; —P(O)2R∘; — P(O)R∘2; —OP(O)R∘2; —OP(O)(OR∘)2; SiR∘3; —(C1-4 straight or branched alkylene)O—N(R∘)2; 42
Attorney Docket No. 01318-0014-00PCT OR-043WO or—(C1-4 straight or branched alkylene)C(O)O—N(R∘)2, wherein each R∘ may be substituted as defined below and is independently hydrogen, C1-6 aliphatic, —CH2Ph, —O(CH2)0-1Ph, —CH2- (5-6 membered heteroaryl ring), or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R∘, taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below. [139] Suitable monovalent substituents on R∘ (or the ring formed by taking two independent occurrences of R∘ together with their intervening atoms), are independently halogen, —(CH2)0-2R●, -(haloR●), —(CH2)0-2OH, —(CH2)0-2OR●, —(CH2)0-2CH(OR●)2; — O(haloR●), —CN, —N3, —(CH2)0-2C(O)R●, —(CH2)0-2C(O)OH, —(CH2)0-2C(O)OR●, —(CH2)0- 2SR●, —(CH2)0-2SH, —(CH2)0-2NH2, —(CH2)0-2NHR●, —(CH2)0-2NR●2, —NO2, —SiR● 3, — OSiR● 3, —C(O)SR●, —(C1-4 straight or branched alkylene)C(O)OR●, or—SSR● wherein each R● is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently selected from C1-4 aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R∘ include ═O and ═S. [140] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include the following: ═O, ═S, ═NNR*2, ═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)2R*, ═NR*, ═NOR*, —O(C(R*2))2-3O—, or —S(C(R*2))2-3S—, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an "optionally substituted" group include:—O(CR*2)2-3O—, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. [141] Suitable substituents on the aliphatic group of R* include halogen, —R●, -(haloR●), —OH, —OR●, —O(haloR●), —CN, —C(O)OH, —C(O)OR●, —NH2, —NHR●, —NR● 2, or —NO2, 43
Attorney Docket No. 01318-0014-00PCT OR-043WO wherein each R● is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently C1-4 aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. [142] Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include —R†, —NR† 2, —C(O)R†, —C(O)OR†, —C(O)C(O)R†, —C(O)CH2C(O)R†, —S(O)2R†, — S(O)2NR† 2, —C(S)NR† 2, —C(NH)NR† 2, or —N(R†)S(O)2R†; wherein each R† is independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted—OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R†, taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. [143] Suitable substituents on the aliphatic group of R† are independently halogen, —R●, -(haloR●), —OH, —OR●, —O(haloR●), —CN, —C(O)OH, —C(O)OR●, —NH2, —NHR●, —NR● 2, or —NO2, wherein each R● is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently C1-4 aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. [144] Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. It is understood that "substitution" or "substituted" comprises the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformation, for example, by rearrangement, cyclization, or elimination. [145] In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein. The permissible substituents can be one or more and the same or different for appropriate organic compounds. The heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of 44
Attorney Docket No. 01318-0014-00PCT OR-043WO the heteroatoms. [146] In various embodiments, the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, each of which optionally is substituted with one or more suitable substituents. In some embodiments, the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, wherein each of the alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone can be further substituted with one or more suitable substituents. [147] Examples of substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, thioketone, ester, heterocyclyl, –CN, aryl, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroaralkoxy, azido, alkylthio, oxo, acylalkyl, carboxy esters, carboxamido, acyloxy, aminoalkyl, alkylaminoaryl, alkylaryl, alkylaminoalkyl, alkoxyaryl, arylamino, aralkylamino, alkylsulfonyl, carboxamidoalkylaryl, carboxamidoaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy, aminocarboxamidoalkyl, cyano, alkoxyalkyl, perhaloalkyl, arylalkyloxyalkyl, and the like. In some embodiments, the substituent is selected from cyano, halogen, hydroxyl, and nitro. [148] As used herein, “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1–19. Pharmaceutically acceptable salts include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed 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, 45
Attorney Docket No. 01318-0014-00PCT OR-043WO tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2–hydroxy–ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2–naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3– phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p–toluenesulfonate, undecanoate, valerate salts, and the like. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1–4alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate. [149] In typical embodiments, the present disclosure is intended to encompass the compounds disclosed herein, and the pharmaceutically acceptable salts, pharmaceutically acceptable esters, tautomeric forms, polymorphs, and prodrugs of such compounds. In some embodiments, the present disclosure includes a pharmaceutically acceptable addition salt, a pharmaceutically acceptable ester, a solvate (e.g., hydrate) of an addition salt, a tautomeric form, a polymorph, an enantiomer, a mixture of enantiomers, a stereoisomer or mixture of stereoisomers (pure or as a racemic or non-racemic mixture) of a compound described herein. [150] Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. 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, 46
Attorney Docket No. 01318-0014-00PCT OR-043WO NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p.268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The disclosure additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers. [151] In certain embodiments, the compounds (e.g., ionizable lipids) and the transfer vehicles (e.g., lipid nanoparticles) of which such compounds are a component exhibit an enhanced (e.g., increased) ability to transfect one or more target cells. Accordingly, also provided herein are methods of transfecting one or more target cells. Such methods generally comprise the step of contacting the one or more target cells with the compounds and/or pharmaceutical compositions disclosed herein such that the one or more target cells are transfected with the circular RNA encapsulated therein. [152] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless defined herein and below in the reminder of the specification, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. 2. CIRCULAR RNA, PRECURSOR RNA & DNA TEMPLATE A. CIRCULAR RNA [153] Provided herein are circular RNAs, in some instances produced by the precursor RNA polynucleotides described elsewhere herein. [154] In some embodiments, provided herein is a circular RNA polynucleotide comprising, in the following order, a 3’ self-spliced exon segment, an intervening region, and a 5’ self-spliced exon segment. In some embodiments, provided herein is a circular RNA polynucleotide comprising, in the following order, a 3’ self-spliced exon segment, a coding sequence, and a 5’ self-spliced exon segment. In some embodiments, provided herein is a circular RNA polynucleotide comprising, in the following order, a 3’ self-spliced exon segment, a translation initiation element (TIE), a coding sequence, and a 5’ self-spliced exon segment. In some embodiments, provided herein is a circular RNA polynucleotide comprising, in the following order, a 3’ self-spliced exon segment, a translation initiation element (TIE), a coding sequence with which the TIE is not naturally associated, and a 5’ self-spliced exon segment. In some embodiments, the TIE comprises an IRES that is a synthetic IRES. 47
Attorney Docket No. 01318-0014-00PCT OR-043WO [155] In some embodiments, provided herein is a circular RNA polynucleotide comprising: i) a 5’ combined accessory element; ii) an intervening region; and iii) a 3’ combined accessory element, where the intervening region is between the 5’ combined accessory element and the 3’ combined accessory element. [156] In some embodiments, the 5’ combined accessory element comprises a 3’ self- spliced exon segment. In some embodiments, the 3’ self-spliced exon segment comprises an exon segment or fragment thereof. In some embodiments, the 3’ self-spliced exon segment comprises a 3ʹ nucleotide of a 3ʹ splice site dinucleotide. In some embodiments, the 3’ self- spliced exon segment comprises an exon segment and a 3’ nucleotide of a 3’ splice site dinucleotide. In some embodiments, the exon segment comprises a natural exon sequence or non-naturally occurring sequence. In some embodiments, the 3' splice site dinucleotides are distinct from the natural splice site dinucleotide(s) associated with a natural Group I or Group II intron sequence. [157] In some embodiments, the 3’self-spliced exon segment comprises a sequence having a percent sequence identity of about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more to a sequence selected from Table A or Table B. In some embodiments, the 3’ self-spliced exon segment is selected from an exon segment disclosed herein, e.g., in Table A or Table B. In some embodiments, the self-spliced exon segment is, e.g., 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, or 15 nucleotides. In some embodiments, the circular RNA comprises a self-spliced exon segment that is 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, or 15 nucleotides from the exonic sequences of Table A or is e.g., 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides from the exonic sequences of Table B. [158] In some embodiments, the 3’ combined accessory element comprises a 5’ self- spliced exon segment. In some embodiments, the 5’ self-spliced exon segment comprises an exon segment or fragment thereof. In some embodiments, the 5’ self-spliced exon segment comprises a 5ʹ nucleotide of a 5ʹ splice site dinucleotide. In some embodiments, the 5’ self- spliced exon segment comprises an exon segment and a 5’ nucleotide of a 5’ splice site dinucleotide. In some embodiments, the exon segment comprises a natural exon sequence or non-naturally occurring sequence. In some embodiments, the 5’splice site dinucleotides are distinct from the natural splice site dinucleotide(s) associated with a natural Group I or Group 48
Attorney Docket No. 01318-0014-00PCT OR-043WO II intron sequence. [159] In some embodiments, the 5’self-spliced exon segment comprises a sequence having a percent sequence identity of about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more to a sequence selected from, e.g., in Table A or Table B. In some embodiments, the 5’ self-spliced exon segment is selected from an exon segment disclosed herein, e.g., in Table A or Table B. See, e.g., supra. In some embodiments, the self- spliced exon segment is e.g., 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, or 15 nucleotides. In some embodiments, the circular RNA comprises a self-spliced exon segment that is 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, or 15 nucleotides from the exonic sequences of Table A or is e.g., 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides from the exonic sequences of Table B. In some embodiments, as set forth herein, the intervening region comprises a noncoding region or a coding region. In some embodiments, the intervening region comprises at least one translation initiation element (TIE). In some embodiments, the TIE comprises a viral or eukaryotic internal ribosome entry site (IRES) and a noncoding or coding region. In some embodiments, the IRES is a synthetic IRES, as described in further detail herein. In some embodiments, the IRES comprises a sequence selected from the sequences in Table 2 or a fragment thereof or a sequence selected from SEQ ID NOS: 1-2989, 3282-3303, and 14067- 24829 (GIRES 0-10762). In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence selected from the sequences in Table 2 or a fragment thereof or a sequence selected from SEQ ID NOS: 1-2989, 3282-3303, and 14067-24829 (GIRES 0- 10762). See, e.g., infra. In some embodiments, the synthetic IRES comprises a sequence set forth in Table 1 herein. [160] In some embodiments the TIE comprises a coding sequence with which the TIE is not naturally associated. [161] In some embodiments, the intervening region comprises an untranslated region (UTR). In some embodiments, the UTR comprises one or more noncoding elements. In some embodiments, the one or more noncoding elements are selected from, e.g., a natural 3ʹ Untranslated Region (UTR), a natural 5ʹ Untranslated Region (UTR), a synthetic spacer sequence, an aptamer, and lncRNA, miRNA, and a miRNA sponge. In some embodiments, 49
Attorney Docket No. 01318-0014-00PCT OR-043WO the noncoding element is or comprises the TIE. [162] In some embodiments, the intervening region comprises a comprises a coding element or coding region. In some embodiments, the coding element comprises a sequence encoding at least one therapeutic protein. In some embodiments, the coding element encodes two or more polypeptides. In some embodiments, the coding element or coding region comprises a sequence encoding, for example, a therapeutic protein, cytokine, immune checkpoint inhibitor, an agonist, a chimeric antigen receptor, an inhibitory receptor agonist or inhibitory receptor, an inhibitory receptor antagonist, one or more TCR chains, a secreted T cell or immune cell engager, a transcription factor, an immunosuppressive enzyme, or a TvHd, as set forth in detail herein. In some embodiments, the coding element or coding region comprises one or more expression sequences or portions thereof, e.g., Table 3, infra. [163] In some embodiments, provided herein are circular RNA polynucleotides comprising, in the following order, i) a 5’ combined accessory element comprising a 3’ self- spliced exon segment; ii) an intervening region; and iii) a 3’ combined accessory element comprising a 5’ self-spliced exon segment. In some embodiments, the 3’ self-spliced exon segment and/or the 5’ self-spliced exon segment is selected from an exon segment disclosed herein, e.g., in Table A or Table B. [164] In some embodiments, provided herein are circular RNA polynucleotides comprising, in the following order, i) a 5’ combined accessory element comprising a 3’ self- spliced exon segment, wherein the 3’ self-spliced exon segment comprises an exon segment; ii) an intervening region; and iii) a 3’ combined accessory element comprising a 5’ self-spliced exon segment, wherein the 5’ self-spliced exon segment comprises an exon segment. In some embodiments, the 3’ self-spliced exon segment and/or the 5’ self-spliced exon segment is selected from an exon segment disclosed herein, e.g., in Table A or Table B. [165] In some embodiments, provided herein are circular RNA polynucleotides comprising, in the following order, i) a 5’ combined accessory element comprising a 3’ self- spliced exon segment, wherein the 3’ self-spliced exon segment comprises an exon segment and a 3’ nucleotide of a 3’ splice site dinucleotide; ii) an intervening region; and iii) a 3’ combined accessory element comprising a 5’ self-spliced exon segment, wherein the 5’ self- spliced exon segment comprises an exon segment and a 5’ nucleotide of a 5’ splice site dinucleotide. In some embodiments, the 3’ self-spliced exon segment and/or the 5’ self-spliced exon segment is selected from an exon segment disclosed herein, e.g., in Table A or Table B. [166] A circular RNA polynucleotide comprising, in the following order, a 3’ self-spliced 50
Attorney Docket No. 01318-0014-00PCT OR-043WO exon segment, an intervening region, and a 5’ self-spliced exon segment, wherein at least one of the 3’ or 5’ self-spliced exon segments is selected from an exon segment comprising a sequence selected from Table A or Table B. [167] As a non-limiting example, a circular RNA polynucleotide comprises the following elements operably connected and arranged in the following sequence: (a) a 3ʹ exon segment comprising a Group I or Group II exon 3ʹ nucleotide of a 3ʹ splice site dinucleotide; (b) an intervening region; and (c) a 5ʹ exon segment comprising a Group I or Group II exon 5ʹ nucleotide of a 5ʹ splice site dinucleotide. [168] As set forth in detail herein, in some embodiments, a circular RNA polynucleotide comprises a retained portion of a monotron element. See, e.g., supra. In some embodiments, a circular RNA polynucleotide comprises: a 5’ internal spacer, a 5’ internal duplex, at least a portion of a terminal element (or sequence or segment), at least a portion of a monotron element (or sequence or segment), a 3’ internal duplex, a 3’ internal spacer, a coding or noncoding region, and an intervening region. In some embodiments, the circular RNA polynucleotide comprises a coding region and the intervening region comprises an IRES. In some embodiments, the IRES is a synthetic IRES, as described in further detail herein. In some embodiments, the monotron element present in the precursor RNA polynucleotide, of which a portion is retained in the circular RNA polynucleotide, comprises a polynucleotide sequence that has a percent sequence identity of about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more to a sequence selected from Table A or Table B. [169] In some embodiments, the circular RNA polynucleotide comprises the following elements operably connected and arranged in the following sequence: (a) a 5’ internal spacer, (b) a 5’ internal duplex, (c) at least a portion of a terminal element, (d) at least a portion of a monotron element, (e) a 3’ internal duplex, (f) a 3’ internal spacer, and (g) an intervening region, optionally comprising a coding region, and IRES. In some embodiments, the IRES is a synthetic IRES, as described in further detail herein. [170] In some embodiments, the circular RNA polynucleotide comprises the following 51
Attorney Docket No. 01318-0014-00PCT OR-043WO elements operably connected and arranged in the following sequence: (a) a 5’ internal spacer, (b) a 5’ internal duplex, (c) at least a portion of a monotron element, (d) at least a portion of a terminal element, (e) a 3’ internal duplex, (f) a 3’ internal spacer, and (g) an intervening region, optionally comprising a coding region, and IRES. In some embodiments, the IRES is a synthetic IRES, as described in further detail herein. [171] As a further non-limiting example, a circular RNA polynucleotide comprises the following elements operably connected and arranged in the following sequence: (a) at least a portion of a terminal element, (b) a 3ʹ exon segment comprising a 3ʹ nucleotide of a 3ʹ splice site dinucleotide, (c) an intervening region, (d) a 5ʹ exon segment comprising a 5ʹ nucleotide of a 5ʹ splice site dinucleotide, and (e) at least a portion of a monotron element; wherein the 5' and/or 3' splice site dinucleotides are distinct from the natural splice site dinucleotide(s) associated with a natural Group I or Group II intron sequence. [172] In some embodiments, element (d) comprises the first nucleotide of a 5ʹ Group I or Group II splice site dinucleotide and a natural exon sequence. In some embodiments, element (b) comprises the second nucleotide of a 3ʹ Group I or Group II exon splice site dinucleotide and a natural exon sequence. [173] In some embodiments, in the circular RNA polynucleotide, the 5ʹ exon element comprises the second nucleotide of a 3ʹ Group I or Group II exon splice site dinucleotide and a natural exon sequence. In some embodiments, the 3ʹ exon element fragment comprises the first nucleotide of a 5ʹ Group I or Group II splice site dinucleotide and a natural exon sequence. In some embodiments, the 5ʹ exon element comprises a 5ʹ internal duplex; and the 3ʹ exon element comprises a 3ʹ internal duplex. In some embodiments, the 5ʹ exon element comprises a 5ʹ internal spacer. In some embodiments, the 3ʹ exon element comprises a 3ʹ internal spacer. [174] In some embodiments, the circular RNA polynucleotide comprises a 5’ internal duplex and a 3’ internal duplex. See, e.g., supra. [175] In some embodiments, the circular RNA polynucleotide comprises a 5’ internal homology region and/or a 3’ internal homology region. See, e.g., supra. 52
Attorney Docket No. 01318-0014-00PCT OR-043WO [176] In some embodiments, the circular RNA polynucleotide comprises internal spacers (IS) of different lengths, e.g., a 5’internal spacer and/or a 3’ internal spacer. See, e.g., supra. [177] In some embodiments, the circular RNA polynucleotide retains portions of the precursor RNA polynucleotides, described elsewhere herein in detail. In some embodiments, portions of the precursor RNA polynucleotide are removed upon circularization. For example, in some embodiments, the circular RNA polynucleotide does not comprise a 5’ external spacer and/or a 3’ external spacer. In some embodiments, the circular RNA polynucleotide does not comprise a 5’ intron segment and/or 3’ intron segment. In some embodiments, the circular RNA polynucleotide does not comprise affinity tags. In some embodiments, the circular RNA polynucleotide does not retain a portion of a monotron element. In certain embodiments, the circular RNA polynucleotide does not retain a monotron element. [178] In some embodiments, and as described in more detail elsewhere herein, the circular RNA polynucleotide comprises modified nucleotides and/or modified nucleosides, namely comprising at least one modified A, C, G, or U/T nucleotide or nucleoside. Exemplary modifications are described in detail elsewhere herein. See, e.g., infra. In some embodiments, a circular RNA polynucleotide comprises modified nucleotides and/or modified nucleosides where between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90%, or 90% and 100% of the nucleotides or nucleosides are modified. As exhibited by the exemplary nucleotide or nucleotide modification presented in more detail, such modifications differ from mutations selected from insertions, deletions, addition, or subtraction of nucleotides, for example, the mutations in a permuted Group I and Group II intron segment; or the additions, deletions, or substitutions of a nucleotide, domain, or motif present in a synthetic IRES (as compared to the naturally occurring IRES). [179] In some embodiments, portions of the polynucleotide comprise between 1% and 10% modification of the nucleotides or nucleosides. In some embodiments, portions of the circular RNA polynucleotide comprise less than 10% modification. In some embodiments, portions of the polynucleotide or the polynucleotide in its entirety comprise no nucleotide or nucleoside modifications. In some embodiments, a circular RNA polynucleotide may lack modifications, where the linear precursors used to produce the circular RNA polynucleotide contained modifications (e.g., in the introns). In some embodiments, incorporation of a nucleotide or nucleoside modification to a precursor RNA polynucleotide hinders or lowers the 53
Attorney Docket No. 01318-0014-00PCT OR-043WO capacity of the circular RNA to circularize, splice, or express. [180] In some embodiments, the circular RNA polynucleotide is from about 50 nucleotides to about 15 kilobases in length. [181] In some embodiments, the circular RNA polynucleotide has an in vivo duration of therapeutic effect in a subject of at least about 10 hours. In some embodiments, the circular RNA polynucleotide has a functional half-life of at least about 10 hours. In some embodiments, the circular RNA polynucleotide has a duration of therapeutic effect in a cell greater than or equal to that of an equivalent linear RNA polynucleotide comprising the same expression sequence. In some embodiments, the circular RNA polynucleotide has a functional half-life in a cell greater than or equal to that of an equivalent linear RNA polynucleotide comprising the same expression sequence. In some embodiments, the circular RNA polynucleotide has an in vivo duration of therapeutic effect in a subject greater than that of an equivalent linear RNA polynucleotide having the same expression sequence. In some embodiments, the circular RNA polynucleotide has an in vivo functional half-life in a subject greater than that of an equivalent linear RNA polynucleotide having the same expression sequence. [182] In some embodiments, provided herein is a non-naturally occurring RNA polynucleotide comprising a translation initiation element (TIE), a coding sequence (e.g., with which the TIE is not naturally associated), and a means for self-splicing. In some embodiments, provided herein is a non-naturally occurring RNA polynucleotide comprising a translation initiation element (TIE), a coding sequence (e.g., with which the TIE is not naturally associated), and a means for self-circularization. In some embodiments, provided herein is provided herein is a non-naturally occurring RNA polynucleotide comprising a translation initiation element (TIE), a coding sequence (e.g., with which the TIE is not naturally associated), and an autocatalytic intron-exon means for self-splicing. In some embodiments, provided herein is a non-naturally occurring RNA polynucleotide comprising a translation initiation element (TIE), a coding sequence (e.g., with which the TIE is not naturally associated), and an autocatalytic intron-exon means for self-circularization. In some embodiments, provided herein is a non-naturally occurring RNA polynucleotide comprising, in the following order, a 3’ exon segment means for self-splicing, a translation initiation element, a coding sequence, and a 5’ exon segment means for self-splicing. In some embodiments, provided herein is a non-naturally occurring RNA polynucleotide comprising, in the following order, a 3’ exon segment means for self-circularization, a translation initiation element, a coding sequence, and a 5’ exon segment means for self-circularization. In some 54
Attorney Docket No. 01318-0014-00PCT OR-043WO embodiments, provided herein is a non-naturally occurring RNA polynucleotide comprising, in the following order, a 3’ exon segment, a translation initiation element, a coding sequence, and a 5’ exon segment, wherein the exon segments are means for self-splicing. In some embodiments, provided herein is a non-naturally occurring RNA polynucleotide comprising, in the following order, a 3’ exon segment, a translation initiation element, a coding sequence, and a 5’ exon segment, wherein the exon segments are means for self-circularization. In some embodiments, provided herein is a circular RNA polynucleotide comprising, in the following order, a 3’ exon segment means for self-circularization, a translation initiation element, a coding sequence, and a 5’ exon segment means for self-circularization. In some embodiments, provided herein is a circular RNA polynucleotide comprising, in the following order, a 3’ exon segment, a translation initiation element, a coding sequence, and a 5’ exon segment, wherein the exon segments are means for self-splicing. B. INTERVENING REGION [183] In various embodiments, a provided polynucleotide (e.g., a DNA template, a linear precursor RNA polynucleotide, or a circular RNA polynucleotide) comprises an intervening region. a. TRANSLATION INITIATION ELEMENT and INTERNAL RIBOSOME ENTRY SITE [184] In some embodiments, the DNA template, linear precursor RNA polynucleotide, and circular RNA polynucleotide comprise an intervening region and/or core functional element. In some embodiments, the intervening region and/or core functional element comprises a coding and/or noncoding element. In some embodiments, the intervening region and/or core functional element further comprises a translation initiation element (TIE) upstream to the coding or noncoding element, and/or a termination element. [185] In some embodiments, the polynucleotide comprises a translation initiation element (TIE). In some embodiments, the intervening region comprises at least one TIE. In some embodiments, the TIE is upstream to a coding element. In some embodiments, TIEs are designed to allow translation efficiency of an encoded protein. Accordingly, in some embodiments, an intervening region comprising one or more coding elements further comprises one or more TIEs. In other embodiments, an intervening region comprising only noncoding elements lacks any TIEs. [186] In some embodiments, a TIE comprises an aptamer complex, synthetic IRES, or other engineered TIE capable of initiating translation of a linear RNA or circular RNA 55
Attorney Docket No. 01318-0014-00PCT OR-043WO polynucleotide. In some embodiments, a TIE comprises an internal ribosome entry site (IRES). In certain embodiments, the TIE provided herein comprise a viral or eukaryotic internal ribosome entry site (IRES) or a fragment or variant thereof. In some embodiments, inclusion of an IRES permits the translation of one or more open reading frames from a circular RNA (e.g., open reading frames that form the expression sequences). In some embodiments, IRES attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation. See, e.g., PCT Application No. WO202261490, which is incorporated herein by reference in its entirety. In some embodiments, the IRES is a synthetic IRES. [187] In some embodiments, the TIE additionally comprises an aptamer complex wherein one or more aptamer sequences are capable of binding to a component of a eukaryotic initiation factor to either enhance or initiate translation. In some embodiments, an aptamer may be used to enhance translation in vivo and in vitro by promoting specific eukaryotic initiation factors (eIF) (e.g., certain aptamers disclosed in International Pat. Appl. No. PCT/EP2018/078794 are capable of binding to eukaryotic initiation factor 4F (eIF4F)). In some embodiments, an aptamer or a complex of aptamers may be capable of binding to EIF4G, EIF4E, EIF4A, EIF4B, EIF3, EIF2, EIF5, EIF1, EIF1A, 40S ribosome, PCBP1 (polyC binding protein), PCBP2, PCBP3, PCBP4, PABP1 (polyA binding protein), PTB, Argonaute protein family, HNRNPK (heterogeneous nuclear ribonucleoprotein K), or La protein. i. Synthetic Internal Ribosome Entry Sites (IRES) and TIEs [188] In some embodiments herein, the circular RNA comprises a sequence, e.g., encoding for a therapeutic protein (i.e. the payload). In certain embodiments, as provided herein, the payload encoded by the circular RNA polynucleotide may be optimized through use of a specific internal ribosome entry sites (IRES), including a synthetic IRES, within the translation initiation element (TIE). In some embodiments, IRES specificity within a circular RNA can promote or significantly enhance expression of specific proteins encoded within the coding element. In some embodiments, the IRES comprises a viral IRES or eukaryotic IRES. In some embodiments, the IRES is a synthetic IRES. [189] In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide therein. In some embodiments, at least one addition, deletion, or substitution thereof yields a synthetic IRES with increased or improved function and/or expression and/or stability as compared to the naturally occurring IRES. Synthetic IRESs, and domains or motifs thereof, have been designed to increase expression of operably linked expression sequences as compared to naturally occurring counterparts. In 56
Attorney Docket No. 01318-0014-00PCT OR-043WO some embodiments, the synthetic IRESs, and domains or motifs thereof, have improved expression, function, and/or stability in hepatocytes, immune cells (e.g., lymphocytes, T cells), muscle cells (e.g., myotubes), for example, as compared to a naturally occurring IRES, the naturally occurring counterpart, or a CVB3 IRES or comparative IRES 1, described below. In some embodiments, the synthetic IRES increases expression of a coding sequence operably linked to the IRES as compared to the naturally occurring IRES. In some embodiments, the synthetic IRES is capable of enhancing expression of the therapeutic protein as compared to a naturally occurring IRES. [190] A multitude of IRES sequences are available and include sequences derived from a wide variety of viruses, such as from leader sequences of picornaviruses such as the encephalomyocarditis virus (EMCV) UTR (Jang et al., J. Virol. (1989) 63: 1651-1660), the polio leader sequence, the hepatitis A virus leader, the hepatitis C virus IRES, human rhinovirus type 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(25): 15125- 15130), an IRES element from the foot and mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697- 2700), a giardiavirus IRES (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397), and the like. Five different “types” of IRESs (Types I, II, III, IV, V) have been classified based on evolutionary conserved sequences. Each type harbors a common RNA structure core maintained by evolutionary conserved covariant substitutions. (See Salas-Martinez 2018, incorporated by reference herein.) For example, Type I IRES elements occur in RNA genome of enterovirus, including poliovirus (PV), coxsackievirus B3 (CVB3), enterovirus 71 (EV71), and human rhinovirus (HRV). Type II IRES elements occur in cardiovirus (EMCV) and aphthovirus (FMDV) RNAs. Type I and Type II IRESs require the C-terminal region of eIF4G, eIF4A, eIF2, and eIF3 to assemble 48S initiation complexes in vitro, but are independent of eIF4E. Translation initiation driven by Type III, present in hepatitis A virus (HAV) RNA, was reported to depend on the integrity of eIF4G. Type IV IRES elements are eIF4G-dependent but depend on eIF2 and eIF3. As reported by Salas-Martinez 2018, the coxsackievirus B3 (CVB3) Type I IRES noted above is well-studied. This exemplary Type I IRES has 7 domains and various motifs within these domains. As set forth in, e.g., Martinez-Salas at Figure 4, and is known in the art, certain domains and motifs are conserved across the Types I, II, III, IV, V IRESs. [191] Inclusion of an IRES permits the translation of one or more open reading frames from a circular RNA (e.g., open reading frames that form the expression sequences). The IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation 57
Attorney Docket No. 01318-0014-00PCT OR-043WO initiation. See, e.g., Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20: 102-110; Kobayashi et al., BioTechniques (1996) 21 :399-402; and Mosser et al., BioTechniques 1997 22 150-161. In some embodiments, the IRES is capable of facilitating expression of a protein encoded by the precursor RNA in a cell. In some embodiments, the IRES is capable of facilitating expression of the protein, such that the expression level of the protein is comparable to or higher than when a control IRES is used. In some embodiments, where the IRES is a synthetic IRES, the control IRES is the naturally occurring or wild-type IRES, namely a version of the IRES that has not been engineered to comprise any comprise any additions, deletions, or substitutions. In other embodiments where the synthetic IRES comprises a modification in a particular domain or motif, the control IRES can be either naturally occurring or synthetic so long as it comprises a naturally occurring or wild-type version of the domain or motif. In other embodiments, where the synthetic IRES comprises a substitution of a domain or motif with a domain or motif from a second IRES, the control IRES can comprise, for example, the IRES without the substitution of the domain or motif (i.e. naturally occurring domain or motif) or the control IRES can comprise the second IRES. [192] Different IRES sequences, including synthetic IRES sequences, have varying ability to drive protein expression, and the ability of any particular identified or predicted IRES sequence to drive protein expression from linear mRNA or circular RNA constructs is unknown and unpredictable. As demonstrated herein, such IRES sequences may have differing effects on protein expression capability depending on cell type, for example in T cells, B cells, NK cells, blood cells, whole blood cells, peripheral blood cells, spleen cells, bone marrow cells, liver cells, or muscle cells. In some embodiments, the novel synthetic IRES sequences described herein may have at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or 100 fold increased expression in a particular cell type compared to control IRES sequences. In some embodiments, the novel synthetic IRES sequences described herein may have at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or 100 fold increased expression in hepatocytes, immune cells (e.g., lymphocytes, T cells), muscle cells (e.g., myotubes) compared to control IRES sequences. In some embodiments, the control IRES sequence can be a previously described IRES sequence. In some embodiments for a synthetic IRES, the control IRES is the naturally occurring or wild- type IRES, namely a version of the IRES that has not been engineered to comprise any comprise any additions, deletions, or substitutions. In other embodiments where the synthetic IRES comprises a modification in a particular domain or motif, the control IRES can be either 58
Attorney Docket No. 01318-0014-00PCT OR-043WO naturally occurring or synthetic so long as it comprises a naturally occurring or wild-type version of the domain or motif. In other embodiments where the synthetic IRES comprises a substitution of a domain or motif with a domain or motif from a second IRES, the control IRES can comprise, for example, the IRES without the substitution of the domain or motif (i.e. naturally occurring domain or motif) or the control IRES can comprise the second IRES. [193] In some embodiments, for driving protein expression, a polynucleotide (e.g., a DNA template, a linear precursor RNA polynucleotide, or a circular RNA polynucleotide) comprises an IRES (naturally occurring or synthetic) operably linked to a protein coding sequence. [194] In some embodiments, the synthetic IRES is a Type I IRES, a Type II IRES, a Type III IRES, a Type IV IRES, or Type V IRES. In some embodiments, the synthetic IRES contains Domain I, Domain II, Domain III, Domain IV, Domain V, Domain VI, and Domain VII. The structure of the different types of IRESs are known in the art, as well as what domains and motifs are conserved across the Type II, III, IV, V IRESs. (See Salas-Martinez 2018.) [195] In some embodiments, the synthetic IRES is a Type I IRES. In some embodiments, the Type I IRES is derived from enterovirus, such as a poliovirus (PV), coxsackievirus B3 (CVB3), enterovirus 71 (EV71), and human rhinovirus (HRV). In some embodiments, the Type I IRES is a coxsackievirus B3 (CVB3) Type I IRES that has 7 domains and various known motifs within the domains. In some embodiments, the Type II IRES is derived from cardiovirus (EMCV) and aphthovirus (FMDV) RNAs. In some embodiments, the Type III IRES is derived from hepatitis A virus. [196] In some embodiments, the IRES (naturally occurring or synthetic) is derived from Aalivirus, Ailurivirus, Ampivirus, Anativirus, Aphthovirus, Aquamavirus, Avihepatovirus, Avisivirus, Boosepivirus, Bopivirus, Caecilivirus, Cardiovirus, Cosavirus, Crahelivirus, Crohivirus, Danipivirus, Dicipivirus, Diresapivirus, Enterovirus, Erbovirus, Felipivirus, Fipivirus, Gallivirus, Gruhelivirus, Grusopivirus, Harkavirus, Hemipivirus, Hepatovirus, Hunnivirus, Kobuvirus, Kunsagivirus, Limnipivirus, Livupivirus, Ludopivirus, Malagasivirus, Marsupivirus, Megrivirus, Mischivirus, Mosavirus, Mupivirus, Myrropivirus, Orivirus, Oscivirus, Parabovirus, Parechovirus, Pasivirus, Passerivirus, Pemapivirus, Poecivirus, Potamipivirus, Pygoscepivirus, Rabovirus, Rafivirus, Rajidapivirus, Rohelivirus, Rosavirus, Sakobuvirus, Salivirus, Sapelovirus, Senecavirus, Shanbavirus, Sicinivirus, Symapivirus, Teschovirus, Torchivirus, Tottorivirus, Tremovirus, Tropivirus, Hepacivirus, Pegivirus, 59
Attorney Docket No. 01318-0014-00PCT OR-043WO Pestivirus, or Flavivirus. In some embodiments herein, the IRES is selected from an Enterovirus, Kobuvirus, Parechovirus, Hunnivirus, Passerivirus, Mischivirus, and Cardiovirus. [197] In some embodiments, the IRES (naturally occurring or synthetic) is an IRES sequence derived from Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus- 1, Human Immunodeficiency Virus type 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna- like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1/RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2/c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E/D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV- PK15C, SF573 Dicistrovirus, Hubei Picorna-like Virus, CRPV, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or an aptamer to eIF4G. [198] In some embodiments, the IRES (naturally occurring or synthetic) comprises in whole or in part a eukaryotic or cellular IRES. In certain embodiments, the IRES is an IRES 60
Attorney Docket No. 01318-0014-00PCT OR-043WO sequence derived from a human gene, wherein the human gene is ABCF1, ABCG1, ACAD10, ACOT7, ACSS3, ACTG2, ADCYAP1, ADK, AGTR1, AHCYL2, AHI1, AKAP8L, AKR1A1, ALDH3A1, ALDOA, ALG13, AMMECR1L, ANGPTL4, ANK3, AOC3, AP4B1, AP4E1, APAF1, APBB1, APC, APH1A, APOBEC3D, APOM, APP, AQP4, ARHGAP36, ARL13B, ARMC8, ARMCX6, ARPC1A, ARPC2, ARRDC3, ASAP1, ASB3, ASB5, ASCL1, ASMTL, ATF2, ATF3, ATG4A, ATP5B, ATP6V0A1, ATXN3, AURKA, AURKA, AURKA, AURKA, B3GALNT1, B3GNTL1, B4GALT3, BAAT, BAG1, BAIAP2, BAIAP2L2, BAZ2A, BBX, BCAR1, BCL2, BCS1L, BET1, BID, BIRC2, BPGM, BPIFA2, BRINP2, BSG, BTN3A2, C12orf43, C14orf93, C17orf62, C1orf226, C21orf62, C2orf15, C4BPB, C4orf22, C9orf84, CACNA1A, CALCOCO2, CAPN11, CASP12, CASP8AP2, CAV1, CBX5, CCDC120, CCDC17, CCDC186, CCDC51, CCN1, CCND1, CCNT1, CD2BP2, CD9, CDC25C, CDC42, CDC7, CDCA7L, CDIP1, CDK1, CDK11A, CDKN1B, CEACAM7, CEP295NL, CFLAR, CHCHD7, CHIA, CHIC1, CHMP2A, CHRNA2, CLCN3, CLEC12A, CLEC7A, CLECL1, CLRN1, CMSS1, CNIH1, CNR1, CNTN5, COG4, COMMD1, COMMD5, CPEB1, CPS1, CRACR2B, CRBN, CREM, CRYBG1, CSDE1, CSF2RA, CSNK2A1, CSTF3, CTCFL, CTH, CTNNA3, CTNNB1, CTNNB1, CTNND1, CTSL, CUTA, CXCR5, CYB5R3, CYP24A1, CYP3A5, DAG1, DAP3, DAP5, DAXX, DCAF4, DCAF7, DCLRE1A, DCP1A, DCTN1, DCTN2, DDX19B, DDX46, DEFB123, DGKA, DGKD, DHRS4, DHX15, DIO3, DLG1, DLL4, DMD UTR, DMD ex5, DMKN, DNAH6, DNAL4, DUSP13, DUSP19, DYNC1I2, DYNLRB2, DYRK1A, ECI2, ECT2, EIF1AD, EIF2B4, EIF4G1, EIF4G2, EIF4G3, ELANE, ELOVL6, ELP5, EMCN, ENO1, EPB41, ERMN, ERVV- 1, ESRRG, ETFB, ETFBKMT, ETV1, ETV4, EXD1, EXT1, EZH2, FAM111B, FAM157A, FAM213A, FBXO25, FBXO9, FBXW7, FCMR, FGF1, FGF1, FGF1A, FGF2, FGF2, FGF-9, FHL5, FMR1, FN1, FOXP1, FTH1, FUBP1, G3BP1, GABBR1, GALC, GART, GAS7, gastrin, GATA1, GATA4, GFM2, GHR, GJB2, GLI1, GLRA2, GMNN, GPAT3, GPATCH3, GPR137, GPR34, GPR55, GPR89A, GPRASP1, GRAP2, GSDMB, GSTO2, GTF2B, GTF2H4, GUCY1B2, HAX1, HCST, HIGD1A, HIGD1B, HIPK1, HIST1H1C, HIST1H3H, HK1, HLA-DRB4, HMBS, HMGA1, HNRNPC, HOPX, HOXA2, HOXA3, HPCAL1, HR, HSP90AB1, HSPA1A, HSPA4L, HSPA5, HYPK, IFFO1, IFT74, IFT81, IGF1, IGF1R, IGF1R, IGF2, IL11, IL17RE, IL1RL1, IL1RN, IL32, IL6, ILF2, ILVBL, INSR, INTS13, IP6K1, ITGA4, ITGAE, KCNE4, KERA, KIAA0355, KIAA0895L, KIAA1324, KIAA1522, KIAA1683, KIF2C, KIZ, KLHL31, KLK7, KRR1, KRT14, KRT17, KRT33A, KRT6A, KRTAP10-2, KRTAP13-3, KRTAP13-4, KRTAP5-11, KRTCAP2, LACRT, LAMB1, 61
Attorney Docket No. 01318-0014-00PCT OR-043WO LAMB3, LANCL1, LBX2, LCAT, LDHA, LDHAL6A, LEF1, LINC-PINT, LMO3, LRRC4C, LRRC7, LRTOMT, LSM5, LTB4R, LYRM1, LYRM2, MAGEA11, MAGEA8, MAGEB1, MAGEB16, MAGEB3, MAPT, MARS, MC1R, MCCC1, METTL12, METTL7A, MGC16025, MGC16025, MIA2, MIA2, MITF, MKLN1, MNT, MORF4L2, MPD6, MRFAP1, MRPL21, MRPS12, MSI2, MSLN, MSN, MT2A, MTFR1L, MTMR2, MTRR, MTUS1, MYB, MYC, MYCL, MYCN, MYL10, MYL3, MYLK, MYO1A, MYT2, MZB1, NAP1L1, NAV1, NBAS, NCF2, NDRG1, NDST2, NDUFA7, NDUFB11, NDUFC1, NDUFS1, NEDD4L, NFAT5, NFE2L2, NFE2L2, NFIA, NHEJ1, NHP2, NIT1, NKRF, NME1-NME2, NPAT, NR3C1, NRBF2, NRF1, NTRK2, NUDCD1, NXF2, NXT2, ODC1, ODF2, OPTN, OR10R2, OR11L1, OR2M2, OR2M3, OR2M5, OR2T10, OR4C15, OR4F17, OR4F5, OR5H1, OR5K1, OR6C3, OR6C75, OR6N1, OR7G2, p53, P2RY4, PAN2, PAQR6, PARP4, PARP9, PC, PCBP4, PCDHGC3, PCLAF, PDGFB, PDZRN4, PELO, PEMT, PEX2, PFKM, PGBD4, PGLYRP3, PHLDA2, PHTF1, PI4KB, PIGC, PIM1, PKD2L1, PKM, PLCB4, PLD3, PLEKHA1, PLEKHB1, PLS3, PML, PNMA5, PNN, POC1A, POC1B, POLD2, POLD4, POU5F1, PPIG, PQBP1, PRAME, PRPF4, PRR11, PRRT1, PRSS8, PSMA2, PSMA3, PSMA4, PSMD11, PSMD4, PSMD6, PSME3, PSMG3, PTBP3, PTCH1, PTHLH, PTPRD, PUS7L, PVRIG, QPRT, RAB27A, RAB7B, RABGGTB, RAET1E, RALGDS, RALYL, RARB, RCVRN, REG3G, RFC5, RGL4, RGS19, RGS3, RHD, RINL, RIPOR2, RITA1, RMDN2, RNASE1, RNASE4, RNF4, RPA2, RPL17, RPL21, RPL26L1, RPL28, RPL29, RPL41, RPL9, RPS11, RPS13, RPS14, RRBP1, RSU1, RTP2, RUNX1, RUNX1T1, RUNX1T1, RUNX2, RUSC1, RXRG, S100A13, S100A4, SAT1, SCHIP1, SCMH1, SEC14L1, SEMA4A, SERPINA1, SERPINB4, SERTAD3, SFTPD, SH3D19, SHC1, SHMT1, SHPRH, SIM1, SIRT5, SLC11A2, SLC12A4, SLC16A1, SLC25A3, SLC26A9, SLC5A11, SLC6A12, SLC6A19, SLC7A1, SLFN11, SLIRP, SMAD5, SMARCAD1, SMN1, SNCA, SNRNP200, SNRPB2, SNX12, SOD1, SOX13, SOX5, SP8, SPARCL1, SPATA12, SPATA31C2, SPN, SPOP, SQSTM1, SRBD1, SRC, SREBF1, SRPK2, SSB, SSB, SSBP1, ST3GAL6, STAB1, STAMBP, STAU1, STAU1, STAU1, STAU1, STAU1, STK16, STK24, STK38, STMN1, STX7, SULT2B1, SYK, SYNPR, TAF1C, TAGLN, TANK, TAS2R40, TBC1D15, TBXAS1, TCF4, TDGF1, TDP2, TDRD3, TDRD5, TESK2, THAP6, THBD, THTPA, TIAM2, TKFC, TKTL1, TLR10, TM9SF2, TMC6, TMCO2, TMED10, TMEM116, TMEM126A, TMEM159, TMEM208, TMEM230, TMEM67, TMPRSS13, TMUB2, TNFSF4, TNIP3, TP53, TP53, TP73, TRAF1, TRAK1, TRIM31, TRIM6, TRMT1, TRMT2B, TRPM7, TRPM8, TSPEAR, TTC39B, TTLL11, 62
Attorney Docket No. 01318-0014-00PCT OR-043WO TUBB6, TXLNB, TXNIP, TXNL1, TXNRD1, TYROBP, U2AF1, UBA1, UBE2D3, UBE2I, UBE2L3, UBE2V1, UBE2V2, UMPS, UNG, UPP2, USMG5, USP18, UTP14A, UTRN, UTS2, VDR, VEGFA, VEGFA, VEPH1, VIPAS39, VPS29, VSIG10L, WDHD1, WDR12, WDR4, WDR45, WDYHV1, WRAP53, XIAP, XPNPEP3, YAP1, YWHAZ, YY1AP1, ZBTB32, ZNF146, ZNF250, ZNF385A, ZNF408, ZNF410, ZNF423, ZNF43, ZNF502, ZNF512, ZNF513, ZNF580, ZNF609, ZNF707, or ZNRD1. [199] In some embodiments, the cell is a myotube. In some embodiments, the IRES (naturally occurring or synthetic) is derived from Bopivirus, Oscivirus, Hunnivirus, Passerivirus, Mischivirus, Kobuvirus, Enterovirus, Cardiovirus, Salivirus, Rabovirus, Parechovirus, Gallivirus, or Sicinivirus. In some embodiments, the IRES is derived from Hunnivirus, Passerivirus, Kobuvirus, Bopivirus, or Enterovirus. In some embodiments, the IRES is derived from Enterovirus I, Enterovirus F, Enterovirus E, Enterovirus J, Enterovirus C, Enterovirus A, Enterovirus B, Aichivirus B, Parechovirus A, Cardiovirus F, Cardiovirus B, or Cardiovirus E. [200] In some embodiments, the cell is a hepatocyte. In some embodiments, the IRES (naturally occurring or synthetic) is derived from Enterovirus, Bopivirus, Mischivirus, Gallivirus, Oscivirus, Cardiovirus, Kobuvirus, Rabovirus, Salivirus, Parechovirus, Hunnivirus, Tottorivirus, Passerivirus, Cosavirus, or Sicinivirus. In some embodiments, the IRES is derived from Enterovirus, Mischivirus, Kobuvirus, Bopivirus, or Gallivirus. In some embodiments, the IRES is derived from Enterovirus B, Enterovirus A, Enterovirus D, Enterovirus J, Enterovirus C, Rhinovirus B, Enterovirus H, Enterovirus I, Enterovirus E, Enterovirus F, Aichivirus B, Aichivirus A, Parechovirus A, Cardiovirus F, Cardiovirus E, or Cardiovirus B. [201] In some embodiments, the cell is a T cell. In some embodiments, the IRES (naturally occurring or synthetic) is derived from Passerivirus, Bopivirus, Hunnivirus, Mischivirus, Enterovirus, Kobuvirus, Rabovirus, Tottorivirus, Salivirus, Cardiovirus, Parechovirus, Megrivirus, Allexivirus, Oscivirus, or Shanbavirus. In some embodiments, the IRES is derived from Passerivirus, Hunnivirus, Mischivirus, Enterovirus, or Kobuvirus. In some embodiments, the IRES is derived from Enterovirus I, Enterovirus D, Enterovirus C, Enterovirus A, Enterovirus J, Enterovirus H, Aichivirus B, Parechovirus A, or Cardiovirus B. ii. Synthetic IRES: engineered additions, deletions, or substitutions [202] In certain embodiments, a TIE provided herein is a synthetic TIE and/or the IRES provided herein is a synthetic IRES. In some embodiments, the synthetic IRES is a Type I IREs, a Type II IRES, a Type III IRES, a Type IV IRES, or Type V IRES. 63
Attorney Docket No. 01318-0014-00PCT OR-043WO [203] In some embodiments, the TIE comprises a synthetic IRES engineered to comprise at least one addition, deletion, or substitution in a nucleotide, domain, or motif, as compared to a naturally occurring IRES. In some embodiments, the synthetic IRES contains at least one substitution or deletion of entire domains or motifs, or regions thereof. Where the addition, deletion, or substitution includes substituting an entire domain or motif, or region thereof, the newly substituted domain or motif or region can be from a second IRES. In some embodiments, the second IRES is a second higher-expressing, naturally occurring, IRES. [204] As exhibited by the exemplary synthetic IRES sequences presented herein, at least one addition, deletion, or substitution of a nucleotide, domain, or motif in a synthetic IRES (as compared to the naturally occurring IRES) differ from the mutations in a permuted Group I and/or Group II intron segment presented herein and the modifications that make up, for example, a modified nucleotide or nucleoside described in detail herein, e.g., present in intronic or exonic elements, e.g., where the modified nucleoside is, e.g., a 5-methylcytidine, 5- methoxyuridine, 1-methyl-pseudouridine, N6-methyladenosine, and/or pseudouridine. Those modifications seek to improve circularization efficiency or splicing efficiency, whereas the changes engineered into the sequences of synthetic IRESs seek to improve function and/or expression and/or stability, as compared to a naturally occurring IRES. [205] In some embodiments, for example, the synthetic IRES is an EMCV IRES that contains two Pol III terminal signals, and comprises an alteration in at least one of the Pol III termination elements. In some embodiments, conserved Pol III terminal signals in the PTB binding motif are modified and/or stop codons are changed in the synthetic IRES. For example, in some embodiments, the synthetic IRES comprises replacing a “UCUUU” or “UUUAU” sequence that binds to polypyrimidine binding tract (PTB) with a “UUCUCU” or “UCUCU” or “UCUAU” PTB binding motif. See Unti, Cell Chemical Biology 31, 163–176, January 18, 2024 (incorporated by reference herein in its entirety). In some embodiments, the Pol III termination signal is replaced with a related sequence from a falcon picornavirus, which does not have a PolIII termination element in the corresponding region. See Unti 2024. In some embodiments, a viral P2A sequence was included, which induces ribosome skipping and cleaves the polypeptide chain. In some embodiments an IRES can also include point mutations and the circular RNA can include stop codon insertions. [206] In some embodiments the oRNA comprises a synthetic IRES. In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a nucleotide or motif in at least one domain. In some embodiments, the synthetic IRES 64
Attorney Docket No. 01318-0014-00PCT OR-043WO comprises at least one addition, deletion, or substitution of a nucleotide or motif in at least one of Domain I, Domain II, Domain III, Domain IV, Domain V, and Domain VI, as compared to a naturally occurring corresponding domain. [207] In certain instances where the oRNA comprises a synthetic IRES comprising a substitution of an entire domain or motif, the replacement domain or motif may be derived from a different IRES, e.g., from a second higher-expressing, naturally occurring, IRES. Replacing the domain or motif with that of a different IRES renders the initial IRES synthetic, even if the domain or motif does not contain any nucleotide modifications and retains the sequence as present in the second, naturally occurring, IRES. [208] In some embodiments, the oRNA comprises a synthetic IRES comprising at least one addition, deletion, or substitution of a nucleotide or motif in Domain I as compared to a naturally occurring Domain I, or comprises a deletion or substitution of Domain I in whole or in part. In some embodiments, the synthetic IRES comprises a deletion of Domain I in whole or in part. In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a nucleotide or motif in Domain I or comprises a deletion or substitution of Domain I in whole or in part as compared to a naturally occurring Domain I, while the other domains are retained. In some embodiments, the synthetic IRES comprises a naturally occurring Domain I. In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a nucleotide or motif in at least one domain, wherein the domain is not Domain I and the naturally occurring sequence of Domain I is conserved. [209] In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a nucleotide or motif in Domain I, and comprises a naturally occurring Domain II, Domain III, Domain IV, and Domain V. In some embodiments, the synthetic IRES comprises a deletion of Domain I whole or in part, and comprises a naturally occurring Domain II, Domain III, Domain IV, and Domain V. In some embodiments, engineering the synthetic IRES to comprise at least one addition, deletion, or substitution of a nucleotide or motif in Domain I is shown to improve IRES function as compared to a naturally occurring IRES or an IRES with a naturally occurring Domain I. In some embodiments, retaining Domain II is shown to improve IRES function, as compared to a synthetic Domain II. In some embodiments, retaining Domain III is shown to improve IRES function, as compared to a synthetic Domain III. In some embodiments, retaining Domain IV is shown to improve IRES function, as compared to a synthetic Domain IV. In some embodiments, retaining Domain V is shown to improve IRES function, as compared to a synthetic Domain V. 65
Attorney Docket No. 01318-0014-00PCT OR-043WO [210] In some embodiments, the synthetic IRES comprises a combination of domains from multiple naturally occurring IRESs. For example, a synthetic IRES may comprise a combination of Domains I, II, and III from a first naturally occurring IRES and Domains IV, V, VI, and VII from a different second naturally occurring IRES. In certain embodiments, combining domains from a first and second naturally occurring IRES retains the IRES function of the synthetic IRES. [211] In some embodiments, the oRNA comprises a synthetic IRES comprising at least one addition, deletion, or substitution of a nucleotide or motif in Domain II as compared to a naturally occurring Domain II, or comprises a deletion or substitution of Domain II in whole or in part. In some embodiments, the synthetic IRES comprises a deletion of Domain II in whole or in part. In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a nucleotide or motif in Domain II or comprises a deletion or substitution of Domain II in whole or in part as compared to a naturally occurring Domain II, while the other domains are retained. In some embodiments, the synthetic IRES comprises a naturally occurring Domain II. In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a nucleotide or motif in at least one domain, wherein the domain is not Domain II and the naturally occurring sequence of Domain II is conserved. [212] In some embodiments, the oRNA comprises a synthetic IRES comprising at least one addition, deletion, or substitution of a nucleotide or motif in Domain III as compared to a naturally occurring Domain III, or comprises a deletion or substitution of Domain III in whole or in part. In some embodiments, the synthetic IRES comprises a deletion of Domain III in whole or in part. In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a nucleotide or motif in Domain III or comprises a deletion or substitution of Domain III in whole or in part as compared to a naturally occurring Domain III, while the other domains are retained. In some embodiments, the synthetic IRES comprises a naturally occurring Domain III. In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a nucleotide or motif in at least one domain, wherein the domain is not Domain III and the naturally occurring sequence of Domain III is conserved. [213] In some embodiments, the oRNA comprises a synthetic IRES comprising at least one addition, deletion, or substitution of a nucleotide or motif in Domain IV as compared to a naturally occurring Domain IV, or comprises a deletion or substitution of Domain IV in whole or in part. In some embodiments, the synthetic IRES comprises a deletion of Domain IV in whole or in part. In some embodiments, the synthetic IRES comprises at least one addition, 66
Attorney Docket No. 01318-0014-00PCT OR-043WO deletion, or substitution of a nucleotide or motif in Domain IV or comprises a deletion or substitution of Domain IV in whole or in part as compared to a naturally occurring Domain IV, while the other domains are retained. In some embodiments, the synthetic IRES comprises a naturally occurring Domain IV. In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a nucleotide or motif in at least one domain, wherein the domain is not Domain IV and the naturally occurring sequence of Domain IV is conserved. [214] In some embodiments, the oRNA comprises a synthetic IRES comprising at least one addition, deletion, or substitution of a nucleotide or motif in Domain IV as compared to a naturally occurring Domain IV, or comprises a deletion or substitution of Domain IV in whole or in part. In some embodiments, the synthetic IRES comprises a substitution of Domain IV as a whole, wherein Domain IV is replaced with a Domain IV from a second IRES. In some embodiments, the entirety of Domain IV is replaced with a Domain IV from a second, higher- expressing, naturally occurring, IRES. See WO2023182948A1, which is incorporated by reference herein in its entirety (describing a domain IV substitution). [215] Domain IV can be conserved between IRES types. Domain IV of a Type 1 IRES, for example, is known to contain a GNRA tetra loop (where N stands for any nucleotide, and R for purine), a C-rich loop, and a EIF2/EIF4G binding site. (See Breyne, PNAS, vol.106: 23, 2009, 9197-9202 and Mailliot & Martin, WIREs RNA 2018, 9:e1458, both incorporated by reference herein). In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a nucleotide in the GNRA tetra loop, C-rich loop, or EIF2/EIF4G binding site in Domain IV. [216] In some embodiments, the at least one addition, deletion, or substitution in Domain IV comprises an addition of an EIF4 aptamer sequence at the 5’ end of Domain IV. In some embodiments, the at least one addition, deletion, or substitution in Domain IV comprises an addition of an EIF3/EIF4G aptamer sequence to the 2 nucleotide bulge and/or 3 nucleotide bulge of Domain IV. Exemplary aptamer EIF4E sequences are Aptamer EIF4E sequence 1: TGTTCAACCAGAGTGAAACCACTAACGGGTCAGAGCCCC (SEQ ID NO: 24901) and Aptamer EIF4E sequence 2: GCCAGAGCAACAACCTTCCGAGCCGCGGGATAAAACCGAG (SEQ ID NO: 24902). [217] In some embodiments where the synthetic IRES comprises at least one addition, deletion, or substitution in Domain IV, the synthetic IRES comprises a naturally occurring Domain VII. For example, in some embodiments, the synthetic IRES comprises an addition of an EIF4 or EIF3/EIF4G aptamer sequence in Domain IV, and does not comprise a 67
Attorney Docket No. 01318-0014-00PCT OR-043WO replacement of nucleotides from the 5’ end of the terminal stem of Domain VII to the 3’ end of the IRES sequence with a EIF4 aptamer sequence. [218] In some embodiments, at least one addition, deletion, or substitution in Domain IV comprises an addition of a Polypyrimidine tract (PPT) in Domain IV. In some embodiments, the PPT tract of the synthetic IRES is replaced with a PPT tract from a second, higher- expressing, naturally occurring, IRES. For example, where the starting IRES (i.e. synthetic IRES before engineered changes) has lower and/or comparable expression levels, replacing the PPT sequence with a donor PPT sequence from a higher-expressing IRES improves the IRES function of the resultant synthetic IRES. In some embodiments, the higher-expressing IRES is a higher-expressing, naturally occurring, IRES. In other embodiments, the synthetic IRES comprises a naturally occurring whole PPT sequence in Domain IV. In some embodiments, the synthetic IRES does not contain an added PTBP1 consensus sequence. In such embodiments, adding a PTBP1 consensus sequence could worsen IRES function. [219] In some embodiments, the at least one addition, deletion, or substitution in Domain IV comprises an addition, deletion, or substitution in a GNRA tetra loop in Domain IV. The native function of the GNRA tetra loop is EIF recruitment and additions, deletions, or substitutions in the GNRA tetra loop in Domain IV are shown to enhance EIF recruitment leading to improved IRES function. In some embodiments, substitution of a GNRA consensus sequence may improve IRES function. [220] In some embodiments, the at least one addition, deletion, or substitution in Domain IV comprises an addition, deletion, or substitution in a negative IRES transacting factor (“- ITAF”), optionally comprising a mutation or deletion of a FBP2/KHSRP binding region, optionally wherein the mutation or deletion is in frame. The FBP2/KHSRP binding region in Domain IV is an internal mechanism to attenuate expression. A synthetic IRES comprising a negative ITAF (“-ITAF”) mutation or deletion of FBP2/KHSRP binding region, preferably in frame, improves IRES function. [221] In some embodiments where the synthetic IRES comprises at least one addition, deletion, or substitution in Domain IV, the C-Loop Region of Domain IV recognized by either PCBP1 or PCBP2 of the synthetic IRES is naturally occurring. Retaining the C-Loop region recognized by either PCBP1 or PCBP2, improves IRES function. The C-Loop at the outer edge of the IRES is required for exposure to protein PCBP1/2 to attract ribosomes. Accordingly, deleting or otherwise mutating the C-loop region recognized by PCBP1 or PCBP2 is shown to worsen IRES function in Domain IV. 68
Attorney Docket No. 01318-0014-00PCT OR-043WO [222] In some embodiments, the oRNA comprises a synthetic IRES comprising at least one addition, deletion, or substitution of a nucleotide or motif in Domain V as compared to a naturally occurring Domain V, or comprises a deletion or substitution of Domain V in whole or in part. In some embodiments, the synthetic IRES comprises a deletion of Domain V in whole or in part. In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a nucleotide or motif in Domain V or comprises a deletion or substitution of Domain V in whole or in part as compared to a naturally occurring Domain V, while the other domains are retained. In some embodiments, the synthetic IRES comprises a naturally occurring Domain V. In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a nucleotide or motif in at least one domain, wherein the domain is not Domain V and the naturally occurring sequence of Domain V is conserved. In some embodiments, retaining Domain V is shown to improve IRES function. [223] In some embodiments, the oRNA comprises a synthetic IRES comprising at least one addition, deletion, or substitution of a nucleotide or motif therein, but the synthetic IRES does not comprise a J-K/L Loop of a Type II IRES in Domain V. [224] In some embodiments, the oRNA comprises a synthetic IRES comprising at least one addition, deletion, or substitution of a nucleotide or motif in Domain VI as compared to a naturally occurring Domain VI, or comprises a deletion or substitution of Domain VI in whole or in part. In some embodiments, the synthetic IRES comprises a deletion of Domain VI in whole or in part. In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a nucleotide or motif in Domain VI or comprises a deletion or substitution of Domain VI in whole or in part as compared to a naturally occurring Domain VI, while the other domains are retained. In some embodiments, the synthetic IRES comprises a naturally occurring Domain VI. In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a nucleotide or motif in at least one domain, wherein the domain is not Domain VI and the naturally occurring sequence of Domain VI is conserved. In some embodiments, retaining Domain VI is shown to improve IRES function. [225] In some embodiments, the oRNA comprises a synthetic IRES comprising at least one addition, deletion, or substitution of a nucleotide or motif in Domain VII as compared to a naturally occurring Domain VII, or comprises a deletion or substitution of Domain VII in whole or in part. In some embodiments, the synthetic IRES comprises a deletion of Domain VII in whole or in part. In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a nucleotide or motif in Domain VII or comprises a deletion or 69
Attorney Docket No. 01318-0014-00PCT OR-043WO substitution of Domain VII in whole or in part as compared to a naturally occurring Domain VII, while the other domains are retained. In some embodiments, the synthetic IRES comprises a naturally occurring Domain VII. In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a nucleotide or motif in at least one domain, wherein the domain is not Domain VII and the naturally occurring sequence of Domain VII is conserved. In some embodiments, retaining Domain VII is shown to improve IRES function. [226] In some embodiments, the oRNA comprises a synthetic IRES comprising at least one addition, deletion, or substitution of a nucleotide or motif in the post-Domain VII terminal loop as compared to a naturally occurring post-Domain VII terminal loop, or comprises a deletion or substitution of the post-Domain VII terminal loop in whole or in part. In some embodiments, the synthetic IRES comprises a deletion of a naturally occurring post-Domain VII terminal stem and an addition of a 15-nt scanning tract comprising the sequence AUN- AUN-AUN-AUN-AUN, wherein N is a U or A. Deleting the post-Domain VII terminal stem and adding a 15-nt scanning tract is shown to improve IRES function as compared to IRES that retains the native (naturally occurring) post-Domain VII terminal stem. [227] In some embodiments, the synthetic IRES comprises a deletion of a naturally occurring post-Domain VII terminal stem and an addition of a 9-nt Kozak sequence in Domain VII. Synthetically engineering the IRES to delete the post-Domain VII terminal stem and adding the 9-nt Kozak sequence is shown to improve IRES function as compared to IRES that retains the native (naturally occurring) post-Domain VII terminal stem. Notably, such a synthetic IRES differs from a naturally occurring IRES having a Kozak sequence in its native form. (See WO2019118919A1.) Such a synthetic IRES also differs from, e.g., a circular RNA simply adding a Kozak sequence, for example, the IRES described in, e.g., WO2022037692A1, where the Kozak sequence is not a replacement of the terminal stem of Domain VII, but rather is simply an addition following the 3’ end of the IRES. In some embodiments, the Kozak sequences that are added to Domain VII can comprise alterations whereby the last three nucleotides of the Kozak sequence can comprise an “AUG” sequence. [228] In some embodiments, the synthetic IRES comprises a deletion of a naturally occurring post-Domain VII terminal stem and an addition of an 11-nt consensus sequence comprising “AACACAACAAA.” (SEQ ID NO: 24903) In some embodiments, the synthetic IRES comprises replacing the naturally occurring post-Domain VII terminal stem with an 11- nt consensus sequence selected from comprising “AACACAACAAA.” (SEQ ID NO: 24903) Synthetically engineering the IRES to replace the post-Domain VII terminal stem with the 11- 70
Attorney Docket No. 01318-0014-00PCT OR-043WO nt consensus sequence comprising “AACACAACAAA” (SEQ ID NO: 24903) is shown to improve IRES function as compared to IRES that retains the native (naturally occurring) post- Domain VII terminal stem. [229] In some embodiments, the synthetic IRES comprises a naturally occurring Domain VII. In some embodiments, retaining Domain VII is shown to improve IRES function. In some embodiments, the nucleotides from the 5’ end of the terminal stem of Domain VII to the 3’ end of the synthetic IRES are retained, and, e.g., are not replaced with an EIF4 aptamer sequence. [230] In some embodiments, the oRNA comprises a synthetic IRES, wherein the synthetic IRES does not contain an addition, deletion, or substitution of a nucleotide in any cryptic codons or contain a deletion or substitution of any cryptic codons in whole or in part. In some embodiments, the synthetic IRES does not contain a deletion of any cryptic codons in part or in whole anywhere in the IRES. A cryptic initiation codon can refer to any AUG anywhere along the IRES and is neither specific to any particular type of IRES nor confined to a specific domain. Retaining cryptic codons is shown to maintain or improve IRES function compared to IRES in which cryptic codons are deleted in part or whole. A cryptic initiation codon can also comprise other non-AUG initiation codons for translation. [231] In some embodiments, the synthetic IRES comprises at least one addition of a cryptic codon in any domain. In some embodiments, the synthetic IRES comprises at least one addition of a cryptic codon in Domain VII. Adding cryptic codon in, e.g., Domain VII, can maintain or improve IRES function. These embodiments wherein the IRESs are synthetically engineered to have an “AUG” cryptic codon sequence in, e.g., Domain VII, differ from IRESs that have naturally occurring AUG cryptic codons present in the IRESs’ native (naturally occurring) form. See WO2019118919A1. [232] IRESs derived computationally from native UTRs have non-canonical stem loop regions (“SL region”). In some embodiments, the synthetic IRES comprises naturally occurring non-canonical SL regions, wherein the SL regions neither contain any additions deletions, or substitutions of a nucleotide in the SL regions nor contain a deletion or substitution of any SL regions in whole or in part. In some embodiments, the synthetic IRES does not contain a: (a) deletion of SL1 in whole or in part, (b) deletion of SL2 in whole or in part, (c) deletion of SL3 in whole or in part, and/or (d) deletion of SL4 in whole or in part. In some embodiments, the synthetic IRES does not contain an addition of a SL4 to an IRES natively lacking a SL4. 71
Attorney Docket No. 01318-0014-00PCT OR-043WO [233] In some embodiments, alterations or deletions of the SL regions in a synthetic IRES can hinder the function of the synthetic IRES. For example, some synthetic IRESs are shown to have a SL1, SL2, and SL3 in replacement of Domain I and/or upstream to Domain II, a SL4 in between Domain IV and V, and a SL5 following domain VII retained from the naturally occurring IRES as compared to a more canonical Type I IRES, such as CVB3. In some embodiments, deletion of one or more SL regions in these synthetic IRES is shown to hinder overall IRES function [234] If lower expression is desirable, e.g., for stability of gene expression, a synthetic IRES can be engineered to retain Domain I, and/or provide at least one addition, deletion, or substitution of a nucleotide or motif in Domain II in whole or in part, and/or provide at least one addition, deletion, or substitution of a nucleotide or motif in Domain III in whole or in part, and/or provide at least one addition, deletion, or substitution of a nucleotide or motif in Domain IV in whole or in part, and/or provide at least one addition, deletion, or substitution of a nucleotide or motif in Domain V in whole or in part. In some embodiments, the synthetic Ires is engineered to retain Domain I, and/or to delete Domain II in whole or in part, and/or to delete Domain III in whole or in part, and/or to delete Domain V in whole or in part, and/or to substitute Domain V with a J-K/L Loop of a Type II IRES. [235] In some embodiments, the oRNA comprises a synthetic IRES comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an IRES sequence in Table 1, or a fragment thereof. In some embodiments, the oRNA comprises a synthetic IRES comprising a sequence in Table 1, or a fragment thereof. In some embodiments, the precursor RNA polynucleotide, circular RNA constructs and related pharmaceutical compositions disclosed herein comprise a synthetic IRES sequence comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an IRES sequence in Table 1, or a fragment thereof. In some embodiments, the precursor RNA polynucleotide, circular RNA constructs and related pharmaceutical compositions disclosed herein comprise a synthetic IRES sequence comprising a sequence in Table 1, or a fragment thereof. Table 1: Synthetic IRES Sequences
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[236] In some embodiments, the oRNA comprising a synthetic IRES, or domains or motifs thereof, comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an IRES sequence in Table 1, or a fragment thereof exhibits improved expression and/or function and/or stability in hepatocytes, immune cells (e.g., lymphocytes, T cells), muscle cells (e.g., myotubes), for example, as compared to a naturally occurring IRES, the naturally occurring counterpart, or a CVB3 IRES or comparative IRES 1. In some embodiments, the oRNA comprising a synthetic IRES, or domains or motifs thereof, comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an IRES sequence comprising IRES 307-314 from Table 1 (SEQ ID NOs: 25452-25459), or a fragment thereof exhibits improved expression and/or function and/or stability in hepatocytes, immune cells (e.g., lymphocytes, T cells), muscle cells (e.g., myotubes), for example, as compared to a naturally occurring IRES, the naturally occurring counterpart, or a CVB3 IRES or comparative IRES 1. In some embodiments, the synthetic IRES increases expression of a coding sequence operably linked to the IRES as compared to the naturally occurring IRES. In some embodiments, the synthetic IRES is capable of enhancing expression of the therapeutic protein as compared to a naturally occurring IRES. iii. Exemplary Corresponding Naturally Occurring IRES Sequences [237] In some embodiments, for driving protein expression, a provided circular RNA comprises an IRES operably linked to a protein coding sequence. In some embodiments, the IRES is synthetic. In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence selected from SEQ ID NOS: 1-2989, 3282-3303, and 14067-24829 (GIRES 0- 10762) or Table 2 below, or SEQ ID NOs: 1-2983 and 3282-3287 of PCT Application No. US2022/33091 (WO202261490) or a fragment thereof. In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide 161
Attorney Docket No. 01318-0014-00PCT OR-043WO as compared to an IRES comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 1-2989, 3282-3303, and 14067-24829 or Table 2 below, or a sequence selected from SEQ ID NOs: 1-2983 and 3282-3287 of PCT Application No. US2022/33091 (WO202261490) or a fragment thereof. In some embodiments, the circular RNA disclosed herein comprises a synthetic IRES sequence comprising at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 1-2989, 3282-3303, and 14067-24829 or Table 2 below, or a sequence selected from SEQ ID NOs: 1-2983 and 3282-3287 of PCT Application No. US2022/33091 (WO202261490) or a fragment thereof. In some embodiments, the circular RNA disclosed herein comprises a synthetic IRES sequence comprising at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence selected from SEQ ID NOs: 1-2989, 3282-3303, and 14067-24829 or Table 2 below or a fragment thereof, or SEQ ID NOs: 1-2983 and 3282-3287 of PCT Application No. US2022/33091 (WO202261490) or a fragment thereof. In some embodiments, such circular RNAs, or domains or motifs thereof, exhibit improved expression and/or function and/or stability in hepatocytes, immune cells (e.g., lymphocytes, T cells), muscle cells (e.g., myotubes), for example, as compared to a naturally occurring IRES, the naturally occurring counterpart, or a CVB3 IRES or comparative IRES 1. [238] Further exemplary naturally occurring IRES sequences are provided in Table 2. In some embodiments, the precursor RNA polynucleotide, circular RNA constructs and related pharmaceutical compositions disclosed herein comprise a synthetic IRES sequence comprising at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an IRES sequence in Table 2, or a fragment thereof. In some embodiments, the precursor RNA polynucleotide, circular RNA constructs and related pharmaceutical compositions disclosed herein comprise a synthetic IRES sequence comprising at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence in Table 2. Table 2: IRES Sequences
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[239] In some embodiments, the IRES comprises comparative IRES 1, wherein the comparative IRES 1 consists of a sequence of: TTTGCTCAGCGTAACTTCTCCGGGTTACGTGGAGACCAAAAGGCTACGGAGACTC GGGCTACGGCCCTGGAGCACCTAGGTGCTCCTAAAGACGTTAGAAGTTGTACAA ACTCGCCCAATAGGGCCCCCCAACCAGGGGGGTAGCGGGCAAGCACTTCTGTTT CCCCGGTATGATCTCATAGGCTGTACCCACGGCTGAAAGAGAGATTATCGTTACC CGCCTCACTACTTCGAGAAGCCCAGTAATGGTTCATGAAGTTGATCTCGTTGACC CGGTGTTTCCCCCACACCAGAAACCTGTGATGGGGGTGGTCATCCCGGTCATGGC GACATGACGGACCTCCCCGCGCCGGCACAGGGCCTCTTCGGAGGACGAGTGACA TGGATTCAACCGTGAAGAGCCTATTGAGCTAGTGTTGATTCCTCCGCCCCCGTGA ATGCGGCTAATCCCAACTCCGGAGCAGGCGGGCCCAAACCAGGGTCTGGCCTGT CGTAACGCGAAAGTCTGGAGCGGAACCGACTACTTTCGGGAAGGCGTGTTTCCTT TTATTTTTATCATGGCTTTTTATGGTGACAACTCCTGGTAGACGTTTTATTGCGTTT ATTGAGAGATTTCCAACAATTGAACAGACTAGAACCACTTGTTTTATCAAACCCT CACAGAATAAGATAACA. (SEQ ID NO: 25228) [240] As set forth in detail above, synthetic IRESs can be engineered to include at least one addition, deletion, or substitution in a nucleotide, domain, or motif, as compared to a naturally occurring IRES, to increase or reduce IRES activities. Nonlimiting examples of changes that can be engineered include, for example, truncating the 5’ and/or 3’ ends of an IRES, adding a spacer 5’ to an IRES, modifying the 6 nucleotides 5’ to the translation initiation site (Kozak sequence), changing alternative translation initiation sites, and creating chimeric/hybrid IRES sequences. In some embodiments, the synthetic IRES sequence in the 168
Attorney Docket No. 01318-0014-00PCT OR-043WO polynucleotide disclosed herein comprises one or more of these changes relative to a natural or native IRES. Additional additions, deletions, or substitutions as compared to naturally occurring IRESs are described above. [241] In some embodiments, the IRES is a synthetic IRES sequence comprising at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence selected from the sequences in Table 2 or a fragment thereof or a sequence selected from SEQ ID NOS: 1-2989 and 3282-3303 or a fragment thereof. In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the sequences in Table 2 or a sequence selected from SEQ ID NOS: 1-2989 and 3282-3303, or a fragment thereof. See also, e.g., PCT Application No. US2022/33091 (WO202261490), which is incorporated herein by reference in its entirety. [242] In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence of any one of SEQ ID NOS: 14067-24829 (GIRES-1 through GIRES-10762) or a fragment thereof. In some embodiments, the synthetic IRES comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence set forth in any one of SEQ ID NOS: 14067-24829 (GIRES-1 through GIRES-10762), or a fragment thereof, optionally barcoded with a barcode sequence selected from SEQ ID NOs: 3304-14066 (e.g., the IRES of SEQ ID NO: 14067 is barcoded with the barcode sequence of SEQ ID NO: 3304, the IRES of SEQ ID NO: 14068 is barcoded with the barcode sequence of SEQ ID NO: 3305, the IRES of SEQ ID NO: 14069 is barcoded with the barcode sequence of SEQ ID NO: 3306, and sequentially thereon). In some embodiments, the unmodified or naturally occurring TIE or IRES comprises a consensus sequence as set forth in the Table of Exemplary Consensus Sequences herein (SEQ ID NOs: 24867-24892), wherein N is any nucleotide (e.g., pursuant to IUPAC). In some embodiments, the naturally occurring TIE comprises at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, or at least 700 nucleotides (e.g., contiguous nucleotides) of said consensus sequence. 169
Attorney Docket No. 01318-0014-00PCT OR-043WO [243] Disclosed herein, in certain embodiments, is a circular RNA polynucleotide (oRNA), comprising a core functional element, and a pharmaceutically acceptable salt, buffer, diluent, or combination thereof; wherein the core functional element comprises a translation initiation element (TIE), wherein the TIE comprises a synthetic IRES comprising at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to a sequence having at least 85% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-2989, 3282-3303, and 14067-24829, or a fragment thereof. Disclosed herein, in certain embodiments, is a circular RNA polynucleotide (oRNA), comprising a core functional element, and a pharmaceutically acceptable salt, buffer, diluent, or combination thereof; wherein the core functional element comprises a translation initiation element (TIE), wherein the TIE comprises a synthetic IRES comprising at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence having at least 85% sequence identity to a sequence set forth in Table 2, or a fragment thereof. [244] Disclosed herein, in certain embodiments, is a circular RNA polynucleotide (oRNA), comprising a core functional element, and a pharmaceutically acceptable salt, buffer, diluent, or combination thereof; wherein the core functional element comprises a translation initiation element (TIE), wherein the TIE comprises a synthetic IRES that comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence having at least 85% sequence identity to a sequence set forth in any one of SEQ ID NOs: 793, 876, 1017, 1216, and 3291, wherein the oRNA is capable of expressing a therapeutic protein. [245] Disclosed herein, in certain embodiments, is a circular RNA polynucleotide (oRNA), comprising a core functional element, and a pharmaceutically acceptable salt, buffer, diluent, or combination thereof; wherein the core functional element comprises a translation initiation element (TIE), wherein the TIE comprises a synthetic IRES that comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence having at least 85% sequence identity to a sequence set forth in any one of SEQ ID NOs: 785, 823, 840, 857, 861, 862, 864, 983, 1023, 1168, 1169, 1171, 1179, 1192, 1284, 1287, 2285, 2742, 2777, 2778, 3283, 3290, 3293, and 3302, wherein the oRNA is capable of expressing a therapeutic protein. [246] Disclosed herein, in certain embodiments, is a circular RNA polynucleotide (oRNA), comprising a core functional element, and a pharmaceutically acceptable salt, buffer, diluent, or combination thereof; wherein the core functional element comprises a translation 170
Attorney Docket No. 01318-0014-00PCT OR-043WO initiation element (TIE), wherein the TIE a synthetic IRES that comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence having at least 85% sequence identity to a sequence set forth in any one of SEQ ID NOs: 75, 77, 137, 532, 566, 580, 648, 693, 752, 787, 791, 820, 839, 843, 852, 863, 871, 874, 922, 959, 984, 1015, 1026, 1041, 1047, 1059, 1068, 1134, 1177, 1178, 1180, 1189, 1193, 1198, 1263, 1276, 1280, 1282, 2601, 2615, 2616, 2617, 2618, 2627, 2667, 2681, 2746, 2758, 3284, 3285, 3289, 3292, 3294, 3295, 3296, 3297, 3298, 3299, and 3301, wherein the oRNA is capable of expressing a therapeutic protein. [247] In certain embodiments, the TIE comprises an internal ribosome entry site (IRES). In some embodiments, the IRES is a synthetic IRES that comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence of any one of SEQ ID NOS: 14067-24829 or a fragment thereof. In some embodiments, the IRES comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence of any one of the IRES sequences SEQ ID NOS: 14067-24829 or a fragment thereof. In some embodiments, the circular RNA disclosed herein comprises an IRES that comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence of any one of SEQ ID NOS: 14067-24829 or a fragment thereof. In some embodiments, the circular RNA disclosed herein comprises an IRES that comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence of any one of SEQ ID NOS: 14067-24829 or a fragment thereof. [248] In some embodiments, the IRES comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES a sequence set forth in any one of SEQ ID NOs: 793, 876, 1017, 1216, and 3291 or a fragment thereof. In some embodiments, the IRES comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence set forth in any one of SEQ ID NOs: 793, 876, 1017, 1216, and 3291 or a fragment thereof. In some embodiments, the circular RNA disclosed herein comprises an IRES comprising at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES 171
Attorney Docket No. 01318-0014-00PCT OR-043WO comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 793, 876, 1017, 1216, and 3291 or a fragment thereof. In some embodiments, the circular RNA disclosed herein comprises an IRES that comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence set forth in any one of SEQ ID NOs: 793, 876, 1017, 1216, and 3291 or a fragment thereof. In some embodiments, the IRES comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence set forth in any one of SEQ ID NOs: 785, 823, 840, 857, 861, 862, 864, 983, 1023, 1168, 1169, 1171, 1179, 1192, 1284, 1287, 2285, 2742, 2777, 2778, 3283, 3290, 3293, and 3302 or a fragment thereof. In some embodiments, the IRES comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence set forth in any one of SEQ ID NOs: 785, 823, 840, 857, 861, 862, 864, 983, 1023, 1168, 1169, 1171, 1179, 1192, 1284, 1287, 2285, 2742, 2777, 2778, 3283, 3290, 3293, and 3302 or a fragment thereof. In some embodiments, the circular RNA disclosed herein comprises an IRES that comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence set forth in any one of SEQ ID NOs: 785, 823, 840, 857, 861, 862, 864, 983, 1023, 1168, 1169, 1171, 1179, 1192, 1284, 1287, 2285, 2742, 2777, 2778, 3283, 3290, 3293, and 3302 or a fragment thereof. In some embodiments, the circular RNA disclosed herein comprises an IRES that comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence set forth in any one of SEQ ID NOs: 785, 823, 840, 857, 861, 862, 864, 983, 1023, 1168, 1169, 1171, 1179, 1192, 1284, 1287, 2285, 2742, 2777, 2778, 3283, 3290, 3293, and 3302 or a fragment thereof. In some embodiments, the IRES comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence set forth in any one of SEQ ID NOs: 75, 77, 137, 532, 566, 580, 648, 693, 752, 787, 791, 820, 839, 843, 852, 863, 871, 874, 922, 959, 984, 1015, 1026, 1041, 1047, 1059, 1068, 1134, 1177, 1178, 1180, 1189, 1193, 1198, 1263, 1276, 1280, 1282, 2601, 2615, 2616, 2617, 2618, 2627, 2667, 2681, 2746, 2758, 3284, 3285, 3289, 3292, 3294, 3295, 3296, 3297, 3298, 3299, and 3301 or a fragment thereof. In some embodiments, the IRES comprises at least one addition, deletion, or substitution of a domain, 172
Attorney Docket No. 01318-0014-00PCT OR-043WO motif, or nucleotide as compared to an IRES comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence set forth in any one of SEQ ID NOs: 75, 77, 137, 532, 566, 580, 648, 693, 752, 787, 791, 820, 839, 843, 852, 863, 871, 874, 922, 959, 984, 1015, 1026, 1041, 1047, 1059, 1068, 1134, 1177, 1178, 1180, 1189, 1193, 1198, 1263, 1276, 1280, 1282, 2601, 2615, 2616, 2617, 2618, 2627, 2667, 2681, 2746, 2758, 3284, 3285, 3289, 3292, 3294, 3295, 3296, 3297, 3298, 3299, and 3301 or a fragment thereof. In some embodiments, the circular RNA disclosed herein comprises an IRES that comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to set forth in any one of SEQ ID NOs: 75, 77, 137, 532, 566, 580, 648, 693, 752, 787, 791, 820, 839, 843, 852, 863, 871, 874, 922, 959, 984, 1015, 1026, 1041, 1047, 1059, 1068, 1134, 1177, 1178, 1180, 1189, 1193, 1198, 1263, 1276, 1280, 1282, 2601, 2615, 2616, 2617, 2618, 2627, 2667, 2681, 2746, 2758, 3284, 3285, 3289, 3292, 3294, 3295, 3296, 3297, 3298, 3299, and 3301 or a fragment thereof. In some embodiments, the circular RNA disclosed herein comprises an IRES that comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence set forth in any one of SEQ ID NOs: 75, 77, 137, 532, 566, 580, 648, 693, 752, 787, 791, 820, 839, 843, 852, 863, 871, 874, 922, 959, 984, 1015, 1026, 1041, 1047, 1059, 1068, 1134, 1177, 1178, 1180, 1189, 1193, 1198, 1263, 1276, 1280, 1282, 2601, 2615, 2616, 2617, 2618, 2627, 2667, 2681, 2746, 2758, 3284, 3285, 3289, 3292, 3294, 3295, 3296, 3297, 3298, 3299, and 3301 or a fragment thereof. [249] In some embodiments, the IRES comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence selected from SEQ ID NOs: 1-2983 and 3282-3287 or a fragment thereof. In some embodiments, the IRES comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 1-2983 and 3282-3287 or a fragment thereof. In some embodiments, the circular RNA disclosed herein comprises an IRES that comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 1-2983 and 3282-3287 or a fragment 173
Attorney Docket No. 01318-0014-00PCT OR-043WO thereof. In some embodiments, the circular RNA disclosed herein comprises an IRES that comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide as compared to an IRES comprising a sequence selected from SEQ ID NOs: 1-2983 and 3282- 3287 or a fragment thereof. [250] Modifications of IRES sequences are disclosed in further detail elsewhere herein to increase or reduce IRES activities, for example, by truncating the 5’ and/or 3’ ends of the IRES, adding a spacer 5’ to the IRES, modifying the 6 nucleotides 5’ to the translation initiation site (Kozak sequence), modification of alternative translation initiation sites, and creating chimeric/hybrid IRES sequences. In some embodiments, the IRES sequence in the circular RNA disclosed herein comprises one or more of these modifications relative to a naturally occurring IRES (e.g., SEQ ID NOS: 1-2989, 3282-3303, and 14067-24829). In some embodiments, the IRES sequence in the circular RNA disclosed herein comprises one or more of these modifications in a domain or motif relative to a native domain or motif. iv. IRES CONSENSUS SEQUENCES [251] In some embodiments, a TIE disclosed herein comprises a naturally occurring and/or synthetic IRES sequence and includes an IRES consensus sequence or synthetic IRES designed from the IRES consensus sequence. In some embodiments, the IRES comprises a consensus sequence as set forth in the Table of Exemplary Consensus Sequences, below, wherein N is any nucleotide (e.g., pursuant to IUPAC) or a synthetic IRES comprising at least one addition, deletion, or substitution of the consensus sequence. In some embodiments, the TIE or IRES comprises at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, or at least 700 nucleotides (e.g., contiguous nucleotides) of said consensus sequence. Table of Exemplary Consensus Sequences
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b. CODING AND NONCODING ELEMENT [252] In some embodiments, the intervening region and/or core functional element comprises one or more coding elements. In some embodiments, the intervening region and/or core functional element comprises a combination of coding and noncoding elements. In some embodiments, the intervening region and/or core functional element comprises noncoding elements, e.g., microRNA binding site, IRES transacting factor region, restriction site, a RNA editing region, structural or sequence element, a granule site, a zip code element, or an RNA trafficking element. In some embodiments, the coding region is a part of the core functional 182
Attorney Docket No. 01318-0014-00PCT OR-043WO element or intervening region located between the 5’ end and 3’ end of the linear precursor RNA polynucleotide and resultant circular RNA. [253] In some embodiments, the coding element comprises an expression sequence. In some embodiments, the coding element comprises a sequence encoding at least one therapeutic protein. In some embodiments, the coding element encodes two or more polypeptides. In some embodiments, the sequences encoding the two or more polypeptides are separated by a ribosomal skipping element or a nucleotide sequence encoding a protease cleavage site. In certain embodiments, the ribosomal skipping element encodes thosea-asigna virus 2A peptide (T2A), porcine teschovirus-1 2 A peptide (P2A), foot-and-mouth disease virus 2 A peptide (F2A), equine rhinitis A vims 2A peptide (E2A), cytoplasmic polyhedrosis vims 2A peptide (BmCPV 2A), or flacherie vims of B. mori 2A peptide (BmIFV 2A). Coding elements or regions and payloads are described in further detail elsewhere herein. [254] In some embodiments, the intervening region comprises at least one translation initiation element (TIE). TIEs are designed to allow translation efficiency of an encoded protein. In some embodiments, core functional elements comprising one or more coding elements will further comprise one or more TIEs. In some embodiments, a translation initiation element (TIE) comprises a synthetic TIE. In some embodiments, a synthetic TIE comprises aptamer complexes, synthetic IRES or other engineered TIEs capable of initiating translation of a linear RNA or circular RNA polynucleotide. In certain embodiments, the TIE provided herein comprise an internal ribosome entry site (IRES). In certain embodiments, the TIE provided herein comprise a viral or eukaryotic internal ribosome entry site (IRES) or a fragment or variant thereof. In certain embodiments, the IRES comprises one or more modified nucleotides compared to the wild-type viral IRES or eukaryotic IRES. See, e.g., PCT Application No. US2022/33091, which is incorporated herein by reference in its entirety. In some embodiments, the TIE comprises a synthetic IRES engineered to comprise at least one addition, deletion, or substitution in a nucleotide, domain, or motif, as compared to a naturally occurring IRES. As set forth in detail elsewhere herein, the synthetic IRES maintains or improves IRES expression and/or function and/or stability as compared to an unmodified or naturally occurring IRES or an IRES that otherwise does not comprise certain engineered modifications. In some embodiments, the synthetic IRES maintain IRES function as compared to an unmodified or a naturally occurring IRES. [255] In some embodiments, the intervening region comprises one or more noncoding elements. In some embodiments, the noncoding element comprises an untranslated region 183
Attorney Docket No. 01318-0014-00PCT OR-043WO (UTR) or fragment thereof. In some embodiments, the noncoding element is a natural 5ʹ UTR. In some embodiments, the noncoding element is a natural 3ʹ UTR. In some embodiments, the noncoding element is a synthetic spacer sequence. In some embodiments, the noncoding element is an aptamer. In some embodiments, the noncoding element is or comprises a translation initiation element (TIE). In some embodiments, the noncoding element comprises a lncRNA, miRNA, or a miRNA sponge. [256] In some embodiments, the intervening region comprises a TIE comprising an untranslated region (UTR) or a fragment thereof, an aptamer complex or a fragment thereof, or a combination thereof. In certain embodiments, the TIE contains modified nucleotides or nucleosides. [257] In some embodiments, the noncoding element comprises an untranslated region (UTR). In some embodiments, the noncoding element is a natural 5’ UTR. In some embodiments, the noncoding element is a natural 3’ UTR. In some embodiments, the noncoding element is a synthetic spacer sequence. In some embodiments, the noncoding element is an aptamer or synthetic aptamer. In some embodiments, the noncoding element is or comprises a translation initiation element (TIE). c. STOP CODON OR STOP CASSETTE [258] In various embodiments, the intervening region and/or core functional element comprises a stop codon or stop cassette. In some embodiments, the sequence is located downstream to a TIE and coding element. In some embodiments, the sequence is located downstream to a coding element and upstream to a TIE. In some embodiments, the intervening region comprises a stop codon. In one embodiment, the intervening region comprises a stop cassette. In some embodiments, the stop cassette comprises at least 2 stop codons. In some embodiments, the stop cassette comprises at least 2 frames of stop codons. In the same embodiment, the frames of the stop codons in a stop cassette each comprise 1, 2 or more stop codons. In some embodiments, the stop cassette comprises a LoxP or a RoxStopRox, or frt- flanked stop cassette. In the same embodiment, the stop cassette comprises a lox-stop-lox stop cassette. C. INTRON ELEMENTS, EXON ELEMENTS & TERMINAL ELEMENTS [259] Polynucleotides provided herein (e.g., DNA templates, precursor RNA polynucleotides, or circular RNA polynucleotides) may comprise one or more intron elements, exon elements, and/or terminal elements. In some embodiments, each intron element, exon 184
Attorney Docket No. 01318-0014-00PCT OR-043WO element, and terminal element may independently comprise one or more spacers, intron segments, exon segments, duplex regions, affinity sequences, and/or untranslated elements. These sequence elements within the intron elements, exon elements, or terminal elements are arranged to optimize circularization and/or protein expression. a. INTRON AND EXON ELEMENTS [260] In various embodiments, an intron element (e.g., 3’ intron element or 5’ intron element) comprises a permuted intron segment. In some embodiments, a 3’ permuted intron segment is a contiguous sequence at least 75% homologous (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous) to a 3’ proximal fragment of a natural intron (e.g., a group I or group II intron) including the 5’ nucleotide of the 3’ splice site dinucleotide. In some embodiments, a 5’ permuted intron fragment is a contiguous sequence at least 75% homologous (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous) to a 5’ proximal fragment of a natural intron (e.g., a group I or group II intron) including the 3’ nucleotide of the 5’ splice site dinucleotide. Exemplary splice site dinucleotides are described in the Table herein. [261] In some embodiments, an intron element comprises an intron derived from a trans- splicing ribozyme. In some embodiments, the intron element comprises a Group I trans- splicing ribozyme (e.g., a Tetrahymena trans-splicing ribozyme) segment. In some embodiments, the trans-splicing ribozyme segment along with an exon segment that may cleave a target site (e.g., a sequence of interest and/or a coding element) and subsequently ligate cleaved targe site to a 3’ exon to form a circular RNA product. [262] In various embodiments, a provided polynucleotide (e.g., a DNA template or a precursor RNA polynucleotide) comprises a 5’ exon element located upstream to the intervening region. In some embodiments, a provided polynucleotide comprises a 3’ intron element located downstream to the intervening region. In various embodiments, a provided polynucleotide comprises a 3’ exon element located upstream to the intervening region. In some embodiments, a provided polynucleotide comprises a 3’ intron element located upstream to the intervening region. [263] According to the present disclosure, the 3’ exon element and 5’ exon element each comprise an exon segment. In some embodiments, the 5’ exon element comprises a 3’ exon segment. In some embodiments, the 3’ exon element comprises a 5’ exon segment. In some embodiments, the 3’ and/or 5’ exon segment is a self-spliced or self-splicing exon segment. In some embodiments, the self-spliced and/or self-splicing exon segment comprises in part or in 185
Attorney Docket No. 01318-0014-00PCT OR-043WO whole a naturally occurring exon sequence from a virus, bacterium or eukaryotic DNA vector. In other embodiments, the self-spliced and/or self-splicing exon segment comprises in part or in whole a non-naturally occurring sequence. [264] In some embodiments, a 3’ exon segment is a contiguous sequence at least 75% homologous (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous) to the 5’-proximal end of an exon adjacent a 3’ intron segment as described herein, including the 3’ nucleotide of the splice site dinucleotide. In some embodiments, a 5’ exon segment is a contiguous sequence at least 75% homologous (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous) to the 3’-proximal end of an exon adjacent a 5’ intron segment as described herein, including the 5’ nucleotide of the splice site dinucleotide. [265] In some embodiments, at least one of the exon segments is less than 15 nucleotides in length. In some embodiments, the 3ʹ exon segment and/or 5ʹ exon segment comprises a Group I exon segment or a Group II exon segment less than 15 nucleotides in length. [266] In some embodiments, the circular RNA comprises a self-spliced exon segment that is 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, or 15 nucleotides. In some embodiments, the circular RNA comprises a self-spliced exon segment that is 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, or 15 nucleotides from the exonic sequences of Table A (in which sequences are shown as 15-nucleotide exonic sequence, intronic sequence, 15-nucleotide exonic sequence), e.g., contiguous nucleotides from the 5’ or 3’ end of the exonic sequences of Table A. In some embodiments, the circular RNA comprises a self-spliced exon segment that is 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides from the exonic sequences of Table B (in which sequences are shown as 10- nucleotide exonic sequence, intronic sequence, 10-nucleotide exonic sequence), e.g., contiguous nucleotides from the 5’ or 3’ end of the exonic sequences of Table B. [267] In some embodiments, the intron segment is a Group I intron and the exon segment comprises a Group I self-splicing exon segment. In some embodiments, the intron segment is a Group II intron and the exon segment comprises a Group II self-splicing exon segment. [268] In some embodiments, the exon element comprises a sequence directed to a native Group I intron-adjacent exon segment sequence or Group II intron-adjacent exon segment sequence, or fragment thereof. In some embodiments, the exon element comprises at least one 186
Attorney Docket No. 01318-0014-00PCT OR-043WO mutation of a native Group I intron-adjacent exon segment sequence or Group II intron- adjacent exon segment sequence, or fragment thereof. In some embodiments, the exon element comprises at least one deletion of a native Group I intron-adjacent exon segment sequence or Group II intron-adjacent exon segment sequence, or fragment thereof. In some embodiments, the exon element comprises at least one insertion of a native Group I intron-adjacent exon segment sequence or Group II intron-adjacent exon segment sequence, or fragment thereof. In some embodiments, the native Group I intron segment or Group II intron segment sequences are selected from a sequence in Table A or Table B, below. b. TERMINAL ELEMENTS [269] In various embodiments, a provided polynucleotide (e.g., a DNA template or a precursor RNA polynucleotide) comprises a terminal element. In some embodiments, the terminal element is located upstream to the intervening region. In some embodiments, the terminal element is non-intronic. In some embodiments, the terminal element lacks one or both nucleotides of a natural splice site dinucleotide associated with a natural Group I or Group II intron sequence. In some embodiments, a portion or the entire terminal element is excised after circularization of a precursor RNA polynucleotide comprising said terminal element. [270] In some embodiments, a polynucleotide comprises a terminal element, an intervening region, and a monotron. In some embodiments, the polynucleotide comprises, in the following order, a terminal element, an intervening region, and a monotron. In some embodiments, the polynucleotide comprises, in the following order, a monotron, an intervening region, and a terminal element. In some embodiments, the terminal element comprises a splice site nucleotide capable of engaging in a transesterification reaction with the monotron. [271] In some embodiments, the terminal element comprises an excised terminal segment and a retained terminal segment. In the same embodiments, the retained terminal segment is retained after circularization of a precursor RNA polynucleotide comprising such a terminal element. In the same embodiments, the exercised terminal segment is not retained after circularization of a precursor RNA polynucleotide comprising such a terminal element. In still the same embodiments, the nucleotide sequence of the terminal element is non-natural or synthetic. [272] In some embodiments, the terminal element comprises a natural exon or a fragment thereof. In some embodiments, the terminal element is retained after circularization of a precursor RNA polynucleotide comprising said terminal element. [273] In some embodiments, the terminal element is capable of binding to a 3’ intron 187
Attorney Docket No. 01318-0014-00PCT OR-043WO element (e.g., the 3’ intron element comprised in the same polynucleotide). In some embodiments, the terminal element is capable of directing or functionalizing the splicing activity of a 3’ intron element (e.g., the 3’ intron element comprised in the same polynucleotide). c. EXEMPLARY INTRON ELEMENTS, EXON ELEMENTS & TERMINAL ELEMENTS [274] For means of example and not intended to be limiting, in some embodiments, a 5’ intron element comprises, in the following 5’ to 3’ order: a 5’ leading sequence, an optional 5’ external duplex, a 5’ affinity tag, a 5’ external spacer, and a 3’ permuted intron segment. In the same embodiments, the 5’ exon element comprises, in the following 5’ to 3’ order: a 3’ exon segment, an optional 5’ internal duplex, and a 5’ internal spacer. In the same embodiments, the 3’ exon element comprises, in the following 5’ to 3’ order: a 3’ internal spacer, an optional 3’ internal duplex, and a 5’ exon segment. In still the same embodiments, the 3’ intron element comprises, in the following 5’ to 3’ order: a 5’ permuted intron segment, a 3’ external spacer, an optional 3’ external duplex, a 3’ affinity tag, and a 3’ lagging sequence. [275] As another exemplary embodiment, a terminal element comprises, in the following 5’ to 3’ order: a 5’ leading sequence, a 5’ external spacer, an excised terminal segment, a retained terminal segment, an optional 5’ internal duplex, and a 5’ internal spacer. In the same embodiments, the 3’ exon element comprises, in the following 5’ to 3’ order: a 3’ internal spacer, an optional 3’ internal duplex, and a 5’ exon segment. In still the same embodiments, the 3’ intron element comprises, in the following 5’ to 3’ order: a 5’ permuted intron segment, a 3’ external spacer, an optional 3’ external duplex, and a 3’ lagging sequence. [276] In some embodiments, the terminal element sequence has a percent sequence identity of about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more to an exon fragment of a sequence selected from Tables A or B. [277] For means of example and not intended to be limiting, in some embodiments, a 3’ intron element comprises in the following 5’ to 3’ order: a leading untranslated sequence, a 5’ affinity tag, an optional 5’ external duplex region, a 5’ external spacer, and a 3’ intron fragment. In the same embodiments, the 3’ exon element comprises in the following 5’ to 3’ order: a 3’ exon fragment, an optional 5’ internal duplex region, an optional 5’ internal duplex region, and a 5’ internal spacer. In the same embodiments, the 5’ exon element comprises in the following 5’ to 3’ order: a 3’ internal spacer, an optional 3’ internal duplex region, and a 5’ exon fragment. In still the same embodiments, the 3’ intron element comprises in the following 5’ to 3’ order: a 5’ intron fragment, a 3’ external spacer, an optional 3’ external duplex region, a 3’ affinity 188
Attorney Docket No. 01318-0014-00PCT OR-043WO tag, and a trailing untranslated sequence. In some embodiments, the affinity tag is a polyA affinity tag. [278] In some embodiments, the 5ʹ intron element is located 5ʹ to the 5ʹ exon element. In some embodiments, the 5ʹ intron element is adjacent to the 5ʹ exon element. In some embodiments, the 3ʹ intron element is located 3ʹ to the 3ʹ exon element. In some embodiments, the 3ʹ intron element is adjacent to the 3ʹ exon element. [279] In some embodiments, the 5ʹ exon element comprises a 5ʹ internal duplex sequence located 3ʹ to the 3ʹ exon segment. In some embodiments, the 3ʹ exon element comprises a 3ʹ internal duplex sequence located 5ʹ to the 5ʹ exon segment. In some embodiments, the 5ʹ intron element comprises a 5ʹ external duplex sequence located 5ʹ to the 3ʹ permuted intron segment. In some embodiments, the 3ʹ intron element comprises a 3ʹ external duplex sequence located 3ʹ to the 5ʹ permuted intron segment. In some embodiments, the 5ʹ intron element is adjacent to the 5ʹ exon element. In some embodiments, the 3ʹ intron element is located 3ʹ to the 3ʹ exon element. In some embodiments, the 3ʹ intron element is adjacent to the 3ʹ exon element. [280] In some embodiments, the 5ʹ intron comprises a 5ʹ affinity tag, a 5ʹ external spacer, and the 3ʹ permuted intron segment. In some embodiments, the 5ʹ exon comprises the 3ʹ exon segment, a 5ʹ internal duplex sequence, and a 5ʹ internal spacer. In some embodiments, the 5ʹ affinity tag is adjacent to the 5ʹ external spacer. In some embodiments, the 5ʹ affinity tag is located 5ʹ to the 5ʹ external spacer. In some embodiments, the 5ʹ internal duplex sequence is adjacent to the 5ʹ internal spacer. In some embodiments, the 5ʹ internal duplex sequence is located 5ʹ to the 5ʹ internal spacer. In some embodiments, the 3ʹ exon comprises a 3ʹ internal spacer, 3ʹ internal duplex sequence, and the 5ʹ exon segment. In some embodiments, the 3ʹ intron comprises the 5ʹ permuted intron segment, a 3ʹ external spacer, and a 3ʹ affinity tag. In some embodiments, the 3ʹ affinity tag is adjacent to the 3ʹ external spacer. In some embodiments, the 3ʹ affinity tag is located 3ʹ to the 3ʹ external spacer. In some embodiments, the 3ʹ internal duplex sequence is adjacent to the 3ʹ internal spacer. In some embodiments, the 3ʹ internal duplex sequence is located 3ʹ to the 3ʹ internal spacer. In some embodiments, the affinity tag is a polyA affinity tag. [281] In some embodiments, the 5ʹ exon comprises a 5ʹ internal duplex sequence located between the 3ʹ exon segment and the intervening region. In some embodiments, the 3ʹ exon comprises a 3ʹ internal duplex sequence positioned between the intervening region and the 5ʹ exon segment. In some embodiments, the polynucleotide comprises a 5ʹ internal duplex sequence and a 3ʹ internal duplex sequence. 189
Attorney Docket No. 01318-0014-00PCT OR-043WO [282] In some embodiments, the 3ʹ and 5ʹ permuted intron segments each independently comprise a Group I intron segment, a Group II intron segment, a synthetic intron segment, or a variant thereof. In some embodiments, the 3ʹ permuted intron segment comprises a 3ʹ Group I intron segment or a variant thereof. In some embodiments, the 5ʹ permuted intron segment comprises a 5ʹ Group I intron segment or a variant thereof. In some embodiments, the 3ʹ permuted intron segment comprises a 3ʹ Group II intron segment or a variant thereof. In some embodiments, the 5ʹ permuted intron segment comprises a 5ʹ Group II intron segment or a variant thereof. [283] In some embodiments, the 3ʹ permuted intron segment or element, 5ʹ permuted intron segment or element, or both the 3ʹ and 5ʹ permuted intron segments or elements are at least 100, at least 90, at least 80, at least 70, at least 60, and/or at least 50 nucleotides in length. In some embodiments, the 3ʹ permuted intron element, 5ʹ permuted intron element, or both the 3ʹ and 5ʹ permuted intron elements are at least 50 nucleotides in length. In some embodiments, the 3ʹ permuted intron element, 5ʹ permuted intron element, or both the 3ʹ and 5ʹ permuted intron elements have a percent sequence identity of about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more to a naturally occurring intron. [284] In some embodiments, the 3ʹ permuted intron element, 5ʹ permuted intron element, or both the 3ʹ and 5ʹ permuted intron elements comprise a native Group I intron segment or Group II intron segment sequence. In some embodiments, the 3ʹ permuted intron element, 5ʹ permuted intron element, or both the 3ʹ and 5ʹ permuted intron elements comprise one or more nucleotide substitutions of a native Group I intron segment or Group II intron segment sequence. In some embodiments, the 3ʹ permuted intron element, 5ʹ permuted intron element, or both the 3ʹ and 5ʹ permuted intron elements comprise one or more nucleotide insertions of a native Group I intron segment or Group II intron segment sequence. In some embodiments, the 3ʹ permuted intron element, 5ʹ permuted intron element, or both the 3ʹ and 5ʹ permuted intron elements comprise one or more nucleotide deletions of a native Group I intron segment or Group II intron segment sequence. In some embodiments, the 3ʹ permuted intron element, 5ʹ permuted intron element, or both the 3ʹ and 5ʹ permuted intron elements comprise a nucleotide substitution of one or both the dinucleotide of a native Group I or Group II intron splice site dinucleotide. In some embodiments, the 3ʹ Group I or Group II intron segment or the 5ʹ Group I or Group II intron segment comprises one, two, three, four, five, six, seven, eight, nine, ten, or more mutations of a native Group I intron or Group II intron sequence. In some embodiments, the mutations are selected from insertion, deletion, mutation, addition, and 190
Attorney Docket No. 01318-0014-00PCT OR-043WO subtraction. In some embodiments, the mutations are deletions of two or more nucleotides of the 3ʹ Group I or Group II intron segment or the 5ʹ Group I or Group II intron segment, or combinations thereof. In some embodiments, the mutations are two or more deletions of the 5ʹ Group I intron segment at the 3ʹ end or two or more deletions of the 3ʹ Group I intron segment at the 5ʹ end. [285] In some embodiments, the native Group I intron segment or Group II intron segment sequences are selected from a sequence in Table A or Table B, below. In some embodiments, the 3ʹ and/or 5ʹ permuted intron element comprise a polynucleotide sequence having a percent sequence identity of about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more to a naturally occurring intron selected from a sequence set forth in Table A or Table B, below, or a fragment or segment thereof. In some embodiments, the 3ʹ and/or 5ʹ permuted intron element comprise a polynucleotide sequence selected from a sequence set forth in Table A or Table B. In some embodiments, the 3ʹ and/or 5ʹ permuted intron element comprise a polynucleotide sequence selected from a sequence set forth in Table A or Table B. [286] In some embodiments, the 3ʹ permuted intron segment comprises a 3ʹ Group I or Group II intron segment derived from a gene selected from a genus and/or species selected from column 2 of Tables A or B; and/or the 5ʹ permuted intron segment comprises a 5ʹ Group I or Group II intron segment derived from a gene selected from a genus and/or species selected from column 2 of Tables A or B. [287] In some embodiments, the 3ʹ Group I or Group II intron segment or the 5ʹ Group I or Group II intron segment are derived from a gene selected from a species selected from: Cyanobacterium Anabaena sp., T4 phage, Hypocrea pallida, Bulbithecium hyalosporum, Myoarachis inversa, Geosmithia argillacea, Coxiella burnetii, Agrobacterium tumefaciens, Azoarcus, Nostoc, Cordyceps capitata, Prochlorothrix hollandica, Tilletiopsis orzyzicola, Tetrahymena thermophila, and Staphylococcus phage Twort. Table A: Group I introns (flanked by 15nt exons)
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Table B: Group II introns (flanked by 10nt exons)
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[288] In some embodiments, the 3ʹ or 5’ intron segments and/or 3’ or 5’ exon segments are derived from a gene selected from a species selected from: Cyanobacterium Anabaena sp., T4 phage, Coxiella burnetii, Azoarcus, Tetrahymena thermophila, and Staphylococcus phage Twort. In some embodiments, the 3’ or 5’ intron segment and/or 3’ or 5’ exon segment are developed from permuting at a position along a Cyanobacterium Anabaena sp., T4 phage, Coxiella burnetii, Azoarcus, Tetrahymena thermophila, and Staphylococcus phage Twort intron and/or exon sequence. In some embodiments, the 5’ or 3 monotron element are derived from a gene selected from a species selected from: Cyanobacterium Anabaena sp., T4 phage, 229
Attorney Docket No. 01318-0014-00PCT OR-043WO Coxiella burnetii, Azoarcus, Tetrahymena thermophila, and Staphylococcus phage Twort. In some embodiments, the 3’ or 5’ intron segment and/or 3’ or 5’ exon segment are developed from permuting at a position along a Cyanobacterium Anabaena sp., T4 phage, Coxiella burnetii, Azoarcus, Tetrahymena thermophila, and Staphylococcus phage Twort intron and/or exon sequence. [289] In some embodiments, the intron segments and/or exon segments of a provided polynucleotide are derived from a gene from the same species (e.g., a polynucleotide comprises Azoarcus 3’ and 5’ exon segments and Azoarcus 3’ and 5’ intron segments). In other embodiments, the 3’ or 5’ intron segments or 3’ or 5’ exon segments of a provided polynucleotide are derived from genes of different species (e.g., a polynucleotide comprises an Anabaena intron segment and Staphylococcus phage Twort exon segment). In certain embodiments, the monotron element of a provided polynucleotide is derived from a gene of a different species than the 3’ or 5’ intron segments and/or 3’ or 5’ exon segments (e.g., a polynucleotide comprises a Staphylococcus phage Twort montron element and an Anabaena intron segment). In some embodiments, use of genes of one species of an intron segment and/or exon segment may allow for more efficient or effective circularization or self-splicing of one or more polynucleotides as compared to another gene of a different species. In certain embodiments, the gene used of one species develop an intron segment may more efficiently promote the interaction between an intron segment and a nucleophile (e.g., form a more efficient or effective binding pocket that promotes the transesterification reaction of a splice site nucleotide) as compared to an intron segment developed from a gene of a different species. In some embodiments, the gene of one species from which an intron segment is derived may be more efficient in forming a binding pocket for a nucleophile as compared to a different gene of the same species. In some embodiments, the species of gene from which the intron segment is derived may be more efficient in forming a binding pocket for a nucleophile as compared to a species of genes comprising the same and/or homologous sequence from a different species. [290] As described herein, in some embodiments, a provided polynucleotide comprises an intron segment and/or exon segment derived from permuting at a position along a Group I or Group II gene selected from Table A or Table B. Location or position of the permutation sites may enhance the ability of an intron segment to effectively splice and/or circularize in a provided polynucleotide. In some embodiments, the Group I or Group II genes are permuted at a position that enhances splicing or circularization activity of an intron segment of a provided polynucleotide as compared to a different permutation site. In certain embodiments, the Group 230
Attorney Docket No. 01318-0014-00PCT OR-043WO I or II genes are permuted at a position in an intron segment of a provided polynucleotide that enhances the provided polynucleotide’s ability to self-circularize as compared to a different permutation site. In some embodiments, the Group I or II genes are permuted at a position that enhances or promotes the splicing activity of an intron segment to another intron segment, monotron element and/or exon segment. In some embodiments, the Group I or II genes are permuted at a position that allows the intron segment to more efficiently splice or self-splice than an intron segment permuted at a different position. In certain embodiments, a position of a permutation site may promote the interaction between an intron segment and a nucleophile (e.g., form a more efficient or effective binding pocket that promotes the transesterification reaction of a splice site nucleotide). [291] As described herein, permutation sites positions are described to mean that the permutation of the natural or synthetic intron occurs at the junction between the listed amino acid and the adjacent downstream amino acid (e.g., an Anabaena position 189 permutation site corresponds herein to a permutation site between amino acids 189 and 190). [292] In some embodiments, a provided polynucleotide comprises an intron segment derived from permuting at a position along a gene selected from a species selected from: Cyanobacterium Anabaena sp., T4 phage, Coxiella burnetii, Azoarcus, Tetrahymena thermophila, and Staphylococcus phage Twort. In certain embodiments, a provided polynucleotide comprises an intron segment derived from permuting a Cyanobacterium Anabaena sp. gene. In these embodiments, the permutation site of the Cyanobacterium Anabaena sp. gene may be downstream relative to amino acid positions 1, 2, 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 231
Attorney Docket No. 01318-0014-00PCT OR-043WO 260, 261, 262, 263, 264, or 265 of the Cyanobacterium Anabaena sp. gene. In certain embodiments, a provided polynucleotide comprises an intron segment derived from permuting an Azoarcus gene. In these embodiments, the permutation site of the Azoarcus gene may be downstream relative to amino acid positions 1, 2, 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, or 221 of the Azoarcus gene. In certain embodiments, a provided polynucleotide comprises an intron segment derived from permuting an Coxiella burnetii gene. In these embodiments, the permutation site of the Coxiella burnetii gene may be downstream relative to amino acid positions 1, 2, 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 232
Attorney Docket No. 01318-0014-00PCT OR-043WO 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, or 390 of the Coxiella burnetii gene. In certain embodiments, a provided polynucleotide comprises an intron segment derived from permuting a Tetrahymena thermophila gene. In these embodiments, the permutation site of the Tetrahymena thermophila gene may be downstream relative to amino acid positions 1, 2, 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, or 436 of a Tetrahymena thermophila gene. In certain embodiments, a provided polynucleotide comprises an intron segment derived from permuting an T4 phage (td) gene. In these embodiments, the permutation site of the T4 phage (td) gene may be downstream relative to amino acid positions 1, 2, 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, 50, 51, 52, 53, 233
Attorney Docket No. 01318-0014-00PCT OR-043WO 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, or 289 of the T4 phage (td) gene. In certain embodiments, a provided polynucleotide comprises an intron segment derived from permuting a Staphylococcus phage Twort gene. In these embodiments, the permutation site of the Staphylococcus phage Twort gene may be downstream relative to amino acid positions 1, 2, 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, or 281 of the Staphylococcus phage Twort gene. [293] Also provided herein are methods of identifying an exon and/or intron element or identifying a combined accessory element comprising a mutated Group I or Group II exon and/or intron segment (as described herein) that allows production of a circular RNA that is 234
Attorney Docket No. 01318-0014-00PCT OR-043WO translatable or biologically active inside a eukaryotic cell. In some embodiments, such a method comprises: (i) inserting 5ʹ and 3ʹ Group I or Group II intronic sequences derived from a database of native intronic sequence into a precursor RNA polynucleotide; (ii) transcribing the polynucleotide into RNA in vitro or allowing the polynucleotide to be transcribed into RNA by a cell; and (iii) determining the circularization efficiency of the RNA produced by the polynucleotide by identifying the amount of circularized RNA, the amount of excised intronic sequences, the amount of precursor RNA remaining after circularization, and combinations thereof. [294] In some embodiments, the mutated Group I or Group II exon and/or intron element or segment comprises a deletion, insertion or substitution of at least one nucleotide, including but not limited to a nucleotide substitution of one or both the dinucleotides of the 5ʹ and/or 3ʹ Group I splice site dinucleotides. In some embodiments, the 5ʹ or 3ʹ Group I or Group II intronic sequences, or combinations thereof are sequenced. In some embodiments, the method further comprises comparing the circularization efficiency of the polynucleotide with a polynucleotide comprising a native intronic sequence, or a parent polynucleotide. [295] Also provided herein are methods of identifying or determining a polynucleotide sequence that improves RNA circularization efficiency compared to a polynucleotide comprising a native intronic sequence or to a parent polynucleotide with a known sequence, the method comprising modifying a DNA sequence encoding the precursor RNA polynucleotide described herein comprising: (i) modifying at least one nucleotide and/or altering the length of the 5ʹ intron element and/or 3ʹ intron element of the DNA sequence encoding the precursor RNA polynucleotide described herein; (ii) altering the length of the 5ʹ and/or 3ʹ internal and/or external spacer sequence of the DNA sequence encoding precursor RNA polynucleotide; (iii) altering the length of the 5ʹ and/or 3ʹ internal duplex sequence of the DNA sequence encoding the precursor RNA polynucleotide; (iv) altering the length of the 5ʹ and/or 3ʹ exon sequence of the DNA sequence encoding the precursor RNA polynucleotide; or (v) combinations thereof; and 235
Attorney Docket No. 01318-0014-00PCT OR-043WO transcribing the polynucleotide comprising the DNA sequence into RNA in vitro or allowing the polynucleotide comprising the DNA sequence to be transcribed into RNA by a cell; and determining the circularization efficiency of the RNA produced by the polynucleotide comprising the DNA sequence by identifying the amount of circularized RNA, the amount of excised intronic sequences, the amount of precursor RNA remaining after circularization, and combinations thereof. In some embodiments, the method further comprises comparing the circularization efficiency of the polynucleotide with a polynucleotide comprising a native intronic sequence, or a parent polynucleotide. d. SPACER [296] In various embodiments, a provided polynucleotide (e.g., a DNA template, a precursor RNA polynucleotide, or a circular RNA polynucleotide) comprises one or more spacers. [297] In certain embodiments, the DNA template, precursor linear RNA polynucleotide and circular RNA provided herein comprise a 5’ and/or a 3’ spacer. In some embodiments, the polynucleotide comprises one or more spacers in the intron elements. In some embodiments, the polynucleotide comprises one or more spacers in the exon elements. In some embodiments, the polynucleotide comprises a spacer in the 3’ intron fragment (also referred to as “5’ external spacer”). In some embodiments, the polynucleotide comprises a spacer in the 5’ intron fragment (also referred to as “3’ external spacer”). In some embodiments, the polynucleotide comprises a spacer in the 3’ exon fragment (also referred to as “5’ internal spacer”). In some embodiments, the polynucleotide comprises a spacer in the 5’ exon fragment (also referred to as “3’ internal spacer”). [298] In certain embodiments, the polynucleotide comprises a spacer in the 3’ intron fragment and/or a spacer in the 5’ intron fragment. In some embodiments, the 5ʹ external spacer is located 5ʹ to the 3ʹ permuted intron segment. In some embodiments, the 5ʹ internal spacer is located 3ʹ to the 3ʹ exon segment. In some embodiments, the 3ʹ external spacer is located 3ʹ to the 5ʹ permuted intron segment. In some embodiments, the 3ʹ external spacer is located 5ʹ to the 5ʹ exon segment. [299] In certain embodiments, the polynucleotide comprises a 5ʹ external spacer located between a leading untranslated sequence and the 5ʹ or 3ʹ intron element. In certain embodiments, the polynucleotide comprises a 3ʹ external spacer located between the 3ʹ or 5ʹ intron element and a lagging untranslated sequence. [300] In certain embodiments where the polynucleotide comprises a monotron, the 236
Attorney Docket No. 01318-0014-00PCT OR-043WO polynucleotide can comprise an internal spacer sequence positioned between the terminal element and the intervening region, and/or between the intervening region and the monotron element. In certain embodiments, the polynucleotide can comprise an external spacer positioned adjacent to the terminal element and/or an external spacer positioned adjacent to the monotron element. [301] In certain embodiments, the spacers aid with circularization or protein expression due to symmetry created in the overall sequence of the precursor RNA polynucleotide. In certain embodiments, including a 5’ internal spacer and/or including a spacer between the 3’ group I intron fragment and the intervening region may conserve secondary structures in those regions by preventing them from interacting, thus increasing splicing efficiency. In certain embodiments, there is a spacer, for example, between the 3’ permuted intron segment and the intervening region, wherein the spacer may prevent structured regions of an IRES or aptamer of a TIE comprised in the intervening region from interfering with the folding of the 3’ permuted intron segment or reduces the extent to which this occurs. [302] In some embodiments, the polynucleotide further comprises an aptamer. In some embodiments, the aptamer is synthetic. [303] In some embodiments, the first spacer (e.g., between the 3’ group I or II intron fragment and intervening region) and second spacer (e.g., between the two expression sequences and intervening region) comprise additional base pairing regions that are predicted to base pair with each other and not to the first and second duplex regions. In other embodiments, the first spacer (e.g., between 3’ group I or II intron fragment and intervening region) and second spacer (e.g., between the one of the intervening region and 5’ group I or II intron fragment) comprise additional base pairing regions that are predicted to base pair with each other and not to the first and second duplex regions. [304] In certain embodiments, the polynucleotide comprises a first (5’) and a second (3’) spacer. In some embodiments, the polynucleotide comprises a 5’ external spacer and a 3’ external spacer, wherein the spacers comprise additional base pairing regions that are predicted to base pair with each other and not to the first and second duplex regions. In other embodiments, the polynucleotide comprises a 5’ internal spacer and a 3’ internal spacer, wherein the spacers comprise additional base pairing regions that are predicted to base pair with each other and not to the first and second duplex regions. In some embodiments, such spacer base pairing brings the permuted intron segments in close proximity to each other, which may increase splicing efficiency. Additionally, in some embodiments, the combination of base 237
Attorney Docket No. 01318-0014-00PCT OR-043WO pairing between the first and second duplex regions, and separately, base pairing between the first and second spacers, promotes the formation of a splicing bubble containing the permuted intron segments flanked by adjacent regions of base pairing. [305] Typical spacers are contiguous sequences with one or more of the following qualities: (1) predicted to avoid interfering (e.g., forming duplex) with proximal structures, for example, the IRES, expression sequence, aptamer, or intron; (2) is at least 5 nt long and no longer than 100 nt; (3) is located adjacent to the permuted intron segment; and (4) contains one or more of the following: (a) an unstructured region at least 5 nt long, (b) a region of base pairing at least 5 nt long to a distal sequence, such as another spacer, and (c) a structured region at least 5 nt long limited in scope to the sequence of the spacer. In various embodiments, a spacer is not predicted to form a duplex of more than 8 nucleotides in length with any sequences within 250 nucleotides in either direction. In some embodiments, the spacer is not predicted to form a duplex of more than 8 nucleotides in length with any sequences within 1000 nucleotides in either direction. In some embodiments, the spacer comprises an unstructured, structured or randomly generated polynucleotide sequence. Spacers may have several regions, including an unstructured region, a base pairing region, a hairpin/structured region, and combinations thereof. In an embodiment, the spacer has a structured region with high GC content. In an embodiment, a region within a spacer base pairs with another region within the same spacer. In an embodiment, a region within a spacer base pairs with a region within another spacer. In an embodiment, a spacer comprises one or more hairpin structures. In an embodiment, a spacer comprises one or more hairpin structures with a stem of 4 to 12 nucleotides and a loop of 2 to 10 nucleotides. [306] In some embodiments, a spacer sequence is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides in length. In some embodiments, a spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides in length. In some embodiments, a spacer sequence is between 5 and 50, 10 and 50, 20 and 50, 20 and 40, and/or 25 and 35 nucleotides in length. In certain embodiments, a spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In some embodiments, the spacer sequence is at least 5 nucleotides in length, and/or about 5 to about 60 nucleotides in length. [307] In some embodiments, a spacer sequence is a polyA sequence. In some embodiments, a spacer sequence is a polyAC sequence. In some embodiments, a spacer 238
Attorney Docket No. 01318-0014-00PCT OR-043WO comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polyAC content. In some embodiments, a spacer comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polypyrimidine (C/T or C/U) content. e. DUPLEX [308] In various embodiments, a provided polynucleotide (e.g., a DNA template, a precursor RNA polynucleotide, or a circular RNA polynucleotide) comprises one or more duplexes. [309] In some embodiments, the polynucleotide comprises a 5’ external duplex located within the 3’ intron fragment. In some embodiments, the polynucleotide comprises a 3’ external duplex located within the 5’ intron fragment. In some embodiments, the polynucleotide comprises a 5ʹ internal duplex sequence and a 3ʹ internal duplex sequence. In some embodiments, the polynucleotide comprises a 5’ internal duplex located within the 3’ exon fragment. In some embodiments, the 5ʹ internal duplex sequence is positioned between the 5ʹ exon element and the intervening region. In some embodiments, the polynucleotide comprises a 3’ internal duplex located within the 5’ exon fragment. In some embodiments, the 3ʹ internal duplex sequence is positioned between the intervening region and the 3ʹ exon element. In certain embodiments, the polynucleotide comprises a 5’ external duplex located within the 3’ intron fragment and a 3’ external duplex located within the 5’ intron fragment. In some embodiments, the polynucleotide comprises a 5’ internal duplex located within the 3’ exon fragment and a 3’ internal duplex located within the 5’ exon fragment. In some embodiments, the polynucleotide comprises a 5’ external duplex, 5’ internal duplex, a 3’ internal duplex region, and a 3’ external duplex. [310] In some embodiments, the polynucleotide comprises a monotron element, intervening region, and terminal element, and a 5ʹ internal duplex sequence and a 3ʹ internal duplex sequence. In some embodiments, if the terminal element is upstream of the monotron element, the 5ʹ internal duplex sequence is positioned between the terminal element and the intervening region, and the 3ʹ internal duplex sequence is positioned between the intervening region and the monotron element. In some embodiments, if the monotron element is upstream of the terminal element, the 5ʹ internal duplex sequence is positioned between monotron and the intervening region, and the 3ʹ internal duplex sequence is positioned between the intervening region and the terminal element. In some embodiments, the 5’ or 3’ internal duplex is positioned adjacent to a 5’ or 3’ internal spacer. 239
Attorney Docket No. 01318-0014-00PCT OR-043WO [311] In some embodiments, the polynucleotide comprises a first (5’) duplex and a second (3’) duplex (e.g., a 5’ external duplex region and a 3’ external duplex region). In certain embodiments, the first and second duplex regions may form perfect or imperfect duplexes. Thus, in certain embodiments, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the first duplex and second duplex may be base paired with one another. In some embodiments, the duplexes regions are predicted to have less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%) base pairing with unintended sequences in the RNA (e.g., non-duplex sequences). In some embodiments, the 5ʹ internal duplex sequence and 3ʹ internal duplex sequence are at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary. In some embodiments, including such first duplex and second duplex on the 5’ and 3’ ends of the precursor RNA strand, respectively, and adjacent or very close to the permuted intron segment, bring the permuted intron segments in close proximity to each other, increasing splicing efficiency. [312] In some embodiments, a duplex, whether, e.g., a 5’ internal duplex sequence or 3’ internal duplex sequence, is 3-100 nt in length (e.g., 3-75 nt in length, 3-50 nt in length, 20-50 nt in length, 35-50 nt in length, 5-25 nt in length, 5-20 nt in length, 9-19 nt in length). In some embodiments, a duplex has a length of about 9 to about 50 nt. In one embodiment, a duplex has a length of about 9 to about 19 nt. In one embodiment, a duplex has a length of about 5 to about 20 nt nucleotides in length, inclusive. In one embodiment, the 5ʹ internal duplex sequence and 3ʹ internal duplex sequence are each independently about 9 to about 50 nt, about 9 to about 19 nt, or about 5 to about 20 nt nucleotides in length, inclusive. In one embodiment, a duplex has a length of about 20 to about 40 nt. In some embodiments, a duplex 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 nt in length. In certain embodiments, a duplex has a length of about 30 nt. In certain embodiments, the 5' and 3' internal duplex sequences are predicted to form a contiguous duplex. In some embodiments, the contiguous duplex has a length of 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 nt in length. In some embodiments, the contiguous duplex has a length of no longer than 35 nucleotides. In some embodiments, at least one of the exon segments is less than 15 nucleotides in length. [313] In some embodiments, the 5ʹ internal duplex sequence and/or 3ʹ internal duplex 240
Attorney Docket No. 01318-0014-00PCT OR-043WO sequence each have a GC content of at least 10%. [314] In other embodiments, the polynucleotide does not comprise of any duplex to optimize translation or circularization. f. AFFINITY SEQUENCES [315] In various embodiments, a provided polynucleotide (e.g., a DNA template, a precursor linear RNA polynucleotide, or a circular RNA polynucleotide) may comprise an affinity sequence (or affinity tag) In some embodiments, a precursor RNA polynucleotide comprises at least one affinity tag. In some embodiments, the affinity tag is located in the 3’ intron element. In some embodiments, the affinity tag is located in the 5’ intron element. In some embodiments, both (3’ and 5’) intron elements each comprise an affinity tag. In some embodiments, the 5ʹ affinity tag is located 5ʹ to the 3ʹ permuted intron segment. In some embodiments, the 3ʹ affinity tag is located 3ʹ to the 5ʹ permuted intron segment. [316] In some embodiments, the polynucleotide comprises a monotron element comprising an affinity tag and/or terminal element comprising an affinity tag. In some embodiments, the terminal element comprises (a) a 5ʹ affinity tag if the terminal element is located upstream of the monotron element, wherein the 5ʹ affinity tag is located 5ʹ to the terminal element; or (b) a 3ʹ affinity tag if the monotron element is located upstream of the terminal element, wherein the 3ʹ affinity tag is located 3ʹ to the terminal element. In some embodiments, the monotron element comprises (a) a 3ʹ affinity tag if the terminal element is located upstream of the monotron element, wherein the 3ʹ affinity tag is located 3ʹ to the monotron element; or (b) a 5ʹ affinity tag if the monotron element is located upstream of the terminal element, wherein the 5ʹ affinity tag is located 5ʹ to the monotron element. In some embodiments, if the precursor RNA polynucleotide comprises an external spacer, the 5’ or 3’ affinity tag is positioned adjacent to the external spacer. [317] In one embodiment, an affinity tag of the 3’ intron element is the length as an affinity tag in the 5’ intron element. In some embodiments, an affinity tag of the 3’ intron element is the same sequence as an affinity tag in the 5’ intron element. In some embodiments, the affinity sequence is placed to optimize oligo-dT purification. [318] In some embodiments, the one or more affinity tags present in a precursor RNA polynucleotide are removed upon circularization. See, for example, Figures 97A and 97B from WO2022261490, which are incorporated by reference herein in entirety. In some embodiments, affinity tags are added to remaining linear RNA after circularization of precursor RNA is performed. In some such embodiments, affinity tags are added enzymatically to linear RNA. 241
Attorney Docket No. 01318-0014-00PCT OR-043WO The presence of one or more affinity tags in linear RNA and their absence from circular RNA can facilitate purification of circular RNA. In some embodiments, such purification is performed using a negative selection or affinity-purification method. In some embodiments, such purification is performed using a binding agent that preferentially or specifically binds to the affinity tag. [319] In some embodiments, an affinity sequence, such as biotin, is added to linear RNA by ligation. In some embodiments, an oligonucleotide comprising an affinity sequence is ligated to linear RNA. In some embodiments, an oligonucleotide conjugated to an affinity handle is ligated to the linear RNA. In some embodiments, a solution comprising the linear RNA ligated to the affinity sequence or handle and the circular RNA that does not comprise an affinity sequence or handle are contacted with a binding agent comprising a solid support conjugated to an oligonucleotide complementary to the affinity sequence or to a binding partner of the affinity handle, such that the linear RNA binds to the binding agent, and the circular RNA is eluted or separated from the solid support. [320] In some embodiments, an affinity tag comprises a polyA sequence or is a polyA affinity tag. In some embodiments the polyA sequence is at least 15, 30, or 60 nt in length. In some embodiments, the affinity tag comprising a polyA sequence is present in two places in a precursor linear RNA. In some embodiments, one or both polyA sequences are 15-50 nt in length. In some embodiments, one or both polyA sequences are 20-25 nt in length. In some embodiments, the polyA sequence(s) is removed upon circularization. Thus, an oligonucleotide hybridizing with the polyA sequence, such as a deoxythymidine oligonucleotide (oligo(dT)) conjugated to a solid surface (e.g., a resin), can be used to separate circular RNA from its precursor RNA. [321] Any purification method for circular RNA described herein may comprise one or more buffer exchange steps. In some embodiments, buffer exchange is performed after in vitro transcription (IVT) and before additional purification steps. In some such embodiments, the IVT reaction solution is buffer exchanged into a buffer comprising Tris. In some embodiments, the IVT reaction solution is buffer exchanged into a buffer comprising greater than 1 mM or greater than 10 mM one or more monovalent salts, such as NaCl or KCl, and optionally comprising EDTA. In some embodiments, buffer exchange is performed after purification of circular RNA is complete. In some embodiments, buffer exchange is performed after IVT and after purification of circular RNA. In some embodiments, the buffer exchange that is performed after purification of circular RNA comprises exchange of the circular RNA into water or 242
Attorney Docket No. 01318-0014-00PCT OR-043WO storage buffer. In some embodiments, the storage buffer comprises 1mM sodium citrate, pH 6.5. g. LEADING SEQUENCES & LAGGING SEQUENCES [322] In various embodiments, provided polynucleotide (e.g., a DNA template, a precursor linear RNA polynucleotide, or a circular RNA polynucleotide) comprises a leading untranslated sequence. In some embodiments, the leading untranslated sequence is located at the 5’ end in the 3’ intron fragment (also referred to as “5’ leading sequence”). In some embodiments, the leading untranslated sequence comprises the last nucleotide of a transcription start site (TSS). In some embodiments, the TSS is chosen from a viral, bacterial, or eukaryotic DNA template. In one embodiment, the leading untranslated sequence comprises the last nucleotide of a TSS and 0 to 100 additional nucleotides. In some embodiments, the TSS is a spacer. In some embodiments, the leading untranslated sequence contains a guanosine at the 5’ end. [323] In various embodiments, provided polynucleotide (e.g., a DNA template, a precursor linear RNA polynucleotide, or a circular RNA polynucleotide) comprises a lagging untranslated sequence (also referred to as “trailing sequence”). In some embodiments, the lagging untranslated sequence is located at the 3’ end. In some embodiments, the polynucleotide comprises a 3ʹ external spacer located between the 3ʹ intron element and a lagging untranslated sequence. In some embodiments, the polynucleotide a leading untranslated sequence at the 5ʹ end. In some embodiments, the polynucleotide comprises a 5ʹ external spacer located between a leading untranslated sequence and the 5ʹ intron element. [324] In some embodiments, the polynucleotide comprises a monotron element and a leading untranslated sequence. In some embodiments, the polynucleotide comprises a 5ʹ external spacer positioned between a leading untranslated sequence and either the terminal element or monotron element. In some embodiments, the polynucleotide comprises a monotron element and a lagging untranslated sequence. In some embodiments, the polynucleotide comprises a 3’ external spacer positioned between the lagging untranslated sequence and either the monotron element or terminal element. [325] In some embodiments, the lagging untranslated sequence comprises a restriction site sequence or a fragment thereof. In certain embodiments, the restriction site sequence or fragment thereof is used to linearize the polypeptide (e.g., DNA template). In some embodiments, the restriction site sequence is derived from a natural viral, bacterial or eukaryotic DNA template. 243
Attorney Docket No. 01318-0014-00PCT OR-043WO D. MONOTRON ELEMENT [326] Provided herein is a precursor RNA polynucleotide comprising a monotron (also called a monotron element or monotron sequence) and a terminal element (also called a terminal sequence). In some embodiments, the monotron has ribozymatic activity that allows it to enzymatically self-cleave. In some embodiments, the monotron is capable of forming a phosphodiester bond with a terminal sequence, i.e., a sequence containing a splice site dinucleotide and optionally a natural exon sequence or fragment thereof. In some embodiments, the precursor RNA polynucleotide comprises a terminal element; an intervening region, and a monotron element. [327] In some embodiments, the precursor RNA polynucleotide comprises, in the following order, (a) a terminal element; (b) an intervening region, and (c) a monotron element. In some embodiments, the terminal sequence is upstream of the monotron sequence in the precursor RNA polynucleotide. In such embodiments: (i) the terminal element comprises a splice site nucleotide, (ii) the monotron element comprises a splice site dinucleotide at or near the 5’ end of the monotron, and (iii) the monotron element is capable of interacting with a nucleophile that is capable of cleaving at the splice site dinucleotide at or near the 5’ end of the monotron, where the cleavage product of (iii) comprises a 5’ splice site nucleotide that is capable of cleaving at the splice site nucleotide of the terminal element. In some embodiments, the nucleophile is a free nucleophile that is introduced to the precursor RNA polynucleotide, e.g., not in cis and/or covalently linked to the precursor RNA polynucleotide. In some embodiments, the nucleophile is a guanosine that is capable of cleaving at the splice site dinucleotide at or near the 5’ end of the monotron. In some embodiments, the guanosine is a free guanosine that is introduced to the precursor RNA polynucleotide, e.g., not in cis and/or covalently linked to the precursor RNA polynucleotide. In some embodiments, the cleavage product of (iii) comprises a 5’ splice site nucleotide having a 3’ hydroxyl group that is capable of cleaving at the splice site nucleotide of the terminal element. [328] In some embodiments, the precursor RNA polynucleotide comprises, in the following order, (a) a monotron element; (b) an intervening region, and (c) terminal element. In some embodiments, the monotron sequence is upstream of the terminal sequence in the precursor RNA polynucleotide. In such embodiments: (i) the monotron element comprises a splice site dinucleotide at or near the 3’ end of the monotron, (ii) the terminal element comprises a splice site nucleotide, and (iii) the monotron element is capable of interacting with a nucleophile that is capable of cleaving at the splice site nucleotide of the terminal element, 244
Attorney Docket No. 01318-0014-00PCT OR-043WO where the cleavage product of (iii) comprises a 5’ splice site nucleotide that is capable of cleaving at the splice site dinucleotide at or near the 3’ end of the monotron. In some embodiments, the nucleophile is a free nucleophile that is introduced to the precursor RNA polynucleotide, e.g., not in cis and/or covalently linked to the precursor RNA polynucleotide. In some embodiments, the nucleophile is a guanosine that is capable of cleaving at the splice site nucleotide of the terminal element. In some embodiments, the guanosine is a free guanosine that is introduced to the precursor RNA polynucleotide, e.g., not in cis and/or covalently linked to the precursor RNA polynucleotide. In some embodiments, the cleavage product of (iii) comprises a 5’ splice site nucleotide having a 3’ hydroxyl group that is capable of cleaving at the splice site nucleotide of the terminal element. [329] In some embodiments where the terminal sequence is upstream to the monotron, the monotron can perform two transesterification reactions. The monotron can (a) self-cleave and (b) form a phosphodiester bond with the terminal sequence. In some embodiments, the reactions (a) and (b) are sequential. In some embodiments, (a) the monotron is capable of interacting with a nucleophile that is capable of cleaving at the splice site dinucleotide at or near the 5’ end of the monotron, and (b) the cleavage product of (a), i.e., the 5’ splice site nucleotide, e.g., having a 3’ hydroxyl group, engages in a transesterification reaction (cleaves) at the splice site nucleotide of the terminal sequence, yielding a circular RNA or oRNA. In these embodiments, the monotron interacts with the nucleophile by forming a binding pocket with the nucleophile, and the linear precursor is capable of adopting a conformation in which the nucleophile is in proximity to and is capable of cleaving at the splice site dinucleotide at or near the 5’ end of the monotron. In some embodiments, the nucleophile can be a guanosine, e.g., a free guanosine that is introduced to the precursor RNA polynucleotide. [330] In some embodiments where the monotron sequence is upstream of the terminal sequence, the monotron can also perform two transesterification reactions. In some embodiments, (a) the monotron is capable of interacting with a nucleophile that is capable of cleaving at the splice site nucleotide of the terminal element, and (b) the cleavage product of (a), i.e., the 5’ splice site nucleotide, e.g., having a 3’ hydroxyl group, engages in a transesterification reaction (cleaves) at the splice site dinucleotide at or near the 3’ end of the monotron, yielding a circular RNA or oRNA. In these embodiments, the monotron interacts with the nucleophile by forming a binding pocket with the nucleophile, and the linear precursor is capable of adopting a conformation in which the nucleophile is in proximity to and is capable of cleaving the splice site nucleotide of the terminal element. In some embodiments, the 245
Attorney Docket No. 01318-0014-00PCT OR-043WO nucleophile can be a guanosine, e.g., a free guanosine that is introduced to the precursor RNA polynucleotide. [331] In some embodiments, the monotron comprises a 5’ proximal end of a natural group I or group II intron including the splice site dinucleotide and optionally a natural exon sequence or fragment thereof. In some embodiments, the 5’ end of the monotron refers to nucleotides within the 5’ half of the monotron. In some embodiments, the 3’ end of the monotron refers to nucleotides within the 3’ half of the monotron. In some embodiments, at or near the 5’ end of the monotron refers to within the 5’ half of the monotron. In some embodiments, at or near the 5’ end of the monotron refers to within the first ten 5’ positions in the monotron. In some embodiments, at the 5’ end of the monotron refers to the first 5’ position(s) in the monotron. In some embodiments, at or near the 3’ end of the monotron refers to within the 3’ half of the monotron. In some embodiments, at or near the 3’ end of the monotron refers to within the last ten 3’ positions in the monotron. In some embodiments, at the 3’ end of the monotron refers to last 3’ position(s) in the monotron. [332] In some embodiments, the splice site nucleotide of the terminal element is not a natural splice site dinucleotide associated with a natural Group I or Group II intron sequence. In some embodiments, the terminal element comprises at least a portion of a natural exon or a fragment of a natural exon. In some embodiments, the natural exon is a Group I or Group II exon. In some embodiments, the natural exon or fragment thereof is 10-20 nucleotides in length. In some embodiments, the terminal element comprises a synthetic derivative of a natural exon or fragment thereof. In some embodiments, the terminal element comprises an exon or synthetic nucleotides that are longer than the splice site nucleotide that can help with splicing. [333] In some embodiments, the terminal element sequence has a percent sequence identity of about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more to an exon fragment of a sequence selected from Tables A or B. In some embodiments, the terminal element sequence comprises an exon fragment comprising one, two, three, four, five, six, seven, eight, nine, ten, or more mutations to a sequence selected from Tables A or B. The mutations are, for example, selected from insertions, deletions, mutations, additions, and subtractions. In some embodiments, the terminal element or exon fragment thereof comprises a polynucleotide sequence selected from a sequence set forth in Table A or Table B. [334] In some embodiments, the terminal element is less than 500, less than 450, less than 246
Attorney Docket No. 01318-0014-00PCT OR-043WO 400, less than 350, less than 300, less than 250, less than 200, less than 150, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, or less than 10 nucleotides in length. [335] In some embodiments, the terminal element is capable of directing or functionalizing the splicing activity of the monotron element. [336] In some embodiments, a portion of the terminal segment is retained upon circularization. In some embodiments, a portion of the terminal segment is excised upon circularization. In some embodiments, all or a portion of the terminal element is excised post- circularization. In some embodiments, the terminal element is not excised upon cleavage and is retained post-cleavage. [337] In some embodiments, the monotron element comprises at least a portion of a Group I or Group II intron. In some embodiments, Group I or Group II intron is selected from a genus and/or species described in Tables A or B. In some embodiments, the Group I or Group II intron is from a gene selected from Cyanobacterium Anabaena sp., T4 phage, Hypocrea pallida, Bulbithecium hyalosporum, Myoarachis inversa, Geosmithia argillacea, Coxiella burnetii, Agrobacterium tumefaciens, Azoarcus, Nostoc, Cordyceps capitata, Prochlorothrix hollandica, and Tilletiopsis orzyzicola. In some embodiments, the monotron element or a fragment thereof has a percent sequence identity of about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more to a sequence selected from Tables A or B. In some embodiments, the monotron element sequence or fragment thereof comprises one, two, three, four, five, six, seven, eight, nine, ten, or more mutations to a sequence selected from Tables A or B. The mutations are, for example, selected from insertions, deletions, additions, and subtractions. In some embodiments the monotron element sequence or fragment thereof comprises a polynucleotide sequence having a percent sequence identity of about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more to a portion of a sequence set forth in Table A or Table B. [338] In some embodiments, the Group I or Group II intron or introns, or portion thereof, are at least 10 nucleotides in length. [339] In some embodiments, the monotron element comprises at least one mutation of a native Group I intron-adjacent exon sequence or Group II intron-adjacent exon sequence. In some embodiments, the at least one mutation is at least one substation mutation of a native Group I intron-adjacent exon sequence or Group II intron-adjacent exon sequence. In some embodiments, the at least one mutation is at least one deletion of a native Group I intron- 247
Attorney Docket No. 01318-0014-00PCT OR-043WO adjacent exon sequence or Group II intron-adjacent exon sequence. In some embodiments, at least one of the exon segments is less than 15 nucleotides in length. In some embodiments, the monotron element comprises a 3ʹ exon segment and/or 5ʹ exon segment, wherein the 3’ or 5’ exon segment comprises a Group I exon segment or a Group II exon segment less than 15 nucleotides in length. In some embodiments, the 3ʹ exon segments and/or 5ʹ exon segments, have a percent sequence identity of about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more to a native Group I intron-adjacent exon sequence or Group II intron-adjacent exon sequence. [340] In some embodiments, the monotron element is less than 500 nucleotides in length. [341] In some embodiments, the monotron element is capable of inducing circularization when it interacts with the terminal element. In some embodiments, a portion of the monotron element is excised post-circularization. In some embodiments, the monotron element is fully excised post-circularization. In some embodiments, a portion of the monotron element and a portion of the terminal element are retained and excised post-circularization. [342] In some embodiments, the precursor RNA polynucleotide comprises at least one affinity tag or affinity sequence. Affinity sequences are described in further detail herein. In some embodiments, the affinity tag comprises a polyA sequence or is a polyA affinity tag. In some embodiments the terminal element comprises an affinity tag. In some embodiments, the terminal element comprises a 5ʹ affinity tag or a 3ʹ affinity tag. In some embodiments, the monotron element comprises an affinity tag. In some embodiments, the monotron element comprises a 3ʹ affinity tag or a 5ʹ affinity tag. [343] In some embodiments, the precursor RNA polynucleotide comprises an internal and/or external spacer. Spacers of the present disclosure are described in further detail herein. In some embodiments, the precursor RNA polynucleotide comprises an internal spacer sequence positioned between the terminal element and the intervening region. In some embodiments, the precursor RNA polynucleotide comprises an internal spacer sequence positioned between the intervening region and the monotron element. In some embodiments, the precursor RNA polynucleotide comprises an external spacer. In some embodiments, the external spacer is positioned adjacent to the terminal element. In some embodiments, the external spacer is positioned adjacent to the monotron element. In some embodiments, the precursor RNA polynucleotide comprises internal spacers and/or external spacers. The internal spacers and external spacers can each comprise an unstructured, structured or randomly generated polynucleotide sequence. In some embodiments, the internal spacers and external 248
Attorney Docket No. 01318-0014-00PCT OR-043WO spacers are at least 5 nucleotides in length and can be about 5 - 60 nucleotides in length. In some embodiments, the internal and external spacers are 5 - 60 nucleotides in length, inclusive. [344] In some embodiments, the precursor RNA polynucleotide comprises one or more duplexes. Duplexes of the present disclosure are described in further detail herein. In some embodiments, the precursor RNA polynucleotide comprises a 5ʹ internal duplex sequence and a 3ʹ internal duplex sequence. In embodiments where the terminal element is upstream of the monotron element, the 5ʹ internal duplex sequence is positioned between the terminal element and the intervening region, and the 3ʹ internal duplex sequence is positioned between the intervening region and the monotron element. In embodiments where the monotron element is upstream of the terminal element, the 5ʹ internal duplex sequence is positioned between monotron and the intervening region, and the 3ʹ internal duplex sequence is positioned between the intervening region and the terminal element. In some embodiments, the 5ʹ internal duplex sequence and 3ʹ internal duplex sequence are at least 80% complementary. In some embodiments, a duplex is 3-100 nucleotides in length. In some embodiments, a duplex is 5-20 nucleotides in length, inclusive. In some embodiments, the 5' and 3' internal duplex sequences are capable of forming, and are predicted to form, a contiguous duplex. In some embodiments, the continuous duplex has a length of no longer than about 35 nucleotides. In some embodiments, the 5ʹ internal duplex sequence and/or 3ʹ internal duplex sequence each have a GC content of at least 10%. [345] In some embodiments, the precursor RNA polynucleotide comprises at least one affinity tag and at least one external spacer. In some embodiments, the precursor RNA polynucleotide comprises at least one internal duplex and at least one internal spacer, for example a 5’ affinity tag and 5’ internal spacer and/or a 3’ affinity tag and 3’ internal spacer. In embodiments where the polynucleotide comprises a 5’ affinity tag, the 5ʹ affinity tag is positioned adjacent to the 5ʹ external spacer, and in certain embodiments is positioned 5ʹ to the 5ʹ external spacer. In some embodiments where the polynucleotide comprises a 3ʹ affinity tag, the 3ʹ affinity tag is positioned adjacent to the 3’ external spacer, and in certain embodiments is positioned 3ʹ to the 3ʹ external spacer. [346] In some embodiments, the precursor RNA polynucleotide comprises at least one duplex and at least one internal spacer. In some embodiments, the precursor RNA polynucleotide comprises at least one internal duplex and at least one internal spacer, for example a 5’ internal duplex and a 5’ internal spacer and/or a 3’ internal duplex and a 3’ internal spacer. In some embodiments where the polynucleotide comprises a 5ʹ internal duplex, the 5ʹ 249
Attorney Docket No. 01318-0014-00PCT OR-043WO internal duplex is positioned adjacent to the 5ʹ internal spacer, and in certain embodiments is positioned 5ʹ to the 5ʹ internal spacer. In some embodiments where the polynucleotide comprises a 3ʹ internal duplex, the 3ʹ internal duplex is positioned adjacent to the 3ʹ internal spacer, and in certain embodiments, the 3ʹ internal duplex is positioned 3ʹ to the 3ʹ internal spacer. [347] In some embodiments, the precursor polynucleotide comprises a 3’ and/or 5’ exon segment. In some embodiments, at least a portion of the 3’ and/or 5’ exon segment is codon optimized. [348] In some embodiments, the precursor RNA polynucleotide described above further comprises a leading untranslated sequence and/or a lagging untranslated sequence. For example, the precursor RNA polynucleotide can comprise a 5ʹ external spacer that is positioned between a leading untranslated sequence and the terminal element if the terminal element is upstream of the monotron element; or between a leading untranslated sequence and the monotron element if the monotron element is upstream of the terminal element. In some embodiments, the precursor RNA polynucleotide comprises a 3ʹ external spacer that is positioned between the monotron element and a lagging untranslated sequence if the terminal element is upstream of the monotron element; or between the terminal element and a lagging untranslated sequence if the monotron element is upstream of the terminal element. [349] As described in detail elsewhere herein, the intervening region of the precursor RNA polynucleotide can comprise sequences directed to, for example, an aptamer, a coding element, a stop codon or stop cassette, an intervening region comprising an untranslated region, a noncoding element. As set forth in detail herein, the intervening region can comprise a coding element where the coding element comprises, for example, a sequence encoding a therapeutic protein. In some embodiments, the intervening region comprises an untranslated region, which can comprise one or more non-coding element, including but not limited to, a natural 5ʹ Untranslated Region (UTR), a natural 3ʹ Untranslated Region (UTR), a synthetic spacer sequence, an aptamer, TIE, a viral or eukaryotic IRES, or sequences selected from, e.g., lncRNA, miRNA, or a miRNA sponge. [350] In some embodiments, the polynucleotide comprises at least one mutation of a native Group I intron-adjacent exon sequence or Group II intron-adjacent exon sequence. The mutation can be, for example, at least one mutation of a native Group I or native Group II intron-adjacent exon sequence. For example, the mutation can be one substitution, at least one deletion, and/or at least one insertion of a native Group I or Group II intron-adjacent exon 250
Attorney Docket No. 01318-0014-00PCT OR-043WO sequence. In some embodiments, at least one of the exon segments is less than 15 nucleotides in length. In some embodiments, the 3ʹ exon segment and/or 5ʹ exon segment comprises a Group I exon segment or a Group II exon segment. In some embodiments, the at least one exon segment is less than 15 nucleotides in length. In some embodiments, the at least one exon segment has a percent sequence identity of about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more to a native Group I intron-adjacent exon sequence or Group II intron-adjacent exon sequence. In some embodiments, the at least one segment is selected from a 3ʹ exon segment, 5ʹ exon segment, or a 3ʹ and 5ʹ permuted exon segment. In some embodiments, the exon sequence or fragment is in the terminal element. [351] Also provided herein are polynucleotides encoding the precursor RNA polynucleotides described above that comprise a monotron and terminal element. Polynucleotides, for example, DNA templates comprising sequences encoding the precursor RNAs described above, and their uses in related methods are described elsewhere herein. Polynucleotides of the present disclosure can comprise, for example, an expression vector, DNA plasmid, a cosmid, a PCR product, dbDNA close-ended DNA (ceDNA), and a viral polynucleotide. In some embodiments, the polynucleotides can comprises a promoter segment, for example a T7 promoter, SP6 promoter or a fragment thereof. [352] Also provided herein are circular RNA polynucleotides produced by the precursor RNAs described above that comprise a monotron and terminal element. Circular RNAs are described in detail elsewhere herein. In some embodiments, a circular RNA polynucleotide comprises: at least a portion of a terminal element, an intervening region, and at least a portion of a monotron element. In some embodiments, a circular RNA comprises: (a) at least a portion of a terminal element, (b) a 3ʹ exon segment comprising a 3ʹ nucleotide of a 3ʹ splice site dinucleotide, (c) an intervening region, (d) a 5ʹ exon segment comprising a 5ʹ nucleotide of a 5ʹ splice site dinucleotide, and (e) at least a portion of a monotron element. In some embodiments, (d) comprises the first nucleotide of a 5ʹ Group I or Group II splice site dinucleotide and a natural exon sequence and (b) comprises the second nucleotide of a 3ʹ Group I or Group II exon splice site dinucleotide and a natural exon sequence. In some embodiments, the 5' and/or 3' splice site dinucleotides are distinct from the natural splice site dinucleotide(s) associated with a natural Group I or Group II intron sequence. In some embodiments, the circular RNA polynucleotides comprise additional elements, including but not limited to, a 5ʹ internal duplex and/or 3ʹ internal duplex; a 5ʹ internal spacer and/or 3ʹ internal spacer. In some embodiments, the circular RNA polynucleotide is from about 50 nucleotides to about 15 251
Attorney Docket No. 01318-0014-00PCT OR-043WO kilobases in length. [353] Related cells comprising the precursor RNA polynucleotides, delivery or transfer vehicles, and pharmaceutical compositions thereof are described elsewhere herein in further detail. Related methods of producing circularized RNA and related methods of treating a subject in need thereof are also provided herein. [354] Also provided herein are methods of identifying a monotron element and terminal element pair that allows production of a circular RNA that is translatable or biologically active inside a eukaryotic cell, comprising, for example: (i) inserting a mutated 5ʹ and 3ʹ Group I or Group II intron sequence derived from a database of native intronic sequence to form a monotron element into a precursor RNA polynucleotide described above; (ii) inserting a synthetic polynucleotide sequence to form a terminal element into a precursor RNA polynucleotide described above; (iii) transcribing the polynucleotide into RNA in vitro or allowing the polynucleotide to be transcribed into RNA by a cell; and (iv) determining the circularization efficiency of the RNA produced by the polynucleotide by identifying the amount of circularized RNA, the amount of excised intronic sequences, the amount of precursor RNA remaining after circularization, and combinations thereof. In some embodiments, the mutated 5ʹ and 3ʹ Group I or Group II intron sequence comprises at least one deletion, insertion or substitution of at least one nucleotide. In some embodiments, the 5ʹ or 3ʹ Group I or Group II intronic sequences, or combinations thereof are sequenced. [355] Also provided herein are methods for determining a polynucleotide sequence that improves RNA circularization efficiency compared to a polynucleotide comprising a native intronic sequence or to a parent polynucleotide with a known sequence, the method comprising modifying a DNA sequence encoding the precursor RNA polynucleotide described above, the modifying comprising: (i) mutating at least one nucleotide and/or altering the length of the terminal element and/or monotron element of the DNA sequence encoding the precursor RNA polynucleotide described above; (ii) altering the length of the 5ʹ and/or 3ʹ internal and/or external spacer sequence of the DNA sequence encoding precursor RNA polynucleotide described above; (iii) altering the length of the 5ʹ and/or 3ʹ internal duplex sequence of the DNA sequence encoding the precursor RNA polynucleotide described above; (iv) altering the length of the 5ʹ and/or 3ʹ exon sequence of the DNA sequence encoding the precursor RNA polynucleotide described above; (iv) or combinations thereof; and transcribing the polynucleotide comprising the DNA sequence into RNA in vitro or allowing the polynucleotide comprising the DNA sequence to be transcribed into RNA by a cell; and determining the 252
Attorney Docket No. 01318-0014-00PCT OR-043WO circularization efficiency of the RNA produced by the polynucleotide comprising the DNA sequence by identifying the amount of circularized RNA, the amount of excised intronic sequences, the amount of precursor RNA remaining after circularization, and combinations thereof. In some embodiments, the methods further comprise comparing the circularization efficiency of the polynucleotide with a polynucleotide comprising a native intronic sequence, or a parent polynucleotide. E. ADDITIONAL ELEMENTS [356] In various embodiments, a provided polynucleotide (e.g., a DNA template, a linear precursor RNA polynucleotide, or a circular RNA polynucleotide) further comprises one or more elements for enhancing circularization, translation, or both. In certain embodiments, these elements are located with specificity between or within the intron elements, exon elements, or intervening region of the polynucleotide. [357] As an example, but not intended to be limiting, a polynucleotide, a precursor RNA polynucleotide, or circular RNA can comprise an IRES transacting factor region, a miRNA binding site, a restriction site, an RNA editing region, a structural or sequence element, a granule site, a zip code element, and/or an RNA trafficking element or another specialized sequence as found in the art that enhances promotes circularization and/or translation of the protein encoded within the circular RNA polynucleotide. [358] In some embodiments, the polynucleotide, precursor RNA polynucleotide, or circular RNA comprises an IRES transacting factor (ITAF) region. In some embodiments, the IRES transacting factor region modulates the initiation of translation through binding to PC- P1 - PCBP4 (polyC binding protein), PABP1 (polyA binding protein), PTB (polyprimidine tract binding), Argonaute protein family, HNRNPK (Heterogeneous nuclear ribonucleoprotein K protein), or La protein. In some embodiments, the IRES transacting factor region comprises a polyA, polyC, polyAC, or polyprimidine track. In some embodiments, the ITAF region is located within the intervening region or core functional element. In some embodiments, the ITAF region is located within the TIE. [359] In certain embodiments, the polynucleotide, precursor RNA polynucleotide, or circular RNA comprises a lncRNA, miRNA, or a miRNA sponge. In certain embodiments, at least one miRNA binding site is included. In some embodiments the miRNA binding site is located within the 5’ intron element, 5’ exon element, intervening region or core functional element, 3’ exon element, and/or 3’ intron element. In some embodiments, the miRNA binding site is located within the spacer within the intron element or exon element. In certain 253
Attorney Docket No. 01318-0014-00PCT OR-043WO embodiments, the miRNA binding site comprises the entire spacer regions. In some embodiments, the 5’ intron element and 3’ intron elements each comprise identical miRNA binding sites. In another embodiment, the miRNA binding site of the 5’ intron element comprises a different, in length or nucleotides, miRNA binding site than the 3’ intron element. In one embodiment, the 5’ exon element and 3’ exon element comprise identical miRNA binding sites. In other embodiments, the 5’ exon element and 3’ exon element comprise different, in length or nucleotides, miRNA binding sites. In some embodiments, the miRNA binding sites are located adjacent to each other within the circular RNA construct, linear RNA polynucleotide precursor, and/or DNA template. In certain embodiments, the first nucleotide of one of the miRNA binding sites follows the first nucleotide last nucleotide of the second miRNA binding site. In some embodiments, the miRNA binding site is located within a translation initiation element (TIE) of an intervening region or core functional element. In one embodiment, the miRNA binding site is located before, trailing or within an internal ribosome entry site (IRES). In another embodiment, the miRNA binding site is located before, trailing, or within an aptamer complex. [360] Incorporation of miRNA sequences can permit tissue-specific expression of a coding sequence within an intervening region or core functional element. For example, in a circular RNA intended to express a protein in immune cells, miRNA binding sequences resulting in expression suppression in tissues such as the liver or kidney may be desired. Such miRNA binding sequences may be selected based on the cell or tissue expression of miRNAs. The unique sequences defined by the miRNA nomenclature are widely known and accessible to those working in the microRNA field. For example, they can be found in the miRDB public database. As a non-limiting example, one or more miR-122 target sites can be inserted in the circular RNA. [361] In some embodiments, the miR-122 site can comprise the following sequence: CAAACACCATTGTCACACTCCAA (SEQ ID NO: 25429). F. Modified nucleotides or nucleosides [362] In certain embodiments, a provided polynucleotide (e.g., a precursor RNA polynucleotide, a circular RNA polynucleotide, or a DNA template) comprises modified nucleotides and/or modified nucleosides, namely comprising at least one modified A, C, G, or U/T nucleotide or nucleoside. As exhibited by the exemplary nucleotide or nucleotide modification presented below, such modifications differ from mutations selected from 254
Attorney Docket No. 01318-0014-00PCT OR-043WO insertions, deletions, addition, or subtraction of nucleotides, for example, the mutations in a permuted Group I and Group II intron segment; or the additions, deletions, or substitutions of a nucleotide, domain, or motif present in a synthetic IRES (as compared to the naturally occurring IRES). [363] In some embodiments, the polynucleotide is a precursor RNA polynucleotide and comprises at least one modified A, C, G, or U nucleotide or nucleoside. In some embodiments, the precursor RNA polynucleotide is linear. In some embodiments, the precursor RNA polynucleotide is capable of producing a circular RNA comprising at least one modified nucleotide or nucleoside after splicing. In some embodiments, the precursor RNA polynucleotide comprising one or more modified nucleotide or nucleoside is capable of circularizing when incubated in the presence of one or more guanosine nucleotides or nucleoside (e.g., GTP) and a divalent cation (e.g., Mg2+). In some embodiments, the polynucleotide is a circular RNA polynucleotide and comprises at least one modified A, C, G, or U nucleotide or nucleoside modifications. [364] In some embodiments, modified nucleotides or nucleosides occur throughout a precursor RNA polynucleotide. In some embodiments, the RNA polynucleotide comprises 5’ and 3’ combined accessory elements comprising one or more modified nucleotides. In some embodiments, the RNA polynucleotide comprises an intron element and/or exon element comprising one or more modified nucleotide or nucleoside. [365] In some embodiments, portions of the 3’ and/or 5’ intron and/or exon segments in a linear precursor RNA polynucleotide of the present disclosure contain modified nucleotides or nucleosides. In some embodiments, the secondary structures of at least the intron and/or exon segments are preserved. In some embodiments, the terminal element comprises at least one modified nucleotide or nucleoside. In some embodiments, the terminal element, intervening region, and/or monotron comprises at least one modified nucleotide or nucleoside. In certain embodiments, the RNA polynucleotide comprises a spacer comprising at least one modified nucleotide or nucleoside. In certain embodiments, the RNA polynucleotide comprises a duplex comprising at least one modified nucleotide or nucleoside. In certain embodiments, the RNA polynucleotide comprises an affinity sequence comprising at least one modified nucleotide or nucleoside. In certain embodiments, the RNA polynucleotide comprises a leading and/or lagging strand comprising at least one modified nucleotide or nucleoside. In some embodiments, the RNA polynucleotide comprises a coding or a noncoding element comprising at least one modified nucleotide or nucleoside. In some embodiments, the 255
Attorney Docket No. 01318-0014-00PCT OR-043WO RNA polynucleotide comprises a translation initiation element (TIE) comprising at least one modified nucleotide or nucleoside. In certain embodiments, the polynucleotide comprises a stop codon and/or stop cassette comprising one or more modified nucleotide or nucleoside. [366] In some embodiments, a precursor RNA polynucleotide comprising at least one modified A, C, G, or U nucleotide or nucleoside comprises at least a portion of each of: a. a 5’ combined accessory element, comprising: i. a 3’ intron segment, ii. a 3’ exon segment, b. an intervening region comprising an internal ribosome entry site (IRES) and a noncoding or coding region, c. a 3’ combined accessory element, comprising: i. a 5’ exon segment, and ii. a 5’ intron segment. [367] In some embodiments, a circular RNA comprising at least one modified A, C, G, or U nucleotide or nucleoside comprises at least a portion of each of: a. a post-splicing 3’ exon segment, b. optionally a 5’ internal homology region, c. optionally a 5’ spacer, d. an intervening region comprising an internal ribosome entry site (IRES) and a noncoding or coding region, e. optionally a 3’ spacer, f. optionally a 3’ internal homology region, and g. a post-splicing 5’ exon segment. [368] In some embodiments, the modified nucleoside is m5C (5-methylcytidine). In another embodiment, the modified nucleoside is m5U (5-methyluridine). In another embodiment, the modified nucleoside is m6A (N6-methyladenosine). In another embodiment, the modified nucleoside is s2U (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 m1A (1- methyladenosine); m2A (2-methyladenosine); Am (2’-O-methyladenosine); ms2 m6A (2- methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio- N6 isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2- methylthio-N6-(cis-hydroxyisopentenyl)adenosine); g6A (N6-glycinylcarbamoyladenosine); 256
Attorney Docket No. 01318-0014-00PCT OR-043WO t6A (N6-threonylcarbamoyladenosine); ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); hn6A(N6- hydroxynorvalylcarbamoyladenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2’-O-ribosyladenosine (phosphate)); I (inosine); m1I (1- methylinosine); m1Im (1,2’-O-dimethylinosine); m3C (3-methylcytidine); Cm (2’-O- methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); f5C (5-formylcytidine); m5Cm (5,2′-O-dimethylcytidine); ac4Cm (N4-acetyl-2’-O-methylcytidine); k2C (lysidine); m1G (1- methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2′-O- methylguanosine); m2 2G (N2,N2-dimethylguanosine); m2Gm (N2,2’-O-dimethylguanosine); m2 2Gm (N2,N2,2’-O-trimethylguanosine); Gr(p) (2’-O-ribosylguanosine(phosphate)); yW (wybutosine); o2yW (peroxywybutosine); oHyW (hydroxywybutosine); OhyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQ0 (7- cyano-7-deazaguanosine); preQ1 (7-aminomethyl-7-deazaguanosine); G+ (archaeosine); D (dihydrouridine); m5Um (5,2’-O-dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2- thiouridine); s2Um (2-thio-2’-O-methyluridine); acp3U (3-(3-amino-3- carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U (5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5- (carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonylmethyluridine); mcm5Um (5-methoxycarbonylmethyl-2’-O- methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5S2U (5- aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); mnm5s2U (5- methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2′-O-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cmnm5Um (5-carboxymethylaminomethyl- 2′-O-methyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m6 2A (N6,N6-dimethyladenosine); Im (2’-O-methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2’-O-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6,2’-O-dimethyladenosine); m6 2Am (N6,N6,O-2’- trimethyladenosine); m2,7G (N2,7-dimethylguanosine); m2,2,7G (N2,N2,7-trimethylguanosine); m3Um (3,2’-O-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2’-O- methylcytidine); m1Gm (1,2’-O-dimethylguanosine); m1Am (1,2’-O-dimethyladenosine); 257
Attorney Docket No. 01318-0014-00PCT OR-043WO τm 5U (5-taurinomethyluridine); τm5s2U (5-taurinomethyl-2-thiouridine)); imG-14 (4- demethylwyosine); imG2 (isowyosine); N1-methylpseudouridine; or ac6A (N6- acetyladenosine). [369] In some embodiments, the modified nucleoside may include a compound selected from the group of: 258yridine-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2- thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl- pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2- thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4- thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza- pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2- methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-m ethoxy-2-thio- pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5- formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza- pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2- methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl- pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza- adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6- isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6- dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine, 1- methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio- guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6- thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2- methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1- methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N1-methylpseudouridine; and N2,N2- dimethyl-6-thio-guanosine. 258
Attorney Docket No. 01318-0014-00PCT OR-043WO [370] In another embodiment, the modifications are independently selected from 5- methylcytosine, pseudouridine and 1-methylpseudouridine. [371] In some embodiments, the modified ribonucleosides include 5-methylcytidine, 5- methoxyuridine, 1-methyl-pseudouridine, N6-methyladenosine, and/or pseudouridine. [372] In some embodiments, the modified nucleoside is N1-methylpseudouridine. [373] In some embodiments, the modified nucleotide or nucleoside is selected from one or more of: 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N,- dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3- methyluridine, 5-methylcytidine, 5-methyluridine, 5-(2-amino)propyl uridine, 5- halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3- methyicytidine, 6- methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2- dimethylguanosine, 5- methylaminoethyluridine, 5-methyloxyuridine, 7-deaza-adenosine, 6- azouridine, 6- azocytidine, 6-azothymidine, 5-methyl-2-thiouridine, 2-thiouridine, 4- thiouridine, 2- thiocytidine, dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl substituted naphthyl groups, an O- and N-alkylated purines and pyrimidines, N6- methyladenosine, 5- methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, pyridine-2-one, aminophenol, 2,4,6-trimethoxy benzene, modified cytosines that act as G- clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. [374] Additional modified nucleotides and nucleosides can be selected from clinically validated modified nucleotides described in the art. See, e.g., US20190345503A1 (m6A- modified circRNA); US20220288176A1 (m6A modification of circRNA); US20220251578A1 (at least one N6-methyladenosine (m6A)); WO2022271965A2 (N6-methyladenosine, 2- thiouridine, and 2’-O-methylcytidine), which are each incorporated by reference in their entireties. [375] In some embodiments, a first and second precursor polynucleotide are provided, where the first precursor RNA polynucleotide comprises a 3’ intron fragment of a first intron (Intron 1), a 5’ intron fragment of a second intron (Intron 2), a translation initiation element, a fragment of a sequence of interest (e.g., coding region), and two exon fragments that correspond with the intron fragments; and the second precursor comprises a 3’ intron fragment of the second intron (Intron 2) and a 5’ intron fragment of the first intron (Intron 1), a fragment of the sequence of interest of the first precursor, and exon fragments corresponding to those in 259
Attorney Docket No. 01318-0014-00PCT OR-043WO the first precursor. In these embodiments, the first and second linear precursor RNA polynucleotides are capable of forming a circular RNA. In some embodiments, the first precursor comprises no nucleotide or nucleoside modifications and the second precursor comprises nucleotide or nucleoside modifications. In some embodiments, the first precursor comprises nucleotide or nucleoside modifications and the second precursor comprises no nucleotide or nucleoside modifications. In some embodiments, the first precursor and the second precursor comprise no nucleotide or nucleoside modifications. In some embodiments, the first precursor and the second precursor comprise nucleotide or nucleoside modifications. [376] Indeed, contrary to publications contending that, for example, “[i]ncorporation of m6A modification into circRNA does not affect splicing to form circRNA” (see, e.g., Chen et al., 2019, Mol Cell, N6-Methyladenosine Modification Controls Circular RNA Immunity), the disclosures herein demonstrate that the incorporation of certain nucleotide and/or nucleoside modifications to a precursor RNA polynucleotide can affect the circularization and/or splicing of the circular RNA. (See Kariko et al., 2005, Immunity, Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA; Kariko et al., 2005, Mol Ther, Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability; Wesselhoeft et al., 2019, Mol Cell, RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In Vivo; Chen et al., 2022, Nature Biotechnology, Engineering circular RNA for enhanced protein production). Modified nucleotide or nucleosides may exhibit different physical properties to their unmodified counterparts. In some embodiments, the presence of a modified nucleotide or nucleoside can affect the folding patterns and/or function of an accessory element, translation initiation element (TIE), and/or coding element within the circular RNA or linear precursor. Position and composition of a nucleotide or nucleoside modification in a polynucleotide are impacted by the nucleotide or nucleoside composition (i.e., A, C, G, or U nucleotide or nucleoside) of the accessory elements, TIE, or coding elements. [377] In some embodiments, in a provided polynucleotide (e.g., a precursor RNA polynucleotide, or a circular RNA polynucleotide, described in more detail elsewhere herein), between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90%, or 90% and 100% of the nucleotides or nucleosides are unmodified. In some embodiments, a provided 260
Attorney Docket No. 01318-0014-00PCT OR-043WO polynucleotide (e.g., a precursor RNA polynucleotide, or a circular RNA polynucleotide, described in more detail elsewhere herein) comprises modified nucleotides and/or modified nucleosides where between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90%, or 90% and 100% of the nucleotides or nucleosides are modified. In some embodiments, between 1% and 10% of the nucleotides or nucleosides are modified. [378] In some embodiments, in portions of the polynucleotide (e.g., a precursor RNA polynucleotide, or a circular RNA polynucleotide, described in more detail elsewhere herein), between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90%, or 90% and 100% of the nucleotides or nucleosides are modified. For example, in some embodiments, between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90%, or 90% and 100% of the nucleotides or nucleosides in the intervening region are modified. In some embodiments, between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90%, or 90% and 100% of the nucleotides or nucleosides in the IRES are modified. In some embodiments, between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90%, or 90% and 100% of the nucleotides or nucleosides in the noncoding or coding region are modified. In some embodiments, between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90%, or 90% and 100% of the nucleotides or nucleosides in the 5’ intron segment and/or 3’ intron segment are modified. In some embodiments, between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90%, or 90% and 100% of the nucleotides or nucleosides in the 5’ 261
Attorney Docket No. 01318-0014-00PCT OR-043WO exon segment or post-splicing exon segment and/or 3’ exon segment or post-splicing exon segment are modified. In some embodiments, between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90%, or 90% and 100% of the nucleotides or nucleosides in the 5’ spacer and/or 3’ spacer are modified. In some embodiments, between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90%, or 90% and 100% of the nucleotides or nucleosides in the 5’ homology region and/or 3’ homology region are modified. In some embodiments, the secondary structures of at least the intron and/or exon segments are preserved. In some embodiments, the secondary structure of the coding or noncoding region is preserved. In some embodiments, the IRES is unmodified or substantially unmodified to preserve secondary structure needed to initiate translation. [379] In some embodiments, between 1% and 10% of the nucleotides or nucleosides are modified in a polynucleotide of the present disclosure (e.g., a precursor RNA polynucleotide, or a circular RNA polynucleotide, described in more detail elsewhere herein). In some embodiments, portions of the polynucleotide comprise between 1% and 10% modification of the nucleotides or nucleosides. For example, in some embodiments, between 1% and 10% of the nucleotides or nucleosides in the intervening region are modified. In some embodiments, between 11% and 10% of the nucleotides or nucleosides in the IRES are modified. In some embodiments, between 1% and 10% of the nucleotides or nucleosides in the noncoding or coding region are modified. In some embodiments, between 1% and 10% of the nucleotides or nucleosides in the 5’ intron segment and/or 3’ intron segment are modified. In some embodiments, between 11% and 10% of the nucleotides or nucleosides in the 5’ exon segment or post-splicing exon segment and/or 3’ exon segment or post-splicing exon segment are modified. In some embodiments, between 1% and 10% of the nucleotides or nucleosides in the 5’ spacer and/or 3’ spacer are modified. In some embodiments, between 1% and 10% of the nucleotides or nucleosides in the 5’ homology region and/or 3’ homology region are modified. [380] In some embodiments, the polynucleotides comprising modified nucleotides and/or modified nucleosides provide additional stability and resistance to immune activation. In some embodiments, polynucleotides comprising modified nucleotides and/or modified nucleosides 262
Attorney Docket No. 01318-0014-00PCT OR-043WO maintain stability and resistance to immune activation as compared to a corresponding polynucleotide comprising no modified nucleotides and/or modified nucleosides. [381] In some embodiments, a precursor RNA polynucleotide with modified nucleotides and/or nucleosides improves circularization as compared to a corresponding linear precursor RNA polynucleotide comprising no nucleotide or nucleoside modifications or other appropriate control. In other embodiments, a precursor RNA polynucleotide with modified nucleotides and/or nucleosides maintains the same circularization as compared to a corresponding precursor RNA polynucleotide comprising no nucleotide or nucleoside modifications or other appropriate control. In some embodiments, the precursor polynucleotides comprising modified nucleotides and/or modified nucleosides maintain circularization at greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 100% (i.e., improves circularization), as compared to a corresponding precursor polynucleotide comprising no nucleotide or nucleoside modifications. In some embodiments, the precursor polynucleotides maintain circularization at greater than 70% as compared to a corresponding precursor polynucleotide comprising no nucleotide or nucleoside modifications. In some embodiments, the precursor polynucleotides maintain circularization at greater than 80% as compared to a corresponding precursor polynucleotide comprising no nucleotide or nucleoside modifications. In some embodiments, the precursor polynucleotides maintain circularization at greater than 90% as compared to a corresponding precursor polynucleotide comprising no nucleotide or nucleoside modifications. In some embodiments, the precursor polynucleotides exhibit greater than 100% circularization (i.e., improved circularization) as compared to a corresponding precursor polynucleotide comprising no nucleotide or nucleoside modifications. [382] In some embodiments a circular RNA with modified nucleotides and/or nucleosides reduces immunogenicity and/or improves translation of the coding region as compared to a corresponding circular RNA comprising no nucleotide or nucleoside modifications. In other embodiments, a circular RNA polynucleotide with modified nucleotides and/or nucleosides maintains the same immunogenicity and/or translation of the coding region as compared to a corresponding circular RNA comprising no nucleotide or nucleoside modifications. For example, in some embodiments, the circular RNAs described herein comprising at least one modified A, C, G, or U nucleotide or nucleoside exhibit reduced immunogenicity, without losing circularization and/or translation. In some embodiments, the circular RNAs described herein exhibit immunogenicity that is reduced by about 10% to about 99%, for example 263
Attorney Docket No. 01318-0014-00PCT OR-043WO reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as compared to a corresponding circular RNA comprising no nucleotide or nucleoside modifications. In some embodiments, the polynucleotides comprising modified nucleosides provide additional stability and resistance to immune activation. [383] In some embodiments, portions of the polynucleotide (e.g., a precursor RNA polynucleotide, or a circular RNA polynucleotide) or the polynucleotide in its entirety comprises no nucleotide or nucleoside modifications. In some embodiments, portions of the polynucleotide (e.g., a precursor RNA polynucleotide, or a circular RNA polynucleotide, described in more detail elsewhere herein) comprise less than 10% modification. For example, in some embodiments, the intervening region comprises no nucleotide or nucleoside modifications or less than 10% of the nucleotides or nucleosides therein are modified. In some embodiments, the IRES comprises no nucleotide or nucleoside modifications or less than 10% of the nucleotides or nucleosides therein are modified. In some embodiments, the noncoding or coding region comprises no nucleotide or nucleoside modifications or less than 10% of the nucleotides or nucleosides therein are modified. In some embodiments, the 5’ intron segment and/or 3’ intron segment comprises no nucleotide or nucleoside modifications or less than 10% of the nucleotides or nucleosides therein are modified. In some embodiments, the 5’ exon segment or post-splicing exon segment and/or 3’ exon segment or post-splicing exon segment comprises no nucleotide or nucleoside modifications or less than 10% of the nucleotides or nucleosides therein are modified. In some embodiments, the 5’ spacer and/or 3’ spacer comprises no nucleotide or nucleoside modifications or less than 10% of the nucleotides or nucleosides therein are modified. In some embodiments, the 5’ homology region, 3’ homology region comprises no nucleotide or nucleoside modifications or less than 10% of the nucleotides or nucleosides therein are modified. [384] In some embodiments herein, between 1% and 10% of the nucleotides or nucleosides are modified in a linear precursor RNA polynucleotide or circular RNA of the present disclosure. In some embodiments in a linear precursor RNA polynucleotide or circular RNA of the present disclosure, the intervening region comprises no nucleotide or nucleoside modifications or is less than 10% modified; the IRES comprises no nucleotide or nucleoside modifications or is less than 10% modified; the noncoding or coding region comprises no nucleotide or nucleoside modifications or is less than 10% modified; the 5’ intron segment and/or 3’ intron segment comprises no nucleotide or nucleoside modifications or is less than 264
Attorney Docket No. 01318-0014-00PCT OR-043WO 10% modified; the 5’ exon segment or post-splicing exon segment and/or 3’ exon segment or post-splicing exon segment comprises no nucleotide or nucleoside modifications or is less than 10% modified; the 5’ spacer and/or 3’ spacer comprises no nucleotide or nucleoside modifications or is less than 10% modified; and/or the 5’ homology region, 3’ homology region comprises no nucleotide or nucleoside modifications or is less than 10% modified. [385] In some embodiments, modified nucleotides or nucleotides occur throughout a precursor RNA polynucleotide. In other embodiments, portions of the 3’ and/or 5’ intron and/or exon segments in a linear precursor RNA polynucleotide of the present disclosure contain modified nucleotides or nucleosides, but the remaining portions of the linear precursor do not comprise nucleotide or nucleoside modifications. In some embodiments, portions of the 3’ and/or 5’ intron and/or exon segments in a linear precursor RNA polynucleotide of the present disclosure contain modified nucleotides or nucleosides, but the remaining portions of the linear precursor comprise minimal nucleotide or nucleoside modifications. In some embodiments, portions of the 3’ and/or 5’ intron and/or exon segments in a linear precursor RNA polynucleotide of the present disclosure contain modified nucleotides or nucleosides, but the remaining portions of the linear precursor comprise less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% modified nucleotides or nucleosides. [386] In some embodiments, where the circular RNA is produced from a linear precursor and where the linear precursor is modified at the 3’ and/or 5’ ends only, the circular RNA contains only the modified nucleotide or nucleosides that remain after circularization. [387] In certain embodiments, a circular RNA is prepared by providing modified nucleotides or nucleosides to precursor RNA comprising: a. a 5’ combined accessory element, comprising: i. a 3’ intron segment, ii. a 3’ exon segment, b. an intervening region comprising an internal ribosome entry site (IRES) and a noncoding or coding region, c. a 3’ combined accessory element, comprising: i. a 5’ exon segment, and ii. a 5’ intron segment. [388] In certain embodiments, a circular RNA is prepared by providing a first and second linear precursor RNA polynucleotide, wherein the first and second linear precursor RNA polynucleotides are capable of forming a circular RNA. In some embodiments, either the first 265
Attorney Docket No. 01318-0014-00PCT OR-043WO precursor or the second precursor but not both precursors comprises at least one modified A, C, G, or U nucleotide or nucleoside. In some embodiments, the first precursor comprises at least one modified A, C, G, or U nucleotide or nucleoside and the second precursor comprises no modified nucleotides or nucleosides. In some embodiments, the second precursor comprises least one modified A, C, G, or U nucleotide or nucleoside and the first precursor comprises no modified nucleotides or nucleosides. [389] In some embodiments, the first precursor comprises a 3’ intron fragment of a first intron (Intron 1), a 5’ intron fragment of a second intron (Intron 2), a translation initiation element, a fragment of a sequence of interest (e.g., coding region), and two exon fragments that correspond with the intron fragments. In some embodiments, the second precursor comprises a 3’ intron fragment of the second intron (Intron 2) and a 5’ intron fragment of the first intron (Intron 1), a fragment of the sequence of interest of the first precursor, and exon fragments corresponding to those in the first precursor. [390] In some embodiments, the TIE of the first precursor RNA polynucleotides comprises an IRES. In some embodiments, the first precursor RNA polynucleotide comprises a noncoding or coding region. [391] In some embodiments, the first and second precursor RNA polynucleotides further comprise spacers and/or homology arms. [392] In some embodiments, between 0% and 99%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 99% of the nucleotides or nucleosides in the first linear precursor are modified. In some embodiments, between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 100% of the nucleotides or nucleosides in the first linear precursor are modified. In some embodiments, between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 100% of the nucleotides or nucleosides in the first linear precursor are unmodified. [393] In some embodiments, in portions of the first linear polynucleotide, between 0% and 99%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 266
Attorney Docket No. 01318-0014-00PCT OR-043WO 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 99% of the nucleotides or nucleosides are modified. For example, in some embodiments, between 0% and 99%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 99% of the nucleotides or nucleosides in the intervening region of the first linear polynucleotide are modified. In some embodiments, between 0% and 99%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 99% of the nucleotides or nucleosides in the TIE (e.g., IRES) of the first linear polynucleotide are modified. In some embodiments, between 0% and 99%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 99% of the nucleotides or nucleosides in the noncoding or coding region of the first linear polynucleotide are modified. In some embodiments, between 0% and 99%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 99% of the nucleotides or nucleosides in the 5’ and/or 3’ intron fragment of the first linear polynucleotide are modified. In some embodiments, between 0% and 99%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 99% of the nucleotides or nucleosides in the 5’ and/or 3’ exon fragment of the first linear polynucleotide are modified. In some embodiments, between 0% and 99%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 99% of the nucleotides or nucleosides in the spacer of the first linear polynucleotide are modified. In some embodiments, between 0% and 99%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 99% of the nucleotides or nucleosides in the internal and/or external homology region of the first linear 267
Attorney Docket No. 01318-0014-00PCT OR-043WO polynucleotide are modified. [394] In some embodiments, in portions of the first linear polynucleotide, less than 10% of the nucleotides or nucleosides are modified. For example, in some embodiments, less than 10% of the nucleotides or nucleosides in the intervening region of the first linear polynucleotide are modified. In some embodiments, less than 10% of the nucleotides or nucleosides in the TIE (e.g., IRES) of the first linear polynucleotide are modified. In some embodiments, less than 10% of the nucleotides or nucleosides in the noncoding or coding region of the first linear polynucleotide are modified. In some embodiments, less than 10% of the nucleotides or nucleosides in the 5’ and/or 3’ intron fragment of the first linear polynucleotide are modified. In some embodiments, less than 10% of the nucleotides or nucleosides in the 5’ and/or 3’ exon fragment of the first linear polynucleotide are modified. In some embodiments, less than 10% of the nucleotides or nucleosides in the spacer of the first linear polynucleotide are modified. In some embodiments, less than 10% of the nucleotides or nucleosides in the internal and/or external homology region of the first linear polynucleotide are modified. [395] In some embodiments, the intervening region of the first linear polynucleotide comprises no nucleotide or nucleoside modifications. In some embodiments, the TIE (e.g, IRES) of the first linear polynucleotide comprises no nucleotide or nucleoside modifications. In some embodiments, the noncoding or coding region of the first linear polynucleotide comprises no nucleotide or nucleoside modifications. In some embodiments, the 5’ and/or 3’ intron fragment of the first linear polynucleotide comprises no nucleotide or nucleoside modifications. In some embodiments, the 5’ and/or 3’ exon fragment of the first linear polynucleotide comprises no nucleotide or nucleoside modifications. In some embodiments, the spacer of the first linear polynucleotide comprises no nucleotide or nucleoside modifications. In some embodiments, the internal and/or external homology region of the first linear polynucleotide comprises no nucleotide or nucleoside modifications. [396] In some embodiments, between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 100% of the nucleotides or nucleosides in the second linear precursor are modified. In some embodiments, between 0% and 99%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 99% of the nucleotides or nucleosides in the second 268
Attorney Docket No. 01318-0014-00PCT OR-043WO linear precursor are modified. In some embodiments, between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 100% of the nucleotides or nucleosides in the second linear precursor are unmodified. [397] In some embodiments, in portions of the second linear polynucleotide, between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 100% of the nucleotides or nucleosides are modified. In some embodiments, between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 100% of the nucleotides or nucleosides in the noncoding or coding region of the second linear polynucleotide are modified. In some embodiments, between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 100% of the nucleotides or nucleosides in the 5’ and/or 3’ intron fragment of the second linear polynucleotide are modified. In some embodiments, between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 100% of the nucleotides or nucleosides in the 5’ and/or 3’ exon fragment of the second linear polynucleotide are modified. In some embodiments, between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 100% of the nucleotides or nucleosides in the spacer of the second linear polynucleotide are modified. In some embodiments, between 1% and 100%, 1% and 2%, 1% and 3%, 1% and 4%, 1% and 5%, 5% and 6%, 5% and 7%, 5% and 8%, 5% and 9%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 100% of the nucleotides or nucleosides in the internal and/or external homology region of the second linear polynucleotide are modified. [398] In some embodiments, in portions of the second linear polynucleotide, less than 269
Attorney Docket No. 01318-0014-00PCT OR-043WO 10% of the nucleotides or nucleosides are modified. For example, in some embodiments, less than 10% of the nucleotides or nucleosides in the noncoding or coding region of the second linear polynucleotide are modified. In some embodiments, less than 10% of the nucleotides or nucleosides in the 5’ and/or 3’ intron fragment of the second linear polynucleotide are modified. In some embodiments, less than 10% of the nucleotides or nucleosides in the 5’ and/or 3’ exon fragment of the second linear polynucleotide is modified. In some embodiments, less than 10% of the nucleotides or nucleosides in the spacer of the second linear polynucleotide are modified. In some embodiments, less than 10% of the nucleotides or nucleosides in the internal and/or external homology region of the second linear polynucleotide are modified. [399] For example, in some embodiments, the noncoding or coding region of the second linear polynucleotide comprises no nucleotide or nucleoside modifications. In some embodiments, the 5’ intron fragment and/or 3’ intron fragment of the second linear polynucleotide comprises no nucleotide or nucleoside modifications. In some embodiments, the 5’ exon fragment and/or 3’ exon fragment of the second linear polynucleotide comprises no nucleotide or nucleoside modifications. In some embodiments, the spacer of the second linear polynucleotide comprises no nucleotide or nucleoside modifications. In some embodiments, the internal and/or external homology region of the second linear polynucleotide comprises no nucleotide or nucleoside modifications. [400] In some embodiments, in a first linear precursor of the present disclosure, the intervening region comprises no nucleotide or nucleoside modifications or is less than 10% modified; the TIE (e.g., IRES) comprises no nucleotide or nucleoside modifications or is less than 10% modified; the noncoding or coding region comprises no nucleotide or nucleoside modifications or is less than 10% modified; the 5’ and/or 3’ intron fragment comprises no nucleotide or nucleoside modifications or is less than 10% modified; the 5’ and/or 3’ exon fragment comprises no nucleotide or nucleoside modifications or is less than 10% modified; the spacer comprises no nucleotide or nucleoside modifications or is less than 10% modified; and/or the internal and/or external homology region comprises no nucleotide or nucleoside modifications or is less than 10% modified. [401] In some embodiments, in a second linear precursor of the present disclosure, the noncoding or coding region comprises no nucleotide or nucleoside modifications or is less than 10% modified; the 5’ intron fragment and/or 3’ intron fragment comprises no nucleotide or nucleoside modifications or is less than 10% modified; the 5’ exon fragment and/or 3’ exon 270
Attorney Docket No. 01318-0014-00PCT OR-043WO fragment comprises no nucleotide or nucleoside modifications or is less than 10% modified; the spacer comprises no nucleotide or nucleoside modifications or is less than 10% modified; and/or the internal and/or external homology region comprises no nucleotide or nucleoside modifications or is less than 10% modified. [402] In some embodiments, incorporation of a nucleotide or nucleoside modification to a precursor RNA polynucleotide hinders or lowers the capacity of the circular RNA to circularize, splice, or express. In some embodiments, the precursor polynucleotide comprising no modified nucleotides and/or nucleosides maintains or improves circularization as compared to a precursor polynucleotide comprising one or more nucleotide or nucleoside modification. In some embodiments, the precursor polynucleotide comprising no modified nucleotides or nucleosides maintains circularization at greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 100% (i.e., improves circularization), as compared to a corresponding precursor polynucleotide comprising one or more nucleotide or nucleoside modification. [403] In some embodiments, the polynucleotides comprising no nucleotide or nucleoside modifications, for an example a circular RNA, has comparable or reduced immunogenicity as compared to a polynucleotide comprising one or more nucleotide or nucleoside modification. In some embodiments, the circular RNAs described herein (i.e., a circular RNA polynucleotide comprising no nucleotide or nucleoside modification) exhibit immunogenicity that is reduced by about 10% to about 99%, for example reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as compared to a corresponding circular RNA comprising one or more nucleotide or nucleoside modifications. In some embodiments, the polynucleotides comprising no modified nucleotides and/or modified nucleosides, for example a circular RNA, maintain or improve translation of a coding region as compared to a corresponding polynucleotide comprising one or more nucleotide or nucleoside modifications. In some embodiments, the polynucleotides comprising no modified nucleosides provide additional stability and resistance to immune activation. In some embodiments, for example, the polynucleotide comprising no modified A, C, G, or U nucleotide or nucleoside maintains expression at greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 100%, as compared to a corresponding precursor polynucleotide comprising one or more nucleotide or nucleoside modifications. In some embodiments, the non-modified polynucleotides maintain expression at greater than 70% as compared to a corresponding polynucleotide comprising one or more 271
Attorney Docket No. 01318-0014-00PCT OR-043WO nucleotide or nucleoside modifications. In some embodiments, the non-modified polynucleotides maintain expression at greater than 80% as compared to a corresponding polynucleotide comprising one or more nucleotide or nucleoside modifications. In some embodiments, the non-modified polynucleotides maintain expression at greater than 90% as compared to a corresponding polynucleotide comprising one or more nucleotide or nucleoside modifications. In some embodiments, the non-modified polynucleotides exhibit greater than 100% expression (i.e., improved expression) as compared to a corresponding polynucleotide comprising one or more nucleotide or nucleoside modifications. In some embodiments, the non-modified polynucleotides exhibit greater purification efficacy as compared to a corresponding polynucleotide comprising one or more nucleotide or nucleoside modifications. In some embodiments, for example, the polynucleotide comprising no modified A, C, G, or U nucleotide or nucleoside exhibits greater purification efficacy at greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 100%, as compared to a corresponding precursor comprising one or more nucleotide or nucleoside modification. G. Codon Optimization [404] In particular embodiments, polynucleotides may be codon-optimized. A codon- optimized sequence may be one in which codons in a polynucleotide encoding a polypeptide have been substituted in order to increase the expression, stability and/or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, (iii) systematic variation of codons including context, (iv) variation of codons according to their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence, (ix) prior knowledge about the function of the DNA sequences upon which design of the codon substitution set is to be based, and/or (x) systematic variation of codon sets for each amino acid. In some embodiments, a codon optimized polynucleotide may minimize ribozyme collisions and/or limit structural interference between the expression sequence and the core functional element. Codon optimization can be performed by methods known in the art. 272
Attorney Docket No. 01318-0014-00PCT OR-043WO H. PRECURSOR RNA [405] Provided herein are also precursor RNAs comprising both 5ʹ intron and exon elements and 3ʹ exon and intron elements or comprising only 3ʹ exon and intron elements for producing circular RNAs with enhanced circularization efficiency. [406] Accordingly, provided herein is a precursor RNA polynucleotide capable of producing a circular RNA polynucleotide after splicing, wherein the precursor RNA polynucleotide comprises both 5ʹ intron and exon elements and 3’ exon and intron elements (e.g., combined accessory elements). Also provided is a precursor RNA polynucleotide capable of producing a circular RNA polynucleotide after splicing, wherein the precursor RNA polynucleotide comprises only 3ʹ exon and intron elements. [407] In some embodiments, a provided precursor RNA polynucleotide comprises (i) 3ʹ permuted intron segment comprising a 5ʹ nucleotide of a 3ʹ splice site dinucleotide; and (ii) a 3ʹ exon segment comprising a 3ʹ nucleotide of a 3ʹ splice site dinucleotide. In some embodiments, a provided precursor RNA polynucleotide comprises (i) a 5ʹ exon segment comprising a 5ʹ nucleotide of a 5ʹ splice site dinucleotide; and (ii) a 5ʹ permuted intron segment comprising a 3ʹ nucleotide of a 5ʹ splice site dinucleotide. In some embodiments, a provided precursor RNA polynucleotide comprises a terminal element comprising (a) an excised terminal segment and a retained terminal segment or (b) a natural exon or a fragment thereof. [408] In some embodiments, a provided precursor RNA polynucleotide comprises (i) a 5ʹ intron element comprising a 3ʹ permuted intron segment comprising a 5ʹ nucleotide of a 3ʹ splice site dinucleotide; (ii) a 5ʹ exon element comprising a 3ʹ exon segment comprising a 3ʹ nucleotide of a 3ʹ splice site dinucleotide; (iii) a 3ʹ exon element comprising a 5ʹ exon segment comprising a 5ʹ nucleotide of a 5ʹ splice site; and (iv) a 3ʹ intron element comprising a 5ʹ permuted intron segment comprising a 3ʹ nucleotide of a 5ʹ splice site dinucleotide. In some embodiments, a provided precursor RNA polynucleotide comprises (i) a terminal element comprising (a) an excised terminal segment and/or a retained terminal segment or (b) a natural exon or a fragment thereof; (ii) a 5ʹ intron element comprising a 3ʹ permuted intron segment comprising a 5ʹ nucleotide of a 3ʹ splice site dinucleotide; and (iii) a 5ʹ exon element comprising a 3ʹ exon segment comprising a 3ʹ nucleotide of a 3ʹ splice site dinucleotide. [409] In some embodiments, a provided precursor RNA polynucleotide comprises a 5’ combined accessory element comprising (i) a 3ʹ permuted intron segment comprising a 5ʹ nucleotide of a 3ʹ splice site dinucleotide; and (ii) a 3ʹ exon segment comprising a 3ʹ nucleotide of a 3ʹ splice site dinucleotide. In some embodiments, element (ii) is located upstream to the 273
Attorney Docket No. 01318-0014-00PCT OR-043WO intervening region. In some embodiments, the 5ʹ combined accessory element comprises a 3ʹ exon segment comprising a Group I or Group II exon 3ʹ nucleotide of a 3ʹ splice site dinucleotide. [410] In some embodiments, a provided precursor RNA polynucleotide comprises a 3ʹ combined accessory element comprising (i) a 5ʹ exon segment comprising a 5ʹ nucleotide of a 5ʹ splice site dinucleotide; and (ii) a 5ʹ permuted intron segment comprising a 3ʹ nucleotide of a 5ʹ splice site dinucleotide. In some embodiments, element (ii) is located downstream to the intervening region. In some embodiments, a 3ʹ combined accessory element comprises a 5ʹ exon segment comprising a Group I or Group II exon 5ʹ nucleotide of a 5ʹ splice site dinucleotide. [411] In some embodiments, a provided precursor RNA polynucleotide comprises a 5’ combined accessory element, an intervening region, and a 3ʹ combined accessory element. In some embodiments, (a) the 5’ combined accessory element comprises (i) a 3ʹ permuted intron segment comprising a 5ʹ nucleotide of a 3ʹ splice site dinucleotide; and (ii) a 3ʹ exon segment comprising a 3ʹ nucleotide of a 3ʹ splice site dinucleotide; and (b) the 3ʹ combined accessory element comprising (i) a 5ʹ exon segment comprising a 5ʹ nucleotide of a 5ʹ splice site dinucleotide; and (ii) a 5ʹ permuted intron segment comprising a 3ʹ nucleotide of a 5ʹ splice site dinucleotide. In some embodiments, the 5ʹ nucleotide of a 3ʹ splice site dinucleotide, 3ʹ nucleotide of a 3ʹ splice site dinucleotide, 5ʹ nucleotide of a 5ʹ splice site dinucleotide and 3ʹ nucleotide of a 5ʹ splice site dinucleotide are optionally a combination of nucleotides or a portion of a sequence selected from Table A or Table B. [412] In some embodiments, the 5’ combined accessory element is located 5ʹ to the intervening region; and the intervening region is located is 5ʹ to the 3ʹ combined accessory element. [413] In some embodiments, a provided precursor RNA polynucleotide comprises a terminal element, an intervening region, and a monotron element. In some embodiments, the monotron element is located 5’ to the intervening region, which is located 5’ to the terminal element. In other embodiments, the monotron element is located 3’ to the intervening region, which is located 3’ to the terminal element. As set forth in further detail below, in some embodiments, the terminal element comprises a splice site nucleotide and the monotron element comprises a splice site dinucleotide and a splice site nucleotide. [414] In some embodiments, the precursor RNA polynucleotide is linear. [415] In some embodiments, permuted intron-exon splicing results in circularization of 274
Attorney Docket No. 01318-0014-00PCT OR-043WO the precursor RNA polynucleotide. During splicing, a transesterification reaction can occur at the 5’ splice site and a second transesterification reaction can occur at the 3’ splice site. In some embodiments, splicing of the precursor RNA polynucleotide results in the removal of the 3ʹ intron element and the 5ʹ intron element. Accordingly, the circular RNA polynucleotide produced after splicing of the precursor RNA polynucleotide lacks the 3ʹ intron segment and the 5ʹ intron segment, but retains the 3ʹ exon segment and the 5ʹ exon segment. [416] In some embodiments, the precursor RNA polynucleotide is capable of circularizing when incubated in the presence of one or more guanosine nucleotides or nucleoside (e.g., GTP) and a divalent cation (e.g., Mg2+). [417] In some embodiments, the precursor RNA polynucleotide is between 300 and 10000, between 400 and 9000, between 500 and 8000, between 600 and 7000, between 700 and 6000, between 800 and 5000, between 900 and 5000, between 1000 and 5000, between 1100 and 5000, between 1200 and 5000, between 1300 and 5000, between 1400 and 5000, or between 1500 and 5000 nucleotides (nt) in length. In some embodiments, the precursor RNA polynucleotide is at least 300 nt, at least 400 nt, at least 500 nt, at least 600 nt, at least 700 nt, at least 800 nt, at least 900 nt, at least 1000 nt, at least 1100 nt, at least 1200 nt, at least 1300 nt, at least 1400 nt, at least 1500 nt, at least 2000 nt, at least 2500 nt, at least 3000 nt, at least 3500 nt, at least 4000 nt, at least 4500 nt, or at least 5000 nt in length. In some embodiments, the precursor RNA polynucleotide is no more than 3000 nt, no more than 3500 nt, no more than 4000 nt, no more than 4500 nt, no more than 5000 nt, no more than 6000 nt, no more than 7000 nt, no more than 8000 nt, no more than 9000 nt, or no more than 10000 nt in length. In some embodiments, the precursor RNA polynucleotide is about 300 nt, about 400 nt, about 500 nt, about 600 nt, about 700 nt, about 800 nt, about 900 nt, about 1000 nt, about 1100 nt, about 1200 nt, about 1300 nt, about 1400 nt, about 1500 nt, about 2000 nt, about 2500 nt, about 3000 nt, about 3500 nt, about 4000 nt, about 4500 nt, about 5000 nt, about 6000 nt, about 7000 nt, about 8000 nt, about 9000 nt, or about 10000 nt in length. [418] In various embodiments, provided herein are DNA templates that transcribe into precursor RNA polynucleotides of the disclosure. Accordingly, provided herein are DNA templates comprising sequences encoding the precursor RNAs of the disclosure. In some embodiments, the DNA template or polynucleotide of the present disclosure comprises a vector, a PCR product, a plasmid, a minicircle DNA, a cosmid, an artificial chromosome, a complementary DNA (cDNA), an extrachromosomal DNA (ecDNA), a doggybone DNA (dbDNA), a close-ended DNA (ceDNA), a viral polynucleotide, or a fragment thereof. In some 275
Attorney Docket No. 01318-0014-00PCT OR-043WO embodiments, the polynucleotide of the present disclosure is selected from a DNA plasmid, a cosmid, a PCR product, dbDNA, close-ended DNA (ceDNA), and a viral polynucleotide. In some embodiments, the polynucleotide further comprises a promoter segment or sequence. In some embodiments, the DNA template is linearized. In other embodiments, the DNA template is non-linearized. In some embodiments, the DNA template is single-stranded. In some embodiments, the DNA template is double-stranded. In some embodiments, the DNA template comprises in whole or in part from a viral, bacterial or eukaryotic vector. [419] In various embodiments, provided herein is a circular RNA polynucleotide produced by circularization of a precursor RNA polynucleotide described herein. [420] In some embodiments, the circular RNA polynucleotide is produced inside a cell. In some embodiments, a provided precursor RNA is transcribed using a DNA template in the cytoplasm (e.g., by a bacteriophage RNA polymerase) or nucleus (e.g., by host RNA polymerase II) and then circularized. [421] In some embodiments, the circular RNA polynucleotide is between 300 and 10000, between 400 and 9000, between 500 and 8000, between 600 and 7000, between 700 and 6000, between 800 and 5000, between 900 and 5000, between 1000 and 5000, between 1100 and 5000, between 1200 and 5000, between 1300 and 5000, between 1400 and 5000, or between 1500 and 5000 nucleotides (nt) in length. In some embodiments, the circular RNA polynucleotide is at least 300 nt, at least 400 nt, at least 500 nt, at least 600 nt, at least 700 nt, at least 800 nt, at least 900 nt, at least 1000 nt, at least 1100 nt, at least 1200 nt, at least 1300 nt, at least 1400 nt, at least 1500 nt, at least 2000 nt, at least 2500 nt, at least 3000 nt, at least 3500 nt, at least 4000 nt, at least 4500 nt, or at least 5000 nt in length. In some embodiments, the circular RNA polynucleotide is no more than 3000 nt, no more than 3500 nt, no more than 4000 nt, no more than 4500 nt, no more than 5000 nt, no more than 6000 nt, no more than 7000 nt, no more than 8000 nt, no more than 9000 nt, or no more than 10000 nt in length. In some embodiments, the circular RNA polynucleotide is about 300 nt, about 400 nt, about 500 nt, about 600 nt, about 700 nt, about 800 nt, about 900 nt, about 1000 nt, about 1100 nt, about 1200 nt, about 1300 nt, about 1400 nt, about 1500 nt, about 2000 nt, about 2500 nt, about 3000 nt, about 3500 nt, about 4000 nt, about 4500 nt, about 5000 nt, about 6000 nt, about 7000 nt, about 8000 nt, about 9000 nt, or about 10000 nt in length. [422] Circular RNA polynucleotides lack the free ends necessary for exonuclease- mediated degradation, causing them to be resistant to several mechanisms of RNA degradation and granting extended half-lives when compared to an equivalent linear RNA. Circularization 276
Attorney Docket No. 01318-0014-00PCT OR-043WO may allow for the stabilization of RNA polynucleotides that generally suffer from short half- lives and may improve the overall efficacy of exogenous mRNA in a variety of applications. [423] In some embodiments, the circular RNA polynucleotide has 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 polynucleotide has a functional half-life of 5-80, 10-70, 15-60, or 20-50 hours. In some embodiments, the circular RNA polynucleotide has a functional half-life greater (e.g., at least 1.5-fold greater or at least 2-fold greater) than that of an equivalent linear RNA polynucleotide comprising the same expression sequence. In some embodiments, the circular RNA polynucleotide, or a pharmaceutical composition thereof, has a functional half-life in a human cell greater than or equal to that of a pre-determined threshold value. In some embodiments, the functional half-life is determined by a functional protein assay. For example, in some embodiments, the functional half-life is determined by an in vitro luciferase assay, wherein the activity of Gaussia luciferase (GLuc) is measured in the media of human cells (e.g., HepG2) expressing the circular RNA polynucleotide every 1, 2, 6, 12, or 24 hours over 1, 2, 3, 4, 5, 6, 7, or 14 days. In some embodiments, the functional half-life is determined by an in vivo assay, wherein levels of a protein encoded by the expression sequence of the circular RNA polynucleotide are measured in patient serum or tissue samples every 1, 2, 6, 12, or 24 hours over 1, 2, 3, 4, 5, 6, 7, or 14 days. In some embodiments, the pre-determined threshold value is the functional half-life of a reference linear RNA polynucleotide comprising the same expression sequence as the circular RNA polynucleotide. In some embodiment, the functional half-life of a circular RNA polynucleotides provided herein in eukaryotic cells (e.g., mammalian cells, such as human cells) as assessed by protein synthesis is at least 20 hours (e.g., at least 80 hours). [424] In some embodiments, the circular RNA polynucleotide provided herein has higher functional stability than an mRNA comprising the same expression sequence. In some embodiments, the circular RNA provided herein has higher functional stability than an mRNA comprising the same expression sequence, 5moU modifications, optimized UTR, cap, and/or polyA tail. [425] In some embodiments, a provided circular RNA polynucleotide may have a higher magnitude of expression, e.g., a higher magnitude of expression 24 hours after administration of RNA to cells, than an equivalent linear mRNA. In some embodiments, the circular RNA polynucleotide has a higher magnitude of expression than an mRNA comprising the same expression sequence, 5moU modifications, optimized UTR, cap, and/or polyA tail. 277
Attorney Docket No. 01318-0014-00PCT OR-043WO [426] In some embodiments, a provided circular RNA polynucleotide is transfected into a cell. In some embodiments, the DNA template, which transcribes into the precursor RNA polynucleotide from which the circular RNA polynucleotide is produced, is transfected into a cell and subsequently transcribed in the cell. Transcription of the circular RNA from the transfected DNA template may be induced via polymerases. In some embodiments, the polymerases are endogenous polymerases of the cell. In some embodiments, the polymerases are added to the cell. In some other embodiments, the polymerases are encoded by one or more nucleic acids transfected into the cell. [427] In some embodiments, the circular RNA polynucleotide is administered to an animal (e.g., a human) such that a polypeptide (e.g., an adjuvant, an adjuvant-like molecule, or an immunomodulatory molecule) encoded by the circular RNA polynucleotide is expressed inside the animal. [428] In some embodiments, a provided circular RNA is less immunogenic than an equivalent mRNA when exposed to an immune system of an organism or a certain type of immune cell. In some embodiments, the circular RNA is associated with modulated production of cytokines when exposed to an immune system of an organism or a certain type of immune cell. For example, in some embodiments, the circular RNA is associated with reduced production of IFN-β1, RIG-I, IL-2, IL-6, IFNγ, and/or TNFα when exposed to an immune system of an organism or a certain type of immune cell as compared to mRNA comprising the same expression sequence. In some embodiments, the circular RNA is associated with less IFN-β1, RIG-I, IL-2, IL-6, IFNγ, and/or TNFα transcript induction when exposed to an immune system of an organism or a certain type of immune cell as compared to mRNA comprising the same expression sequence. In some embodiments, the circular RNA is less immunogenic than mRNA comprising the same expression sequence. In some embodiments, the circular RNA is less immunogenic than mRNA comprising the same expression sequence, 5moU modifications, an optimized UTR, a cap, and/or a polyA tail. [429] Various circular RNA, circular RNA constructs, compositions comprising circular RNA, precursor RNA, and related methods are described, for example in US patent application 17/853,576, WO2019236673, WO2020237227, WO2021113777, WO2021226597, WO2021189059, WO2021236855, WO2022261490, WO2023056033, WO2023081526, WO2023141586, WO2023250375, and WO2024102677, which are each incorporated by reference in their entireties. 3. PAYLOADS – CODING REGIONS 278
Attorney Docket No. 01318-0014-00PCT OR-043WO [430] In various embodiments, a provided polynucleotide (e.g., a DNA template, a linear precursor RNA polynucleotide, or a circular RNA polynucleotide) comprises one or more expression sequences or portions thereof. In some embodiments, the precursor RNA polynucleotide and circular RNA constructs comprise at least one expression sequence encoding a binding molecule. In certain embodiments, the precursor RNA polynucleotide and the circular RNA constructs comprise at least one expression sequence encoding a therapeutic protein and an IRES, wherein the IRES can facilitate expression of the protein when delivered in vivo. In some embodiments, the IRES is synthetic, wherein the synthetic IRES comprises at least one addition, deletion, or substitution of a domain, motif, or nucleotide therein. In some embodiments, the at least one addition, deletion, or substitution thereof yields a synthetic IRES with increased or improved function and/or expression and/or stability as compared to the naturally occurring IRES. In some embodiments, the synthetic IRESs described herein exhibit improved expression and/or function and/or stability in hepatocytes, immune cells (e.g., lymphocytes, T cells), muscle cells (e.g., myotubes), for example, as compared to a naturally occurring IRES, the naturally occurring counterpart, or a CVB3 IRES or comparative IRES 1. Synthetic IRESs have been designed to increase expression of operably linked expression sequences (coding sequences) as compared to naturally occurring counterparts. In some embodiments, the synthetic IRES is capable of enhancing expression of the therapeutic protein as compared to a naturally occurring IRES. [431] In some embodiments, the coding (or non-coding region) is a part of the intervening region or core functional element located in between the 5’ end and 3’ end of a linear precursor RNA polynucleotide and resultant circular RNA. [432] In some embodiments, the precursor RNA polynucleotide and circular RNA may encode for various therapeutic proteins, cytokines, immune checkpoint inhibitors, agonists, chimeric antigen receptors (CARs), inhibitory receptor agonists, one or more T-Cell Receptors, and/or B- cell Receptors that are available in the art. The chimeric proteins may also include, for example, recombinant fusion proteins, chimeric mutant protein, or other fusion proteins. In some embodiments, the circular RNA comprising a synthetic IRES comprises at least one expression sequence encoding a therapeutic protein. In some embodiments, the sequence encoding for a therapeutic protein is selected from a chimeric antigen receptor (CAR), T-cell receptor (TCR), B-cell receptor (BCR), immune cell activation or inhibitory receptor, recombinant fusion protein, chimeric mutant protein, fusion protein, an antibody, nanobody, non-antibody protein, immune modulatory ligand, receptor, 279
Attorney Docket No. 01318-0014-00PCT OR-043WO structural protein, growth factor ligand or receptor, hormone or hormone receptor, transcription factor, checkpoint inhibitor or agonist, Fc fusion protein, anticoagulant, blood clotting factor, chaperone protein, antimicrobial protein, structural protein, biochemical enzyme, tight junction protein, mitochondrial stress response, cytoskeletal protein, metal- binding protein, or small molecule. [433] In some embodiments, the circular RNA comprises more than 1 expression sequence, e.g., 2, 3, 4, or 5 expression sequences. In some embodiments, the circular RNA is a bicistronic RNA. In some embodiments, the bicistronic RNA is codon optimized. Exemplary bicistronic circular RNA are described in WO2021/189059A2, which is incorporated by reference herein in its entirety. [434] In some embodiments, the precursor RNA polynucleotide and circular RNA constructs comprise at least one expression sequence encoding an antigen, adjuvant, or adjuvant-like protein, e.g., from an infectious agent. In these embodiments, the circular RNA construct may be used as a vaccine. [435] In some embodiments, the expression sequence encodes a therapeutic protein. Nonlimiting examples of therapeutic proteins are listed in Table 3. In some embodiments, the scFv, heavy variable domain, light variable domain, heavy CDR sequences, and/or light CDR sequences of the therapeutic proteins listed in Table 3 may be used. Table 3: Exemplary therapeutic proteins
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[436] In some embodiments, the therapeutic protein is selected from a CD19-targted chimeric antigen receptor (CAR), a BCMA-targeted CAR, MAGE-A4 T-cell receptor (TCR), NY-ESO TCR, erythropoietin (EPO), phenylalanine hydroxylase (PAH), carbamoyl phosphate synthetase I (CPS1), Cas9, ADAMTS13, FOXP3, IL-10, or IL-2. Exemplary sequences are provided herein in Table 3. In some embodiments, the target amino acid has an amino acid sequence that is identical to or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from Table 3, or a fragment thereof. [437] In some embodiments, the expression sequence encodes a therapeutic protein. In some embodiments, the expression sequence encodes a cytokine, e.g., IL-12p70, IL-15, IL-2, IL-18, IL-21, IFN-α, IFN- β, IL-10, TGF-beta, IL-4, or IL-35, or a functional fragment thereof. In some embodiments, the expression sequence encodes an immune checkpoint inhibitor. In some embodiments, the expression sequence encodes an agonist (e.g., a TNFR family member such as CD137L, OX40L, ICOSL, LIGHT, or CD70). In some embodiments, the expression sequence encodes a chimeric antigen receptor. In some embodiments, the expression sequence encodes an inhibitory receptor agonist (e.g., PDL1, PDL2, Galectin-9, VISTA, B7H4, or MHCII) or inhibitory receptor (e.g., PD1, CTLA4, TIGIT, LAG3, or TIM3). In some embodiments, the expression sequence encodes an inhibitory receptor antagonist. In some embodiments, the expression sequence encodes one or more TCR chains (alpha and beta chains 286
Attorney Docket No. 01318-0014-00PCT OR-043WO or gamma and delta chains). In some embodiments, the expression sequence encodes a secreted T cell or immune cell engager (e.g., a bispecific antibody such as BiTE, targeting, e.g., CD3, CD137, or CD28 and a tumor-expressed protein e.g., CD19, CD20, or BCMA etc.). In some embodiments, the expression sequence encodes a transcription factor (e.g., FOXP3, HELIOS, TOX1, or TOX2). In some embodiments, the expression sequence encodes an immunosuppressive enzyme (e.g., IDO or CD39/CD73). In some embodiments, the expression sequence encodes a GvHD (e.g., anti-HLA-A2 CAR-Tregs). [438] In some embodiments, the circular RNA encodes one or more wild type or engineered proteins, peptides or polypeptides (e.g., antigens, adjuvant, or adjuvant-like proteins) that is suitable for use in a circular RNA vaccine. The expression sequence may be an antigen or adjuvant derived from an infectious agent, e.g., a virus, bacterium, fungus, protozoan, and/or parasite. In some embodiments, the antigen is a viral antigen. In an embodiment, the antigen is a SARS-CoV-2 antigen. In an embodiment, the antigen is SARS- CoV-2 spike protein. In an embodiment, the antigen is derived from the group consisting of rotavirus, foot and mouth disease virus, influenza A virus, influenza B virus, influenza C virus, H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, human parainfluenza type 2, herpes simplex virus, Epstein-Barr virus, varicella virus, porcine herpesvirus 1, cytomegalovirus, lyssavirus, Bacillus anthracis, anthrax PA and derivatives, poliovirus, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis E, distemper virus, venezuelan equine encephalomyelitis, feline leukemia virus, reovirus, respiratory syncytial virus, Lassa fever virus, polyoma tumor virus, canine parvovirus, papilloma virus, tick borne encephalitis virus, rinderpest virus, human rhinovirus species, Enterovirus species, Mengovirus, paramyxovirus, avian infectious bronchitis virus, human T-cell leukemia-lymphoma virus 1, human immunodeficiency virus-1, human immunodeficiency virus-2, norovirus, lymphocytic choriomeningitis virus, parvovirus B19, adenovirus, rubella virus, yellow fever virus, dengue virus, bovine respiratory syncitial virus, corona virus, Bordetella pertussis, Bordetella bronchiseptica, Bordetella parapertussis, Brucella abortis, Brucella melitensis, Brucella suis, Brucella ovis, Brucella species, Escherichia coli, Salmonella species, Salmonella typhi, Streptococci, Vibrio cholera, Vibrio parahaemolyticus, Shigella, Pseudomonas, tuberculosis, avium, Bacille Calmette Guerin, Mycobacterium leprae, Pneumococci, Staphlylococci, Enterobacter species, Rochalimaia henselae, Pasteurella haemolytica, Pasteurella multocida, Chlamydia trachomatis, Chlamydia psittaci, Lymphogranuloma venereum, Treponema pallidum, Haemophilus species, Mycoplasma bovigenitalium, Mycoplasma pulmonis, 287
Attorney Docket No. 01318-0014-00PCT OR-043WO Mycoplasma species, Borrelia burgdorferi, Legionalla pneumophila, Colstridium botulinum, Corynebacterium diphtheriae, Yersinia entercolitica, Rickettsia rickettsii, Rickettsia typhi, Rickettsia prowsaekii, Ehrlichia chaffeensis, Anaplasma phagocytophilum, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Schistosomes, trypanosomes, Leishmania species, Filarial nematodes, trichomoniasis, sarcosporidiasis, Taenia saginata, Taenia solium, Leishmania, Toxoplasma gondii, Trichinella spiralis, coccidiosis, Eimeria tenella, Cryptococcus neoformans, Candida albican, Aspergillus fumigatus, coccidioidomycosis, Neisseria gonorrhoeae, malaria circumsporozoite protein, malaria merozoite protein, trypanosome surface antigen protein, pertussis, alphaviruses, adenovirus, diphtheria toxoid, tetanus toxoid, meningococcal outer membrane protein, streptococcal M protein, influenza hemagglutinin, cancer antigen, tumor antigens, toxins, clostridium perfringens epsilon toxin, ricin toxin, pseudomonas exotoxin, exotoxins, neurotoxins, cytokines, cytokine receptors, monokines, monokine receptors, plant pollens, animal dander, and dust mites. In some embodiments, the adjuvant is selected from or derived from the group consisting of BCSP31, MOMP, FomA, MymA, ESAT6, PorB, PVL, Porin, OmpA, PepO, OmpU, Lumazine synthase, Omp16, Omp19, CobT, RpfE, Rv0652, HBHA, NhhA, DnaJ, Pneumolysin, Falgellin, IFN-alpha, IFN-gamma, IL-2, IL-12, IL-15, IL-18, IL-21, GM-CSF, IL-1b, IL-6, TNF-a, IL-7, IL-17, IL-1Beta, anti-CTLA4, anti-PD1, anti-41BB, PD-L1, Tim-3, Lag-3, TIGIT, GITR, and anti-CD3. [439] In some embodiments, a provided polynucleotide encodes a protein that is made up of subunits that 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 that more than one provided polynucleotides (e.g., circular RNA polynucleotides) are delivered in the transfer vehicle and each polynucleotide encodes a separate subunit of the protein. In certain embodiments, polynucleotides encoding the individual subunits may be administered in separate transfer vehicles. Alternatively, a single polynucleotide (e.g., circular RNA polynucleotide) may be engineered to encode more than one subunit. A. ANTIGEN-RECOGNITION RECEPTORS a. CHIMERIC ANTIGEN RECEPTORS (CARs) [440] In some embodiments, a provided polynucleotide (e.g., a DNA template, a linear precursor RNA polynucleotide, or a circular RNA polynucleotide) encodes one or more chimeric antigen receptors (CARs). CARs are genetically-engineered receptors. These 288
Attorney Docket No. 01318-0014-00PCT OR-043WO engineered receptors may be inserted into and expressed by immune cells, including T cells via circular RNA as described herein. With a CAR, a single receptor may be programmed to both recognize a specific antigen and, when bound to that antigen, activate the immune cell to attack and destroy the cell bearing that antigen. When these antigens exist on tumor cells, an immune cell that expresses the CAR may target and kill the tumor cell. In some embodiments, the CAR encoded by the polynucleotide comprises (i) an antigen-binding molecule that specifically binds to a target antigen, (ii) a hinge domain, a transmembrane domain, and an intracellular domain, and (iii) an activating domain. [441] In some embodiments, an orientation of the CARs in accordance with the disclosure comprises an antigen binding domain (such as an scFv) in tandem with a costimulatory domain and an activating domain. The costimulatory domain may comprise one or more of an extracellular portion, a transmembrane portion, and an intracellular portion. In other embodiments, multiple costimulatory domains may be utilized in tandem. i. Antigen binding domain [442] CARs may be engineered to bind to an antigen (such as a cell-surface antigen) by incorporating an antigen binding molecule that interacts with that targeted antigen. In some embodiments, the antigen binding molecule is an antibody fragment thereof, e.g., one or more single chain antibody fragment (scFv). An scFv is a single chain antibody fragment having the variable regions of the heavy and light chains of an antibody linked together. See U.S. Patent Nos. 7,741,465, and 6,319,494, as well as Eshhar et al., Cancer Immunol Immunotherapy (1997) 45: 131-136. An scFv retains the parent antibody's ability to specifically interact with target antigen. scFvs are useful in chimeric antigen receptors because they may be engineered to be expressed as part of a single chain along with the other CAR components. Id. See also Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161 : 2791-2797. It will be appreciated that the antigen binding molecule is typically contained within the extracellular portion of the CAR such that it is capable of recognizing and binding to the antigen of interest. Bispecific and multispecific CARs are contemplated within the scope of the disclosure, with specificity to more than one target of interest. [443] In some embodiments, the antigen binding molecule comprises a single chain, wherein the heavy chain variable region and the light chain variable region are connected by a linker. In some embodiments, the VH is located at the N terminus of the linker and the VL is located at the C terminus of the linker. In other embodiments, the VL is located at the N 289
Attorney Docket No. 01318-0014-00PCT OR-043WO terminus of the linker and the VH is located at the C terminus of the linker. In some embodiments, the linker comprises at least about 5, at least about 8, at least about 10, at least about 13, at least about 15, at least about 18, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 amino acids. [444] In some embodiments, the antigen binding molecule comprises a nanobody. In some embodiments, the antigen binding molecule comprises a DARPin. In some embodiments, the antigen binding molecule comprises an anticalin or other synthetic protein capable of specific binding to target protein. [445] In some embodiments, the CAR comprises an antigen binding domain specific for an antigen selected from the group CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin- like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3, TNF receptor family member B cell maturation (BCMA), Tn antigen ((Tn Ag) or (GaINAca-Ser/Thr)), prostate-specific membrane antigen (PSMA), Receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-Like Tyrosine Kinase 3 (FLT3), Tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, Carcinoembryonic antigen (CEA), Epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), Interleukin-13 receptor subunit alpha-2, mesothelin, Interleukin 11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), Protease Serine 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis(Y) antigen, CD24, Platelet-derived growth factor receptor beta (PDGFR-beta), Stage-specific embryonic antigen-4 (SSEA-4), CD20, Folate receptor alpha, HER2, HER3, Mucin 1, cell surface associated (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), Prostase, prostatic acid phosphatase (PAP), elongation factor 2 mutated (ELF2M), Ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2), glycoprotein 100 (gp100), oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl), tyrosinase, ephrin type-A receptor 2 (EphA2), Fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight-melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), Folate receptor beta, tumor endothelial marker 1 (TEM1/CD248), tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), thyroid stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, 290
Attorney Docket No. 01318-0014-00PCT OR-043WO member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), Polysialic acid, placenta-specific 1 (PLAC1), hexasaccharide portion of globoH glycoceramide (GloboH), mammary gland differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), Hepatitis A virus cellular receptor 1 (HAVCR1), adrenoceptor beta 3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K 9 (LY6K), Olfactory receptor 51E2 (OR51E2), TCR Gamma Alternate Reading Frame Protein (TARP), Wilms tumor protein (WT1), Cancer/testis antigen 1 (NY-ESO-1), Cancer/testis antigen 2 (LAGE-1a), MAGE family members (including MAGE-A1, MAGE-A3 and MAGE-A4), ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X Antigen Family, Member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie 2), melanoma cancer testis antigen- 1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), Fos-related antigen 1, tumor protein p53 (p53), p53 mutant, prostein, surviving, telomerase, prostate carcinoma tumor antigen-1, melanoma antigen recognized by T cells 1, Rat sarcoma (Ras) mutant, human Telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoints, melanoma inhibitor of apoptosis (ML-IAP), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-Acetyl glucosaminyl-transferase V (NA17), paired box protein Pax-3 (PAX3), Androgen receptor, Cyclin B1, v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN), Ras Homolog Family Member C (RhoC), Tyrosinase-related protein 2 (TRP-2), Cytochrome P4501B1 (CYP1B1), CCCTC-Binding Factor (Zinc Finger Protein)-Like, Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3), Paired box protein Pax-5 (PAX5), proacrosin binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A kinase anchor protein 4 (AKAP-4), synovial sarcoma, X breakpoint 2 (SSX2), Receptor for Advanced Glycation Endproducts (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), legumain, human papilloma virus E6 (HPV E6), human papilloma virus E7 (HPV E7), intestinal carboxyl esterase, heat shock protein 70-2 mutated (mut hsp70-2), CD79a, CD79b, CD72, Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), Glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), MUC16, 5T4, 8H9, ανβθ integrin, αvβ6 integrin, alphafetoprotein (AFP), B7-H6, ca-125, CA9, 291
Attorney Docket No. 01318-0014-00PCT OR-043WO CD44, CD44v7/8, CD52, E-cadherin, EMA (epithelial membrane antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB4, epithelial tumor antigen (ETA), folate binding protein (FBP), kinase insert domain receptor (KDR), k-light chain, L1 cell adhesion molecule, MUC18, NKG2D, oncofetal antigen (h5T4), tumor/testis-antigen 1B, GAGE, GAGE-1, BAGE, SCP-1, CTZ9, SAGE, CAGE, CT10, MART-1, immunoglobulin lambda-like polypeptide 1 (IGLL1), Hepatitis B Surface Antigen Binding Protein (HBsAg), viral capsid antigen (VCA), early antigen (EA), EBV nuclear antigen (EBNA), HHV-6 p41 early antigen, HHV-6B U94 latent antigen, HHV-6B p98 late antigen, cytomegalovirus (CMV) antigen, large T antigen, small T antigen, adenovirus antigen, respiratory syncytial virus (RSV) antigen, haemagglutinin (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 lympotrophic virus (HTLV-1) antigen, Merkel cell polyoma virus small T antigen, Merkel cell polyoma virus large T antigen, Kaposi sarcoma-associated herpesvirus (KSHV) lytic nuclear antigen and KSHV latent nuclear antigen. [446] In some embodiments, the circular RNA constructs and related pharmaceutical compositions comprise the expression sequences described in Table 3, above. In some embodiments, the circular RNA constructs and related pharmaceutical compositions disclosed herein comprise an expression sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence in Table 3, wherein the codon sequence produces a protein having the desired sequence. ii. Hinge / spacer domain [447] In some embodiments, a CAR of the instant disclosure comprises a hinge or spacer domain. In some embodiments, the hinge/spacer domain may comprise a truncated hinge/spacer domain (THD) the THD domain is a truncated version of a complete hinge/spacer domain (“CHD”). In some embodiments, an extracellular domain is from or derived from (e.g., comprises all or a fragment of) ErbB2, glycophorin A (GpA), CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8a, CD8[T CDl la (IT GAL), CDl lb (IT GAM), CDl lc (ITGAX), CDl ld (IT 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), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B-cell antigen receptor complex-associated alpha chain), CD79B (B-cell antigen receptor complex-associated beta 292
Attorney Docket No. 01318-0014-00PCT OR-043WO chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (0X40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-zeta), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK- p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRT AM), CD357 (TNFRSF18), inducible T cell co-stimulator (ICOS), LFA-1 (CDl la/CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1/CBP, a CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, a Toll ligand receptor, and fragments or combinations thereof. A hinge or spacer domain may be derived either from a natural or from a synthetic source. [448] In some embodiments, a hinge or spacer domain is positioned between an antigen binding molecule (e.g., an scFv) and a transmembrane domain. In this orientation, the hinge/spacer domain provides distance between the antigen binding molecule and the surface of a cell membrane on which the CAR is expressed. In some embodiments, a hinge or spacer domain is from or derived from an immunoglobulin. In some embodiments, a hinge or spacer domain is selected from the hinge/spacer regions of IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or a fragment thereof. In some embodiments, a hinge or spacer domain comprises, is from, or is derived from the hinge/spacer region of CD8 alpha. In some embodiments, a hinge or spacer domain comprises, is from, or is derived from the hinge/spacer region of CD28. In some embodiments, a hinge or spacer domain comprises a fragment of the hinge/spacer region of CD8 alpha or a fragment of the hinge/spacer region of CD28, wherein the fragment is anything less than the whole hinge/spacer region. In some embodiments, the fragment of the CD8 alpha hinge/spacer region or the fragment of the CD28 hinge/spacer region comprises an amino acid sequence that excludes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids at the N- terminus or C-Terminus, or both, of the CD8 alpha hinge/spacer region, or of the CD28 hinge/spacer region. 293
Attorney Docket No. 01318-0014-00PCT OR-043WO iii. Transmembrane domain [449] The CAR of the present disclosure may further comprise a transmembrane domain and/or an intracellular signaling domain. The transmembrane domain may be designed to be fused to the extracellular domain of the CAR. It may similarly be fused to the intracellular domain of the CAR. In some embodiments, the transmembrane domain that naturally is associated with one of the domains in a CAR is used. In some instances, the transmembrane domain may be selected or modified (e.g., by an amino acid substitution) to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. [450] Transmembrane regions may be derived from (i.e. comprise) a receptor tyrosine kinase (e.g., ErbB2), glycophorin A (GpA), 4-1BB/CD137, activating NK cell receptors, an immunoglobulin protein, B7-H3, BAFFR, BFAME (SEAMF8), BTEA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96 (Tactile), CD1 la, CD1 lb, CD1 lc, CD1 Id, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (EIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IE-2R beta, IE-2R gamma, IE-7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA4, ITGA6, IT GAD, ITGAE, ITGAE, IT GAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, EAT, LFA-1, LFA-1, a ligand that specifically binds with CD83, LIGHT, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CDl-la/CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or a fragment, truncation, or a combination thereof. [451] In some embodiments, suitable intracellular signaling domain include, but are not limited to, activating Macrophage/Myeloid cell receptors CSFR1, MYD88, CD14, TIE2, TLR4, CR3, CD64, TREM2, DAP10, DAP12, CD169, DECTIN1, CD206, CD47, CD163, 294
Attorney Docket No. 01318-0014-00PCT OR-043WO CD36, MARCO, TIM4, MERTK, F4/80, CD91, C1QR, LOX-1, CD68, SRA, BAI-1, ABCA7, CD36, CD31, Lactoferrin, or a fragment, truncation, or combination thereof. [452] In some embodiments, a receptor tyrosine kinase may be derived from (e.g., comprise) Insulin receptor (InsR), Insulin-like growth factor I receptor (IGF1R), Insulin receptor-related receptor (IRR), platelet derived growth factor receptor alpha (PDGFRa), platelet derived growth factor receptor beta (PDGFRfi). KIT proto-oncogene receptor tyrosine kinase (Kit), colony stimulating factor 1 receptor (CSFR), fms related tyrosine kinase 3 (FLT3), fms related tyrosine kinase 1 (VEGFR-1), kinase insert domain receptor (VEGFR-2), fms related tyrosine kinase 4 (VEGFR-3), fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), fibroblast growth factor receptor 4 (FGFR4), protein tyrosine kinase 7 (CCK4), neurotrophic receptor tyrosine kinase 1 (trkA), neurotrophic receptor tyrosine kinase 2 (trkB), neurotrophic receptor tyrosine kinase 3 (trkC), receptor tyrosine kinase like orphan receptor 1 (ROR1), receptor tyrosine kinase like orphan receptor 2 (ROR2), muscle associated receptor tyrosine kinase (MuSK), MET proto-oncogene, receptor tyrosine kinase (MET), macrophage stimulating 1 receptor (Ron), AXL receptor tyrosine kinase (Axl), TYR03 protein tyrosine kinase (Tyro3), MER proto-oncogene, tyrosine kinase (Mer), tyrosine kinase with immunoglobulin like and EGF like domains 1 (TIE1), TEK receptor tyrosine kinase (TIE2), EPH receptor A1 (EphAl), EPH receptor A2 (EphA2), (EPH receptor A3) EphA3, EPH receptor A4 (EphA4), EPH receptor A5 (EphA5), EPH receptor A6 (EphA6), EPH receptor A7 (EphA7), EPH receptor A8 (EphA8), EPH receptor A10 (EphAlO), EPH receptor B1 (EphBl), EPH receptor B2 (EphB2), EPH receptor B3 (EphB3), EPH receptor B4 (EphB4), EPH receptor B6 (EphB6), ret proto oncogene (Ret), receptor-like tyrosine kinase (RYK), discoidin domain receptor tyrosine kinase 1 (DDR1), discoidin domain receptor tyrosine kinase 2 (DDR2), c-ros oncogene 1, receptor tyrosine kinase (ROS), apoptosis 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). iv. Costimulatory domain [453] In certain embodiments, the CAR comprises a costimulatory domain. In some embodiments, the costimulatory domain comprises 4-1BB (CD137), CD28, or both, and/or an intracellular T cell signaling domain. In a preferred embodiment, the costimulatory domain is human CD28, human 4-1BB, or both, and the intracellular T cell signaling domain is human CD3 zeta (ζ).4-1BB, CD28, CD3 zeta may comprise less than the whole 4-1BB, CD28 or CD3 295
Attorney Docket No. 01318-0014-00PCT OR-043WO zeta, respectively. Chimeric antigen receptors may incorporate costimulatory (signaling) domains to increase their potency. See U.S. Patent Nos. 7,741,465, and 6,319,494, as well as Krause et al. and Finney et al. (supra), Song et al., Blood 119:696-706 (2012); Kalos et al., Sci Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Amur. Rev. Pharmacol. Toxicol. 56:59-83 (2016). [454] In some embodiments, a costimulatory domain comprises the amino acid sequence of the following sequences: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL or QVQLVQSGAEVEKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPN SGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCASGWDFDYWGQGT LVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQSPSSLSASVGDRVTITCRASQSIRY YLSWYQQKPGKAPKLLIYTASILQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCL QTYTTPDFGPGTKVEIK. See, e.g., PCT Application No. US2022/33091 (WO202261490), which is incorporated herein by reference in its entirety. v. Intracellular signaling domain [455] The intracellular (signaling) domain of the engineered T cells disclosed herein may provide signaling to an activating domain, which then activates at least one of the normal effector functions of the immune cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. [456] In some embodiments, suitable intracellular signaling domain comprise, but are not limited to 4-1BB/CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD 19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96 (Tactile), CD1 la, CD1 lb, CD1 lc, CD1 1d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand that specifically binds with CD83, LIGHT, LTBR, Ly9 (CD229), Lyl08, lymphocyte function-associated antigen- 1 (LFA-1; CDl-la/CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMF1; 296
Attorney Docket No. 01318-0014-00PCT OR-043WO CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or a fragment, truncation, or a combination thereof. [457] CD3 is an element of the T cell receptor on native T cells, and has been shown to be an important intracellular activating element in CARs. In some embodiments, the CD3 is CD3 zeta. In some embodiments, the activating domain comprises an amino acid sequence at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR. See, e.g., PCT Application No. US2022/33091, which is incorporated herein by reference in its entirety. b. T-CELL RECEPTORS (TCR) [458] In some embodiments, a provided polynucleotide (e.g., a DNA template, a linear precursor RNA polynucleotide, or a circular RNA polynucleotide) encodes a T-cell receptor. TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature, and links to the IMGT public database of TCR sequences. Native alpha-beta heterodimeric TCRs have an alpha chain and a beta chain. Broadly, each chain may comprise variable, joining and constant regions, and the beta chain also usually contains a short diversity region between the variable and joining regions, but this diversity region is often considered as part of the joining region. Each variable region may comprise three CDRs (Complementarity Determining Regions) embedded in a framework sequence, one being the hypervariable region named CDR3. There are several types of alpha chain variable (Vα) regions and several types of beta chain variable (Vβ) regions distinguished by their framework, CDR1 and CDR2 sequences, and by a partly defined CDR3 sequence. The Vα types are referred to in IMGT nomenclature by a unique TRAV number. Thus “TRAV21” defines a TCR Vα region having unique framework and CDR1 and CDR2 sequences, and a CDR3 sequence which is partly defined by an amino acid sequence which is preserved from TCR to TCR but which also includes an amino acid sequence which varies from TCR to TCR. In the same way, “TRBV5-1” defines a TCR Vβ region having unique framework and CDR1 and CDR2 sequences, but with only a partly defined CDR3 sequence. [459] The joining regions of the TCR are similarly defined by the unique IMGT TRAJ 297
Attorney Docket No. 01318-0014-00PCT OR-043WO and TRBJ nomenclature, and the constant regions by the IMGT TRAC and TRBC nomenclature. [460] The beta chain diversity region is referred to in IMGT nomenclature by the abbreviation TRBD, and, as mentioned, the concatenated TRBD/TRBJ regions are often considered together as the joining region. [461] The unique sequences defined by the IMGT nomenclature are widely known and accessible to those working in the TCR field. For example, they can be found in the IMGT public database. The “T cell Receptor Factsbook”, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8 also discloses sequences defined by the IMGT nomenclature, but because of its publication date and consequent time-lag, the information therein sometimes needs to be confirmed by reference to the IMGT database. [462] Native TCRs exist in heterodimeric αβ or γδ forms. However, recombinant TCRs consisting of αα or ββ homodimers have previously been shown to bind to peptide MHC molecules. Therefore, the TCR of the present disclosure may be a heterodimeric αβ TCR or may be an αα or ββ homodimeric TCR. [463] For use in adoptive therapy, an αβ heterodimeric TCR may, for example, be transfected as full length chains having both cytoplasmic and transmembrane domains. In certain embodiments TCRs of the present disclosure may have an introduced disulfide bond between residues of the respective constant domains, as described, for example, in WO 2006/000830. [464] TCRs of the present disclosure, particularly alpha-beta heterodimeric TCRs, may comprise an alpha chain TRAC constant domain sequence and/or a beta chain TRBC1 or TRBC2 constant domain sequence. The alpha and beta chain constant domain sequences may be mutated by truncation or substitution to delete the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2. The alpha and/or beta chain constant domain sequence(s) may also be mutated by substitution of cysteine residues for Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2, the said cysteines forming a disulfide bond between the alpha and beta constant domains of the TCR. [465] Binding affinity (inversely proportional to the equilibrium constant KD) and binding half-life (expressed as T½) can be determined by any appropriate method. It will be appreciated that doubling the affinity of a TCR results in halving the KD. T½ is calculated as ln 2 divided by the off-rate (koff). So doubling of T½ results in a halving in koff. KD and koff values for TCRs are usually measured for soluble forms of the TCR, i.e. those forms which are truncated 298
Attorney Docket No. 01318-0014-00PCT OR-043WO to remove cytoplasmic and transmembrane domain residues. Therefore it is to be understood that a given TCR has an improved binding affinity for, and/or a binding half-life for the parental TCR if a soluble form of that TCR has the said characteristics. Preferably the binding affinity or binding half-life of a given TCR is measured several times, for example, 3 or more times, using the same assay protocol, and an average of the results is taken. [466] Since the TCRs of the present disclosure have utility in adoptive therapy, the disclosure includes a non-naturally occurring and/or purified and/or or engineered cell, especially a T-cell, presenting a TCR of the present disclosure. There are a number of methods suitable for the transfection of T cells with nucleic acid (such as DNA, cDNA or RNA) encoding the TCRs of the disclosure (see for example Robbins et al., (2008) J Immunol. 180: 6116-6131). T cells expressing the TCRs will be suitable for use in adoptive therapy-based treatment of cancers such as those of the pancreas and liver. As will be known to those skilled in the art, there are a number of suitable methods by which adoptive therapy can be carried out (see for example Rosenberg et al., (2008) Nat Rev Cancer 8(4): 299-308). [467] As is well-known in the art TCRs of the present disclosure may be subject to post- translational modifications when expressed by transfected cells. Glycosylation is one such modification, which may comprise the covalent attachment of oligosaccharide moieties to defined amino acids in the TCR chain. For example, asparagine residues, or serine/threonine residues are well-known locations for oligosaccharide attachment. The glycosylation status of a particular protein depends on a number of factors, including protein sequence, protein conformation and the availability of certain enzymes. Furthermore, glycosylation status (i.e., oligosaccharide type, covalent linkage and total number of attachments) can influence protein function. Therefore, when producing recombinant proteins, controlling glycosylation is often desirable. Glycosylation of transfected TCRs may be controlled by mutations of the transfected gene (Kuball J et al. (2009), J Exp Med 206(2):463-475). Such mutations are also encompassed herein. [468] A TCR may be specific for an antigen in the group MAGE-A1, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-A13, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE- Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (AGE-B4), tyrosinase, brain glycogen phosphorylase, Melan-A, MAGE-C1, MAGE-C2, NY-ESO-1, LAGE-1, SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, alpha-actinin-4, Bcr-Abl fusion 299
Attorney Docket No. 01318-0014-00PCT OR-043WO protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6- AML1 fusion protein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-2, and 3, neo-PAP, myosin class I, OS-9, pml-RARa fusion protein, PTPRK, K-ras, N-ras, Triosephosphate isomeras, 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 antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 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\cyclophilin C-associated protein), TAAL6, TAG72, TLP, and TPS. c. B-CELL RECEPTORS (BCR) [469] In some embodiments, a provided polynucleotide (e.g., a DNA template, a linear precursor RNA polynucleotide, or a circular RNA polynucleotide) encodes one or more B-cell receptors (BCRs). BCRs (or B-cell antigen receptors) are immunoglobulin molecules that form a type I transmembrane protein on the surface of a B cell. A BCR is capable of transmitting activatory signal into a B cell following recognition of a specific antigen. Prior to binding of a B cell to an antigen, the BCR will remain in an unstimulated or “resting” stage. Binding of an antigen to a BCR leads to signaling that initiates a humoral immune response. [470] A BCR is expressed by mature B cells. These B cells work with immunoglobulins (Igs) in recognizing and tagging pathogens. The typical BCR comprises a membrane-bound immunoglobulin (e.g., mIgA, mIgD, mIgE, mIgG, and mIgM), along with associated and Igα/Igβ (CD79a/CD79b) heterodimers (α/β). These membrane-bound immunoglobulins are tetramers consisting of two identical heavy and two light chains. Within the BCR, the membrane bound immunoglobulins is capable of responding to antigen binding by signal transmission across the plasma membrane leading to B cell activation and consequently clonal expansion and specific antibody production (Friess M et al. (2018), Front. Immunol.2947(9)). The Igα/Igβ heterodimers is responsible for transducing signals to the cell interior. [471] A Igα/Igβ heterodimer signaling relies on the presence of immunoreceptor tyrosine- 300
Attorney Docket No. 01318-0014-00PCT OR-043WO based activation motifs (ITAMs) located on each of the cytosolic tails of the heterodimers. ITAMs comprise two tyrosine residues separated by 9-12 amino acids (e.g., tyrosine, leucine, and/or valine). Upon binding of an antigen, the tyrosine of the BCR’s ITAMs become phosphorylated by Src-family tyrosine kinases Blk, Fyn, or Lyn (Janeway C et al., Immunobiology: The Immune System in Health and Disease (Garland Science, 5th ed.2001)). d. OTHER CHIMERIC PROTEINS [472] In addition to the chimeric proteins provided above, a provided polynucleotide (e.g., a DNA template, a linear precursor RNA polynucleotide, or a circular RNA polynucleotide) may encode for a various number of other chimeric proteins available in the art. The chimeric proteins may include recombinant fusion proteins, chimeric mutant protein, or other fusion proteins. B. IMMUNE MODULATORY LIGANDS AND CYTOKINES [473] In some embodiments, a provided polynucleotide (e.g., a DNA template, a linear precursor RNA polynucleotide, or a circular RNA polynucleotide) encodes for an immune modulatory ligand. In certain embodiments, the immune modulatory ligand may be immunostimulatory; while in other embodiments, the immune modulatory ligand may be immunosuppressive. [474] In some embodiments, the circular RNA polynucleotide encodes for a cytokine or a functional fragment thereof, including but not limited to interferons, chemokines, interleukins, growth factors, and other cytokines known in the art. In some embodiments, the cytokine comprises a chemokine, interferon, interleukin, lymphokine, and/or tumor necrosis factor. Chemokines are chemotactic cytokine produced by a variety of cell types in acute and chronic inflammation that mobilizes and activates white blood cells. An interferon comprises a family of secreted α-helical cytokines induced in response to specific extracellular molecules through stimulation of TLRs (Borden, Molecular Basis of Cancer (Fourth Edition) 2015). Interleukins are cytokines expressed by leukocytes. [475] Descriptions and/or amino acid sequences of IL-2, IL-7, IL-10, IL-12, IL-15, IL- 18, IL-27β, IFNγ, and/or TGFβ1 are provided herein and at the www.uniprot.org database at 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). 301
Attorney Docket No. 01318-0014-00PCT OR-043WO C. TRANSCRIPTION FACTORS [476] Regulatory T cells (Treg) are important in maintaining homeostasis, controlling the magnitude and duration of the inflammatory response, and in preventing autoimmune and allergic responses. [477] In general, Tregs are thought to be mainly involved in suppressing immune responses, functioning in part as a “self-check” for the immune system to prevent excessive reactions. In particular, Tregs are involved in maintaining tolerance to self-antigens, harmless agents such as pollen or food, and abrogating autoimmune disease. [478] Tregs are found throughout the body including, without limitation, the gut, skin, lung, and liver. Additionally, Treg cells may also be found in certain compartments of the body that are not directly exposed to the external environment such as the spleen, lymph nodes, and even adipose tissue. Each of these Treg cell populations is known or suspected to have one or more unique features and additional information may be found in Lehtimaki and Lahesmaa, Regulatory T cells control immune responses through their non-redundant tissue specific features, 2013, FRONTIERS IN IMMUNOL., 4(294): 1-10, the disclosure of which is hereby incorporated in its entirety. [479] Typically, Tregs are known to require TGF-β and IL-2 for proper activation and development. Tregs, expressing abundant amounts of the IL-2 receptor (IL-2R), are reliant on IL-2 produced by activated T cells. Tregs are known to produce both IL-10 and TGF-β, both potent immune suppressive cytokines. Additionally, Tregs are known to inhibit the ability of antigen presenting cells (APCs) to stimulate T cells. One proposed mechanism for APC inhibition is via CTLA-4, which is expressed by Foxp3+ Tregs. It is thought that CTLA-4 may bind to B7 molecules on APCs and either block these molecules or remove them by causing internalization resulting in reduced availability of B7 and an inability to provide adequate co- stimulation for immune responses. Additional discussion regarding the origin, differentiation and function of Tregs may be found in Dhamne et al., Peripheral and thymic Foxp3+ regulatory T cells in search of origin, distinction, and function, 2013, Frontiers in Immunol., 4 (253): 1- 11, the disclosure of which is hereby incorporated in its entirety. D. CHECKPOINT INHIBITORS & AGONISTS [480] In some embodiments, a provided polynucleotide (e.g., a DNA template, a linear precursor RNA polynucleotide, or a circular RNA polynucleotide) encodes one or more checkpoint inhibitors or agonists. 302
Attorney Docket No. 01318-0014-00PCT OR-043WO [481] In some embodiments, the immune checkpoint inhibitor is an inhibitor of Programmed Death-Ligand 1 (PD-L1, also known as B7-H1, CD274), Programmed Death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell costimulator), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with collageneous structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1, or any combinations thereof. In some embodiments, the immune checkpoint inhibitor is an inhibitor of IDO1, CTLA4, PD-1, LAG3, PD-L1, TIM3, or combinations thereof. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4. In some embodiments, the immune checkpoint inhibitor is an inhibitor of LAG3. In some embodiments, the immune checkpoint inhibitor is an inhibitor of TIM3. In some embodiments, the immune checkpoint inhibitor is an inhibitor of IDO1. [482] As described herein, at least in one aspect, the disclosure encompasses the use of immune checkpoint antagonists. Such immune checkpoint antagonists include antagonists of immune checkpoint molecules such as Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), Programmed Cell Death Protein 1 (PD-1), Programmed Death-Ligand 1 (PDL-1), Lymphocyte- activation gene 3 (LAG-3), and T-cell immunoglobulin and mucin domain 3 (TIM-3). An antagonist of CTLA-4, PD-1, PDL-1, LAG-3, or TIM-3 interferes with CTLA-4, PD-1, PDL-1, LAG-3, or TIM-3 function, respectively. Such antagonists of CTLA-4, PD-1, PDL-1, LAG-3, and TIM-3 can include antibodies which specifically bind to CTLA-4, PD-1, PDL-1, LAG-3, and TIM-3, respectively and inhibit and/or block biological activity and function. E. OTHERS [483] In some embodiments, the payload encoded within one or more of the coding elements is a hormone, FC fusion protein, anticoagulant, blood clotting factor, protein associated with deficiencies and genetic disease, a chaperone protein, an antimicrobial protein, an enzyme (e.g., metabolic enzyme), a structural protein (e.g., a channel or nuclear pore protein), protein variant, small molecule, antibody, nanobody, an engineered non-body antibody, or a combination thereof. 4. PRODUCTION OF POLYNUCLEOTIDES 303
Attorney Docket No. 01318-0014-00PCT OR-043WO A. Circular RNA preparation [484] Thus, in certain embodiments provided herein is a method of making circular RNA. In certain embodiments, the method comprises synthesizing precursor RNA by transcription (e.g., run-off transcription) using a vector provided herein (e.g., a 5’ intron element, a 5’ exon element, an intervening region (e.g., comprising a synthetic IRES) or core functional element, a 3’ exon element, and a 3’ intron element) as a template, and incubating the resulting precursor RNA in the presence of divalent cations (e.g., magnesium ions) and GTP such that it circularizes to form circular RNA. In some embodiments, the precursor RNA disclosed herein is capable of circularizing in the absence of magnesium ions and GTP and/or without the step of incubation with magnesium ions and GTP. In some embodiments, the precursor linear RNA polynucleotide circularizes when incubated in the presence of one or more guanosine nucleotides or nucleoside (e.g., GTP) and a divalent cation (e.g., Mg2+). [485] In certain embodiments, transcription occurs at a Mg2+ concentration of at least 3 mM of magnesium. In certain embodiments, the transcription occurs at a Mg2+ concentration of no more than 100mM of magnesium. In certain embodiments, transcription occurs at a Mg2+ concentration of or about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM, 60 mM, 61 mM, 62 mM, 63 mM, 64 mM, 65 mM, 66 mM, 67 mM, 68 mM, 69 mM, 70 mM, 71 mM, 72 mM, 73 mM, 74 mM, 75 mM, 76 mM, 77 mM, 78 mM, 79 mM, 80 mM, 81 mM, 82 mM, 83 mM, 84 mM, 85 mM, 86 mM, 87 mM, 88 mM, 89 mM, 90 mM, 91 mM, 92 mM, 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM, or 100 mM. In some embodiments, the greater concentration of Mg2+ during transcription of a linear RNA polynucleotide improves circularization and/or splicing as compared to the same linear RNA polynucleotide undergoing transcription at a lower Mg2+ concentration. In some embodiments, the 3’ exon element, 5’ exon element, and/or core functional element in whole or in part promotes the circularization of the precursor linear RNA polynucleotide to form the circular RNA construct provided herein. [486] In other embodiments, the method comprises ligation. In some embodiments, the method comprises chemical ligation. In some embodiments, the method comprises splint mediated ligation. In some embodiments, the ligation is performed with a T4 ligase using splint 304
Attorney Docket No. 01318-0014-00PCT OR-043WO DNA. [487] In some embodiments, the method of preparing a circular RNA comprises providing modified nucleotides or nucleosides to precursor RNA comprising: a. a 5’ combined accessory element, comprising: i. a 3’ intron segment, ii. a 3’ exon segment, b. an intervening region comprising an internal ribosome entry site (IRES) and a noncoding or coding region, c. a 3’ combined accessory element, comprising: i. a 5’ exon segment, and ii. a 5’ intron segment. In some embodiments, the IRES is a synthetic IRES containing at least one addition, deletion, or substitution of a nucleotide, domain, or motif, as described in further detail herein. [488] In some embodiments, the method of preparing a circular RNA comprises providing a first and second linear precursor RNA polynucleotide, wherein the first and second linear precursor RNA polynucleotides are capable of forming a circular RNA (e.g., ligation or permuted introns). [489] In some embodiments, a first precursor and a second precursor may be ligated to form a circular RNA. In some embodiments, the first precursor and the second precursor each comprise a short adapter sequence at their 5’ and 3’ ends. In some embodiments, the adapter sequences comprise homology arms with splints used for circularization. Splint ligation may be performed in the presence of a DNA splint using a suitable ligase to generate a circular RNA polynucleotide. Ligation methods are known in the art. See, e.g., Wesselhoeft et al., 2019. [490] In some embodiments, a first precursor and a second precursor may splice to form a circular RNA comprising a sequence of interest, e.g., a coding region. Each of the first precursor and the second precursor comprises at least one fragment of the sequence of interest, e.g., the first precursor comprises the 5’ fragment of the sequence of interest and the second precursor comprises the 3’ fragment of the sequence of interest. In these embodiments, the 5’ fragment of the sequence of interest, the 3’ fragment of the sequence of interest, and two additional fragments of the sequence of interest (Exon 2A, Exon 2B), together form the sequence of interest. In these embodiments, sequence of interest consists of, e.g., in 5’ to 3’ order, the 5’ fragment of the sequence of interest, an exonic fragment of the sequence of interest (Exon 2A), an exonic fragment of the sequence of interest (Exon 2B), and the 3’ 305
Attorney Docket No. 01318-0014-00PCT OR-043WO fragment of the sequence of interest. [491] In some embodiments, the first precursor comprises the following: (a) two intron fragments (e.g., 3’ intron fragment of a first intron (Intron 1) and a 5’ intron fragment of a second intron (Intron 2)), (b) a translation initiation element (e.g., IRES), (c) a 5’ fragment of the sequence of interest, and (d) two exon fragments that correspond with the intron fragments (e.g., Exon 1B and Exon 2A). In some embodiments, the IRES is a synthetic IRES containing at least one addition, deletion, or substitution of a nucleotide, domain, or motif, as described in further detail herein. [492] In some embodiments, one exon fragment (e.g., Exon 2A) is a part of a sequence of interest, for example in the coding or noncoding region. The coding region is scanned for sequences that are homologous to this exon (Exon 2A) fragment, thereby allowing splicing to occur without altering the resulting coding sequence in the circular RNA. [493] In some embodiments, the second precursor comprises the following: (a) two intron fragments that correspond with those in the first precursor (e.g., 3’ intron fragment of the second intron (Intron 2) and the 5’ intron fragment of the first intron (Intron 1)), and (b) two exon fragments that correspond with those on the first precursor (e.g., Exon 2B, which corresponds to the 5’ fragment of the 3’ fragment of the sequence of interest (e.g., coding region) and Exon 1A), and (c) the 3’ fragment of the sequence of interest. [494] In some embodiments, the coding sequence is scanned for regions that are homologous to this exon (Exon 2B) fragment, thereby allowing splicing to occur without altering the resulting coding sequence in the circular RNA. The first precursor and the second precursor may be incubated together to facilitate splicing between the first precursor and the second precursor in order to generate a circular RNA polynucleotide, which comprises specific modified regions and specific unmodified regions. [495] In some embodiments, a first precursor comprises an optional first external homology region (Arm 1A), a first intron fragment (3’ intron fragment of a first intron (Intron 1)), a first exon fragment (Exon 1B), an optional internal homology region, an optional spacer, a translation initiation element (e.g., synthetic IRES), the 5’ fragment of the sequence of interest (e.g., coding region), a second exon fragment (Exon 2A), a second intron fragment (5’ 306
Attorney Docket No. 01318-0014-00PCT OR-043WO intron fragment of a second intron (Intron 2)), and an optional second external homology region (Arm 2A). In these embodiments, the second precursor comprises an optional first external homology region (Arm 2B), a first intron fragment (3’ intron fragment of the second intron (Intron 2)), a first exon fragment (Exon 2B corresponding to the 5’ fragment of the 3’ fragment of the sequence of interest (e.g., coding region)), the 3’ fragment of the sequence of interest (e.g., coding region), an optional spacer, an optional internal homology region, a second exon fragment (Exon 1A), a second intron fragment (5’ fragment of the first intron (Intron 1)), and an optional second external homology region (Arm 1B). [496] In some embodiments, either the first precursor or the second precursor comprises a monotron. [497] In some embodiments, the first precursor comprises an optional first external homology region (Arm 1A), a first intron fragment (3’ intron fragment of a first intron (Intron 1)), a first exon fragment (Exon 1B), an optional internal homology region, an optional spacer, a translation initiation element (e.g., synthetic IRES), the 5’ fragment of the sequence of interest (e.g., coding region), a second exon fragment (Exon 2A), a terminal element corresponding to a monotron sequence, and an optional second external homology region (Arm 2A). In these embodiments, the second precursor comprises an optional external homology region (Arm 2B), the monotron sequence via Intron 2, a first exon fragment (Exon 2B, which corresponds to the 5’ fragment of the 3’ fragment of the sequence of interest (e.g., coding region)), the 3’ fragment of the sequence of interest (e.g., coding region), an optional spacer, an optional internal homology region, a second exon fragment (Exon 1A), an intron fragment (5’ intron fragment of Intron 2), and an optional second external homology region (Arm 1B). [498] In some embodiments, the first precursor comprises an optional first external homology region (Arm 1A), a monotron sequence via Intron 1, a first exon fragment (Exon 1B), an optional internal homology region, an optional spacer, a translation initiation element (e.g., synthetic IRES), the 5’ fragment of the sequence of interest (e.g., coding region), a second exon fragment (Exon 2A), an intron fragment (5’ intron fragment of Intron 2), and an optional second external homology region. In these embodiments, the second precursor comprises an optional external homology region (Arm 2B), an intron fragment (3’ intron fragment of Intron 2), a first exon fragment (Exon 2B, which corresponds to the 5’ fragment of the 3’ fragment of the sequence of interest (e.g., coding region)), a 3’ fragment of the sequence of interest (e.g., coding region), an optional spacer, an optional internal homology region, a second exon fragment (Exon 1A), a terminal element corresponding to the monotron 307
Attorney Docket No. 01318-0014-00PCT OR-043WO sequence, and an optional second external homology region (Arm 1B). [499] In some embodiments, each of the first precursor and the second precursor comprises a monotron. In some embodiments, the first precursor comprises an optional first external homology region (Arm 1A), a first monotron sequence via Intron 1, a first exon fragment (Exon 1B), an optional internal homology region, an optional spacer, a translation initiation element (e.g., synthetic IRES), the 5’ fragment of the sequence of interest, a second exon fragment (Exon 2A), a terminal element corresponding to a second monotron sequence via Intron 2, and an optional second external homology region (Arm 2A). In these embodiments, the second precursor comprises an optional external homology region (Arm 2B), a second monotron sequence via Intron 2, a first exon fragment (Exon 2B), the 3’ fragment of the sequence of interest (e.g., coding region), an optional spacer, an optional internal homology region, a second exon fragment (Exon 1A), a terminal element corresponding to the first monotron sequence via Intron 1, and an optional second external homology region (Arm 1B). [500] In some embodiments, provided herein are circular RNA that do not comprise modified nucleotides and/or modified nucleosides. Also provided herein are modified circular RNA (i.e., comprising at least one modified nucleotide and/or modified nucleoside) prepared from the methods described herein. In some embodiments, the modified circular RNA affects immunogenicity, circularization, and/or translation as compared to circular RNA prepared with RNA precursor polynucleotides that comprise no nucleotide or nucleoside modifications. [501] It has been discovered that circular RNA has reduced immunogenicity relative to a corresponding mRNA, at least partially because the mRNA contains an immunogenic 5’ cap. When transcribing a DNA vector from certain promoters (e.g., a T7 promoter, SP6 promoter, or a fragment thereof) to produce a precursor RNA, it is understood that the 5’ end of the precursor RNA is G. To reduce the immunogenicity of a circular RNA composition that contains a low level of contaminant linear mRNA, an excess of GMP relative to GTP can be provided during transcription such that most transcripts contain a 5’ GMP, which cannot be capped. Therefore, in some embodiments, transcription is carried out in the presence of an excess of GMP. In some embodiments, transcription is carried out where the ratio of GMP concentration to GTP concentration is within 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. [502] In some embodiments, a composition comprising circular RNA has been purified. Circular RNA may be purified by any known method commonly used in the art, such as column chromatography, gel filtration chromatography, and size exclusion chromatography. In some 308
Attorney Docket No. 01318-0014-00PCT OR-043WO embodiments, purification comprises one or more of the following steps: phosphatase treatment, HPLC size exclusion purification, and RNase R digestion. In some embodiments, purification comprises the following steps in order: RNase R digestion, phosphatase treatment, and HPLC size exclusion purification. In some embodiments, purification comprises reverse phase HPLC. In some embodiments, a purified composition contains less double stranded RNA, DNA splints, triphosphorylated RNA, phosphatase proteins, protein ligases, capping enzymes and/or nicked RNA than unpurified RNA. In some embodiments, purification of circular RNA comprises an affinity-purification or negative selection method described herein. In some embodiments, purification of circular RNA comprises separation of linear RNA from circular RNA using oligonucleotides that are complementary to a sequence in the linear RNA but are not complementary to a sequence in the circular RNA. In some embodiments, a purified composition is less immunogenic than an unpurified composition. In some embodiments, immune cells exposed to a purified composition produce less TNFα, RIG-I, IL-2, IL-6, IFNγ, and/or a type 1 interferon, e.g., IFN-β1, than immune cells exposed to an unpurified composition. [503] In some embodiments, circular RNA is produced by transcribing a DNA polynucleotide sequence that is complementary to a precursory RNA polynucleotide that is described herein. In certain embodiments, circular RNA provided herein is produced in vitro. In certain embodiments, circular RNA provided herein is produced inside a cell. In some embodiments, the cell selected from, for example, an immune cell, muscle cell, neural cell, epithelial cell and a tumor cell. In some embodiments, precursor RNA is transcribed using a DNA template (e.g., in some embodiments, using a vector provided herein) in the cytoplasm by a bacteriophage RNA polymerase, or in the nucleus by host RNA polymerase II and then circularized. [504] Exemplary methods of circularization of precursor RNA can be found in, for example, WO2020/237227, which is incorporated by reference herein in its entirety. WO2020/237227, inter alia, describes using the permuted intron exon (PIE) circularization strategy to circularize long precursor RNA. In it, a 1.1kb sequence containing a full-length encephalomyocarditis virus (EMCV) IRES, a Gaussia luciferase (GLuc) expression sequence, and two short exon fragments of the permuted intron-exon (PIE) construct were inserted between the 3’ and 5’ introns of the permuted group I catalytic intron in the thymidylate synthase (Td) gene of the T4 phage. Precursor RNA was synthesized by run-off transcription. Circularization was attempted by heating the precursor RNA in the presence of magnesium 309
Attorney Docket No. 01318-0014-00PCT OR-043WO ions and GTP, but splicing products were not obtained. Perfectly complementary 9 nucleotide and 19 nucleotide long homology regions were designed and added at the 5’ and 3’ ends of the precursor RNA. The splicing product was treated with RNase R. Sequencing across the putative splice junction of RNase R-treated splicing reactions revealed ligated exons, and digestion of the RNase R-treated splicing reaction with oligonucleotide-targeted RNase H produced a single band in contrast to two bands yielded by RNase H-digested linear precursor. WO2020/237227 further indicates that a series of spacers was designed and inserted between the 3’ PIE splice site and the IRES. These spacers were designed to either conserve or disrupt secondary structures within intron sequences in the IRES (e.g., synthetic IRES), 3’ PIE splice site, and/or 5’ splice site. [505] Further methods for preparing circular RNA are described in PCT Application No. US2022/33091, which is incorporated herein by reference in its entirety. B. Precursor RNA preparation [506] The DNA templates provided herein can be made using standard techniques of molecular biology. For example, the various elements of the vectors provided herein can be obtained using recombinant methods, such as by screening cDNA and genomic libraries from cells, or by deriving the polynucleotides from a DNA template known to include the same. [507] The various elements of the DNA template provided herein can also be produced synthetically, rather than cloned, based on the known sequences. The complete sequence can be assembled from overlapping oligonucleotides prepared by standard methods and assembled into the complete sequence. See, e.g., Edge, Nature (1981) 292:756; Nambair et al., Science (1984) 223 : 1299; and Jay et al., J. Biol. Chem. (1984) 259:6311. [508] Thus, particular nucleotide sequences can be obtained from DNA template harboring the desired sequences or synthesized completely, or in part, using various oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR) techniques where appropriate. One method of obtaining nucleotide sequences encoding the desired DNA template elements is by annealing complementary sets of overlapping synthetic oligonucleotides produced in a conventional, automated polynucleotide synthesizer, followed by ligation with an appropriate DNA ligase and amplification of the ligated nucleotide sequence via PCR. See, e.g., Jayaraman et al., Proc. Natl. Acad. Sci. USA (1991) 88:4084-4088. Additionally, oligonucleotide-directed synthesis (Jones et al., Nature (1986) 54:75-82), oligonucleotide directed mutagenesis of preexisting nucleotide regions (Riechmann et al., Nature (1988) 332:323-327 and Verhoeyen et al., Science 310
Attorney Docket No. 01318-0014-00PCT OR-043WO (1988) 239: 1534-1536), and enzymatic filling-in of gapped oligonucleotides using T4 DNA polymerase (Queen et al., Proc. Natl. Acad. Sci. USA (1989) 86: 10029-10033) can be used. [509] Transcription of a DNA template (e.g., comprising a 3’ intron element, 3’ exon element, an intervening region or core functional element including an IRES (e.g., synthetic IRES) and expression sequence, a 5’ exon element, and a 5’ intron element) results in formation of a precursor linear RNA polynucleotide capable of circularizing. In some embodiments, this DNA template comprises a vector, PCR product, plasmid, minicircle DNA, cosmid, artificial chromosome, complementary DNA (cDNA), extrachromosomal DNA (ecDNA), or a fragment therein. In certain embodiments, the minicircle DNA may be linearized or non-linearized. In certain embodiments, the plasmid may be linearized or non-linearized. In some embodiments, the DNA template may be single-stranded. In other embodiments, the DNA template may be double-stranded. In some embodiments, the DNA template comprises in whole or in part from a viral, bacterial or eukaryotic vector. In some embodiments, the polynucleotide of the present disclosure is an expression vector. [510] The precursor RNA provided herein can be generated by incubating a DNA template provided herein under conditions permissive of transcription of the precursor RNA encoded by the DNA template. For example, in some embodiments a precursor RNA is synthesized by incubating a DNA template provided herein that comprises an RNA polymerase promoter or promoter segment upstream of its 5’ duplex sequence and/or expression sequences with a compatible RNA polymerase enzyme under conditions permissive of in vitro transcription. In some embodiments, the DNA template is incubated inside of a cell by a bacteriophage RNA polymerase or in the nucleus of a cell by host RNA polymerase II. In some embodiments, the polynucleotide of the present disclosure is an expression vector, wherein the expression vector comprises a polymerase promoter sequence or segment. [511] In certain embodiments, provided herein is a method of generating precursor RNA by performing in vitro transcription using a DNA template provided herein as a template (e.g., a vector provided herein with an RNA polymerase promoter or promoter segment positioned upstream of the 5’ duplex region). [512] In some embodiments, the DNA template shares the same sequence as the precursor linear RNA polynucleotide prior to splicing of the precursor linear RNA polynucleotide (e.g., a 3’ intron element, a 3’ exon element, an intervening region core functional element, and a 5’ exon element, a 5’ intron element). In some embodiments, said linear precursor RNA polynucleotide undergoes splicing leading to the removal of the 3’ intron element and 5’ intron 311
Attorney Docket No. 01318-0014-00PCT OR-043WO element during the process of circularization. In some embodiments, the resulting circular RNA polynucleotide lacks a 3’ intron fragment and a 5’ intron fragment, but maintains a 3’ exon fragment, an intervening region or a core functional element, and a 5’ exon element. [513] In certain embodiments, the resulting precursor RNA can be used to generate circular RNA (e.g., a circular RNA polynucleotide provided herein) by incubating it in the presence of magnesium ions and guanosine nucleotide or nucleoside at a temperature at which RNA circularization occurs (e.g., between 20 °C and 60 °C). Precursor RNA are generally described in PCT Application No. US2022/33091, which is incorporated herein by reference in its entirety. 5. TRANSFER VEHICLE & OTHER DELIVERY MECHANISMS A. IONIZABLE LIPIDS [514] In certain embodiments, disclosed herein are ionizable lipids that may be used as a component of a transfer vehicle to facilitate or enhance the delivery and release of circular RNA to one or more target cells (e.g., by permeating or fusing with the lipid membranes of such target cells). In certain embodiments, an ionizable lipid comprises one or more cleavable functional groups (e.g., a disulfide) 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 facilitating a phase transition in the lipid bilayer of the one or more target cells. [515] In some embodiments, an ionizable lipid is as described in international patent application PCT/US2020/038678. In some embodiments, an ionizable lipid is a lipid as represented by formula 1 of or as listed in Tables 1 or 2 of US Patent No.9,708,628, the content of which is herein incorporated by reference in its entirety. In some embodiments, an ionizable lipid is as described in pages 7-13 of US Patent No. 9,765,022 or as represented by formula 1 of US Patent No. 9,765,022, the content of which is herein incorporated by reference in its entirety. In some embodiments, an ionizable lipid is described in pages 12-24 of International Patent Application No. PCT/US2019/016362 or as represented by formula 1 of International Patent Application PCT/US2019/016362, the contents of which are herein incorporated by reference in their entirety. In some embodiments, a lipid or transfer vehicle is a lipid as described in International Patent Application Nos. PCT/US2010/061058, PCT/US2018/058555, PCT/US2018/053569, PCT/US2017/028981, PCT/US2019/025246, PCT/US2019/015913, PCT/US2019/016362, PCT/US2019/016362, US Application Publication Nos. US2019/0314524, US2019/0321489, US2019/0314284, and 312
Attorney Docket No. 01318-0014-00PCT OR-043WO US2019/0091164, the contents of which are herein incorporated by reference in their entireties. Suitable cationic lipids for use in the compositions and methods herein include those described in international patent publication WO 2010/053572 and/or US patent application 15/809,680, e.g., C12-200. In certain embodiments, the compositions and methods herein employ an ionizable cationic lipid described in WO2013149140 (incorporated herein by reference), such as, e.g., (15Z,18Z)—N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien- 1-amine (HGT5000), (15Z,18Z)—N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1- yl)tetracosa-4,15,18-trien-1-amine (HGT5001), and (15Z,18Z)—N,N-dimethyl-6-((9Z,12Z)- octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5002). In certain embodiments, the compositions and methods herein employ an ionizable cationic lipid described US patent publications 2017/0190661 and 2017/0114010, incorporated herein by reference in their entirety. [516] In some embodiments, the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride or “DOTMA” is used. (Felgner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355). DOTMA can be formulated alone or can be combined with the neutral lipid, dioleoylphosphatidyl-ethanolamine or “DOPE” or other cationic or non- cationic lipids into a transfer vehicle or a lipid nanoparticle, and such liposomes can be used to enhance the delivery of nucleic acids into target cells. Other suitable cationic lipids include, for example, 5-carboxyspermylglycinedioctadecylamide or “DOGS,” 2,3-dioleyloxy-N- [2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium or “DOSPA” (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989); U.S. Pat. Nos. 5,171,678; 5,334,761), 1,2-Dioleoyl-3- Dimethylammonium-Propane or “DODAP,” 1,2-Dioleoyl-3-Trimethylammonium-Propane or “DOTAP.” Contemplated cationic lipids also include 1,2-distearyloxy-N,N-dimethyl-3- aminopropane or “DSDMA”, 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or “DODMA,” 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or “DLinDMA,” 1,2-dilinolenyloxy-N,N- dimethyl-3-aminopropane or “DLenDMA,” N-dioleyl-N,N-dimethylammonium chloride or “DODAC,” N,N-distearyl-N,N-dimethylammonium bromide or “DDAB,” N-(1,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide or “DMRIE,” 3- dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12- octadecadienoxy)propane or “CLinDMA,” 2-[5’-(cholest-5-en-3-beta-oxy)-3’-oxapentoxy)-3- dimethy 1-1-(cis,cis-9’, 1-2’-octadecadienoxy)propane or “CpLinDMA,” N,N-dimethyl-3,4- dioleyloxybenzylamine or “DMOBA,” 1,2-N,N’-dioleylcarbamyl-3-dimethylaminopropane or “DOcarbDAP,” 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or “DLinDAP,” 1,2-N,N’- 313
Attorney Docket No. 01318-0014-00PCT OR-043WO Dilinoleylcarbamyl-3-dimethylaminopropane or “DLincarbDAP,” 1,2-Dilinoleoylcarbamyl-3- dimethylaminopropane or “DLinCDAP,” 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]- dioxolane or “DLin-K-DMA,” 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or “DLin- K-XTC2-DMA,” and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N- dimethylethanamine (DLin-KC2-DMA)) (See, WO 2010/042877; Semple et al., Nature Biotech. 28:172-176 (2010)), or mixtures thereof. (Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol. 23(8): 1003-1007 (2005); PCT Publication WO2005/121348A1). [517] The use of cholesterol-based cationic lipids to formulate the transfer vehicles (e.g., lipid nanoparticles) is also contemplated herein. Such cholesterol-based cationic lipids can be used, either alone or in combination with other cationic or non-cationic lipids. Suitable cholesterol-based cationic lipids include, for example, GL67, DC-Chol (N,N-dimethyl-N- ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or ICE. [518] In some embodiments, the one or more of the cationic or ionizable lipids provide increased activity of the nucleic acid and improved tolerability of the compositions in vivo. [519] PCT/US2022/033091 (WO 2022/261490) describes representative cationic lipids of any one of the disclosed embodiments and is incorporated by reference herein in its entirety. [520] In some embodiments, the cationic lipid (or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof) has a structure selected from one of the following that are described in detail on pages 113-118 of WO 2022/261490 and page 113-118 of WO 2023/056033, which are incorporated by reference herein in their entireties:
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[521] In some embodiments, for example, a cationic lipid of any one of the disclosed embodiments has a structure of Formula I and/or is selected from structure numbers I-1 through I-41, set forth at pages 119-130 and in Table 1 of WO 2022/261490; has a structure of Formula II and/or is selected from structure numbers II-1 through II-46, set forth at pages 130-146 and in Table 2 of WO 2022/261490; has a structure of Formula III and/or is selected from structure numbers III-1 through III-49, set forth at pages 146-157 and in Table 3 of WO 2022/261490; has a structure of Formula IV or V and/or is selected from structure numbers IV-1 through IV- 3, set forth at pages 157-174 and in Table 4 of WO 2022/261490; has a structure of Formula VI and/or is selected from structure numbers VI-1 through VI-37, set forth at pages 174-188 and in Table 5 of WO 2022/261490; has a structure of Formula VII and/or is selected from structure numbers VII-1 through VII-11, set forth at pages 188-195 in Table 6 of WO 2022/261490; has a structure of Formula VIII and/or is selected from structure numbers VIII- 1 through VII-12, set forth at pages 195-201 and in Table 7 of WO 2022/261490; has a structure of Formula IX and/or is selected from structure numbers IX-1 through IX-18, set forth at pages 201-208 and in Table 8 of WO 2022/261490; has a structure of Formula X and/or is selected from structure numbers X-1 through X-17, set forth at pages 208-213 and in Table 6 of WO 2022/261490; has a structure of Formula XI and/or is structure number XIa or Formula XII 315
Attorney Docket No. 01318-0014-00PCT OR-043WO and/or is selected from structure numbers XIIA-XIIJ, as described at pages 213-220 in WO 2022/261490. WO 2023/056033 describes similar structures, and is incorporated by reference in its entirety. [522] In some embodiments, an ionizable lipid is a compound of Formula (1), Formula (1-1), Formula (1-2), Formula (2), Formula (3), Formula (3-1), Formula (3-2), Formula (3-3), Formula (5), or Formula (6), in WO 2022/261490, which is incorporated by reference herein in its entirety. WO 2022/261490 provides exemplary reaction schemes that illustrate an exemplary method to make compounds of Formula (1). WO 2023/056033 describes similar structures, and is incorporated by reference in its entirety. [523] In some embodiments, the ionizable lipid has a beta-hydroxyl amine head group. In some embodiments, the ionizable lipid has a gamma-hydroxyl amine head group. [524] In some embodiments, an ionizable lipid of the disclosure is a lipid selected from Table 10a, Table 10b, or Table 10 on pages 235-271- of WO 2022/261490, which is incorporated herein by reference in its entirety. In some embodiments, the ionizable lipid is Lipid 26, 27, 53, 54, 45, 46, 137, 138, 139, 128, or 130 in Table 10a of WO 2022/261490. In some embodiments, an ionizable lipid of the disclosure is Lipid 15 from Table 10b of WO 2022/261490. [525] In an embodiment, the ionizable lipid is described in US patent publication number US20170210697A1. In an embodiment, the ionizable lipid is described in US patent publication number US20170119904A1. [526] In some embodiments, the ionizable lipid has one of the structures set forth in Table 11 of WO 2022/261490, which is incorporated herein by reference in its entirety, certain of which are described in international patent application PCT/US2010/061058. In some embodiments, the ionizable lipids may include a lipid selected from Tables 12, 13, 14, or 15a of WO 2022/261490. [527] In some embodiments, the transfer vehicle comprises Lipid A, Lipid B, Lipid C, and/or Lipid D, described in detail, including methods of synthesis that are known in the art, in WO 2022/261490, WO 2023/056033, and PCT/US2017/028981, which are incorporated herein by reference in their entireties. [528] Also contemplated are ionizable lipids such as the dialkylamino-based, imidazole- based, and guanidinium-based lipids. See, e.g., PCT/US2010/058457, incorporated herein by reference. For example, certain embodiments are directed to a composition comprising one or more imidazole-based ionizable lipids, for example, the imidazole cholesterol ester or “ICE” 316
Attorney Docket No. 01318-0014-00PCT OR-043WO lipid, (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol- 4-yl)propanoate, as represented by structure (XIII) of WO 2022/261490 and WO 2023/056033, which are incorporated herein by reference in their entireties. In an embodiment, a transfer vehicle for delivery of circRNA may comprise one or more imidazole-based ionizable lipids, for example, the imidazole cholesterol ester or “ICE” lipid (3S, 10R, 13R, 17R)-10, 13- dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17- tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, as represented by structure (XIII). In some embodiments, an ionizable lipid is described by US patent publication number 20190314284. [529] In certain embodiments, the ionizable lipid is described by structure (XIV), structure XVII (referred to herein as “HGT4001”), structure XVIII (referred to herein as “HGT4002”), structure XIX (referred to herein as “HGT4003”), structure XX (referred to herein as “HGT4004”), or structure XXI (referred to herein as “HGT4005”) of WO 2022/261490 and WO 2023/056033, which are incorporated herein by reference in their entireties. [530] In some embodiments, the ionizable lipid is selected from a lipid with a structure depicted on 390-457 of WO 2022/261490, which is incorporated herein by reference in its entirety. [531] WO 2023/056033 also describes representative cationic lipids of any one of the disclosed embodiments and is incorporated by reference herein in its entirety. In some embodiments, for example, a cationic lipid of any one of the disclosed embodiments has the structure of Formula (7), (7-1), (7-2), (7-3), (8), (8-1), (8-2), (8-3), (8-4), (9), (10), (11), and/or (12) of WO 2023/056033. In some embodiments, the cationic lipid is selected from a lipid with a structure depicted in any of Tables 10a-10f, Table 11, Tables 12, 13, 14, or 15a of WO 2023/056033. In some embodiments, the ionizable lipid is described by structure (XIV) of WO 2023/056033, and pharmaceutical compositions comprising the compound of structure XIV are envisioned. In some embodiments, the cationic lipid is selected from a lipid with a structure depicted on pages 386-439 of WO 2023/056033. [532] In some embodiments, the one or more of the cationic or ionizable lipids are represented by Formula (I): 317
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Formula (I) wherein: n is an integer between 1 and 4; Ra is hydrogen or hydroxyl; and R1 and R2 are each independently a linear or branched C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 heteroalkyl, optionally substituted by one or more substituents selected from a group consisting of oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl. [533] In some embodiments, Ra is hydrogen. In some embodiments, Ra is hydroxyl. [534] In some embodiments, the ionizable lipid is represented by Formula (Ia -1), Formula (Ia- 2), or Formula (Ia-3):
Formula (Ia-1) Formula (Ia -2) Formula (Ia-3) [535] In some embodiments, the ionizable lipid is represented by Formula (Ib-1), Formula (Ib- 2), or Formula (Ib-3): 318
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Formula (Ib-1) Formula (Ib-2) Formula (Ib-3) [536] In some embodiments, the ionizable lipid is represented by Formula (Ib-4), Formula (Ib- 5), Formula (Ib-6), Formula (Ib-7), Formula (Ib-8), or Formula (Ib-9):
Formula (Ib-7) Formula (Ib-8) Formula (Ib-9) [537] In some embodiments, the one or more of the cationic or ionizable lipids are represented by Formula (I), wherein R1 and R2 are each independently selected from:
, , 319
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[538] In some embodiments, R1 and R2 are the same. In some embodiments, R1 and R2 are different. [539] In various embodiments, the one or more of the cationic or ionizable lipids are represented by Formula (I*):
Formula (I*) wherein: n* is an integer between 1 to 7, 320
Attorney Docket No. 01318-0014-00PCT OR-043WO Ra is hydrogen or hydroxyl, Rb is hydrogen or C1-C6 alkyl, R1 and R2 are each independently a linear or branched C1-C30 alkyl, C2-C30 alkenyl, or C1-C30 heteroalkyl, optionally substituted by one or more substituents selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, alkylcarbonyloxy, alkylcarbonate, alkenyloxycarbonyl, alkenylcarbonyloxy, alkenylcarbonate, alkynyloxycarbonyl, alkynylcarbonyloxy, alkynylcarbonate, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl. [540] In some embodiments, the one or more of the cationic or ionizable lipids are represented by Formula (II):
Formula (II) wherein: each n is independently an integer from 2-15; L1 and L3 are each independently –OC(O)–* or –C(O)O–*, wherein
indicates the attachment point to R1 or R3; R1 and R3 are each independently a linear or branched C9-C20 alkyl or C9-C20 alkenyl, optionally substituted by one or more substituents selected from a group consisting of oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, 321
Attorney Docket No. 01318-0014-00PCT OR-043WO (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; and R2 is selected from a group consisting of: ,
[541] In some embodiments, the ionizable lipid is selected from an ionizable lipid of Formula II, wherein R1 and R3 are each independently selected from a group consisting of:
[542] In some embodiments, R1 and R3 are the same. In some embodiments, R1 and R3 are different. [543] In some embodiments, the one or more of the cationic or ionizable lipids are represented by Formula (II-1) or Formula (II-2): 322
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Formula (II-2). [544] In some embodiments, the ionizable lipid is selected from an ionizable lipid of WO2015/095340 (lipid number 123 of Table 4). In some embodiments, the ionizable lipid is selected from an ionizable lipid of WO2021/021634, WO2020/237227, or WO2019/236673 (lipid numbers 124- 127 of Table 4). In some embodiments, the ionizable lipid is selected from an ionizable lipid of WO2021226597 and WO2021113777 (lipid numbers 128-131 of Table 4). [545] In some embodiments, the transfer vehicle comprises an ionizable lipid selected from an ionizable lipid represented in Table 4. In particular embodiments, where the ionizable lipid is of Formula I, the ionizable lipid is selected from lipid numbers 16, 45, 86, 89, and 90 of Table 4, below. In particular embodiments where the ionizable lipid is an ionizable lipid of Formula II, the ionizable lipid is selected from lipid numbers 128-131 of Table 4, below. [546] In some embodiments, the one or more of the cationic or ionizable lipids are represented by Formula (III):
Formula (III) or a pharmaceutically acceptable salt thereof, wherein L1 is C2-C11 alkylene, C4-C10-alkenylene, or C4-C10-alkynylene; X1 is OR1, SR1, or N(R1)2, where R1 is independently H or unsubstituted C1-C6 alkyl; and R2 and R3 are each independently C6-C30-alkyl, C6-C30-alkenyl, or C6-C30-alkynyl. [547] In some embodiments, the one or more of the cationic or ionizable lipids are represented by Formula (III*): 323
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Formula (III*) or a pharmaceutically acceptable salt thereof, wherein L1 is C2-C11 alkylene, C4-C10-alkenylene, or C4-C10-alkynylene; X1 is OR1, SR1, or N(R1)2, where R1 is independently H or unsubstituted C1-C6 alkyl; and R2 and R3 are each independently a linear or branched C1-C30 alkyl, C2-C30 alkenyl, or C1-C30 heteroalkyl, optionally substituted by one or more substituents selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, alkylcarbonyloxy, alkylcarbonate, alkenyloxycarbonyl, alkenylcarbonyloxy, alkenylcarbonate, alkynyloxycarbonyl, alkynylcarbonyloxy, alkynylcarbonate, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl. Table 4: Exemplary Ionizable Lipid Structures
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[548] In some embodiments, an ionizable lipid is a compound of Formula (15):
, Formula (15) or is a pharmaceutically acceptable salt thereof, wherein: n* is an integer from 1 to 7; Ra is hydrogen or hydroxyl; Rh is hydrogen or C1-C6 alkyl; 359
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R1* and R2* are independently selected from: –(CH2)qC(O)O(CH2)rC(R8)(R9)(R10), –(CH2)qOC(O)(CH2)rC(R8)(R9)(R10), and –(CH2)qOC(O)O(CH2)rC(R8)(R9)(R10); wherein: q is an integer from 0 to 12, r is an integer from 0 to 6, wherein at least one occurrence of r is not 0; R8 is H or R11; R9, R10, and R11 are each independently C1-C20 alkyl or C2-C20-alkenyl; and wherein (i) R1 is R1*, (ii) R2 is R2*, or (iii) R1 is R1* and R2 is R2*. [549] In some embodiments of Formula (15), Ra is hydrogen and the ionizable lipid is of Formula (16):
. Formula (16) or is a pharmaceutically acceptable salt thereof, wherein: n* is an integer from 1 to 7. [550] In some embodiments of Formula (16), the ionizable lipid is of Formula (17):
Formula (17) or a pharmaceutically acceptable salt thereof, wherein: n is an integer from 1 to 7; q and q’ are each independently integers from 0 to 12; r and r’ are each independently integers from 0 to 6, wherein at least one of r or r’ is not 0; ZA and ZB are each independently selected from ^-C(O)O-, ^-OC(O), and -OC(O)O-; where ^ 360
Attorney Docket No. 01318-0014-00PCT OR-043WO denotes the attachment point to -(CH2)q- or -(CH2)q’-; and R9A, R9B, R10A, and R10B are each independently C1-C20 alkyl or C2-C20 alkenyl. [551] In some embodiments of Formula (17), ZA and ZB are ^-C(O)O-, and the ionizable lipid is of Formula (17a-1)
. Formula (17a-1) [552] In some embodiments of Formula (17), ZA and ZB are ^-OC(O)-, and the ionizable lipid is of Formula (17a-2)
. Formula (17a-2) [553] In some embodiments of Formula (17), ZA and ZB are -O(C)(O)O-, and the ionizable lipid is represented by Formula (17a-3):
. Formula (17a-3) [554] In some embodiments of Formula (15), Ra is hydroxyl and the ionizable lipid is of Formula (18): 361
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, Formula (18) or is a pharmaceutically acceptable salt thereof, wherein: n* is an integer from 1 to 7; Rh is hydrogen or C1-C6 alkyl;
R1* and R2* are independently selected from: –(CH2)qC(O)O(CH2)rC(R8)(R9)(R10), –(CH2)qOC(O)(CH2)rC(R8)(R9)(R10), and –(CH2)qOC(O)O(CH2)rC(R8)(R9)(R10); wherein: q is an integer from 0 to 12, r is an integer from 0 to 6, wherein at least one occurrence of r is not 0; R8 is hydrogen or R11; R9, R10, and R11 are each independently C1-C20 alkyl or C2-C20-alkenyl; wherein (i) R1 is R1*, (ii) R2 is R2*, or (iii) R1 is R1* and R2 is R2*; and wherein, for (iii), (a) R1* and R2* are different or (b) R9 and R10 have different numbers of carbon atoms for at least one of R1* and R2* . [555] In some embodiments of Formula (18), the ionizable lipid of is of Formula (19):
, Formula (19) or is a pharmaceutically acceptable salt thereof, wherein: n is an integer from 1 to 7; q and q’ are each independently integers from 0 to 12; r and r’ are each independently integers from 0 to 6, wherein at least one of r or r’ is not 0; ZA and ZB are each independently selected from ^-C(O)O-, ^-OC(O), and -OC(O)O-; where ^ denotes the attachment point to -(CH2)q- or -(CH2)q’;-and 362
Attorney Docket No. 01318-0014-00PCT OR-043WO R9A, R9B, R10A, and R10B are each independently C1-C20 alkyl or C2-C20 alkenyl. [556] In some embodiments of Formula (19), ZA and ZB are ^-C(O)O-, and the ionizable lipid is of Formula (19a-1):
. Formula (19a-1) [557] In some embodiments of Formula (19), ZA and ZB are ^-OC(O)-, and the ionizable lipid is of Formula (19a-2):
. Formula (19a-2) [558] In some embodiments of Formula (19), ZA and ZB are -O(C)(O)O-, and the ionizable lipid is represented by Formula (19a-3):
. Formula (19a-3) [559] In some embodiments of Formula (15), R1 is C1-C30 alkyl, and the ionizable lipid is of Formula (20):
, Formula (20) or is a pharmaceutically acceptable salt thereof, wherein: 363
Attorney Docket No. 01318-0014-00PCT OR-043WO ZA is selected from ^-C(O)O-, ^-OC(O)-, and -OC(O)O-; where ^ denotes the attachment point to -(CH2)q-; R9A and R10A are each independently C1-C20 alkyl or C2-C20 alkenyl; n is an integer from 1 to 7; q is an integer from 0 to 12; and r is an integer from 1 to 6. [560] In some embodiments of Formula (20), ZA is ^-C(O)O-, and the ionizable lipid is of Formula (20a-1):
. Formula (20a-1) [561] In some embodiments of Formula (20), ZA is ^-OC(O)-, and the ionizable lipid is of Formula (20a-2):
. Formula (20a-2) [562] In some embodiments of Formula (20), ZA is -OC(O)O-, and the ionizable lipid is of Formula (20a-3):
. Formula (20a-3) [563] In some embodiments of Formula (15), R2 is C1-C30 alkyl, and the ionizable lipid is of Formula (21): 364
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, Formula (21) or is a pharmaceutically acceptable salt thereof, wherein: ZB is selected from ^-C(O)O-, ^-OC(O)-, and -OC(O)O-; where ^ denotes the attachment point to -(CH2)q’-; R9B and R10B are each independently C1-C20 alkyl or C2-C20 alkenyl; n is an integer from 1 to 7; q’ is an integer from 0 to 12; and r’ is an integer from 1 to 6. [564] In some embodiments of Formula (21), ZB is ^-C(O)O-, and the ionizable lipid is of Formula (21a-1):
. Formula (21a-1) [565] In some embodiments of Formula (21), ZB is ^-OC(O)-, and the ionizable lipid is of Formula (21a-2):
. Formula (21a-2) [566] In some embodiments of Formula (21), ZB is -OC(O)O-, and the ionizable lipid is of Formula (21a-3): 365
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. Formula (21a-3) [567] In some embodiments, an ionizable lipid is selected from the table below:
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[568] In some embodiments, an ionizable lipid of the present disclosure is represented by Formula (22):
, Formula (22) or is a pharmaceutically acceptable salt thereof, wherein: Ra is hydrogen or hydroxyl;
R1* and R2* are independently selected from: –(CH2)qC(O)O(CH2)rC(R4)(R5)(R6), –(CH2)qOC(O)(CH2)rC(R4)(R5)(R6), and –(CH2)qOC(O)O(CH2)rC(R4)(R5)(R6); wherein: q is an integer from 0 to 12, r is an integer from 0 to 6, wherein at least one occurrence of r is not 0; R4 is hydrogen or R7; R5, R6, and R7 are each independently C1-C20 alkyl or C2-C20-alkenyl; wherein (i) R1 is R1*, (ii) R2 is R2*, or (iii) R1 is R1* and R2 is R2*; and 370
Attorney Docket No. 01318-0014-00PCT OR-043WO R3 is L-R’, wherein L is linear or branched C1-C10 alkylene, and R’ is (i) mono- or bicyclic heterocyclyl or heteroaryl, such as imidazolyl, pyrazolyl, 1,2,4-triazolyl, or benzimidazolyl, each optionally substituted at one or more available carbon and nitrogen by C1-C6 alkyl, or (ii) RA, RB, or RC, wherein RA is selected from:
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with the proviso that the ionizable lipid is not: 375
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[569] In some embodiments of Formula (22), R3 is selected from: , ,
[570] In some embodiments of Formula (22), R1 is R1*, R2 is R2*, and the ionizable lipid is of Formula (23):
Formula (23) wherein: q and q’ are each independently integers from 0 to 12; 378
Attorney Docket No. 01318-0014-00PCT OR-043WO r and r’ are each independently integers from 0 to 6, wherein at least one of r or r’ is not 0; ZA and ZB are each independently selected from ^-C(O)O-, ^-OC(O), and -OC(O)O-; where ^ denotes the attachment point to -(CH2)q- or -(CH2)q’-; and R5A, R5B, R6A , and R6B are each independently C1-C20 alkyl or C2-C20 alkenyl. [571] In some embodiments of Formula (23), ZA and ZB are ^-C(O)O-, and the ionizable lipid is of Formula (23a-1):
. Formula (23a-1) [572] In some embodiments of Formula (23), ZA and ZB are ^-OC(O)-, and the ionizable lipid is of Formula (23a-2)
. Formula (23a-2) [573] In some embodiments of Formula (23), ZA and ZB are -O(C)(O)O-, and the ionizable lipid is represented by Formula (23a-3):
. Formula (23a-3) [574] In some embodiments of Formula (22), R2 is C1-C30 alkyl, and the ionizable lipid is of Formula (25): 379
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, Formula (25) or is a pharmaceutically acceptable salt thereof, wherein: ZB is selected from ^-C(O)O-, ^-OC(O)-, and -OC(O)O-; where ^ denotes the attachment point to -(CH2)q’-; R5B and R6B are each independently C1-C20 alkyl or C2-C20 alkenyl; q’ is an integer from 0 to 12; and r’ is an integer from 1 to 6. [575] In some embodiments of Formula (25), ZB is ^-C(O)O-, and the ionizable lipid is of Formula (25a-1):
. Formula (25a-1) [576] In some embodiments of Formula (25), ZB is ^-OC(O)-, and the ionizable lipid is of Formula (25a-2):
. Formula (25a-2) [577] In some embodiments of Formula (25), ZB is -OC(O)O-, and the ionizable lipid is of Formula (25a-3):
. Formula (25a-3) [578] In some embodiments, an ionizable lipid is selected from the table below: 380
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[579] In some embodiments, an ionizable lipid is selected from the table below:
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disclosed in one of US 2023/0053437; US 2019/0240354; US 2010/0130588; US 2021/0087135; WO 2021/204179; US 2021/0128488; US 2020/0121809; US 2017/0119904; US 2013/0108685; US 2013/0195920; US 2015/0005363; US 2014/0308304; US 2013/0053572; WO 2019/232095A1; WO 2021/077067; WO 2019/152557; US 2017/0210697; or WO 2019/089828A1, each of which is incorporated by reference herein in their entirety. [581] In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US Application publication US2017/0119904, which is incorporated by reference herein, in its entirety. [582] In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in PCT Application publication WO2021/204179, which is incorporated by reference herein, in its entirety. [583] In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in PCT Application WO2022/251665A1, which is incorporated by reference herein, in its entirety. [584] In some embodiments, an LNP described herein comprises an ionizable lipid of Table Z: 383
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Series “A” [586] In some embodiments, an LNP of the present disclosure comprises an ionizable lipid disclosed in PCT Application Publication WO2023044343A1, which is incorporated by reference herein, in its entirety. Formula (X) [587] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X):
or a pharmaceutically acceptable salt thereof, wherein each cc is independently selected from 3 to 9; Rxx is selected from hydrogen and optionally substituted C1-C6 alkyl; and (i) ee is 1, each dd is independently selected from 1 to 4; and 385
Attorney Docket No. 01318-0014-00PCT OR-043WO each Rww is independently selected from the group consisting of C4-C14 alkyl, branched C4-C12 alkenyl, C4-C12 alkenyl comprising at least two double bonds, and C9-C12 alkenyl, wherein any –(CH2)2- of the C4-C14 alkyl can be optionally replaced with C2-C6 cycloalkylenyl; (ii) ee is 0, each dd is 1; and each Rww is linear C4-C12 alkyl. [588] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein Rxx is H. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein Rxx is optionally substituted C1-C6 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein Rxx is C1 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein Rxx is C2 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein Rxx is C3 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein Rxx is C4 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein Rxx is C5 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein Rxx is C6 alkyl. [589] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is independently selected from the group consisting of C4-C14 alkyl, branched C4-C12 alkenyl, C4-C12 alkenyl comprising at least two double bonds, and C9-C12 alkenyl, wherein any –(CH2)2- of the C4-C14 alkyl can be optionally replaced with C2-C6 cycloalkylenyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C4-C14 alkyl, wherein any –(CH2)2- of the C4-C14 alkyl can be optionally replaced with C2-C6 cycloalkylenyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C4-C14 alkyl, wherein any –(CH2)2- of the C4-C14 alkyl can be optionally replaced with cyclopropylene. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is branched C4-C12 alkenyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C4-C12 alkenyl comprising at least two double bonds. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C9-C12 alkenyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is linear C4-C12 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is independently selected from the group consisting of C6- C14 alkyl, branched C8-C12 alkenyl, C8-C12 alkenyl comprising at least two double bonds, and C9-C12 alkenyl, wherein any –(CH2)2- of the C6-C14 alkyl can be optionally replaced with cyclopropylene. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C6-C14 alkyl, wherein any –(CH2)2- of the C6-C14 alkyl can be optionally replaced with 386
Attorney Docket No. 01318-0014-00PCT OR-043WO cyclopropylene. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is branched C8-C12 alkenyl, e.g., (linear or branched C3-C5 alkylenyl)- (branched C5-C7alkenyl), e.g., (branched C5 alkylenyl)-(branched C5alkenyl), e.g.,
. [590] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C8-C12 alkenyl comprising at least two double bonds. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C9-C12 alkenyl. [591] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is independently selected from the group consisting of C6-C14 alkyl (e.g., C6, C8, C9, C10, C11, C13 alkyl), wherein any –(CH2)2- of the C6-C14 alkyl can be optionally replaced with cyclopropylene. [592] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is independently branched C8-C12 alkenyl (e.g., branched C10 alkenyl). [593] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is independently C8-C12 alkenyl comprising at least two double bonds (e.g., C9 or C10 alkenyl comprising two double bonds). [594] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is independently (C1 alkylenyl)-(cyclopropylene-C6 alkyl) or (C2 alkylenyl)- (cyclopropylene-C2 alkyl). In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is independently (C1 alkylenyl)-(cyclopropylene-C6 alkyl). In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is independently (C2 alkylenyl)-(cyclopropylene-C2 alkyl). [595] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C4 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C5 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C6 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C7 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C8 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C9 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C10 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C11 alkyl. In some embodiments, ionizable lipids of the present disclosure have 387
Attorney Docket No. 01318-0014-00PCT OR-043WO a structure of Formula (X), wherein each Rww is C12 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C13 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C14 alkyl. [596] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C9 alkenyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C10 alkenyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C11 alkenyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C12 alkenyl. [597] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C8 alkenyl comprising at least two double bonds. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C9 alkenyl comprising at least two double bonds. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C10 alkenyl comprising at least two double bonds. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C11 alkenyl comprising at least two double bonds. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C12 alkenyl comprising at least two double bonds. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C13 alkenyl comprising at least two double bonds. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C14 alkenyl comprising at least two double bonds. [598] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C9 alkyl, wherein one –(CH2)2- of the C9 alkyl is replaced with C2-C6 cycloalkylenyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C9 alkyl, wherein one –(CH2)2- of the C9 alkyl is replaced with cyclopropylene. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C9 alkyl, wherein two –(CH2)2- of the C9 alkyl are replaced with C2-C6 cycloalkylenyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C9 alkyl, wherein two –(CH2)2- of the C9 alkyl are replaced with cyclopropylene. [599] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is linear C4 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is linear C5 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is linear C6 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula 388
Attorney Docket No. 01318-0014-00PCT OR-043WO (X), wherein each Rww is linear C7 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is linear C8 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is linear C9 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is linear C10 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is linear C11 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is linear C12 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is linear C13 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is linear C14 alkyl. [600] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is branched C8 alkenyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is branched C9 alkenyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is branched C10 alkenyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is branched C11 alkenyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is branched C12 alkenyl. [601] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each cc is independently selected from 3 to 7. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each cc is 3. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each cc is 4. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each cc is 5. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each cc is 6. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each cc is 7. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each cc is 8. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each cc is 9. [602] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each dd is independently selected from 1 to 4. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each dd is 1. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each dd is 2. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each dd is 3. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each dd is 4. [603] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula 389
Attorney Docket No. 01318-0014-00PCT OR-043WO (X), wherein ee is 1. [604] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein ee is 0. Formula (X-A) [605] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein the ionizable lipids of the present disclosure have a structure of Formula (X-A):
or a pharmaceutically acceptable salt thereof, wherein each cc is independently selected from 3 to 7; each dd is independently selected from 1 to 4; Rxx is selected from hydrogen and optionally substituted C1-C6 alkyl; and each Rww is independently selected from the group consisting of C4-C14 alkyl or (linear or branched C3-C5 alkylenyl)-(branched C5-C7alkenyl). [606] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein Rxx is hydrogen. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein Rxx is C1 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein Rxx is C2 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein Rxx is C3 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein Rxx is C4 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein Rxx is C5 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein Rxx is C6 alkyl. [607] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each cc is 4, 5, 6, or 7. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each cc is 3. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each cc is 4. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each cc is 5. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each cc is 6. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each cc is 7. 390
Attorney Docket No. 01318-0014-00PCT OR-043WO [608] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each dd is 1 or 3. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each dd is 1. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each dd is 2. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each dd is 3. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each dd is 4. [609] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each Rww is C4-C14 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each Rww is C4 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each Rww is C5 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X- A), wherein each Rww is C6 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each Rww is C7 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each Rww is C8 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each Rww is C9 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each Rww is C10 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each Rww is C11 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each Rww is C12 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each Rww is C13 alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each Rww is C14 alkyl. [610] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein each Rww is (linear or branched C3-C5 alkylenyl)-(branched C5-C7alkenyl), e.g., (branched C5 alkylenyl)-(branched C5alkenyl), e.g.,
. [611] In some embodiments, ionizable lipids of the present disclosure comprise an acyclic core. In some embodiments, ionizable lipids of the present disclosure are selected from any lipid in Table (I- A) below or a pharmaceutically acceptable salt thereof: 391
Attorney Docket No. 01318-0014-00PCT OR-043WO Table (I-A). Non-Limiting Examples of Ionizable Lipids with an Acyclic Core
392
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393
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394
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395
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396
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397
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398
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399
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400
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401
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402
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403
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404
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405
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406
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407
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Series “CY” [612] In some embodiments, an LNP of the present disclosure comprises an ionizable lipid disclosed in PCT Application Publication WO2023044333A1, which is incorporated by reference herein, in its entirety. Formula (CY) [613] In some embodiments, an LNP disclosed herein comprises an ionizable lipid of Formula 408
Attorney Docket No. 01318-0014-00PCT OR-043WO (CY)
(CY), or a pharmaceutically acceptable salt thereof, wherein: R1 is selected from the group consisting of -OH, -OAc, R1a,
Z1 is optionally substituted C1-C6 alkyl; X1 is optionally substituted C2-C6 alkylenyl; X2 is selected from the group consisting of a bond, -CH2- and -CH2CH2-; X2’ is selected from the group consisting of a bond, -CH2- and -CH2CH2-; X3 is selected from the group consisting of a bond, -CH2- and -CH2CH2-; X3’ is selected from the group consisting of a bond, -CH2- and -CH2CH2-; X4 and X5 are independently optionally substituted C2-C14 alkylenyl or optionally substituted C2-C14 alkenylenyl; Y1 and Y2 are independently selected from the group consisting of
, , , , , , , where
each Z2 is independently H or optionally substituted C1-C8 alkyl; each Z3 is indpendently optionally substituted C1-C6 alkylenyl; R2 is selected from the group consisting of optionally substituted C4-C20 alkyl, optionally substituted C2-C14 alkenyl, and –(CH2)pCH(OR6)(OR7); R3 is selected from the group consisting of optionally substituted C4-C20 alkyl, optionally substituted C2-C14 alkenyl, or (CH2)qCH(OR8)(OR9); R1a is: 409
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R2a, R2b, and R2c are independently hydrogen and C1-C6 alkyl; R3a, R3b, and R3c are independently hydrogen and C1-C6 alkyl; R4a, R4b, and R4c are independently hydrogen and C1-C6 alkyl; R5a, R5b, and R5c are independently hydrogen and C1-C6 alkyl; R6, R7, R8, and R9 are independently optionally substituted C1-C14 alkyl, optionally substituted C2-C14 alkenyl, or -(CH2)m-A-(CH2)nH; each A is independently a C3-C8 cycloalkylenyl; each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; p is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, and 7; and q is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, and 7. Formulas (CY-I), (CY-II), (CY-III), (CY-IV), (CY-V), (CY-VI), (CY-VII), (CY-VIII), (CY-IX), (CY-IV-a), (CY-IV-b), (CY-IV-c), (CY-IV-d), (CY-IV-e), and (CY-IV-f) [614] In some embodiments, the present disclosure comprises a compound of any of the below Formulae:
410
Attorney Docket No. 01318-0014-00PCT OR-043WO (CY-VI) (CY-VII)
(CY-IV-a) (CY-IV-b) (CY-IV-c) [615] In some embodiments, the present disclosure includes a compound of Formula (CY-IV-d), (CY-IV-e), or (CY-IV-f)
(CY-IV-d) (CY-IV-e) (CY-IV-f), Formula (CY-IV’) [616] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CY-IV’):
or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, X1, X2, X3, X4, X5, Y1, and Y2 are as defined in connection with Formula (CY-I’). Formula (CY-VI’) [617] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CY-VI’): 411
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or a pharmaceutically acceptable salt thereof, wherein R1, R6, R7, R8, R9, X1, X2, X3, X4, X5, Y1, and Y2 are as defined in connection with Formula (CY-I’). [618] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CY-VI’), or a pharmaceutically acceptable salt thereof, wherein R1 is -OH. [619] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CYVI’), or a pharmaceutically acceptable salt thereof, wherein X1 is C2-C6 alkylenyl. [620] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CY-VI’), or a pharmaceutically acceptable salt thereof, wherein X2 is -CH2CH2-. [621] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CYVI’), or a pharmaceutically acceptable salt thereof, wherein X4 is C2-C6 alkylenyl. [622] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CYVI’), or a pharmaceutically acceptable salt thereof, wherein X5 is C2-C6 alkylenyl. [623] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CY-VI’), or a pharmaceutically acceptable salt thereof, wherein Y1 is:
. [624] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CY-VI’), or a pharmaceutically acceptable salt thereof, wherein Y2 is:
. [625] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CYVI’), or a pharmaceutically acceptable salt thereof, wherein each Z3 is independently optionally substituted C1-C6 alkylenyl. [626] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CY-VI’), or a pharmaceutically acceptable salt thereof, wherein each Z3 is -CH2CH2-. [627] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CY-VI’), or a pharmaceutically acceptable salt thereof, wherein R6 is C5-C14 alkyl. 412
Attorney Docket No. 01318-0014-00PCT OR-043WO [628] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CY-VI’), or a pharmaceutically acceptable salt thereof, wherein R7 is C5-C14 alkyl. [629] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CY-VI’), or a pharmaceutically acceptable salt thereof, wherein R6 is C6-C14 alkenyl. [630] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CY-VI’), or a pharmaceutically acceptable salt thereof, wherein R7 is C6-C14 alkenyl. [631] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CY-VI’), or a pharmaceutically acceptable salt thereof, wherein R8 is C5-C16 alkyl. [632] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CY-VI’), or a pharmaceutically acceptable salt thereof, wherein R9 is C5-C14 alkyl. [633] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CY-VI’), or a pharmaceutically acceptable salt thereof, wherein R8 is C6-C14 alkenyl. [634] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CY-VI’), or a pharmaceutically acceptable salt thereof, wherein R9 is C6-C14 alkenyl. [635] In some embodiments, ionizable lipids of the present disclosure comprise a heterocyclic core, wherein the heteroatom is nitrogen. In some embodiments, the heterocyclic core comprises pyrrolidine or a derivative thereof. In some embodiments, the heterocyclic core comprises piperidine or a derivative thereof. [636] In some embodiments, Lipids of the Present Disclosure are selected from any lipid in Table (I-B) below or a pharmaceutically acceptable salt thereof:
413
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414
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415
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416
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417
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418
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419
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420
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421
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422
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423
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424
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425
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426
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Series “C” [637] In some embodiments, an LNP of the present disclosure comprises an ionizable lipid disclosed in PCT Publication WO2023122752A1, which is incorporated by reference herein, in its entirety. [638] In one embodiment, the disclosure provides a compound of Formula IA: 427
Attorney Docket No. 01318-0014-00PCT OR-043WO
or a pharmaceutically acceptable salt or solvate thereof, wherein: A is selected from the group consisting of -N(R1a)- and -C(R')-OC(=O)(R8a)-; R1a is -L1-R1; L1 is C2-C6 alkylenyl or –(CH2)2-6-OC(=O)-; R1 is selected from the group consisting of -OH,
R2a, R2b, and R2c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R3a, R3b, and R3c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R4a, R4b, and R4c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R5a, R5b, and R5c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R6a, R6b, and R6c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or R6a and R6b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; and R6c is selected from the group consisting of hydrogen and C1C6 alkyl; R7a, R7b, and R7c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or R7a and R7b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; and R7c is selected from the group consisting of hydrogen and C1C6 alkyl; R' is selected from the group consisting of hydrogen and C1-C6 alkyl; R8a is - L2-R8; L2 is C2-C6 alkylenyl; 428
Attorney Docket No. 01318-0014-00PCT OR-043WO R8 is selected from the group consisting
,
R9a and R9b are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or R9a and R9b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; Q1 is C1-C20 alkylenyl; W1 is selected from the group consisting of -C(=O)O-, -OC(=O)-, -C(=O)N(R12a)-, -N(R12a)C(=O)-, - OC(=O)N(R12a)-, - N(R12a)C(=O)O-, and -OC(=O)O-; R12a is selected from the group consisting of hydrogen and C1-C6 alkyl; X1 is optionally substituted C1-C15 alkylenyl; or X1 is a bond; Y1 is selected from the group consisting of -(CH2)m-, -O-, -S-, and -S-S-; m is 0, 1, 2, 3, 4, 5, or 6; Z1 is selected from the group consisting of optionally substituted C4-C12 cycloalkylenyl,
R10 is selected from the group consisting of hydrogen, C1-C20 alkyl, and C2-C20 alkenyl; Q2 is C1-C20 alkylenyl; W2 is selected from the group consisting of -C(=O)O-, -C(=O)N(R12b)-, -OC(=O)N(R12b)-, and - OC(=O)O-; R12b is selected from the group consisting of hydrogen and C1-C6 alkyl; X2 is optionally substituted C1-C15 alkylenyl; or X2 is a bond; Y2 is selected from the group consisting of -(CH2)n-, -O-, -S-, and -S-S-; n is 0, 1, 2, 3, 4, 5, or 6; Z2 is selected from the group consisting of -(CH2)p-, optionally substituted C4-C12 cycloalkylenyl, 429
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p is 0 or 1; and R11 is selected from the group consisting of hydrogen, C1-C10 alkyl, and C2-C10 alkenyl; wherein one or more methylene linkages of X1, X2, Y1, Y2, Z1, Z2, R10, and R11, are optionally and independently replaced with a group selected from -O-, -CH=CH-, -S- and C3-C6 cycloalkylenyl. [639] In one embodiment, the disclosure provides a compound of Formula IB:
or a pharmaceutically acceptable salt or solvate thereof, wherein: A is selected from the group consisting of -N(R1a)- and -C(R')-OC(=O)(R8a)-; R1a is -L1-R1; L1 is C2-C6 alkylenyl or –(CH2)2-6-OC(=O)-; R1 is selected from the group consisting of -OH,
R2a, R2b, and R2c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R3a, R3b, and R3c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R4a, R4b, and R4c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R5a, R5b, and R5c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R6a, R6b, and R6c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or R6a and R6b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; and R6c is selected from the group consisting of hydrogen and C1C6 alkyl; 430
Attorney Docket No. 01318-0014-00PCT OR-043WO R7a, R7b, and R7c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or R7a and R7b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; and R7c is selected from the group consisting of hydrogen and C1C6 alkyl; R' is selected from the group consisting of hydrogen and C1-C6 alkyl; R8a is - L2-R8; L2 is C2-C6 alkylenyl;
R9a and R9b are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or R9a and R9b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; Q1 is C1-C20 alkylenyl; W1 is selected from the group consisting of -C(=O)O-, -OC(=O)-, -C(=O)N(R12a)-, -N(R12a)C(=O)-, - OC(=O)N(R12a)-, - N(R12a)C(=O)O-, and -OC(=O)O-; R12a is selected from the group consisting of hydrogen and C1-C6 alkyl; X1 is optionally substituted C1-C15 alkylenyl; or X1 is a bond; Y1 is selected from the group consisting of -(CH2)m-, -O-, -S-, and -S-S-; m is 0, 1, 2, 3, 4, 5, or 6; Z1 is selected from the group consisting of optionally substituted C5-C12 bridged cycloalkylenyl,
R10 is selected from the group consisting of hydrogen, C1-C20 alkyl, and C2-C20 alkenyl; Q2 is C1-C20 alkylenyl; W2 is selected from the group consisting of -C(=O)O-, -C(=O)N(R12b)-, -OC(=O)N(R12b)-, and - OC(=O)O-; R12b is selected from the group consisting of hydrogen and C1-C6 alkyl; X2 is optionally substituted C1-C15 alkylenyl; or 431
Attorney Docket No. 01318-0014-00PCT OR-043WO X2 is a bond; Y2 is selected from the group consisting of -(CH2)n-, -O-, -S-, and -S-S-; n is 0, 1, 2, 3, 4, 5, or 6; Z2 is selected from the group consisting of -(CH2)p-, optionally substituted C4-C12 cycloalkylenyl,
p is 0 or 1; and R11 is selected from the group consisting of hydrogen, C1-C10 alkyl, and C2-C10 alkenyl; wherein one or more methylene linkages of X1, X2, Y1, Y2, Z1, Z2, R10, and R11, are optionally and independently replaced with a group selected from -O-, -CH=CH-, -S- and C3-C6 cycloalkylenyl. [640] In one embodiment, the disclosure provides a compound of Formula IC:
or a pharmaceutically acceptable salt or solvate thereof, wherein: A is selected from the group consisting of -N(R1a)- and -C(R')-OC(=O)(R8a)-; R1a is -L1-R1; L1 is C2-C6 alkylenyl or –(CH2)2-6-OC(=O)-; R1 is selected from the group consisting of -OH,
R2a, R2b, and R2c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R3a, R3b, and R3c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R4a, R4b, and R4c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; 432
Attorney Docket No. 01318-0014-00PCT OR-043WO R5a, R5b, and R5c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R6a, R6b, and R6c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or R6a and R6b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; and R6c is selected from the group consisting of hydrogen and C1C6 alkyl; R7a, R7b, and R7c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or R7a and R7b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; and R7c is selected from the group consisting of hydrogen and C1C6 alkyl; R' is selected from the group consisting of hydrogen and C1-C6 alkyl; R8a is - L2-R8; L2 is C2-C6 alkylenyl;
R9a and R9b are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or R9a and R9b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; Q1 is C1-C20 alkylenyl; W1 is selected from the group consisting of -C(=O)O-, -OC(=O)-, -C(=O)N(R12a)-, -N(R12a)C(=O)-, - OC(=O)N(R12a)-, - N(R12a)C(=O)O-, and -OC(=O)O-; R12a is selected from the group consisting of hydrogen and C1-C6 alkyl; X1 is optionally substituted branched C1-C15 alkylenyl; or X1 is a bond; Y1 is selected from the group consisting of -(CH2)m-, -O-, -S-, and -S-S-; m is 0, 1, 2, 3, 4, 5, or 6; Z1 is selected from the group consisting of optionally substituted C4-C12 cycloalkylenyl,
R10 is selected from the group consisting of hydrogen, C1-C20 alkyl, and C2-C20 alkenyl; Q2 is C1-C20 alkylenyl; 433
Attorney Docket No. 01318-0014-00PCT OR-043WO W2 is selected from the group consisting of -C(=O)O-, -C(=O)N(R12b)-, -OC(=O)N(R12b)-, and - OC(=O)O-; R12b is selected from the group consisting of hydrogen and C1-C6 alkyl; X2 is optionally substituted C1-C15 alkylenyl; or Y2 is selected from the group consisting of -(CH2)n-, -O-, -S-, and -S-S-; n is 0, 1, 2, 3, 4, 5, or 6; Z2 is of -(CH2)p-; p is 0 or 1; and R11 is C1-C20 branched alkyl; wherein one or more methylene linkages of X1, X2, Y1, Y2, Z1, Z2, R10, and R11, are optionally and independently replaced with a group selected from -O-, -CH=CH-, -S- and C3-C6 cycloalkylenyl. [641] In some embodiments, the disclosure provides a compound of any one of Formulae IA, IB, IC, or a pharmaceutically acceptable salt or solvate thereof, wherein Z1 is optionally substituted C5- C12 bridged cycloalkylenyl. [642] In some embodiments, the disclosure provides a compound of any one of Formulae IA, IB, IC, or a pharmaceutically acceptable salt or solvate thereof, wherein Z1 is not adamantyl. [643] In one embodiment, the disclosure provides a compound of Formula ID:
or a pharmaceutically acceptable salt or solvate thereof, wherein: A is selected from the group consisting of -N(R1a)- and -C(R')-OC(=O)(R8a)-; R1a is -L1-R1; L1 is C2-C6 alkylenyl or –(CH2)2-6-OC(=O)-; R1 is selected from the group consisting of -OH, 434
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R2a, R2b, and R2c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R3a, R3b, and R3c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R4a, R4b, and R4c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R5a, R5b, and R5c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R6a, R6b, and R6c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or R6a and R6b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; and R6c is selected from the group consisting of hydrogen and C1C6 alkyl; R7a, R7b, and R7c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or R7a and R7b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; and R7c is selected from the group consisting of hydrogen and C1C6 alkyl; R' is selected from the group consisting of hydrogen and C1-C6 alkyl; R8a is - L2-R8; L2 is C2-C6 alkylenyl; R8 is selected from the group consisting
,
R9a and R9b are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or R9a and R9b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; Q1 is C1-C20 alkylenyl; W1 is selected from the group consisting of -C(=O)O-, -OC(=O)-, -C(=O)N(R12a)-, -N(R12a)C(=O)-, - OC(=O)N(R12a)-, - N(R12a)C(=O)O-, and -OC(=O)O-; R12a is selected from the group consisting of hydrogen and C1-C6 alkyl; 435
Attorney Docket No. 01318-0014-00PCT OR-043WO X1 is optionally substituted branched C1-C15 alkylenyl; or X1 is a bond; Y1 is selected from the group consisting of -(CH2)m-, -O-, -S-, and -S-S-; m is 0, 1, 2, 3, 4, 5, or 6; Z1 is optionally substituted C5-C12 bridged cycloalkylenyl; R10 is selected from the group consisting of hydrogen, C1-C20 alkyl, and C2-C20 alkenyl; Q2 is C1-C20 alkylenyl; W2 is selected from the group consisting of -C(=O)O-, -C(=O)N(R12b)-, -OC(=O)N(R12b)-, and - OC(=O)O-; R12b is selected from the group consisting of hydrogen and C1-C6 alkyl; X2 is optionally substituted C1-C15 alkylenyl; or Y2 is -(CH2)n-; n is 0, 1, 2, 3, 4, 5, or 6; Z2 is of -(CH2)p-; p is 0 or 1; and R11 is C1-C20 branched alkyl. [644] In some embodiments, the disclosure provides a compound of Formula ID or a pharmaceutically acceptable salt or solvate thereof, wherein Z1 is not adamantyl. [645] In one embodiment, the disclosure provides a compound of Formula I:
, or a pharmaceutically acceptable salt or solvate thereof, wherein: A is selected from the group consisting of -N(R1a)- and -C(R')-OC(=O)(R8a)-; R1a is -L1-R1; L1 is C2-C6 alkylenyl; R1 is selected from the group consisting of -OH, 436
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R2a, R2b, and R2c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R3a, R3b, and R3c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R4a, R4b, and R4c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R5a, R5b, and R5c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; R6a, R6b, and R6c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or R6a and R6b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; and R6c is selected from the group consisting of hydrogen and C1C6 alkyl; R7a, R7b, and R7c are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or R7a and R7b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; and R7c is selected from the group consisting of hydrogen and C1C6 alkyl; R' is selected from the group consisting of hydrogen and C1-C6 alkyl; R8a is - L2-R8; L2 is C2-C6 alkylenyl; R8 is -NR9aR9b; R9a and R9b are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or R9a and R9b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; Q1 is C1-C20 alkylenyl; W1 is selected from the group consisting of -C(=O)O-, -OC(=O)-, -C(=O)N(R12a)-, -N(R12a)C(=O)-, - OC(=O)N(R12a)-, - N(R12a)C(=O)O-, and -OC(=O)O-; R12a is selected from the group consisting of hydrogen and C1-C6 alkyl; X1 is C1-C15 alkylenyl; or X1 is a bond; Y1 is selected from the group consisting of -(CH2)m-, -O-, -S-, and -S-S-; m is 0, 1, 2, 3, 4, 5, or 6; Z1 is selected from the group consisting of C4-C12 cycloalkylenyl, 437
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R10 is selected from the group consisting of hydrogen, C1-C20 alkyl, and C2-C20 alkenyl; Q2 is C1-C20 alkylenyl; W2 is selected from the group consisting of -C(=O)O-, -C(=O)N(R12b)-, -OC(=O)N(R12b)-, and - OC(=O)O-; R12b is selected from the group consisting of hydrogen and C1-C6 alkyl; X2 is C1-C15 alkylenyl; or X2 is a bond; Y2 is selected from the group consisting of -(CH2)n-, -O-, -S-, and -S-S-; n is 0, 1, 2, 3, 4, 5, or 6; Z2 is selected from the group consisting of -(CH2)p-, C4-C12 cycloalkylenyl,
p is 0 or 1; and R11 is selected from the group consisting of hydrogen, C1-C10 alkyl, and C2-C10 alkenyl. [646] In another embodiment, the disclosure provides a compound of Formula II, III, VI, VI’, VI’’, VI’’’, VII, VII’, VII’’, VII’’’, VIII, VIII’, VIII’’, VIII’’’, IX, IX’, IX’’, IX’’’, X, X’, X’’, X’’’, XI, XI’, XI’’, XI’’’, XII, XII’, XII’’, XII’’’, XIII, XIII’, XIII’’, XIII’’’, XIV, XIV’, XIV’’, XIV’’’, XV, XV’, XV’’, XV’’’, XVI, XVI’, XVI’’, XVI’’’, XVII, XVIII, XVIII’, XIX, XX, or XXI, as described in PCT Publication WO2023122752A1:
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439
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440
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Series “CX” [647] In some embodiments, an LNP of the present disclosure comprises an ionizable lipid disclosed in PCT Publication WO2023196931A1, which is incorporated by reference herein, in its entirety. [648] In some embodiments, lipids of the present disclosure comprise a heterocyclic core, wherein the heteroatom is nitrogen. In some embodiments, the heterocyclic core comprises pyrrolidine or a derivative thereof. In some embodiments, the heterocyclic core comprises piperidine or a derivative thereof. 452
Attorney Docket No. 01318-0014-00PCT OR-043WO [649] In some embodiments, a compound of the present disclosure is represented by Formula (CX-I):
(CX-I) or a pharmaceutically acceptable salt thereof, wherein Z is selected from the group consisting of a bond,
, , ,
each Y is independently selected from the group consisting of
R1 is -(CH2)1-6N(Ra)2 or -(CH2)1-6OH; R2 is optionally substituted C1-C36 alkyl or optionally substituted C2-C36 alkenyl, wherein 1-6 methylene units of R2 are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-; R2’ is optionally substituted C1-C36 alkyl or optionally substituted C2-C36 alkenyl, wherein 1-6 methylene units of R2 are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-;each Ra is independently optionally substituted C1-C6 alkyl; or two Ra are taken together, with the nitrogen on which they are attached, to form an optionally substituted 4-7 membered heterocyclyl ring; m is 0, 1, or 2; n is 1 or 2; and 453
Attorney Docket No. 01318-0014-00PCT OR-043WO p is 1 or 2. [650] In some embodiments, a compound of the present disclosure is represented by Formula (CX-i):
(CX-i) or a pharmaceutically acceptable salt thereof, wherein Z is selected from the group consisting of a bond,
, , ,
each Y is independently selected from the group consisting of ,
; R1 is -(CH2)1-6N(Ra)2; R2 is optionally substituted C1-C36 alkyl or optionally substituted C2-C36 alkenyl, wherein 1-6 methylene units of R2 are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-; each Ra is independently optionally substituted C1-C6 alkyl; or two Ra are taken together, with the nitrogen on which they are attached, to form an optionally substituted 4-7 membered heterocyclyl ring; m is 0, 1, or 2; n is 1 or 2; and p is 1 or 2. 454
Attorney Docket No. 01318-0014-00PCT OR-043WO [651] In some embodiments, the present disclosure comprises a compound selected from any lipid in Table (I-D) below or a pharmaceutically acceptable salt thereof: Table (I-D). Non-Limiting Examples of Ionizable Lipids
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[652] In some embodiments, lipids of the present disclosure comprise a heterocyclic core, wherein the heteroatom is nitrogen. In some embodiments, the heterocyclic core comprises pyrrolidine or a derivative thereof. In some embodiments, the heterocyclic core comprises piperidine or a derivative thereof. Series “CZ” [653] In some embodiments, an LNP of the present disclosure comprises an ionizable lipid 461
Attorney Docket No. 01318-0014-00PCT OR-043WO disclosed in PCT Publication WO2023196931A1, which is incorporated by reference herein, in its entirety. [654] In some embodiments, a compound of the present disclosure is represented by Formula (CZ-I)
(CZ-I) or a pharmaceutically acceptable salt thereof, wherein Z is selected from the group consisting of a bond,
, , ,
each Y is independently selected from the group consisting of , ,
R1 is -(CH2)1-6N(Ra)2; each R2 is independently optionally substituted C1-C36 alkyl or optionally substituted C2- C36 alkenyl, wherein 1-6 methylene units of R2 are optionally replaced with a group each independently selected from cyclopropylene, -O-, -OC(O)-, and -C(O)O-; each Ra is independently optionally substituted C1-C6 alkyl; or two Ra are taken together, with the nitrogen on which they are attached, to form an optionally substituted 4-7 membered heterocyclyl ring; m is 0, 1, or 2; n is 1 or 2; and p is 1 or 2. [655] In some embodiments, the present disclosure comprises a compound selected from any lipid in Table (I-E) below or a pharmaceutically acceptable salt thereof: 462
Attorney Docket No. 01318-0014-00PCT OR-043WO Table (I-E). Non-Limiting Examples of Ionizable Lipids
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464
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Series “S” [656] Described below are a number of exemplary ionizable lipids of the present disclosure. In 465
Attorney Docket No. 01318-0014-00PCT OR-043WO certain embodiments, the ionizable lipid is one selected from those disclosed in PCT Application Publication WO2024192277A1, which is incorporated by reference herein, in its entirety. Formula (S-I) [657] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (S-I):
or a pharmaceutically acceptable salt thereof, wherein: X is N or CH; Y is a bond,
, , , wherein bond marked with an “**” is attached to X; each Z is independently selected from the group consisting of:
wherein the bond marked with an "*" is attached to L; each L is independently C2-C10 alkylenyl; ,
each R is independently -H or C1-C6 aliphatic; each R2 is independently selected from optionally substituted C2-14alkyl and C2-14alkenyl, wherein any –(CH2)2- of the C2-C14 alkyl can be optionally replaced with C3-C6 cycloalkylenyl; 466
Attorney Docket No. 01318-0014-00PCT OR-043WO each R3 independently selected from is H and C1-6alkyl; n is selected from 1 to 6; and each p is independently selected from 1 to 6. Formula (S-M) [658] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (S-M):
or a pharmaceutically acceptable salt thereof, wherein: X is N or CH;
Y is a bond, , , or , wherein bond marked with an “**” is attached to X; each Z is independently selected from the group consisting of:
wherein the bond marked with an
is attached to L; each L is independently C2-C10 alkylenyl;
each R is independently -H or C1-C6 aliphatic; each R3 independently selected from is H and C1-6alkyl; R4 is -CH(SR6)(SR7); 467
Attorney Docket No. 01318-0014-00PCT OR-043WO R5 is -CH(OR8)(OR9); -CH(SR8)(SR9); -CH(R8)(R9) or optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-; R6 and R7 are each independently optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5- C12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or - C(O)O-; and R8 and R9 are each independently optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5- C12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or - C(O)O-; n is selected from 1 to 6; and each p is independently selected from 1 to 6. [659] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (S-Ma)
or a pharmaceutically acceptable salt thereof, wherein: n is selected from 1 to 4; each m is independently selected from 2 to 10; and each p is independently selected from 2 to 6. [660] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (S-Mb)
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Attorney Docket No. 01318-0014-00PCT OR-043WO or a pharmaceutically acceptable salt thereof, wherein: each R3 independently selected from is H and C1-6 alkyl; n is selected from 1 to 4; each m is independently selected from 2 to 10; and each p is independently selected from 2 to 6. [661] In some embodiments, ionizable lipids of the present disclosure comprise an acyclic core. In some embodiments, ionizable lipids of the present disclosure are selected from any lipid in Table (I- F) below or a pharmaceutically acceptable salt thereof: Table (I-F). Non-Limiting Examples of Ionizable Lipids
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Series “AT” [662] Described below are a number of exemplary ionizable lipids of the present disclosure. In certain embodiments, the ionizable lipid is one selected from those disclosed in PCT Application Publication WO2024192277A1, which is incorporated by reference herein, in its entirety. [663] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (AT) 471
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(AT), or a pharmaceutically acceptable salt thereof, wherein: i) A is N; Z is a bond; X1 is optionally substituted C1-C6 aliphatic, wherein the optional substituent is not oxo when X1 is C1 aliphatic; and R1 is selected from the group consisting of:
marked with an "*" is attached to X1; X1 is a bond or optionally substituted C1-C6 aliphatic; R1 is selected from the group consisting of:
X4 is a bond or optionally substituted C1-C6 aliphatic; RZ is NR2 or OH; each R is independently -H or C1-C6 aliphatic; X2 and X3 are each independently optionally substituted C1-C12 aliphatic; Y1 and Y2 are independently selected from the group consisting of 472
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wherein the bond marked with
is attached to X2 for Y1 or X3 for Y2; R2 is optionally substituted C1-C6 aliphatic; R3 is optionally substituted C1-C6 aliphatic; R4 is -CH(OR6)(OR7), -CH(SR6)(SR7), -CH(R6)(R7), or optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-; R5 is -CH(OR8)(OR9), -CH(SR8)(SR9), -CH(R8)(R9), or optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-; R6 and R7 are each independently optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5- C12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or - C(O)O-; and R8 and R9 are each independently optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5- C12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or - C(O)O-. Formula (AT-E’) [664] In certain embodiments, ionizable lipids of the present disclosure have a structure of Formula (AT), wherein the ionizable lipids of the present disclosure have a structure of Formula (AT- E’): 473
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(AT-E’), or a pharmaceutically acceptable salt thereof, wherein R1, R, X1, Z, X2, X3, X4, RZ, Y1, Y2, R2, R3, R6, R7, R8, and R9 are as described in Formula (AT) or as otherwise described in any embodiments below. Formula (AT-F’’’) [665] In certain embodiments, ionizable lipids of the present disclosure have a structure of Formula (AT), wherein the ionizable lipids of the present disclosure have a structure of Formula (AT- F’’’):
(AT-F’’’), [666] or a pharmaceutically acceptable salt thereof, wherein R1, R, X1, Z, X2, X3, X4, RZ, R2, R3, R6, R7, R8, and R9 are as described in Formula (AT) or as otherwise described in any embodiments below. Formula (AT-M) [667] In certain embodiments, ionizable lipids of the present disclosure have a structure of Formula (AT), wherein the ionizable lipids of the present disclosure have a structure of Formula (AT- M):
(AT-M), or a pharmaceutically acceptable salt thereof, wherein R1, R, X2, X3, X4, RZ, Y1, Y2, R2, R3, R4, R5, R6, R7, R8, and R9 are as described in Formula (AT) or as otherwise described in any embodiments below. 474
Attorney Docket No. 01318-0014-00PCT OR-043WO Formula (AT-N’) [668] In certain embodiments, ionizable lipids of the present disclosure have a structure of Formula (AT), wherein the ionizable lipids of the present disclosure have a structure of Formula (AT- N’):
(AT-N’), [669] or a pharmaceutically acceptable salt thereof, wherein R1, R, X1, X2, X3, X4, RZ, R2, R3, R4, R5, R6, R7, R8, and R9 are as described in Formula (AT) or as otherwise described in any embodiments below. Formula (AT-O’) [670] In certain embodiments, ionizable lipids of the present disclosure have a structure of Formula (AT), wherein the ionizable lipids of the present disclosure have a structure of Formula (AT- O’):
(AT-O’), or a pharmaceutically acceptable salt thereof, wherein R1, R, X1, X2, X3, Y1, Y2, X4, RZ, R2, R3, R6, R7, R8, and R9 are as described in Formula (AT) or as otherwise described in any embodiments below. Formula (AT-P’’’) [671] In certain embodiments, ionizable lipids of the present disclosure have a structure of Formula (AT), wherein the ionizable lipids of the present disclosure have a structure of Formula (AT- 475
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(AT-P’’’), or a pharmaceutically acceptable salt thereof, wherein R1, R, X1, X2, X3, R2, R3, X4, RZ, R6, R7, R8, and R9 are as described in Formula (AT) or as otherwise described in any embodiments below. [672] In some embodiments, Lipids of the Present Disclosure are selected from any lipid in Table (I-G) below or a pharmaceutically acceptable salt thereof: Table (I-G). Non-Limiting Examples of Ionizable Lipids of the Present Disclosure
476
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477
Attorney Docket No. 01318-0014-00PCT OR-043WO Series “AC” [673] Described below are a number of exemplary ionizable lipids of the present disclosure. In certain embodiments, the ionizable lipid is one selected from those disclosed in PCT Application Publication WO2024192277A1, which is incorporated by reference herein, in its entirety. [674] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (AC)
(AC), or a pharmaceutically acceptable salt thereof, wherein: R1 is selected from the group consisting of -NR2,
each R is independently -H or C1-C6 aliphatic; X1 is a bond or optionally substituted C2-C6 aliphatic;
; wherein the bond marked with an "*" is attached to X1; X2 and X3 are each independently optionally substituted C1-C12 aliphatic; X4 is a bond or C2-C6 aliphatic; Y1 and Y2 are independently selected from the group consisting of 478
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wherein the bond marked with an
" is attached to X2 for Y1 or X3 for Y2; R2 is optionally substituted C1-C6 aliphatic; R3 is optionally substituted C1-C6 aliphatic; R4 is -CH(OR6)(OR7); R5 is -CH(OR8)(OR9), -CH(R8)(R9), or optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3- C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-; R6 and R7 are each independently optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3- C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-; and R8 and R9 are each independently optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3- C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-. Additional Formulae [675] In certain embodiments, ionizable lipids of the present disclosure have a structure of Formula (AC), wherein the ionizable lipids of the present disclosure have a structure of Formula (AC- A), (AC-B), (AC-C), (AC-D), (AC-D1), (AC-D2), (AC-E), (AC-F), (AC-G), (AC-H), or (AC-I):
479
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480
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(AC-I), or a pharmaceutically acceptable salt thereof, wherein R1, R, X1, Z, X2, X3, Y1, Y2, R2, R3, R4, R5, R6, R7, R8, and R9 are as described in Formula (AC) or as otherwise described in any embodiments below. [676] In some embodiments, Lipids of the Present Disclosure are selected from any lipid in Table (I-H) below or a pharmaceutically acceptable salt thereof: Table (I-H). Non-Limiting Examples of Ionizable Lipids of the Present Disclosure
481
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482
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Series “CO” [677] Described below are a number of exemplary ionizable lipids of the present disclosure. In certain embodiments, the ionizable lipid is one selected from those disclosed in PCT Application Publication WO2024192277A1, which is incorporated by reference herein, in its entirety. [678] The present disclosure provides compound of Formula (CO):
(CO), or a pharmaceutically acceptable salt thereof, wherein: R1 is selected from the group consisting of -NR2,
483
Attorney Docket No. 01318-0014-00PCT OR-043WO each R is independently -H or C1-C6 aliphatic; X1 is optionally substituted C2-C6 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-; X2 is selected from the group consisting of a bond, -CH2- and -CH2CH2-; X3 is selected from the group consisting of a bond, -CH2- and -CH2CH2-; X4 and X5 are each independently optionally substituted C1-C10 aliphatic; Y1 and Y2 are each independently
wherein the bond marked with
is attached to X4 or X5; R2 is optionally substituted C1-C6 aliphatic; R3 is optionally substituted C1-C6 aliphatic; R4 is -CH(OR6)(OR7); -CH(SR6)(SR7); -CH(R6)(R7); or optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-; R5 is -CH(OR8)(OR9); -CH(SR8)(SR9); -CH(R8)(R9) or optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-; R6 and R7 are each independently optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5- C12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or - C(O)O-; and R8 and R9 are each independently optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5- C12 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or - C(O)O-. 484
Attorney Docket No. 01318-0014-00PCT OR-043WO [679] In certain embodiments, the compound of Formula (CO) is a compound of any of the below Formulae:
(CO-F’), (CO-G), 485
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(CO-I’), (CO-J), 486
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487
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(CO-O), (CO-O’), or a pharmaceutically acceptable salt thereof, wherein R1, R, X1, X2, X3, X4, X5, Y1, Y2, R2, R3, R4, R5, R6, R7, R8, and R9 are as described in Formula (CO) or as otherwise described in any embodiments below. [680] In some embodiments, Lipids of the Present Disclosure are selected from any lipid in Table (I-I) below or a pharmaceutically acceptable salt thereof: Table (I-I). Non-Limiting Examples of Ionizable Lipids of the Present Disclosure
488
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489
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490
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Series “CC” [681] Described below are a number of exemplary ionizable lipids of the present disclosure. In certain embodiments, the ionizable lipid is one selected from those disclosed in PCT Application Publication WO2024192277A1, which is incorporated by reference herein, in its entirety. [682] The present disclosure provides compound of Formula (CC)
(CC), or a pharmaceutically acceptable salt thereof, wherein: R1 is selected from the group consisting of -OH, -OAc, -NR2,
each R is independently -H or C1-C6 aliphatic; X1 is optionally substituted C2-C6 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-; X2 is selected from the group consisting of a bond, -CH2- and -CH2CH2-; X2’ is selected from the group consisting of a bond, -CH2- and -CH2CH2-; 491
Attorney Docket No. 01318-0014-00PCT OR-043WO X3 is selected from the group consisting of a bond, -CH2- and -CH2CH2-; X3’ is selected from the group consisting of a bond, -CH2- and -CH2CH2-; X4 and X5 are independently optionally substituted C1-C10 aliphatic; Y1 and Y2 are independently selected from the group consisting of
wherein the bond marked with
is attached to X4 or X5; R2 is optionally substituted C1-C6 aliphatic; R3 is optionally substituted C1-C6 aliphatic; R4 is -CH(OR6)(OR7); -CH(SR6)(SR7); -CH(SR8)(SR9); -CH(R6)(R7); -R10; or optionally substituted C1-C14 aliphatic-R10 wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, - NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-; R5 is -CH(OR8)(OR9); -CH(SR8)(SR9); -CH(R8)(R9); optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, - NHC(O)- or -C(O)O-; -R11; or optionally substituted C1-C14 aliphatic-R11, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3- C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-; R6 and R7 are each independently -R10; optionally substituted -C1-C14 aliphatic-R10; wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, - NHC(O)- or -C(O)O-; R8 and R9 are each independently -R11; optionally substituted -C1-C14 aliphatic wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3- C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-; or optionally substituted -C1-C14 aliphatic-R11 wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-; and each R10 and R11 is independently an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl, or two R10 or two R11 taken together form an optionally substituted bridged bicyclic or multicyclic C5-C12 cycloalkylenyl; or 492
Attorney Docket No. 01318-0014-00PCT OR-043WO each R10 and R11 is independently an optionally substituted cyclic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic C4-C14 cycloalkyl or optionally substituted cyclic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic 4-14 membered heterocyclyl, or two R10 or two R11 taken together form an optionally substituted bridged bicyclic or multicyclic C4-C14 cycloalkyl or optionally substituted bridged bicyclic or multicyclic 4-14 membered heterocyclyl. [683] In certain embodiments, the compound of Formula (CC) is a compound of any one of the Formulae below:
(CC-F’), (CC-G), 493
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(CC-L), (CC-M), or a pharmaceutically acceptable salt thereof, wherein R1, R, X1, X4, X5, Y1, Y2, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, are as described in Formula (CC) or as otherwise described in any embodiments below. [684] In some embodiments, Lipids of the Present Disclosure are selected from any lipid in Table (I-J) below or a pharmaceutically acceptable salt thereof: Table (I-J). Non-Limiting Examples of Ionizable Lipids of the Present Disclosure 494
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495
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496
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497
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498
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499
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Series “CT” [685] Described below are a number of exemplary ionizable lipids of the present disclosure. In certain embodiments, the ionizable lipid is one selected from those disclosed in PCT Application Publication WO2024263729A1, which is incorporated by reference herein, in its entirety. 500
Attorney Docket No. 01318-0014-00PCT OR-043WO [686] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CT)
(CT), or a pharmaceutically acceptable salt thereof, wherein:
, wherein the bond marked with an "*" is attached to X1; X1 is optionally substituted C1-C6 aliphatic; and R1 is selected from the group consisting of -OH, -OAc, -NR2,
marked with an "*" is attached to X1; X1 is a bond or optionally substituted C1-C6 aliphatic; R1 is selected from the group consisting of -OH, -OAc, -NR2, 501
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each R is independently -H or C1-C6 aliphatic; X2 and X3 are each independently optionally substituted C1-C12 aliphatic; Y1 and Y2 are independently selected from the group consisting of ,
wherein the bond marked with an
is attached to X2 for Y1 or X3 for Y2; R2 is a bond or optionally substituted C1-C6 aliphatic; R3 is a bond or optionally substituted C1-C6 aliphatic; R4 is -CH(OR6)(OR7), -CH(SR6)(SR7), -CH(R6)(R7), or optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C14 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, - NHC(O)- or -C(O)O-; R5 is -CH(OR8)(OR9), -CH(SR8)(SR9), -CH(R8)(R9), -R8, or optionally substituted -C1-C6 - aliphatic-R8; R6 and R7 are each independently optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C14 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-; R8 is optionally substituted C1-C14 aliphatic, wherein at least one methylene linkage is replaced with an optionally substituted divalent radical of a structure selected from 502
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R9 is optionally substituted C1-C14 aliphatic, wherein one or more methylene linkages are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, an optionally substituted bridged bicyclic or multicyclic C5-C14 cycloalkylenyl, phenyl, -O-, -NH-, - S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-. Additional Formulae [687] In certain embodiments, ionizable lipids of the present disclosure have a structure of Formula (CT), wherein the ionizable lipids of the present disclosure have a structure of any one of the following Formulae:
503
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504
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505
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506
Attorney Docket No. 01318-0014-00PCT OR-043WO (CT-K’), (CT-K’’),
507
Attorney Docket No. 01318-0014-00PCT OR-043WO (CT-L’’’’), (CT-L’’’’’),
508
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509
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510
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511
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(CT-V’’’’), (CT-V’’), or a pharmaceutically acceptable salt thereof, wherein R1, R, X1, Z, X2, X3, X4, RZ, Y1, Y2, R2, R3, R4, R5, R6, R7, R8, and R9 are as described in Formula (CT) or as otherwise described in any embodiments below. [688] In some embodiments, Lipids of the Present Disclosure are selected from any lipid in Table (I-K) below or a pharmaceutically acceptable salt thereof: Table (I-K). Non-Limiting Examples of Ionizable Lipids of the Present Disclosure
512
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513
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514
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515
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516
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517
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518
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where n is selected from integer 0-6. In some embodiments, n is 1. Series “AX” [689] Described below are a number of exemplary ionizable lipids of the present disclosure. Formula (AX) [690] The present disclosure, in some embodiments, provides compounds of Formula (AX’’’’)
(AX’’’’), 519
Attorney Docket No. 01318-0014-00PCT OR-043WO or a pharmaceutically acceptable salt thereof, wherein: A is selected from an optionally substituted bridged carbocyclic bicycle, bridged carbocyclic multicycle, bridged heterocyclic bicycle, or bridged heterocyclic multicycle; n is an integer selected from 0, 1 and 2; m is an integer selected from 1 and 2, such that m plus n is less than or equal to 3; R1 is selected from -OH, -OAc, -NR2, -N(R)RH,
each R is independently -H or C1-C6 aliphatic; each RH is C1-C6 aliphatic-OH; each X1 and XA is independently a bond or optionally substituted C1-C6 aliphatic; each Y1 is independently selected from
, , , , , , ,
, and a bond; wherein the bond marked with an “*” is attached to X1; each X2 and X3 is independently a bond or optionally substituted C1-C12 aliphatic; each Y2 and Y3 is independently selected from
; wherein the bond marked with an “*” is attached to X2 or X3; each X4 and X5 is independently a bond or optionally substituted C1-C6 aliphatic; each Y4 and Y5 is independently selected from a bond, 520
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; wherein the bond marked with an “*” is attached to X4 or X5; each X6 and X7 is independently a bond or optionally substituted C1-C6 aliphatic; R2 is -CH(OR6)(OR7), -CH(SR6)(SR7), -CH(R6)(R7), -CF(R6)(R7), -R10, optionally substituted C5-C18 aliphatic, or optionally substituted C1-C14 aliphatic-R10, wherein one or more methylene linkages of R2 are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; each R3 is independently -CH(OR8)(OR9), -CH(SR8)(SR9), -CH(R8)(R9), -CF(R8)(R9), -R11, optionally substituted C5-C18 aliphatic, or optionally substituted C1-C14 aliphatic-R11, wherein one or more methylene linkages of R3 are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, - NHC(O)-, or -C(O)O-; R6 and R7 are each independently optionally substituted -C1-C14 aliphatic, -R10, or optionally substituted -C1-C14 aliphatic-R10; wherein one or more methylene linkages of R6 and R7 are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R8 and R9 are each independently optionally substituted -C1-C14 aliphatic, -R11, or optionally substituted -C1-C14 aliphatic-R11; wherein one or more methylene linkages of R8 and R9 are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; and each R10 and R11 is independently an optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic, or bridged multicyclic C4-C14 cycloalkyl or optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic, or bridged multicyclic 4-14 membered heterocyclyl, or two R10 or two R11 taken together form an optionally substituted bridged bicyclic or multicyclic C4-C14 cycloalkyl or optionally substituted bridged bicyclic or multicyclic 4-14 membered heterocyclyl; wherein one or more of X1, XA, X2, X3, X4, X5, X6, X7, R2, and R3 is optionally and independently substituted with one or more substituents selected from -F, -Cl, -Br and -I. [691] The present disclosure, in some embodiments, provides compounds of Formula (AX)
521
Attorney Docket No. 01318-0014-00PCT OR-043WO or a pharmaceutically acceptable salt thereof, wherein: A is selected from an optionally substituted bridged carbocyclic or heterocyclic core selected from the group consisting of:
n is and integer selected from 1 or 2; R1 is selected from the group consisting of -OH, -OAc, -NR2, ,
each R is independently -H or C1-C6 aliphatic; X1 and XA are each independently a bond or optionally substituted C1-C6 aliphatic; Y1 is selected from the group consisting of
, , , , , , ,
, and bond; wherein the bond marked with an "*" is attached to X1; each X2 and X3 is independently a bond or optionally substituted C1-C12 aliphatic; each Y2 and Y3 is independently selected from the group consisting of
; wherein the bond marked with an "*" is attached to X2 or X3; each X4 and X5 is independently optionally substituted C1-C6 aliphatic; R2 is -CH(OR6)(OR7), -CH(SR6)(SR7), -CH(R6)(R7), -R10, optionally substituted C5-C18 aliphatic, or optionally substituted C1-C14 aliphatic-R10, wherein one or more methylene linkages of R2 are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; 522
Attorney Docket No. 01318-0014-00PCT OR-043WO each R3 is independently -CH(OR8)(OR9), -CH(SR8)(SR9), -CH(R8)(R9), -R11, optionally substituted C5-C18 aliphatic, or optionally substituted C1-C14 aliphatic-R11, wherein one or more methylene linkages of R3 are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R6 and R7 are each independently optionally substituted -C1-C14 aliphatic, -R10, or optionally substituted -C1-C14 aliphatic-R10; wherein one or more methylene linkages of R6 and R7 are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R8 and R9 are each independently optionally substituted -C1-C14 aliphatic, -R11, or optionally substituted -C1-C14 aliphatic-R11; wherein one or more methylene linkages of R8 and R9 are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; each R10 and R11 is independently an optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic C4-C14 cycloalkyl or optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic 4-14 membered heterocyclyl, or two R10 or two R11 taken together form an optionally substituted bridged bicyclic or multicyclic C4-C14 cycloalkyl or optionally substituted bridged bicyclic or multicyclic 4-14 membered heterocyclyl. [692] The present disclosure, in some embodiments, provides compounds of Formula (AX’’)
(AX’’), or a pharmaceutically acceptable salt thereof, wherein: A is selected from an optionally substituted 4-14 membered bridged carbocyclic bicycle, bridged carbocyclic multicycle, bridged heterocyclic bicycle, or bridged heterocyclic multicycle; each Y4 and Y5 is independently selected from the group consisting of a bond,
; wherein the bond marked with an "*" is attached to X4 or X5; each X6 and X7 is independently a bond or optionally substituted C1-C6 aliphatic; n is and integer selected from 0, 1 or 2; m is an integer selected from 1 or 2, such that m plus n is less than or equal to 3; and the remaining substituents and variables are as defined in Formula (AX). 523
Attorney Docket No. 01318-0014-00PCT OR-043WO Additional Formulae [693] The present disclosure, in some embodiments, provides compounds of any one of the Formulae below:
524
Attorney Docket No. 01318-0014-00PCT OR-043WO (AX-G), (AX-G’),
(AX-I), 525
Attorney Docket No. 01318-0014-00PCT OR-043WO
526
Attorney Docket No. 01318-0014-00PCT OR-043WO
527
Attorney Docket No. 01318-0014-00PCT OR-043WO
528
Attorney Docket No. 01318-0014-00PCT OR-043WO
(AX-P), 529
Attorney Docket No. 01318-0014-00PCT OR-043WO
(AX-R’), (AX-R’’), 530
Attorney Docket No. 01318-0014-00PCT OR-043WO
(AX-U), 531
Attorney Docket No. 01318-0014-00PCT OR-043WO
(AX-W), 532
Attorney Docket No. 01318-0014-00PCT OR-043WO
533
Attorney Docket No.01318-0014-00PCT OR-043WO
- , 534
Attorney Docket No. 01318-0014-00PCT OR-043WO
535
Attorney Docket No. 01318-0014-00PCT OR-043WO
(AX-AF), 536
Attorney Docket No. 01318-0014-00PCT OR-043WO
537
Attorney Docket No. 01318-0014-00PCT OR-043WO
538
Attorney Docket No. 01318-0014-00PCT OR-043WO
539
Attorney Docket No. 01318-0014-00PCT OR-043WO
(AX-AO), (AX-AO’) or a pharmaceutically acceptable salt thereof, wherein R1, R, X1, XA, X2, X3, X4, X5, Y1, Y2, Y3, R3, R6, R7, R8, R9, R10, and R11 are as described in Formula (AX) or as otherwise described in any embodiments below. A [694] As disclosed in Formula (AX), in certain embodiments, A is
, , ,
embodiments, A is a multivalent adamantyl core,
. In certain embodiments, A is
In certain embodiments, A is 540
Attorney Docket No. 01318-0014-00PCT OR-043WO
. certain embodiments, A is a multivalent (1S,5S,7S)-2,4,10-trioxaadamantane core,
. In certain embodiments, A is
. In certain embodiments,
. [695] In certain embodiments, A is
In certain . In certain embodiments,
. certain e
s, A is
, w ere n * is attached to X1. In certain embodiments, A is
. In certain
embodiments,
. certain embodiments, A is , wherein * is attached to X1. In certain embodiments, A is
. In certain embodiments,
. In certain
embodiments, A is , wherein * is attached to X1. In certain embodiments, A is
. In certain embodiments, A is
. In certain embodiments, A is
, attached to X1. In certain embodiments,
541
Attorney Docket No. 01318-0014-00PCT OR-043WO
certain embodiments,
. certain embodiments, A is , wherein * is attached 1
to X . In certain embodiments, A is , wherein * is attached to X1.
, , , , , , , , 542
Attorney Docket No. 01318-0014-00PCT OR-043WO
n and m [697] As disclosed in Formula (AX’’’’) in certain embodiments, n is an integer selected from 0, 1 or 2 and m is an integer selected from 1 or 2, such that m plus n is less than or equal to 3. In certain embodiments, m is 1 and n is 2. In certain embodiments, m is 2 and n 543
Attorney Docket No. 01318-0014-00PCT OR-043WO is 1. In certain embodiments, m is 1 and n is 1. In certain embodiments, m is 1 and n is 0. [698] As disclosed in Formula (AX), in certain embodiments, Y1 is
, , , , , , ,
, or bond; wherein the bond marked with an "*" is attached to X1. In certain embodiments,
In certain embodiments, Y1 is
. In certain embodiments, Y1 is
. In certain embodiments, Y1 is
. In certain embodiments, Y1 is
. In certain embodiments, . In certain embodiments, Y1 is
. In certain embodiments, Y1 is a bond.
[699] As disclosed in Formula (AX), in certain embodiments, X1 is a bond or optionally substituted C1-C6 aliphatic. In certain embodiments, X1 is a bond. In certain embodiments, X1 is a bond or unsubstituted C1-C6 aliphatic. In certain embodiments, X1 is unsubstituted C1-C6 aliphatic. In certain embodiments, X1 is a bond or optionally substituted C1-C6 alkylene. In certain embodiments, X1 is optionally substituted C1-C6 alkylene. In certain embodiments, X1 is a bond or unsubstituted C1-C6 alkylene. In certain embodiments, X1 is unsubstituted C1-C6 alkylene. In certain embodiments, X1 is a bond or unsubstituted C1-C4 alkylene. In certain embodiments, X1 is unsubstituted C1-C4 alkylene. In certain embodiments, X1 is a bond or unsubstituted C1-C2 alkylene. In certain embodiments, X1 is 544
Attorney Docket No. 01318-0014-00PCT OR-043WO unsubstituted C1-C2 alkylene. In certain embodiments, X1 is unsubstituted C2-C6 alkylene. In certain embodiments, X1 is optionally substituted methylene. In certain embodiments, R2 is optionally substituted C2 alkylene. In certain embodiments, X1 is optionally substituted C3 alkylene. In certain embodiments, X1 is optionally substituted C4 alkylene. In certain embodiments, X1 is optionally substituted C5 alkylene. In certain embodiments, X1 is optionally substituted C6 alkylene. In certain embodiments, X1 is –(CH2)-. In certain embodiments, X1 is –(CH2)2-. In certain embodiments, X1 is– (CH2)3-. In certain embodiments, X1 is –(CH2)4-. In certain embodiments, X1 is –(CH2)5-. In certain embodiments, X1 is–(CH2)6-. XA [700] As disclosed in Formula (AX), in certain embodiments XA is a bond or optionally substituted C1-C6 aliphatic. In certain embodiments, XA is a bond. In certain embodiments, XA is a bond or unsubstituted C1-C6 aliphatic. In certain embodiments, XA is unsubstituted C1-C6 aliphatic. In certain embodiments, XA is a bond or optionally substituted C1-C6 alkylene. In certain embodiments, XA is optionally substituted C1-C6 alkylene. In certain embodiments, XA is a bond or unsubstituted C1-C6 alkylene. In certain embodiments, XA is unsubstituted C1-C6 alkylene. In certain embodiments, XA is a bond or unsubstituted C1-C4 alkylene. In certain embodiments, XA is unsubstituted C1-C4 alkylene. In certain embodiments, XA is a bond or unsubstituted C1-C2 alkylene. In certain embodiments, XA is unsubstituted C1-C2 alkylene. In certain embodiments, XA is unsubstituted C2-C6 alkylene. In certain embodiments, XA is optionally substituted methylene. In certain embodiments, R2 is optionally substituted C2 alkylene. In certain embodiments, XA is optionally substituted C3 alkylene. In certain embodiments, XA is optionally substituted C4 alkylene. In certain embodiments, XA is optionally substituted C5 alkylene. In certain embodiments, XA is optionally substituted C6 alkylene. In certain embodiments, XA is –(CH2)-. In certain embodiments, XA is –(CH2)2-. In certain embodiments, XA is –(CH2)3-. In certain embodiments, XA is –(CH2)4-. In certain embodiments, XA is –(CH2)5-. In certain embodiments, XA is –(CH2)6-. [701] As disclosed in Formula (AX), in certain embodiments R1 is selected from -OH, -OAc, - ,
545
Attorney Docket No. 01318-0014-00PCT OR-043WO [702] In certain embodiments,
NR2 or
. In certain embodiments, R1 is -OH. In certain embodiments, R1 is -OAc. In certain embodiments, R1 is -NR2. In certain embodiments, R1 is -NH2. In certain embodiments, R1 is -NMe2. In certain embodiments, R1 is
-NEt2. In certain embodiments,
. certain embodiments, R1 is . In certain embodiments, R1 is
embodiments,
. certain embodiments,
. certain embodiments, R1
In certain embodiments,
. certain embodiments,
. embodiments,
. [703] In certain embodiments, -X1-Y1-XA-R1 is
, , 546
Attorney Docket No. 01318-0014-00PCT OR-043WO
R [704] As disclosed in Formula (AX), in certain embodiments R is -H or C1-C6 aliphatic. In certain embodiments, R is -H. In certain embodiments, R is C1-C6 aliphatic. In certain embodiments, R is or C1- C6 alkyl. In certain embodiments, R is C1-C4 alkyl. In certain embodiments, R is C1-C2 alkyl. In certain embodiments, R is unsubstituted C1-C6 alkyl. In certain embodiments, R is unsubstituted C1-C4 alkyl. In certain embodiments, R is unsubstituted C1-C2 alkyl. In certain embodiments, R is methyl or ethyl. X2 and X3 [705] As disclosed in Formula (AX), in certain embodiments, X2 and X3 are each independently a bond or optionally substituted C1-C12 aliphatic. In certain embodiments, X2 and X3 are the same. In certain embodiments, X2 and X3 are different. [706] In certain embodiments, X2 is a bond. In certain embodiments, X2 is an optionally substituted C1-C12 alkylene. In certain embodiments, X2 is an optionally substituted C1-C12 alkenylene. In certain embodiments, X2 is an optionally substituted C1-C10 aliphatic. In certain embodiments, X2 is an optionally substituted C1-C10 alkylene. In certain embodiments, X2 is an optionally substituted C1- C10 alkenylene. In certain embodiments, X2 is an optionally substituted C1-C8 aliphatic. In certain embodiments, X2 is an optionally substituted C1-C8 alkylene. In certain embodiments, X2 is an optionally substituted C1-C8 alkenylene. In certain embodiments, X2 is an optionally substituted C1-C6 aliphatic. In certain embodiments, X2 is an optionally substituted C1-C6 alkylene. In certain embodiments, X2 is an optionally substituted C1-C6 alkenylene. In certain embodiments, X2 is an optionally substituted C2-C12 aliphatic. In certain embodiments, X2 is an optionally substituted C2-C12 alkylene. In certain embodiments, X2 is an optionally substituted C2-C12 alkenylene. In certain embodiments, X2 is an optionally substituted C4-C12 aliphatic. In certain embodiments, X2 is an optionally substituted C4-C12 alkylene. In certain embodiments, X2 is an optionally substituted C4-C12 alkenylene. In certain embodiments, X2 is an optionally substituted C4-C10 aliphatic. In certain embodiments, X2 is an optionally substituted C4-C10 alkylene. In certain embodiments, X2 is an 547
Attorney Docket No. 01318-0014-00PCT OR-043WO optionally substituted C4-C10 alkenylene. In certain embodiments, X2 is an optionally substituted C6-C8 aliphatic. In certain embodiments, X2 is an optionally substituted C6-C8 alkylene. In certain embodiments, X2 is an optionally substituted C6-C8 alkenylene. In certain embodiments, X2 is a bond or an optionally substituted C1-C4 aliphatic. In certain embodiments, X2 is a bond or an optionally substituted C1-C4 alkylene. In certain embodiments, X2 is a bond or an unsubstituted C1-C4 aliphatic. In certain embodiments, X2 is a bond or an unsubstituted C1-C4 alkylene. In certain embodiments, X2 is a bond or an optionally substituted C1-C2 aliphatic. In certain embodiments, X2 is a bond or an optionally substituted C1-C2 alkylene. In certain embodiments, X2 is a bond or an unsubstituted C1-C2 aliphatic. In certain embodiments, X2 is a bond or an unsubstituted C1-C2 alkylene. In certain embodiments, X2 is – (CH2)-. In certain embodiments, X2 is –(CH2)2-. In certain embodiments, X2 is –(CH2)3-. In certain embodiments, X2 is–(CH2)4-. In certain embodiments, X2 is–(CH2)5-. In certain embodiments, X2 is– (CH2)6-. In certain embodiments, X2 is –(CH2)7-. In certain embodiments, X2 is –(CH2)8-. In certain embodiments, X2 is–(CH2)9-. In certain embodiments, X2 is–(CH2)10-. [707] In certain embodiments, X3 is a bond. In certain embodiments, X3 is an optionally substituted C1-C12 alkylene. In certain embodiments, X3 is an optionally substituted C1-C12 alkenylene. In certain embodiments, X3 is an optionally substituted C1-C10 aliphatic. In certain embodiments, X3 is an optionally substituted C1-C10 alkylene. In certain embodiments, X3 is an optionally substituted C1- C10 alkenylene. In certain embodiments, X3 is an optionally substituted C1-C8 aliphatic. In certain embodiments, X3 is an optionally substituted C1-C8 alkylene. In certain embodiments, X3 is an optionally substituted C1-C8 alkenylene. In certain embodiments, X3 is an optionally substituted C1-C6 aliphatic. In certain embodiments, X3 is an optionally substituted C1-C6 alkylene. In certain embodiments, X3 is an optionally substituted C1-C6 alkenylene. In certain embodiments, X3 is an optionally substituted C2-C12 aliphatic. In certain embodiments, X3 is an optionally substituted C2-C12 alkylene. In certain embodiments, X3 is an optionally substituted C2-C12 alkenylene. In certain embodiments, X3 is an optionally substituted C4-C12 aliphatic. In certain embodiments, X3 is an optionally substituted C4-C12 alkylene. In certain embodiments, X3 is an optionally substituted C4-C12 alkenylene. In certain embodiments, X3 is an optionally substituted C4-C10 aliphatic. In certain embodiments, X3 is an optionally substituted C4-C10 alkylene. In certain embodiments, X3 is an optionally substituted C4-C10 alkenylene. In certain embodiments, X3 is an optionally substituted C6-C8 aliphatic. In certain embodiments, X3 is an optionally substituted C6-C8 alkylene. In certain embodiments, X3 is an optionally substituted C6-C8 alkenylene. In certain embodiments, X3 is a bond or an optionally substituted C1-C4 aliphatic. In certain embodiments, X3 is a bond or an optionally substituted C1-C4 alkylene. In certain embodiments, X3 is a bond or an unsubstituted C1-C4 aliphatic. In certain embodiments, X3 is a bond or an unsubstituted C1-C4 alkylene. In certain embodiments, X3 is a bond or an optionally substituted C1-C2 aliphatic. In certain embodiments, X3 is a bond or an optionally substituted C1-C2 alkylene. In certain embodiments, X3 is a bond or an unsubstituted C1-C2 aliphatic. In 548
Attorney Docket No. 01318-0014-00PCT OR-043WO certain embodiments, X3 is a bond or an unsubstituted C1-C2 alkylene. In certain embodiments, X3 is – (CH2)-. In certain embodiments, X3 is –(CH2)2-. In certain embodiments, X3 is –(CH2)3-. In certain embodiments, X3 is–(CH2)4-. In certain embodiments, X3 is–(CH2)5-. In certain embodiments, X3 is– (CH2)6-. In certain embodiments, X3 is –(CH2)7-. In certain embodiments, X3 is –(CH2)8-. In certain embodiments, X3 is–(CH2)9-. In certain embodiments, X3 is–(CH2)10-. [708] In certain embodiments, X2 and X3 are each independently a bond or an optionally substituted C1-C4 aliphatic. In certain embodiments, X2 and X3 are each independently a bond or an optionally substituted C1-C4 alkylene. In certain embodiments, X2 and X3 are each independently a bond or an unsubstituted C1-C4 aliphatic. In certain embodiments, X2 and X3 are each independently a bond or an unsubstituted C1-C4 alkylene. In certain embodiments, X2 and X3 are each independently a bond or an optionally substituted C1-C2 aliphatic. In certain embodiments, X2 and X3 are each independently a bond or an optionally substituted C1-C2 alkylene. In certain embodiments, X2 and X3 are each independently a bond or an unsubstituted C1-C2 aliphatic. In certain embodiments, X2 and X3 are each independently a bond or an unsubstituted C1-C2 alkylene. [709] In certain embodiments, X2 and X3 are both a bond or an optionally substituted C1-C4 aliphatic. In certain embodiments, X2 and X3 are both a bond or an optionally substituted C1-C4 alkylene. In certain embodiments, X2 and X3 are both a bond or an unsubstituted C1-C4 aliphatic. In certain embodiments, X2 and X3 are both a bond or an unsubstituted C1-C4 alkylene. In certain embodiments, X2 and X3 are both a bond or an optionally substituted C1-C2 aliphatic. In certain embodiments, X2 and X3 are both a bond or an optionally substituted C1-C2 alkylene. In certain embodiments, X2 and X3 are both a bond or an unsubstituted C1-C2 aliphatic. In certain embodiments, X2 and X3 are both a bond or an unsubstituted C1-C2 alkylene. [710] In certain embodiments, X2 and X3 are both bonds. In certain embodiments, X2 and X3 are both –(CH2)-. In certain embodiments, X2 and X3 are both –(CH2)2-. In certain embodiments, X2 and X3 are both –(CH2)3-. Y2 and Y3 [711] As disclosed in Formula (AX), in certain embodiments, Y2 and Y3 are each independently
, wherein the bond marked with an "*" is attached to X2 for Y2 or X3 for Y3 . In certain embodiments, Y2 and Y3 are the same. In certain embodiments, Y2 and Y3 are different. [712] In certain embodiments, Y2 and Y3 are each independently
, , 549
Attorney Docket No. 01318-0014-00PCT OR-043WO
In certain embodiments, Y2 and Y3 are each independently
. In certain embodiments, Y2 and Y3 are each independently
. In certain embodiments, Y2 and Y3 are each independently
. In certain embodiments, Y2 and Y3 are each independently . In certain embodiments, Y2 is
. In certain embodiments, Y2 is
. ce a embodiments, Y2 is
. In certain embodiments, Y2 is
. , . , . In certain embodiments, Y2 is
. In certain embodiments, Y2 is
. In certain embodiments, Y3 is
. In certain embodiments, Y
s . In certain embodiments, Y3 . In certain embodiments, Y3 is
. In certain embodiments, Y3 is
. certain embodiments, Y3 is . In certain embodiments, Y3 is
. In certain embodiments, Y3 is
. In certain embodiments, Y3 and Y2 are both
. In certain embodiments, Y3 and Y2 are both
. In certain embodiments, Y3 and Y2 are both
. , . X4 and X5 550
Attorney Docket No. 01318-0014-00PCT OR-043WO [713] As disclosed in Formula (AX’’’’), in certain embodiments, X4 and X5 are each independently a bond or optionally substituted C1-C6 aliphatic. As disclosed in Formula (AX), in certain embodiments, X4 and X5 are each independently optionally substituted C1-C6 aliphatic. In certain embodiments, X4 and X5 are the same. In certain embodiments, X4 and X5 are different. [714] In certain embodiments, X4 is a bond. In certain embodiments, X4 is an optionally substituted C1-C6 aliphatic. In certain embodiments, X4 is an optionally substituted C1-C6 alkylene. In certain embodiments, X4 is an optionally substituted C1-C6 alkenylene. In certain embodiments, X4 is an optionally substituted C2-C5 aliphatic. In certain embodiments, X4 is an optionally substituted C2-C5 alkylene. In certain embodiments, X4 is an optionally substituted C2-C5 alkenylene. In certain embodiments, X4 is an optionally substituted C3-C4 aliphatic. In certain embodiments, X4 is an optionally substituted C3-C4 alkylene. In certain embodiments, X4 is an optionally substituted C3-C4 alkenylene. In certain embodiments, X4 is –(CH2)-. In certain embodiments, X4 is –(CH2)2-. In certain embodiments, X4 is –(CH2)3-. In certain embodiments, X4 is –(CH2)4-. In certain embodiments, X4 is –(CH2)5-. In certain embodiments, X4 is –(CH2)6-. [715] In certain embodiments, X5 is a bond. In certain embodiments, X5 is an optionally substituted C1-C6 aliphatic. In certain embodiments, X5 is an optionally substituted C1-C6 alkylene. In certain embodiments, X5 is an optionally substituted C1-C6 alkenylene. In certain embodiments, X5 is an optionally substituted C2-C5 aliphatic. In certain embodiments, X5 is an optionally substituted C2-C5 alkylene. In certain embodiments, X5 is an optionally substituted C2-C5 alkenylene. In certain embodiments, X5 is an optionally substituted C3-C4 aliphatic. In certain embodiments, X5 is an optionally substituted C3-C4 alkylene. In certain embodiments, X5 is an optionally substituted C3-C4 alkenylene. In certain embodiments, X5 is –(CH2)-. In certain embodiments, X5 is –(CH2)2-. In certain embodiments, X5 is –(CH2)3-. In certain embodiments, X5 is –(CH2)4-. In certain embodiments, X5 is –(CH2)5-. In certain embodiments, X5 is –(CH2)6-. [716] In certain embodiments, X4 and X5 are each independently an optionally substituted C1-C4 aliphatic. In certain embodiments, X4 and X5 are each independently an optionally substituted C1-C4 alkylene. In certain embodiments, X4 and X5 are each independently an unsubstituted C1-C4 aliphatic. In certain embodiments, X4 and X5 are each independently an unsubstituted C1-C4 alkylene. In certain embodiments, X4 and X5 are each independently an 551
Attorney Docket No. 01318-0014-00PCT OR-043WO optionally substituted C1-C2 aliphatic. In certain embodiments, X4 and X5 are each independently an optionally substituted C1-C2 alkylene. In certain embodiments, X4 and X5 are each independently an unsubstituted C1-C2 aliphatic. In certain embodiments, X4 and X5 are each independently an unsubstituted C1-C2 alkylene. [717] In certain embodiments, X4 and X5 are both an optionally substituted C1-C4 aliphatic. In certain embodiments, X4 and X5 are both an optionally substituted C1-C4 alkylene. In certain embodiments, X4 and X5 are both an unsubstituted C1-C4 aliphatic. In certain embodiments, X4 and X5 are both an unsubstituted C1-C4 alkylene. In certain embodiments, X4 and X5 are both an optionally substituted C1-C2 aliphatic. In certain embodiments, X4 and X5 are both an optionally substituted C1-C2 alkylene. In certain embodiments, X4 and X5 are both an unsubstituted C1-C2 aliphatic. In certain embodiments, X4 and X5 are both an unsubstituted C1-C2 alkylene. [718] In certain embodiments, X4 and X5 are both a bond. In certain embodiments, X4 and X5 are both –(CH2)-. In certain embodiments, X4 and X5 are both –(CH2)2-. In certain embodiments, X4 and X5 are both –(CH2)3-. In certain embodiments, X4 is –(CH2)2- and X5 is –(CH2)-. In certain embodiments, X4 and X5 are both –(CH2)4-. In certain embodiments, X4 and X5 are both –(CH2)5-. In certain embodiments, X4 and X5 are both –(CH2)6-. [719] In certain embodiments, X4 and X5 are each independently substituted with one or more substituents selected from -F, -Cl, -Br and -I. In certain embodiments, X4 and/or X5 are substituted with one or more -F. In certain embodiments, X4 and X5 are substituted with one or more -F on a carbon atom at a position selected from α-position and β-position from Y4 or Y5, respectively. In certain embodiments, X4 and/or X5 are substituted with one or more -F. In certain embodiments, X4 and X5 are substituted with one or more -F on a carbon atom at a position selected from α-position and β-position from Y2 or Y3, respectively. [720] In certain embodiments, wherein the compound comprises two X5 (in other words, when n=2); each X5 is independently selected from any of the embodiments above, and need not be the same. In certain embodiments, wherein the compound comprises two X5, they are each the same. Y4 and Y5 [721] As disclosed in Formula (AX’’’’), in certain embodiments, Y4 and Y5 are each 552
Attorney Docket No. 01318-0014-00PCT OR-043WO independently a bond,
,
, , , wherein the bond marked with an
is attached to X4 for Y4 or X5 for Y5. In certain embodiments, Y4 and Y5 are the same. In certain embodiments, Y4 and Y5 are different. [722] In certain embodiments, Y4 and Y5 are each independently
, ,
, , , . In certain embodiments, Y4 and Y5 are each independently
, , . In certain embodiments, Y2 and Y5 are each independently
. In certain embodiments, Y4 and Y5 are each independently
. In certain embodiments, Y4 and Y5 are each independently
. In certain embodiments, Y4 is
. In certain embodiments,
. certain embodiments, Y4 is
. In certain embodiments,
. In certain embodiments, Y4 is
. In certain embodiments,
. In certain embodiments, Y4 is
. In certain embodiments,
. In certain embodiments,
. In certain embodiments,
. In certain embodiments, Y5 is
. In certain 553
Attorney Docket No. 01318-0014-00PCT OR-043WO embodiments, Y5 is
. In certain embodiments,
. In certain embodiments, Y5 is
. In certain embodiments, Y5 is
. In certain embodiments, Y5 is
. In certain embodiments, Y5 and Y4 are both
. In certain embodiments, Y5 and Y4 are both
. In certain embodiments, Y5 and Y4 are both
. In certain embodiments, Y5 and Y4 are both
. [723] In certain embodiments, wherein the compound comprises two Y5 (in other words, when n=2); each Y5 is independently selected from any of the embodiments above, and need not be the same. In certain embodiments, wherein the compound comprises two Y5, they are each the same. R2 [724] As disclosed in Formula (AX), in certain embodiments, R2 is -CH(OR6)(OR7), - CH(SR6)(SR7), -CH(R6)(R7), -R10, optionally substituted C5-C18 aliphatic, or optionally substituted C1- C14 aliphatic-R10, wherein one or more methylene linkages of R2 are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, - OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-. In certain embodiments, R2 is -CH(OR6)(OR7). In certain embodiments, R2 is -CH(R6)(R7). In certain embodiments, R2 is -CH(SR6)(SR7). In certain embodiments, R2 is -R10. In certain embodiments, R2 is optionally substituted C5-C18 aliphatic. In certain embodiments, R2 is optionally substituted C1-C14 aliphatic-R10. , ,
, , 554
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. R3 [726] As disclosed in Formula (AX), in certain embodiments, R3 is -CH(OR8)(OR9), - CH(SR8)(SR9), -CH(R8)(R9), -R11, optionally substituted C5-C18 aliphatic, or optionally substituted C1- C14 aliphatic-R11, wherein one or more methylene linkages of R3 are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, - OC(O)O-, -OC(O)-, -NHC(O)- or -C(O)O-. In certain embodiments, R3 is -CH(OR8)(OR9). In certain embodiments, R3 is -CH(R8)(R9). In certain embodiments, R3 is -CH(SR8)(SR9). In certain embodiments, R3 is -R11. In certain embodiments, R3 is optionally substituted C5-C18 aliphatic. In certain embodiments, R3 is optionally substituted C1-C14 aliphatic-R11. , , ,
, , 555
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. [728] In certain embodiments, R2 and R3 are the same. In certain embodiments, R2 and R3 are different. R6 and R7 [729] As disclosed in Formula (AX), in certain embodiments, R6 and R7 are each independently optionally substituted -C1-C14 aliphatic, -R10, or optionally substituted -C1-C14 aliphatic-R10; wherein one or more methylene linkages of R6 and R7 are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)- , -NHC(O)- or -C(O)O-. [730] In certain embodiments, R6 and R7 are the same. In certain embodiments, R6 and R7 are different. [731] In certain embodiments, R6 is optionally substituted C1-C14 aliphatic. In certain embodiments, R6 is optionally substituted C1-C14 alkyl. In certain embodiments, R6 is optionally substituted C1-C14 branched alkyl. In certain embodiments, R6 is optionally substituted C1-C14 straight chain alkyl. In certain embodiments, R6 is optionally substituted C1-C14 alkenyl. In certain embodiments, R6 is optionally substituted C1-C14 branched alkenyl. In certain embodiments, R6 is optionally substituted C1-C14 straight chain alkenyl. In certain embodiments, R6 is optionally substituted C4-C10 alkyl. In certain embodiments, R6 is optionally substituted straight chain C4-C10 alkyl. In certain embodiments, R6 is unsubstituted C6-C10 alkyl. In certain embodiments, R6 is unsubstituted straight chain C4-C10 alkyl. In certain embodiments, R6 is optionally substituted C6-C10 alkyl. In certain embodiments, R6 is optionally substituted straight chain C6-C10 alkyl. In certain embodiments, R6 is unsubstituted C4-C10 alkyl. In certain embodiments, R6 is unsubstituted straight chain C6-C10 alkyl. In certain embodiments, R6 is optionally substituted–(CH2)5CH3. In certain embodiments, R6 is optionally substituted –(CH2)6CH3. In certain embodiments, R6 is optionally substituted –(CH2)7CH3. In certain embodiments, R6 is optionally substituted –(CH2)8CH3. In certain embodiments, R6 is optionally substituted–(CH2)9CH3. [732] In certain embodiments, R7 is optionally substituted C1-C14 aliphatic. In certain 556
Attorney Docket No. 01318-0014-00PCT OR-043WO embodiments, R7 is optionally substituted C1-C14 alkyl. In certain embodiments, R7 is optionally substituted C1-C14 branched alkyl. In certain embodiments, R7 is optionally substituted C1-C14 straight chain alkyl. In certain embodiments, R7 is optionally substituted C1-C14 alkenyl. In certain embodiments, R7 is optionally substituted C1-C14 branched alkenyl. In certain embodiments, R7 is optionally substituted C1-C14 straight chain alkenyl. In certain embodiments, R7 is optionally substituted C4-C10 alkyl. In certain embodiments, R7 is optionally substituted straight chain C4-C10 alkyl. In certain embodiments, R7 is unsubstituted C6-C10 alkyl. In certain embodiments, R7 is unsubstituted straight chain C4-C10 alkyl. In certain embodiments, R7 is optionally substituted C6-C10 alkyl. In certain embodiments, R7 is optionally substituted straight chain C6-C10 alkyl. In certain embodiments, R7 is unsubstituted C4-C10 alkyl. In certain embodiments, R7 is unsubstituted straight chain C6-C10 alkyl. In certain embodiments, R7 is optionally substituted–(CH2)5CH3. In certain embodiments, R7 is optionally substituted –(CH2)6CH3. In certain embodiments, R7 is optionally substituted –(CH2)7CH3. In certain embodiments, R7 is optionally substituted –(CH2)8CH3. In certain embodiments, R7 is optionally substituted–(CH2)9CH3. [733] In certain embodiments, each R6 and R7 are each independently selected from ,
R8 and R9 [734] As disclosed in Formula (AX), in certain embodiments, R8 and R9 are each independently optionally substituted -C1-C14 aliphatic, -R11, or optionally substituted -C1-C14 aliphatic-R11; wherein one or more methylene linkages of R8 and R9 are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)- , -NHC(O)- or -C(O)O-. [735] In certain embodiments, R8 and R9 are the same. In certain embodiments, R8 and R9 are different. [736] In certain embodiments, R8 is optionally substituted C1-C14 aliphatic. In certain embodiments, R8 is optionally substituted C1-C14 alkyl. In certain embodiments, R8 is optionally substituted C1-C14 branched alkyl. In certain embodiments, R8 is optionally substituted C1-C14 straight chain alkyl. In certain embodiments, R8 is optionally substituted C1-C14 alkenyl. In certain embodiments, R8 is optionally substituted C1-C14 branched alkenyl. In certain embodiments, R8 is optionally substituted C1-C14 straight chain alkenyl. In certain embodiments, R8 is optionally substituted C4-C10 alkyl. In certain embodiments, R8 is optionally substituted straight chain C4-C10 alkyl. In certain 557
Attorney Docket No. 01318-0014-00PCT OR-043WO embodiments, R8 is unsubstituted C6-C10 alkyl. In certain embodiments, R8 is unsubstituted straight chain C4-C10 alkyl. In certain embodiments, R8 is optionally substituted C6-C10 alkyl. In certain embodiments, R8 is optionally substituted straight chain C6-C10 alkyl. In certain embodiments, R8 is unsubstituted C4-C10 alkyl. In certain embodiments, R8 is unsubstituted straight chain C6-C10 alkyl. In certain embodiments, R8 is optionally substituted–(CH2)5CH3. In certain embodiments, R8 is optionally substituted –(CH2)6CH3. In certain embodiments, R8 is optionally substituted –(CH2)7CH3. In certain embodiments, R8 is optionally substituted –(CH2)8CH3. In certain embodiments, R8 is optionally substituted–(CH2)9CH3. [737] In certain embodiments, R9 is optionally substituted C1-C14 aliphatic. In certain embodiments, R9 is optionally substituted C1-C14 alkyl. In certain embodiments, R9 is optionally substituted C1-C14 branched alkyl. In certain embodiments, R9 is optionally substituted C1-C14 straight chain alkyl. In certain embodiments, R9 is optionally substituted C1-C14 alkenyl. In certain embodiments, R9 is optionally substituted C1-C14 branched alkenyl. In certain embodiments, R9 is optionally substituted C1-C14 straight chain alkenyl. In certain embodiments, R9 is optionally substituted C4-C10 alkyl. In certain embodiments, R9 is optionally substituted straight chain C4-C10 alkyl. In certain embodiments, R9 is unsubstituted C6-C10 alkyl. In certain embodiments, R9 is unsubstituted straight chain C4-C10 alkyl. In certain embodiments, R9 is optionally substituted C6-C10 alkyl. In certain embodiments, R9 is optionally substituted straight chain C6-C10 alkyl. In certain embodiments, R9 is unsubstituted C4-C10 alkyl. In certain embodiments, R9 is unsubstituted straight chain C6-C10 alkyl. In certain embodiments, R9 is optionally substituted–(CH2)5CH3. In certain embodiments, R9 is optionally substituted –(CH2)6CH3. In certain embodiments, R9 is optionally substituted –(CH2)7CH3. In certain embodiments, R9 is optionally substituted –(CH2)8CH3. In certain embodiments, R9 is optionally substituted–(CH2)9CH3. [738] In certain embodiments, each R8 and R9 are each independently selected from ,
R10 and R11 [739] As disclosed in Formula (AX), in certain embodiments, each R10 and R11 are independently an optionally substituted cyclic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic C4-C14 cycloalkyl or optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic 4-14 membered heterocyclyl, or two R10 or two R11 taken together form an optionally substituted bridged bicyclic or multicyclic C4-C14 cycloalkyl or optionally substituted bridged bicyclic or 558
Attorney Docket No. 01318-0014-00PCT OR-043WO multicyclic 4-14 membered heterocyclyl. [740] In certain embodiments, each R10 and R11 are independently an optionally substituted monocyclic C4-C14 cycloalkyl. In certain embodiments, each R10 and R11 are independently an optionally substituted monocyclic C6-C14 cycloalkyl. In certain embodiments, each R10 and R11 are independently an optionally substituted monocyclic C6-C8 cycloalkyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bicyclic C4-C14 cycloalkyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bicyclic C6-C14 cycloalkyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bicyclic C8-C14 cycloalkyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bicyclic C6-C10 cycloalkyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bridged bicyclic C4-C14 cycloalkyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bridged bicyclic C6-C14 cycloalkyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bridged bicyclic C8-C14 cycloalkyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bridged bicyclic C6- C10 cycloalkyl. In certain embodiments, each R10 and R11 are independently an optionally substituted multicyclic C4-C14 cycloalkyl. In certain embodiments, each R10 and R11 are independently an optionally substituted multicyclic C6-C14 cycloalkyl. In certain embodiments, each R10 and R11 are independently an optionally substituted multicyclic C8-C14 cycloalkyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bridged multicyclic C4-C14 cycloalkyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bridged multicyclic C6-C14 cycloalkyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bridged multicyclic C8-C14 cycloalkyl. In certain embodiments, two R10 or two R11 taken together form an optionally substituted bridged bicyclic or multicyclic C4-C14 cycloalkyl. In certain embodiments, two R10 or two R11 taken together form an optionally substituted bridged bicyclic or multicyclic C6-C14 cycloalkyl. In certain embodiments, two R10 or two R11 taken together form an optionally substituted bridged bicyclic or multicyclic C8-C14 cycloalkyl. [741] In certain embodiments, each R10 and R11 are independently an optionally substituted monocyclic 4-14 membered heterocyclyl. In certain embodiments, each R10 and R11 are independently an optionally substituted monocyclic 6-14 membered heterocyclyl. In certain embodiments, each R10 and R11 are independently an optionally substituted monocyclic C6-C8 heterocyclyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bicyclic 4-14 membered heterocyclyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bicyclic 6-14 membered heterocyclyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bicyclic C8-C14 heterocyclyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bicyclic 6-12 membered heterocyclyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bridged bicyclic 4-14 membered 559
Attorney Docket No. 01318-0014-00PCT OR-043WO heterocyclyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bridged bicyclic 6-14 membered heterocyclyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bridged bicyclic C8-C14 heterocyclyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bridged bicyclic 6-12 membered heterocyclyl. In certain embodiments, each R10 and R11 are independently an optionally substituted multicyclic 4-14 membered heterocyclyl. In certain embodiments, each R10 and R11 are independently an optionally substituted multicyclic 6-14 membered heterocyclyl. In certain embodiments, each R10 and R11 are independently an optionally substituted multicyclic C8-C14 heterocyclyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bridged multicyclic 4-14 membered heterocyclyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bridged multicyclic 6-14 membered heterocyclyl. In certain embodiments, each R10 and R11 are independently an optionally substituted bridged multicyclic C8-C14 heterocyclyl. In certain embodiments, two R10 or two R11 taken together form an optionally substituted bridged bicyclic or multicyclic 4-14 membered heterocyclyl. In certain embodiments, two R10 or two R11 taken together form an optionally substituted bridged bicyclic or multicyclic 6-14 membered heterocyclyl. In certain embodiments, two R10 or two R11 taken together form an optionally substituted bridged bicyclic or multicyclic 8-14 membered heterocyclyl. [742] In certain embodiments, each R10 and R11 is independently an optionally substituted monovalent cyclic group selected from
embodiments, each R10 and R11 is independently a structure selected from
, ,
560
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. 561
Attorney Docket No. 01318-0014-00PCT OR-043WO [743] In some embodiments, ionizable lipids of the present disclosure are selected from any lipid in Table (I-L) below or a pharmaceutically acceptable salt, solvate, stereoisomer, or enantiomer thereof: Table (I-L). Non-Limiting Examples of Ionizable lipids of the present disclosure
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564
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565
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566
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567
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568
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569
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570
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571
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572
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573
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574
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575
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576
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577
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578
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579
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580
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581
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582
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583
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584
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585
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586
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587
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588
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589
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590
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591
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592
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Series “TL” [744] Described below are a number of exemplary ionizable lipids of the present disclosure. Formula (TL) [745] The present disclosure, in some embodiments, provides compounds of Formula (TL’’)
(TL’’), or a pharmaceutically acceptable salt thereof, wherein: A1 is selected from CH and N; A2 is selected from CH and N; L1 is optionally substituted C1-C8 aliphatic, wherein one or more methylene linkages of L1 are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, - NHC(O)-, or -C(O)O-; Z1 is selected from the group consisting of:
, , , , 593
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, ,
, wherein the bond marked with an "*" is attached to L1; Z2 is selected from the group consisting of -O-, -NR-, and -S-; Z3 is selected from the group consisting of a bond, -O-, -NR-, -S-, -OC(O)-, -C(O)O-, -NRC(O)-, - C(O)NR-,-NRC(O)O-, or -OC(O)NR-; each Z4 is independently selected from =CR- and =N-; L2 is optionally substituted C1-C8 aliphatic, wherein one or more methylene linkages of L1 are each optionally and independently replaced with -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, - NHC(O)-, or -C(O)O-; R1 is selected from the group consisting of -OH, -OAc, -NR2,
594
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each R is independently -H or C1-C6 aliphatic; XZ is a bond or optionally substituted C1-C6 aliphatic; RZ is NR2 or OH; X1 and XA are each independently a bond or optionally substituted C1-C6 aliphatic; Y1 is selected from the group consisting of
, and bond; wherein the bond marked with an "*" is attached to X1; each of X2, X3, and X6 is independently a bond or optionally substituted C1-C12 aliphatic; each of Y2, Y3, and Y4 is independently selected from the group consisting of
; wherein the bond marked with an "*" is attached to X2, X3, of X6, as appropriate; each of X4, X5, and X7 is independently optionally substituted C1-C6 aliphatic; R2 is -CH(OR6)(OR7), -CH(SR6)(SR7), -CH(R6)(R7), -R10, optionally substituted C5-C18 aliphatic, or optionally substituted C1-C14 aliphatic-R10, wherein one or more methylene linkages of R2 are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R3 is -CH(OR8)(OR9), -CH(SR8)(SR9), -CH(R8)(R9), -R11, optionally substituted C5-C18 aliphatic, or optionally substituted C1-C14 aliphatic-R11, wherein one or more methylene linkages of R3 are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; 595
Attorney Docket No. 01318-0014-00PCT OR-043WO R12 is -CH(OR13)(OR14), -CH(SR13)(SR14), -CH(R13)(R14), -R15, optionally substituted C5-C18 aliphatic, or optionally substituted C1-C14 aliphatic-R15, wherein one or more methylene linkages of R12 are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, -O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R6 and R7 are each independently optionally substituted -C1-C14 aliphatic, -R10, or optionally substituted -C1-C14 aliphatic-R10; wherein one or more methylene linkages of R6 and R7 are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R8 and R9 are each independently optionally substituted -C1-C14 aliphatic, -R11, or optionally substituted -C1-C14 aliphatic-R11; wherein one or more methylene linkages of R8 and R9 are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; R13 and R14 are each independently optionally substituted -C1-C14 aliphatic, -R15, or optionally substituted -C1-C14 aliphatic-R15; wherein one or more methylene linkages of R13 and R14 are each optionally and independently replaced with an optionally substituted C3-C8 cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; each of R10, R11, and R15 is independently an optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic C4-C14 cycloalkyl or optionally substituted cylic, bicyclic, bridged bicyclic, multicyclic or bridged multicyclic 4-14 membered heterocyclyl, or two R10, R11 or R15 are taken together form an optionally substituted bridged bicyclic or multicyclic C4-C14 cycloalkyl or optionally substituted bridged bicyclic or multicyclic 4-14 membered heterocyclyl. Additional Formulae [746] The present disclosure, in some embodiments, provides compounds of any one of the Formulae below:
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599
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, 601
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Wherein
, and R15 are as described in Formula (TL’’). [747] In some embodiments, ionizable lipids of the present disclosure are selected from any lipid in Table (I-K) below or a pharmaceutically acceptable salt, solvate, stereoisomer, or enantiomer thereof: Table (I-K). Non-Limiting Examples of Ionizable lipids of the present disclosure
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[748] In some embodiments, ionizable lipids of the present disclosure are selected from any lipid in Tables (I-A), (I-B), (I-C), (I-D), (I-E), (I-F), (I-G), (I-H), (I-I), (I-J), (I-K), (I-L), or (I-K) above, an enantiomer thereof, or any mixture of enantiomers thereof, or a pharmaceutically acceptable salt of any of the aforementioned. [749] In some embodiments, an ionizable lipid is described in US patent publication number 20190321489. In some embodiments, an ionizable lipid is described in international patent publication WO 2010/053572, incorporated herein by reference. In some embodiments, an ionizable lipid is C12-200, described at paragraph [00225] of WO 2010/053572. [750] Several ionizable lipids have been described in the literature, many of which are commercially available. In certain embodiments, such ionizable lipids are included in the transfer vehicles described herein. Other suitable cationic lipids include, for example, ionizable cationic lipids as described in U.S. provisional patent application 61/617,468, filed Mar. 29, 2012 (incorporated herein by reference), such as, e.g., (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)- octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), (15Z,18Z)-N,N-dimethyl- 6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (HGT5001), and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1- amine (HGT5002), C12-200 (described in WO 2010/053572), 2-(2,2-di((9Z,12Z)-octadeca- 9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLinKC2-DMA)) (See, WO 2010/042877; Semple et al., Nature Biotech.28:172-176 (2010)), 2-(2,2-di((9Z,2Z)-octadeca- 9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2-DMA), (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4- yl)propanoate (ICE), (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa- 15,18-dien-1-amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1- yl)tetracosa-4,15,18-trien-1-amine (HGT5001), (15Z,18 Z)-N,N-dimethyl-6-((9Z,12Z)- octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5002), 5- carboxyspermylglycine-dioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(spermine- 614
Attorney Docket No. 01318-0014-00PCT OR-043WO carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA) (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989); U.S. Pat. No. 5,171,678; 5,334,761), 1,2-Dioleoyl-3- Dimethylammonium-Propane (DODAP), 1,2-Dioleoyl-3-Trimethylammonium-Propane or (DOTAP). Contemplated ionizable lipids also include 1,2-distcaryloxy-N,N-dimethyl-3- aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2- dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethyl- 3-aminopropane (DLenDMA), N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N- distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3- beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5′-(cholest- 5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,1-2′-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N′- dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N- dimethylpropylamine (DLinDAP), 1,2-N,N′-Dilinoleylcarbamyl-3-dimethylamninopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2- dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4- dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA) or GL67, or mixtures thereof. [751] Also contemplated are cationic lipids such as dialkylamino-based, imidazole-based, and guanidinium-based lipids. For example, also contemplated is the use of the ionizable lipid (3S,10R, 13R, 17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4- yl)propanoate (ICE), as disclosed in International Application No. PCT/US2010/058457, incorporated herein by reference. [752] Also contemplated are ionizable lipids such as the dialkylamino-based, imidazole- based, and guanidinium-based lipids. [753] In some embodiments, an ionizable lipid is described by US patent publication number 20190314284. [754] The ionizable lipids include those disclosed in international patent application PCT/US2019/025246, and US patent publications 2017/0190661 and 2017/0114010, incorporated herein by reference in their entirety. [755] In some embodiments, an ionizable lipid is as described in international patent application PCT/US2019/015913. [756] Preparation methods for the above compounds and compositions are described 615
Attorney Docket No. 01318-0014-00PCT OR-043WO herein below and/or known in the art. [757] It will be appreciated by those skilled in the art that in the process described herein the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include, e.g., hydroxyl, amino, mercapto, and carboxylic acid. Suitable protecting groups for hydroxyl include, e.g., trialkylsilyl or diarylalkylsilyl (for example, t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino, and guanidino include, e.g., t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include, e.g., -C(O)-R’’ (where R’’ is alkyl, aryl, or arylalkyl), p- methoxybenzyl, trityl, and the like. Suitable protecting groups for carboxylic acid include, e.g., alkyl, aryl, or arylalkyl esters. Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in, e.g., Green, T. W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one of skill in the art would appreciate, the protecting group may also be a polymer resin such as a Wang resin, Rink resin, or a 2-chlorotrityl-chloride resin. [758] It will also be appreciated by those skilled in the art, although such protected derivatives of compounds described herein may not possess pharmacological activity as such, they may be administered to a mammal and thereafter metabolized in the body to form compounds described herein which are pharmacologically active. Such derivatives may therefore be described as prodrugs. All prodrugs of compounds described herein are included within the scope of the present disclosure. [759] Furthermore, all compounds described herein which exist in free base or acid form can be converted to their pharmaceutically acceptable salts by treatment with the appropriate inorganic or organic base or acid by methods known to one skilled in the art. Salts of the compounds described herein can also be converted to their free base or acid form by standard techniques. [760] Preparation methods for the above compounds and compositions are described herein below and/or known in the art. It is understood that one skilled in the art may be able to make these compounds by similar methods or by combining other methods known to one skilled in the art. a. AMINE LIPIDS [761] In certain embodiments, transfer vehicle compositions for the delivery of circular 616
Attorney Docket No. 01318-0014-00PCT OR-043WO RNA comprise an amine lipid. In certain embodiments, an ionizable lipid is an amine lipid. [762] In some embodiments, an amine lipid is described in international patent application PCT/US2018/053569. In some embodiments, the amine lipid is Lipid E of WO 2022/261490 and WO 2023/056033, which is (9Z, 12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate. Lipid E may be synthesized according to WO2015/095340 (e.g., pp.84-86). In certain embodiments, the amine lipid is an equivalent to Lipid E. In certain embodiments, an amine lipid is an analog of Lipid E. [763] Amine lipids and other biodegradable lipids suitable for use in the transfer vehicles, e.g., lipid nanoparticles, described herein are biodegradable in vivo. The amine lipids described herein have low toxicity (e.g., are tolerated in animal models without adverse effect in amounts of greater than or equal to 10 mg/kg). In certain embodiments, transfer vehicles composing an amine lipid include those where at least 75% of the amine lipid is cleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days. [764] Biodegradable lipids include, for example, the biodegradable lipids of WO2017/173054, WO2015/095340, and WO2014/136086. [765] Lipid clearance may be measured by methods known by persons of skill in the art. See, for example, Maier, M.A., et al. Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics. Mol. Ther. 2013, 21(8), 1570-78. [766] Transfer vehicle compositions comprising an amine lipid can lead to an increased clearance rate. In some embodiments, the clearance rate is a lipid clearance rate, for example the rate at which a lipid is cleared from the blood, serum, or plasma. In some embodiments, the clearance rate is an RNA clearance rate, for example the rate at which a circRNA is cleared from the blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which transfer vehicles are cleared from the blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which transfer vehicles are cleared from a tissue, such as liver tissue or spleen tissue. In certain embodiments, a high rate of clearance leads to a safety profile with no substantial adverse effects. The amine lipids and biodegradable lipids may reduce transfer vehicle accumulation in circulation and in tissues. In some embodiments, a reduction in transfer vehicle accumulation in circulation and in tissues leads to a safety profile with no substantial adverse effects. [767] Lipids may be ionizable depending upon the pH of the medium they are in. For example, in a slightly acidic medium, the lipid, such as an amine lipid, may be protonated and 617
Attorney Docket No. 01318-0014-00PCT OR-043WO thus bear a positive charge. Conversely, in a slightly basic medium, such as, for example, blood, where pH is approximately 7.35, the lipid, such as an amine lipid, may not be protonated and thus bear no charge. [768] The ability of a lipid to bear a charge is related to its intrinsic pKa. In some embodiments, the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.1 to about 7.4. In some embodiments, the bioavailable lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.1 to about 7.4. For example, the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.8 to about 6.5. Lipids with a pKa ranging from about 5.1 to about 7.4 are effective for delivery of cargo in vivo, e.g., to the liver. Further, it has been found that lipids with a pKa ranging from about 5.3 to about 6.4 are effective for delivery in vivo, e.g.,into tumors. See, e.g., WO2014/136086. b. LIPIDS CONTAINING A DISULFIDE BOND [769] In some embodiments, the ionizable lipid is described in US patent 9,708,628. [770] In some embodiments, the lipid may have an —S—S— (disulfide) bond. The production method for such a compound includes, for example, a method including producing R3a—(Ya—R2a)na—Xa—R1a—SH, and R3b—(Yb—R2b)nb—Xb—R1b—SH, and subjecting them to oxidation (coupling) to give a compound containing —S—S—, a method including sequentially bonding necessary parts to a compound containing an —S—S— bond to finally obtain the compound and the like. Preferred is the latter method. [771] An example of the latter method is described on pages 470-472 of WO 2022/261490, which is incorporated by reference herein in its entirety. [772] Exemplary lipids containing a disulfide bond are described in WO 2022/261490, including the lipid represented by structure (XXII) described therein at pages 459-469 and structures 1-15 of Table 15b, which are incorporated by reference herein in its entirety. WO 2023/056033 is also incorporated by reference herein in its entirety. c. FURTHER EXEMPLARY LIPIDS OR LIPID-LIKE COMPOUNDS [773] In some embodiments, an ionizable lipid is described in US patent 9,765,022. [774] In some embodiments, a lipid-like compound is represented by structure (XXIII), and is described in WO 2022/261490 and WO 2023/056033, including the lipid compounds comprising the exemplary hydrophilic heads, hydrophobic tails, linkers, and exemplary lipid- like compounds described therein. 618
Attorney Docket No. 01318-0014-00PCT OR-043WO [775] As described therein, the lipid-like compounds of structure XXIII of WO 2022/261490 and WO 2023/056033 and other lipid-like compounds can be prepared by methods well known the art. See Wang et al., ACS Synthetic Biology, 1, 403-07 (2012); Manoharan, et al., International Patent Application Publication WO 2008/042973; and Zugates et al., US Patent 8,071,082. WO 2022/261490 and WO 2023/056033 describe an exemplary route of synthesis of the lipid-like compounds, and other suitable starting materials and routes of synthesis known in the art. See, for example, R. Larock, Comprehensive Organic Transformations (2nd Ed., VCH Publishers 1999); P. G. M. Wuts and T. W. Greene, Greene's Protective Groups in Organic Synthesis (4th Ed., John Wiley and Sons 2007); L. Fieser and M. Fieser, Fieser and Fieser' s Reagents for Organic Synthesis (John Wiley and Sons 1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis (2nd ed., John Wiley and Sons 2009) and subsequent editions thereof. Certain lipid-like compounds may contain a non- aromatic double bond and one or more asymmetric centers. Thus, they can occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and cis- or trans- isomeric forms. All such isomeric forms are contemplated. [776] As mentioned above, these lipid-like compounds are useful for delivery of pharmaceutical agents. They can be preliminarily screened for their efficacy in delivering pharmaceutical agents by an in vitro assay and then confirmed by animal experiments and clinic trials. Other methods will also be apparent to those of ordinary skill in the art. [777] The above described complexes can be prepared using procedures described in publications such as Wang et al., ACS Synthetic Biology, 1, 403-07 (2012). Generally, they are obtained by incubating a lipid-like compound and a pharmaceutical agent in a buffer such as a sodium acetate buffer or a phosphate buffered saline ("PBS"). d. Hydrophilic groups [778] In certain embodiments, the selected hydrophilic functional group or moiety may alter or otherwise impart properties to the compound or to the transfer vehicle of which such compound is a component (e.g., by improving the transfection efficiencies of a lipid nanoparticle of which the compound is a component). For example, the incorporation of guanidinium as a hydrophilic head-group in the compounds disclosed herein may promote the fusogenicity of such compounds (or of the transfer vehicle of which such compounds are a component) with the cell membrane of one or more target cells, thereby enhancing, for example, the transfection efficiencies of such compounds. It has been hypothesized that the nitrogen from the hydrophilic guanidinium moiety forms a six-membered ring transition state 619
Attorney Docket No. 01318-0014-00PCT OR-043WO which grants stability to the interaction and thus allows for cellular uptake of encapsulated materials. (Wender, et al., Adv. Drug Del. Rev. (2008) 60: 452-472.) Similarly, the incorporation of one or more amino groups or moieties into the disclosed compounds (e.g., as a head-group) may further promote disruption of the endosomal/lysosomal membrane of the target cell by exploiting the fusogenicity of such amino groups. This is based not only on the pKa of the amino group of the composition, but also on the ability of the amino group to undergo a hexagonal phase transition and fuse with the target cell surface, i.e. the vesicle membrane. (Koltover, et al. Science (1998) 281: 78-81.) The result is believed to promote the disruption of the vesicle membrane and release of the lipid nanoparticle contents into the target cell. [779] Similarly, in certain embodiments the incorporation of, for example, imidazole as a hydrophilic head-group in the compounds disclosed herein may serve to promote endosomal or lysosomal release of, for example, contents that are encapsulated in a transfer vehicle (e.g., lipid nanoparticle) of the present disclosure. Such enhanced release may be achieved by one or both of a proton-sponge mediated disruption mechanism and/or an enhanced fusogenicity mechanism. The proton-sponge mechanism is based on the ability of a compound, and in particular a functional moiety or group of the compound, to buffer the acidification of the endosome. This may be manipulated or otherwise controlled by the pKa of the compound or of one or more of the functional groups comprising such compound (e.g., imidazole). Accordingly, in certain embodiments the fusogenicity of, for example, the imidazole-based compounds disclosed herein (e.g., HGT4001 and HGT4004) are related to the endosomal disruption properties, which are facilitated by such imidazole groups, which have a lower pKa relative to other traditional ionizable lipids. Such endosomal disruption properties in turn promote osmotic swelling and the disruption of the liposomal membrane, followed by the transfection or intracellular release of the polynucleotide materials loaded or encapsulated therein into the target cell. This phenomenon can be applicable to a variety of compounds with desirable pKa profiles in addition to an imidazole moiety. Such embodiments also include multi-nitrogen based functionalities such as polyamines, poly-peptide (histidine), and nitrogen- based dendritic structures. [780] Exemplary ionizable and/or cationic lipids are described in International PCT patent publications WO2015/095340, WO2015/199952, WO2018/011633, WO2017/049245, WO2015/061467, WO2012/040184, WO2012/000104, WO2015/074085, WO2016/081029, WO2017/004143, WO2017/075531, WO2017/117528, WO2011/022460, WO2013/148541, 620
Attorney Docket No. 01318-0014-00PCT OR-043WO WO2013/116126, WO2011/153120, WO2012/044638, WO2012/054365, WO2011/090965, WO2013/016058, WO2012/162210, WO2008/042973, WO2010/129709, WO2010/144740, WO20 12/099755, WO2013/049328, WO2013/086322, WO2013/086373, WO2011/071860, WO2009/132131, WO2010/048536, WO2010/088537, WO2010/054401, WO2010/054406, WO2010/054405, WO2010/054384, WO2012/016184, WO2009/086558, WO2010/042877, WO2011/000106, WO2011/000107, WO2005/120152, WO2011/141705, WO2013/126803, WO2006/007712, WO2011/038160, WO2005/121348, WO2011/066651, WO2009/127060, WO2011/141704, WO2006/069782, WO2012/031043, WO2013/006825, WO2013/033563, WO2013/089151, WO2017/099823, WO2015/095346, and WO2013/086354, and US patent publications US2016/0311759, US2015/0376115, US2016/0151284, US2017/0210697, US2015/0140070, US2013/0178541, US2013/0303587, US2015/0141678, US2015/0239926, US2016/0376224, US2017/0119904, US2012/0149894, US2015/0057373, US2013/0090372, US2013/0274523, US2013/0274504, US2013/0274504, US2009/0023673, US2012/0128760, US2010/0324120, US2014/0200257, US2015/0203446, US2018/0005363, US2014/0308304, US2013/0338210, US2012/0101148, US2012/0027796, US2012/0058144, US2013/0323269, US2011/0117125, US2011/0256175, US2012/0202871, US2011/0076335, US2006/0083780, US2013/0123338, US2015/0064242, US2006/0051405, US2013/0065939, US2006/0008910, US2003/0022649, US2010/0130588, US2013/0116307, US2010/0062967, US2013/0202684, US2014/0141070, US2014/0255472, US2014/0039032, US2018/0028664, US2016/0317458, and US2013/0195920, the contents of all of which are incorporated herein by reference in their entirety. International patent application WO 2019/131770 is also incorporated herein by reference in its entirety. B. STABILIZING LIPIDS (e.g., PEG lipids) [781] A stabilizing lipid or surface stabilizing lipid may be used to enhance the structure of the LNP. A stabilizing lipid as contemplated herein may be a polyethylene glycol (PEG)- modified phospholipid. [782] The use and inclusion of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-Octanoyl- Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000] (C8 PEG-2000 ceramide) in the liposomal and pharmaceutical compositions described herein is contemplated, preferably in combination with one or more of the compounds and lipids disclosed herein. Contemplated PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa 621
Attorney Docket No. 01318-0014-00PCT OR-043WO in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length. In some embodiments, the PEG-modified lipid employed in the compositions and methods described herein is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene Glycol (2000 MW PEG) “DMG- PEG2000.” The addition of PEG-modified lipids to the lipid delivery vehicle may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-polynucleotide composition to the target tissues, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No.5,885,613). Particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18). The PEG-modified phospholipid and derivatized lipids may comprise a molar ratio from about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in a liposomal lipid nanoparticle. [783] In some embodiments, the lipid moiety of the PEG-lipids includes those having lengths of from about C14 to about C22, such as from about C14 to about C16. In some embodiments, a PEG moiety, for example a mPEG-NH2, has a size of about 1000, about 2000, about 5000, about 10,000, about 15,000 or about 20,000 daltons [784] In an embodiment, a PEG-modified lipid is described in International Pat. Appl. No. PCT/US2019/015913, which is incorporated herein by reference in their entirety. In an embodiment, a transfer vehicle comprises one or more PEG-modified lipids. [785] Non-limiting examples of PEG-modified lipids include PEG-modified phosphatidylethanolamines and phosphatidic acids, PEG-ceramide conjugates (e.g., PEG- CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2- diacyloxypropan-3-amines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. In some further embodiments, a PEG-modified lipid may be, e.g., PEG- c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, PEG-DAG, PEG- S-DAG, PEG-PE, PEG-S-DMG, PEG-CER, PEG-dialkoxypropylcarbamate, PEG-OR, PEG- OH, PEG-c-DOMG, or PEG-1. [786] In some still further embodiments, the PEG-modified lipid includes, but is not limited to 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG- diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-l,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). 622
Attorney Docket No. 01318-0014-00PCT OR-043WO [787] In one embodiment, the lipid nanoparticles described herein can comprise a lipid modified with a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE. In one embodiment, the lipid nanoparticles herein comprise PEG- DSPC. [788] In some embodiments the PEG-modified lipids are a modified form of PEG-DMG. PEG-DMG has the following structure:
[789] In some embodiments, the PEG lipid is a compound of Formula (P1):
or a salt or isomer thereof, wherein: r is an integer between 1 and 100; R is C10-40 alkyl, C10-40 alkenyl, or C10-40 alkynyl; and optionally one or more methylene groups of R are independently replaced with C3-10 carbocyclylene, 4 to 10 membered heterocyclylene, C6- 10 arylene, 4 to 10 membered heteroarylene, –N(RN)–, –O–, –S–, –C(O)–,–C(O)N(RN)–, –NRNC(O)– , –NRNC(O)N(RN)–, –C(O)O–, –OC(O)–, –OC(O)O– ,–OC(O)N(RN)–, –NRNC(O)O–, –C(O)S–, – SC(O)–, –C(=NRN)–, –C(=NRN)N(RN)–, –NRNC(=NRN)–, –NRNC(=NRN)N(RN)– ,–C(S)–, – C(S)N(RN)–, –NRNC(S)–, –NRNC(S)N(RN)–, –S(O)–, –OS(O)–, –S(O)O–, –OS(O)O–, –OS(O)2–, – S(O)2O–, –OS(O)2O–, –N(RN)S(O)–, –S(O)N(RN)–, –N(RN)S(O)N(RN)–, –OS(O)N(RN)–, – N(RN)S(O)O–, –S(O)2–, –N(RN)S(O)2–, –S(O)2N(RN)–, –N(RN)S(O)2N(RN)–, –OS(O)2N(RN)–, or –N(RN)S(O)2O–; and each instance of RN is independently hydrogen, C1-6 alkyl, or a nitrogen protecting group. [790] For example, R is C17 alkyl. For example, the PEG lipid is a compound of Formula
or a salt or isomer thereof, wherein r is an integer between 1 and 100. [791] In some embodiments the PEG-modified lipids are a modified form of PEG-C18, or PEG-1. PEG-1 has the following structure:
. 623
Attorney Docket No. 01318-0014-00PCT OR-043WO [792] PEG-lipids are known in the art, such as those described in U.S. Pat. No.8,158,601 and International Pat. Publ. No. WO2015/130584 A2, which are incorporated herein by reference in their entirety. In one embodiment, PEG lipids can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, a PEG-OH or hydroxy-PEGylated lipid comprises an –OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment. C. HELPER LIPIDS [793] In some embodiments, the transfer vehicle (e.g., LNP) described herein comprises one or more non-cationic helper lipids. In some embodiments, the helper lipid is a phospholipid. In some embodiments, the helper lipid is a phospholipid substitute or replacement. In some embodiments, the phospholipid or phospholipid substitute can be, for example, one or more saturated or (poly)unsaturated phospholipids, or phospholipid substitutes, or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties. [794] A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin. [795] A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. [796] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin. [797] In some embodiments, the helper lipid is a 1,2-distearoyl-177-glycero-3- phosphocholine (DSPC) analog, a DSPC substitute, oleic acid, or an oleic acid analog. 624
Attorney Docket No. 01318-0014-00PCT OR-043WO [798] In some embodiments, a helper lipid is a non-phosphatidyl choline (PC) zwitterionic lipid, a DSPC analog, oleic acid, an oleic acid analog, or a DSPC substitute. [799] In some embodiments, a helper lipid is described in PCT/US2018/053569. Helper lipids suitable for use in a lipid composition of the disclosure include, for example, a variety of neutral, uncharged or zwitterionic lipids. Such helper lipids are preferably used in combination with one or more of the compounds and lipids disclosed herein. Examples of helper lipids include, but are not limited to, 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), pohsphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoylsn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-paimitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1- stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3- phosphocholine (DEPC), paimitoyioieoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanol amine (DOPE) dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In one embodiment, the helper lipid may be distearoylphosphatidylcholine (DSPC) or dimyristoyl phosphatidyl ethanolamine (DMPE). In another embodiment, the helper lipid may be distearoylphosphatidylcholine (DSPC). Helper lipids function to stabilize and improve processing of the transfer vehicles. Such helper lipids are preferably used in combination with other excipients, for example, one or more of the ionizable lipids disclosed herein. In some embodiments, when used in combination with an ionizable lipid, the helper lipid may comprise a molar ratio of 5% to about 90%, or about 10% to about 70% of the total lipid present in the lipid nanoparticle. D. STRUCTURAL LIPIDS [800] The transfer vehicles described herein comprise one or more structural lipids. Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can include, but are not limited to, cholesterol, 625
Attorney Docket No. 01318-0014-00PCT OR-043WO fecosterol, ergosterol, bassicasterol, tomatidine, tomatine, ursolic, alpha-tocopherol, and mixtures thereof. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In certain embodiments, the structural lipid includes cholesterol and a corticosteroid (such as, for example, prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof. [801] In an embodiment, a structural lipid is described in international patent application PCT/US2019/015913. [802] In some embodiments, the structural lipid is a sterol (e.g., phytosterols or zoosterols). In certain embodiments, the structural lipid is a steroid. For example, sterols can include, but are not limited to, cholesterol, β-sitosterol, fecosterol, ergosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, or alpha-tocopherol. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In certain embodiments, the structural lipid is alpha-tocopherol. [803] The transfer vehicles described herein comprise one or more structural lipids. Incorporation of structural lipids in a transfer vehicle, e.g., a lipid nanoparticle, may help mitigate aggregation of other lipids in the particle. In certain embodiments, the structural lipid includes cholesterol and a corticosteroid (such as, for example, prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof. [804] In some embodiments, a transfer vehicle includes an effective amount of an immune cell delivery potentiating lipid, e.g., a cholesterol analog or an amino lipid or combination thereof, that, when present in a transfer vehicle, e.g., an lipid nanoparticle, may function by enhancing cellular association and/or uptake, internalization, intracellular trafficking and/or processing, and/or endosomal escape and/or may enhance recognition by and/or binding to immune cells, relative to a transfer vehicle lacking the immune cell delivery potentiating lipid. Accordingly, while not intending to be bound by any particular mechanism or theory, in one embodiment, a structural lipid or other immune cell delivery potentiating lipid of the disclosure binds to C1q or promotes the binding of a transfer vehicle comprising such lipid to C1q. Thus, for in vitro use of the transfer vehicles of the disclosure for delivery of a nucleic acid molecule to an immune cell, culture conditions that include C1q are used (e.g., use of culture media that includes serum or addition of exogenous C1q to serum-free media). For in vivo use of the transfer vehicles of the disclosure, the requirement for C1q is supplied by endogenous C1q. [805] In certain embodiments, the structural lipid is cholesterol. In certain embodiments, 626
Attorney Docket No. 01318-0014-00PCT OR-043WO the structural lipid is an analog of cholesterol. [806] In some embodiments, a structural lipid of any one of the disclosed embodiments is selected from the structures set forth in Table 16 of WO 2022/261490 and Table 16 of WO 2023/056033, which are incorporated by reference herein in their entireties. E. LIPID NANOPARTICLE (LNP) FORMULATIONS [807] In certain embodiments, the transfer vehicle comprises a lipid. In certain embodiments, the transfer vehicle comprises an ionizable lipid. In certain embodiments, the transfer vehicle comprises an ionizable lipid in combination with other lipids, e.g., a structural lipid, and/or a PEG-modified lipid. [808] In certain embodiments, the transfer vehicle is a lipid nanoparticle (LNP), which may be capable of delivering the one or more circular RNA constructs to one or more target cells . In certain embodiments the transfer vehicle is capable of delivering the circular RNA construct to a human immune cell present in a human subject, such that the expression sequence encoding a binding molecule (e.g., CAR) is translated in the human immune cell and expressed on the surface of the human immune cell. [809] In certain embodiments, the transfer vehicles are prepared to encapsulate one or more materials or therapeutic agents (e.g., circular RNA). The process of incorporating a desired therapeutic agent (e.g., circular RNA) into a transfer vehicle is referred to herein as or “loading” or “encapsulating” (Lasic, et al., FEBS Lett., 312: 255-258, 1992). The transfer vehicle-loaded or -encapsulated materials (e.g., circular RNA) may be completely or partially located in the interior space of the transfer vehicle, within a bilayer membrane of the transfer vehicle, or associated with the exterior surface of the transfer vehicle. [810] In some embodiments, a transfer vehicle encapsulates circular RNA. In some embodiments, the transfer vehicle encapsulates at least one circular RNA construct and comprises an ionizable lipid. In some embodiments, the transfer vehicle encapsulates at least one circular RNA construct and comprises an ionizable lipid and an additional lipid selected from a structural lipid, a helper lipid, and a PEG-modified lipid. In some embodiments, the transfer vehicle encapsulates at least one circular RNA construct and comprises an ionizable lipid, a structural lipid, a helper lipid, and/or a PEG-modified lipid. In some embodiments, a transfer vehicle encapsulates at least one circular RNA construct and comprises an ionizable lipid, a structural lipid, a PEG-modified lipid, and a helper lipid. In some embodiments, the transfer vehicle is a lipid nanoparticle. 627
Attorney Docket No. 01318-0014-00PCT OR-043WO [811] Without wishing to be bound by theory, it is thought that transfer vehicles described herein shield encapsulated circular RNA from degradation and provide for effective delivery of circular RNA to target cells in vivo and in vitro. [812] In certain embodiments, the transfer vehicles are formulated based in part upon their ability to facilitate the transfection (e.g., of a circular RNA) of a target cell. In another embodiment, the transfer vehicles may be selected and/or prepared to optimize delivery of circular RNA to a target cell, tissue or organ. For example, if the target cell is a hepatocyte, the properties of the compositions (e.g., size, charge and/or pH) may be optimized to effectively deliver such composition (e.g., lipid nanoparticles) to the target cell or organ, reduce immune clearance and/or promote retention in the target cell or organ. Alternatively, if the target tissue is the central nervous system, the selection and preparation of the transfer vehicle must consider penetration of, and retention within. the blood brain barrier and/or the use of alternate means of directly delivering such compositions to such target tissue (e.g., via intracerebrovascular administration). In certain embodiments, the transfer vehicles may be combined with agents that facilitate the transfer of encapsulated materials across the blood brain barrier (e.g., agents which disrupt or improve the permeability of the blood brain barrier and thereby enhance the transfer of circular RNA to the target cells). While the transfer vehicles described herein can facilitate introduction of circular RNA into target cells, the addition of polycations (e.g., poly L-lysine and protamine) as a copolymer to one or more of the lipid nanoparticles that comprise the pharmaceutical compositions can in some instances markedly enhance the transfection efficiency of several types of transfer vehicles by 2-28 fold in a number of cell lines both in vitro and in vivo (See, N. J. Caplen, et al., Gene Ther. 1995; 2: 603; S. Li, et al., Gene Ther. 1997; 4, 891.). [813] Transfer vehicles described herein can allow the encapsulated polynucleotide to reach the target cell or may preferentially allow the encapsulated polynucleotide to reach the target cells or organs on a discriminatory basis. Alternatively, the transfer vehicles may limit the delivery of encapsulated polynucleotides to other non-targeted cells or organs where the presence of the encapsulated polynucleotides may be undesirable or of limited utility. [814] Loading or encapsulating a polynucleotide, e.g., circular RNA, into a transfer vehicle may serve to protect the polynucleotide from an environment (e.g., serum) which may contain enzymes or chemicals that degrade such polynucleotides and/or systems or receptors that cause the rapid excretion of such polynucleotides. Accordingly, in some embodiments, the compositions described herein are capable of enhancing the stability of the encapsulated 628
Attorney Docket No. 01318-0014-00PCT OR-043WO polynucleotide(s), particularly with respect to the environments into which such polynucleotides will be exposed. [815] In certain embodiments, the transfer vehicles described herein are prepared by combining multiple lipid components (e.g., one or more of the compounds disclosed herein) with one or more polymer components. [816] A lipid nanoparticle may be comprised of additional lipid combinations in various ratios. The selection of ionizable lipids, helper lipids, structural lipids, and/or PEG-modified lipids that make up the lipid nanoparticles, as well as the relative molar ratio of such lipids to each other, is based upon the characteristics of the selected lipid(s), the nature of the intended target cells or tissues and the characteristics of the materials or polynucleotides to be delivered by the lipid nanoparticle. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). [817] The formation of a lipid nanoparticle (LNP) described herein may be accomplished by any methods known in the art. See, e.g., U.S. Pat. Pub. No. US2012/0178702 A1, which is incorporated herein by reference in its entirety. Non-limiting examples of lipid nanoparticle compositions and methods of making them are described, for example, in Semple et al. (2010) Nat. Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51:8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (the contents of each of which are incorporated herein by reference in their entirety). Lipid nanoparticles, formulations, and methods of preparation are described in, e.g., International Pat. Pub. No. WO 2011/127255 or WO 2008/103276, U.S. Pat. Pub. No. US2005/0222064 A1, U.S. Pat. Pub. No. US2013/0156845 A1, International Pat. Pub. No. WO2013/093648 A2, WO2012/024526 A2, U.S. Pat. Pub. No. US2013/0164400 A1, and U.S. Pat. No. 8,492,359, all of which are incorporated herein by reference in their entirety. [818] In some embodiments, the lipid nanoparticle comprises one or more cationic lipids, non-cationic lipids, ionizable lipids, or poly β-amino esters. In some embodiments, the nanoparticle comprises one or more non-cationic lipids. In some embodiments, the lipid nanoparticle comprises one or more PEG-modified lipids, polyglutamic acid lipids, or hyaluronic acid lipids. In some embodiments, the lipid nanoparticle comprises cholesterol. In some embodiments, the lipid nanoparticle comprises arachidonic acid, leukotriene, or oleic acid. In some embodiments, the lipid nanoparticle comprises a targeting moiety, wherein the targeting moiety mediates receptor-mediated endocytosis selectively into cells of a selected 629
Attorney Docket No. 01318-0014-00PCT OR-043WO cell population in the absence of cell selection or purification. In some embodiments, the lipid nanoparticle comprises more than one circular RNA construct. [819] Examples of further suitable lipids include the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). Also contemplated is the use of polymers as transfer vehicles, whether alone or in combination with other transfer vehicles. Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, dendrimers and polyethylenimine. [820] A lipid nanoparticle composition may optionally comprise one or more coatings. For example, a nanoparticle composition may be formulated in a capsule, film, or tablet having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness, or density. [821] In one embodiment, the lipid nanoparticles may have a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 to about 100 nm. In one embodiment, the lipid nanoparticles may have a diameter from about 10 to 500 nm. In one embodiment, the lipid nanoparticle may have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm. Each possibility represents a separate 630
Attorney Docket No. 01318-0014-00PCT OR-043WO embodiment. [822] In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter of 10-500 nm, 20-400 nm, 30-300 nm, or 40-200 nm. In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter of 50-150 nm, 50-200 nm, 80-100 nm, or 80- 200 nm. [823] In some embodiments, the lipid nanoparticles described herein can have a diameter from below 0 .1 µm to up to 1 mm such as, but not limited to, less than 0 .1 µm, less than 1.0 µm, less than 5 µm, less than 10 µm, less than 15 µm, less than 20 µm, less than 25 µm, less than 30 µm, less than 35 µm, less than 40 µm, less than 50 µm, less than 55 µm, less than 60 µm, less than 65 µm, less than 70 µm, less than 75 µm, less than 80 µm, less than 85 µm, less than 90 µm, less than 95 µm, less than 100 µm, less than 125 µm, less than 150 µm, less than 175 µm, less than 200 µm, less than 225 µm, less than 250 µm, less than 275 µm, less than 300 µm, less than 325 µm, less than 350 µm, less than 375 µm, less than 400 µm, less than 425 µm, less than 450 µm, less than 475 µm, less than 500 µm, less than 525 µm, less than 550 µm, less than 575 µm, less than 600 µm, less than 625 µm, less than 650 µm, less than 675 µm, less than 700 µm, less than 725 µm, less than 750 µm, less than 775 µm, less than 800 µm, less than 825 µm, less than 850 µm, less than 875 µm, less than 900 µm, less than 925 µm, less than 950 µm, less than 975 µm. [824] In another embodiment, LNPs may have a diameter from about 1 nm to about 100 nm, from about 1 nm to about 10 nm, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5 nm to about 90 nm, about 10 to about 50 nM, from about 20 to about 50 nm, from about 30 to about 50 nm, from about 40 to about 50 nm, from about 20 to about 60 nm, from about 30 to about 60 nm, from about 40 to about 60 nm, from about 20 to about 70 nm, from about 30 to about 70 nm, from about 40 to about 70 nm, from about 50 to about 70 nm, from about 60 to about 70 nm, from about 20 to about 80 nm, from about 30 to about 80 nm, from about 40 to about 80 nm, from about 50 to about 80 nm, from about 60 to about 80 nm, from about 20 to about 90 nm, from about 30 to about 90 nm, from about 40 to about 90 nm, from about 50 to about 90 nm, from about 60 to about 631
Attorney Docket No. 01318-0014-00PCT OR-043WO 90 nm and/or from about 70 to about 90 nm. Each possibility represents a separate embodiment. [825] A nanoparticle composition may be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the lipid nanoparticle compositions. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a nanoparticle composition may be from about 0.10 to about 0.20. Each possibility represents a separate embodiment. [826] The zeta potential of a nanoparticle composition may be used to indicate the electrokinetic potential of the composition. For example, the zeta potential may describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a nanoparticle composition may be from about -20 mV to about +20 mV, from about -20 mV to about +15 mV, from about -20 mV to about +10 mV, from about -20 mV to about +5 mV, from about -20 mV to about 0 mV, from about -20 mV to about -5 mV, from about -20 mV to about -10 mV, from about -20 mV to about -15 mV from about -20 mV to about +20 mV, from about -20 mV to about +15 mV, from about -20 mV to about +10 mV, from about -20 mV to about +5 mV, from about -20 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV. Each possibility represents a separate embodiment. [827] The efficiency of encapsulation of a therapeutic agent describes the amount of therapeutic agent that is encapsulated or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of therapeutic agent in a solution containing the lipid nanoparticle composition before and after breaking up the lipid nanoparticle composition with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free therapeutic agent (e.g., nucleic acids) in a solution. For the lipid nanoparticle 632
Attorney Docket No. 01318-0014-00PCT OR-043WO compositions described herein, the encapsulation efficiency of a therapeutic agent may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%. [828] In some embodiments, the lipid nanoparticle has a polydispersity value of less than 0.4. In some embodiments, the lipid nanoparticle has a net neutral charge at a neutral pH. In some embodiments, the lipid nanoparticle has a mean diameter of 50-200nm. [829] The properties of a lipid nanoparticle formulation may be influenced by factors including, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the selection of the non-cationic lipid component, the degree of noncationic lipid saturation, the selection of the structural lipid component, the nature of the PEGylation, ratio of all components and biophysical parameters such as size. As described herein, the purity of a PEG lipid component is also important to an LNP’s properties and performance. F. METHODS FOR PREPARING LIPID NANOPARTICLES (LNP) FORMULATIONS [830] In one embodiment, a lipid nanoparticle formulation may be prepared by the methods described in International Publication Nos. WO2011127255 or WO2008103276, each of which is herein incorporated by reference in their entirety. In some embodiments, lipid nanoparticle formulations may be as described in International Publication No. WO2019131770, which is herein incorporated by reference in its entirety. [831] In some embodiments, circular RNA is formulated according to a process described in US patent application 15/809,680. In some embodiments, the present disclosure provides a process of encapsulating circular RNA in transfer vehicles comprising the steps of forming lipids into pre-formed transfer vehicles (i.e. formed in the absence of RNA) and then combining the pre-formed transfer vehicles with RNA. In some embodiments, the novel formulation process results in an RNA formulation with higher potency (peptide or protein expression) and higher efficacy (improvement of a biologically relevant endpoint) both in vitro and in vivo with potentially better tolerability as compared to the same RNA formulation prepared without the step of preforming the lipid nanoparticles (e.g., combining the lipids directly with the RNA). [832] For certain cationic lipid nanoparticle formulations of RNA, in order to achieve 633
Attorney Docket No. 01318-0014-00PCT OR-043WO high encapsulation of RNA, the RNA in buffer (e.g., citrate buffer) has to be heated. In those processes or methods, the heating is required to occur before the formulation process (i.e. heating the separate components) as heating post-formulation (post-formation of nanoparticles) does not increase the encapsulation efficiency of the RNA in the lipid nanoparticles. In contrast, in some embodiments of the novel processes of the present disclosure, the order of heating of RNA does not appear to affect the RNA encapsulation percentage. In some embodiments, no heating (i.e. maintaining at ambient temperature) of one or more of the solutions comprising the pre-formed lipid nanoparticles, the solution comprising the RNA and the mixed solution comprising the lipid nanoparticle encapsulated RNA is required to occur before or after the formulation process. [833] RNA may be provided in a solution to be mixed with a lipid solution such that the RNA may be encapsulated in lipid nanoparticles. A suitable RNA solution may be any aqueous solution containing RNA to be encapsulated at various concentrations. For example, a suitable RNA solution may contain an RNA at a concentration of or greater than about 0.01 mg/ml, 0.05 mg/ml, 0.06 mg/ml, 0.07 mg/ml, 0.08 mg/ml, 0.09 mg/ml, 0.1 mg/ml, 0.15 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, or 1.0 mg/ml. In some embodiments, a suitable RNA solution may contain an RNA at a concentration in a range from about 0.01-1.0 mg/ml, 0.01-0.9 mg/ml, 0.01-0.8 mg/ml, 0.01-0.7 mg/ml, 0.01- 0.6 mg/ml, 0.01-0.5 mg/ml, 0.01-0.4 mg/ml, 0.01-0.3 mg/ml, 0.01-0.2 mg/ml, 0.01-0.1 mg/ml, 0.05-1.0 mg/ml, 0.05-0.9 mg/ml, 0.05-0.8 mg/ml, 0.05-0.7 mg/ml, 0.05-0.6 mg/ml, 0.05-0.5 mg/ml, 0.05-0.4 mg/ml, 0.05-0.3 mg/ml, 0.05-0.2 mg/ml, 0.05-0.1 mg/ml, 0.1-1.0 mg/ml, 0.2- 0.9 mg/ml, 0.3-0.8 mg/ml, 0.4-0.7 mg/ml, or 0.5-0.6 mg/ml. [834] Typically, a suitable RNA solution may also contain a buffering agent and/or salt. Generally, buffering agents can include HEPES, Tris, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate or sodium phosphate. In some embodiments, suitable concentration of the buffering agent may be in a range from about 0.1 mM to 100 mM, 0.5 mM to 90 mM, 1.0 mM to 80 mM, 2 mM to 70 mM, 3 mM to 60 mM, 4 mM to 50 mM, 5 mM to 40 mM, 6 mM to 30 mM, 7 mM to 20 mM, 8 mM to 15 mM, or 9 to 12 mM. [835] Exemplary salts can include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, suitable concentration of salts in an RNA solution may be in a range from about 1 mM to 500 mM, 5 mM to 400 mM, 10 mM to 350 mM, 15 mM to 300 mM, 20 mM to 250 mM, 30 mM to 200 mM, 40 mM to 190 mM, 50 mM to 180 mM, 50 mM 634
Attorney Docket No. 01318-0014-00PCT OR-043WO to 170 mM, 50 mM to 160 mM, 50 mM to 150 mM, or 50 mM to 100 mM. [836] In some embodiments, a suitable RNA solution may have a pH in a range from about 3.5-6.5, 3.5-6.0, 3.5-5.5, 3.5-5.0, 3.5-4.5, 4.0-5.5, 4.0-5.0, 4.0-4.9, 4.0-4.8, 4.0-4.7, 4.0- 4.6, or 4.0-4.5. [837] Various methods may be used to prepare an RNA solution suitable for the present disclosure. In some embodiments, RNA may be directly dissolved in a buffer solution described herein. In some embodiments, an RNA solution may be generated by mixing an RNA stock solution with a buffer solution prior to mixing with a lipid solution for encapsulation. In some embodiments, an RNA solution may be generated by mixing an RNA stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation. [838] According to the present disclosure, a lipid solution contains a mixture of lipids suitable to form transfer vehicles for encapsulation of RNA. In some embodiments, a suitable lipid solution is ethanol based. For example, a suitable lipid solution may contain a mixture of desired lipids dissolved in pure ethanol (i.e. 100% ethanol). In another embodiment, a suitable lipid solution is isopropyl alcohol based. In another embodiment, a suitable lipid solution is dimethylsulfoxide-based. In another embodiment, a suitable lipid solution is a mixture of suitable solvents including, but not limited to, ethanol, isopropyl alcohol and dimethylsulfoxide. [839] A suitable lipid solution may contain a mixture of desired lipids at various concentrations. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration in a range from about 0.1-100 mg/ml, 0.5-90 mg/ml, 1.0- 80 mg/ml, 1.0-70 mg/ml, 1.0-60 mg/ml, 1.0-50 mg/ml, 1.0-40 mg/ml, 1.0-30 mg/ml, 1.0-20 mg/ml, 1.0-15 mg/ml, 1.0-10 mg/ml, 1.0-9 mg/ml, 1.0-8 mg/ml, 1.0-7 mg/ml, 1.0-6 mg/ml, or 1.0-5 mg/ml. [840] Nanoparticles can be made in a 1 fluid stream or with mixing processes such as microfluidics and T-junction mixing of two fluid streams, one of which contains the circular RNA and the other has the lipid components. [841] In some embodiments, the lipid nanoparticles described herein may be synthesized using methods comprising, for example, microfluidic mixers, microstructure-induced chaotic advection (MICA), a Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IJMM) from the Institut fur Mikrotechnik Mainz GmbH, Mainz Germany), using a micromixer chip, and/or using technology. Exemplary mixers and methods are known in the art. 635
Attorney Docket No. 01318-0014-00PCT OR-043WO [842] Additional lipid nanoparticle formulations and methods of producing are described in detail in WO2021226597 and WO2021113777, which are incorporated herein by reference in their entireties. For example, disclosed in WO2021226597 and WO2021113777 is a method of preparing lipid nanoparticle formulations of ionizable lipids 128 and 129 of Table 4. Ethanol phase contained ionizable Lipid 128 or Lipid 129 from Table 4, DOPE, Cholesterol, and DSPE- PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or 62:4:33:1 molar ratio combined with an aqueous phase containing circular RNA and 25 mM sodium acetate buffer at pH 5.2. A 3:1 aqueous to ethanol mixing ratio was used. The formulated LNPs were then dialyzed in 1L of water and exchanged 2 times over 18 hours. Dialyzed LNPs were filtered using 0.2 µm filter. Prior to in vivo dosing, LNPs were diluted in PBS. LNP sizes were determined by dynamic light scattering. A cuvette with 1 mL of 20 µg/mL LNPs in PBS (pH 7.4) was measured for Z-average using the Malvern Panalytical Zetasizer Pro. The Z-average and polydispersity index were recorded G. OTHER DELIVERY VEHICLES KNOWN IN THE ART [843] In certain embodiments, other delivery vehicles that are known in the art may be used to transport the circular RNA (i.e., are transfer vehicles encompassed herein). [844] In some embodiments, liposomes or other lipid bilayer vesicles may be used as a component or as the whole transfer vehicle to facilitate or enhance the delivery and release of circular RAN to one or more target cells. Liposomes are usually characterized by having an interior space sequestered from an outer medium by a membrane of one or more bilayers forming a microscopic sack or vesicle. Bilayer membranes of liposomes are typically formed by lipids, i.e. amphiphilic molecules of synthetic or natural origin that comprise spatially separated hydrophobic or hydrophilic domains (Lasic, D, and Papahadjopoulos, D., eds. Medical Applications of Liposomes. Elsevier, Amsterdam, 1998). [845] In certain embodiments, the transfer vehicle for transporting the circular RNA comprises a dendrimer. Use of “dendrimer” describes the architectural motif of the transfer vehicle. In some embodiments, the dendrimer includes but is not limited to containing an interior core and one or more layers (i.e. generations) that extend or attach out from the interior core. In some of the embodiments, the generations may contain one or more branching points and an exterior surface of terminal groups that attach to the outermost generation. The branching points, in certain embodiments, may be mostly monodispersed and contain symmetric branching units built around the interior core. In some embodiments, the interior 636
Attorney Docket No. 01318-0014-00PCT OR-043WO core. Synthesis of the dendrimer may comprise the divergent method, convergent growth, hypercore and branched monomer growth, double exponential growth, lego chemistry, click chemistry and other methods as available in the art (Mendes L. et al., Molecules. 2017.22 (9): 1401 further describes these methods). [846] In certain embodiments, as described herein, the transfer vehicle for the circular RNA construct comprises a polymer nanoparticle. In some embodiments, the polymer nanoparticle includes nanocapsules and nanospheres. Nanocapsules, in some embodiments, are composed of an oily core surrounded by a polymeric shell. In some embodiments, the circular RNA is contained within the core and the polymeric shell controls the release of the circular RNA. On the other hand, nanospheres comprise a continuous polymeric network in which the circular RNA is retained or absorbed onto the surface. In some embodiments, cationic polymers are used to encapsulate the circular RNA due to the favorable electrostatic interaction of the cations to the negatively charged nucleic acids and cell membrane. The polymer nanoparticle may be prepared by various methods. In some embodiments, the polymer nanoparticle may be prepared by nanoprecipitation, emulsion techniques, solvent evaporation, solvent diffusion, reverse salting-out or other methods available in the art. [847] In certain embodiments, as described herein, the transfer vehicle for the circular RNA construct comprises a polymer-lipid hybrid nanoparticle (LPHNP). In some embodiments, the LPHNP comprises a polymer core enveloped within a lipid bilayer. In some embodiments, the polymer core encapsulates the circular RNA construct. In some embodiments, the LPHNP further comprises an outer lipid bilayer. In certain embodiments this outer lipid bilayer comprises a PEG-lipid, helper lipid, cholesterol or other molecule as known in the art to help with stability in a lipid-based nanoparticle. The lipid bilayer closest to the polymer core mitigates the loss of the entrapped circular RNA during LPHNP formation and protects from degradation of the polymer core by preventing diffusion of water from outside of the transfer vehicle into the polymer core (Mukherjee et al., In J. Nanomedicine. 2019; 14: 1937-1952). [848] In certain embodiments, the circular RNA can be transported using a peptide-based delivery mechanism. In some embodiments, the peptide-based delivery mechanism comprises a lipoprotein. Based on the size of the drug to be delivered, the lipoprotein may be either a low- density (LDL) or high-density lipoprotein (HDL). As seen in US8734853B2, high-density lipoproteins are capable of transporting a nucleic acid in vivo and in vitro. In particular embodiments, the lipid component includes cholesterol. In more particular embodiments, the 637
Attorney Docket No. 01318-0014-00PCT OR-043WO lipid component includes a combination of cholesterol and cholesterol oleate. [849] In certain embodiments, the circular RNA construct can be transported using a carbohydrate carrier or a sugar-nanocapsule. In certain embodiments, the carbohydrate carrier comprises a sugar-decorated nanoparticle, peptide- and saccharide-conjugated dendrimer, nanoparticles based on polysaccharides, and other carbohydrate-based carriers available in the art. As described herein, the incorporation of carbohydrate molecules may be through synthetic means. In some embodiments, the carbohydrate carrier comprises polysaccharides. These polysaccharides may be made from the microbial cell wall of the target cell. For example, carbohydrate carriers comprised of mannan carbohydrates have been shown to successfully deliver mRNA (Son et al., Nano Lett. 2020. 20(3): 1499-1509). [850] In certain embodiments, as provided herein, the transfer vehicle for the circular RNA is a glyconanoparticle (GlycoNP). As known in the art, glyconanoparticles comprise a core comprising gold, iron oxide, semiconductor nanoparticles or a combination thereof. In some embodiments, the glyconanoparticle is functionalized using carbohydrates. In certain embodiments, the glyconanoparticle comprises a carbon nanotube or graphene. In one embodiment the glyconanoparticle comprises a polysaccharide-based GlycoNP (e.g., chitosan- based GlycoNP). In certain embodiments, the glyconanoparticle is a glycodendrimer. [851] In certain embodiments, as provided herein, the circular RNA is transferred through use of an exosome, a type of extracellular vesicle. Exosomes naturally are secreted by various types of cells and are used as a transport vesicle for various forms of cargo. During delivery exosomes can contain and protect specific mRNAs, regulatory microRNAs, lipids, and proteins (Luan et al., Acta Pharmacologica Sinica. 2017. 38:754-763). Naturally, exosomes may be 30 nm to 125 nm. [852] In some embodiments, the exosome may be made in part from an immune cell. As shown in Haney et al, use of immune cell derived exosomes are able to avoid mononuclear phagocytes (J Control Release. 2015. 207:18-30). In some embodiments, the exosome may be a dendritic cell, macrophage, T-cell, B-cell or derived from another immune cell. As seen in WO/2021/041473A1, various forms of RNAs of varying lengths may be transported through exosome delivery including messenger RNA (mRNA), microRNA (miRNA), long intergenic non-coding RNA (lincRNA), long non-coding RNA (lncRNA), non-coding RNA (ncRNA), non-messenger RNA (nmRNA), small RNA (sRNA), small non-messenger RNA (smnRNA), DNA damage response RNA (DD RNA), extracellular RNA (exRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and precursor messenger RNA (pre-mRNA). 638
Attorney Docket No. 01318-0014-00PCT OR-043WO [853] In other embodiments, the transfer vehicle may comprise in whole or in part from a fusome. In some embodiments, the fusome is derived from an endoplasmic reticulum of a germline cyst. In certain embodiments, the germline cyst is from a Drosophila ovary. [854] In certain embodiments, the circular RNA construct may be transported using noncellular and instead be through mechanical delivery mechanisms. In some embodiments, this delivery method includes microneedles, electroporation, continuous pumps and/or gene guns. [855] In some embodiments, the transfer vehicle of the circular RNA construct is a solution or diluent comprising of a salt or a buffer. H. TARGETING [856] In some embodiments, the compositions use targeting moieties that may be bound (either covalently or non-covalently) to the transfer vehicles to encourage localization of such transfer vehicle at certain target cells or target tissues. For example, targeting may be mediated by the inclusion of one or more endogenous targeting moieties in or on the transfer vehicle to encourage distribution to the target cells or tissues. Recognition of the targeting moiety by the target tissues actively facilitates tissue distribution and cellular uptake of the transfer vehicle and/or its contents in the target cells and tissues (e.g., the inclusion of an apolipoprotein-E targeting ligand in or on the transfer vehicle encourages recognition and binding of the transfer vehicle to endogenous low density lipoprotein receptors expressed by hepatocytes). [857] As provided herein, the composition can comprise a moiety capable of enhancing affinity of the composition to the target cell. Targeting moieties may be linked to the outer bilayer of the lipid particle during formulation or post-formulation. These methods are well known in the art. In addition, some lipid particle formulations may employ fusogenic polymers such as PEAA, hemagluttinin, other lipopeptides (see U.S. patent application Ser. No. 08/835,281, and 60/083,294, which are incorporated herein by reference) and other features useful for in vivo and/or intracellular delivery. In other some embodiments, the compositions demonstrate improved transfection efficacies, and/or demonstrate enhanced selectivity towards target cells or tissues of interest. Contemplated therefore are compositions which comprise one or more moieties (e.g., peptides, aptamers, oligonucleotides, a vitamin or other molecules) that are capable of enhancing the affinity of the compositions and their nucleic acid contents for the target cells or tissues. Suitable moieties may optionally be bound or linked to the surface of the transfer vehicle. In some embodiments, the targeting moiety may span the surface of a transfer vehicle or be encapsulated within the transfer vehicle. Suitable moieties and are selected based 639
Attorney Docket No. 01318-0014-00PCT OR-043WO upon their physical, chemical or biological properties (e.g., selective affinity and/or recognition of target cell surface markers or features). Cell-specific target sites and their corresponding targeting ligand can vary widely. Suitable targeting moieties are selected such that the unique characteristics of a target cell are exploited, thus allowing the composition to discriminate between target and non-target cells. For example, in some embodiments, compositions may include surface markers (e.g., apolipoprotein-B or apolipoprotein-E) that selectively enhance recognition of, or affinity to hepatocytes (e.g., by receptor-mediated recognition of and binding to such surface markers). As an example, the use of galactose as a targeting moiety would be expected to direct the compositions to parenchymal hepatocytes, or alternatively the use of mannose containing sugar residues as a targeting ligand would be expected to direct the compositions to liver endothelial cells (e.g., mannose containing sugar residues that may bind preferentially to the asialoglycoprotein receptor present in hepatocytes). (See Hillery A M, et al. “Drug Delivery and Targeting: For Pharmacists and Pharmaceutical Scientists” (2002) Taylor & Francis, Inc.) The presentation of such targeting moieties that have been conjugated to moieties present in the transfer vehicle (e.g., a lipid nanoparticle) therefore facilitate recognition and uptake of the compositions in target cells and tissues. Examples of suitable targeting moieties include one or more peptides, proteins, aptamers, vitamins and oligonucleotides. [858] In particular embodiments, a transfer vehicle comprises a targeting moiety. In some embodiments, the targeting moiety mediates receptor-mediated endocytosis selectively into a specific population of cells or tissue. In some embodiments, the targeting moiety is capable of binding to a T cell antigen. In some embodiments, the targeting moiety is capable of binding to a NK, NKT, or macrophage antigen. In some embodiments, the targeting moiety is capable of binding to a protein selected from the group CD3, CD4, CD8, PD-1, 4-1BB, and CD2. In some embodiments, the targeting moiety is a single chain Fv (scFv) fragment, nanobody, peptide, peptide-based macrocycle, minibody, heavy chain variable region, light chain variable region or fragment thereof. In some embodiments, the targeting moiety is selected from T-cell receptor motif antibodies, T-cell α chain antibodies, T-cell β chain antibodies, T-cell γ chain antibodies, T-cell δ chain antibodies, CCR7 antibodies, CD3 antibodies, CD4 antibodies, CD5 antibodies, CD7 antibodies, CD8 antibodies, CD11b antibodies, CD11c antibodies, CD16 antibodies, CD19 antibodies, CD20 antibodies, CD21 antibodies, CD22 antibodies, CD25 antibodies, CD28 antibodies, CD34 antibodies, CD35 antibodies, CD40 antibodies, CD45RA antibodies, CD45RO antibodies, CD52 antibodies, CD56 antibodies, CD62L antibodies, CD68 640
Attorney Docket No. 01318-0014-00PCT OR-043WO antibodies, CD80 antibodies, CD95 antibodies, CD117 antibodies, CD127 antibodies, CD133 antibodies, CD137 (4-1BB) antibodies, CD163 antibodies, F4/80 antibodies, IL-4Rα antibodies, Sca-1 antibodies, CTLA-4 antibodies, GITR antibodies GARP antibodies, LAP antibodies, granzyme B antibodies, LFA-1 antibodies, transferrin receptor antibodies, and fragments thereof. In some embodiments, the targeting moiety is a small molecule binder of an ectoenzyme on lymphocytes. Small molecule binders of ectoenzymes include A2A inhibitors CD73 inhibitors, CD39 or adesines receptors A2aR and A2bR. Potential small molecules include AB928. [859] In some embodiments, transfer vehicles are formulated and/or targeted as described in Shobaki N, Sato Y, Harashima H. Mixing lipids to manipulate the ionization status of lipid nanoparticles for specific tissue targeting. Int J Nanomedicine.2018;13:8395–8410. Published 2018 Dec 10. In some embodiments, a transfer vehicle is made up of 3 lipid types. In some embodiments, a transfer vehicle is made up of 4 lipid types. In some embodiments, a transfer vehicle is made up of 5 lipid types. In some embodiments, a transfer vehicle is made up of 6 lipid types. [860] In some embodiments, the target cells are deficient in a protein or enzyme of interest. In some embodiments, the compositions of the present disclosure transfect the target cells on a discriminatory basis (i.e., do not transfect non-target cells). The compositions of the present disclosure may also be prepared to preferentially target a variety of target cells, which include, but are not limited to, immune cells, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells (e.g., meninges, astrocytes, motor neurons, cells of the dorsal root ganglia and anterior horn motor neurons), photoreceptor cells (e.g., rods and cones), retinal pigmented epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells. [861] The compositions of the present disclosure may be prepared to preferentially distribute to target cells, including but not limited to the heart, lungs, kidneys, liver, and spleen, ocular, or cells in the central nervous system. In some embodiments, the compositions of the present disclosure distribute into the cells of the liver or spleen to facilitate the delivery and the subsequent expression of the circRNA comprised therein by the cells of the liver (e.g., hepatocytes) or the cells of spleen (e.g., immune cells). The targeted cells may function as a biological “reservoir” or “depot” capable of producing, and systemically excreting a functional 641
Attorney Docket No. 01318-0014-00PCT OR-043WO protein or enzyme. [862] In some embodiments, the transfer vehicles comprise circRNA which encode a deficient protein or enzyme. Upon distribution of such compositions to the target tissues and the subsequent transfection of such target cells, the exogenous circRNA loaded into the transfer vehicle (e.g., a lipid nanoparticle) may be translated in vivo to produce a functional protein or enzyme encoded by the exogenously administered circRNA (e.g., a protein or enzyme in which the subject is deficient). Accordingly, the compositions of the present disclosure exploit a subject's ability to translate exogenously- or recombinantly-prepared circRNA to produce an endogenously-translated protein or enzyme, and thereby produce (and where applicable excrete) a functional protein or enzyme. The expressed or translated proteins or enzymes may also be characterized by the in vivo inclusion of native post-translational modifications which may often be absent in recombinantly-prepared proteins or enzymes, thereby further reducing the immunogenicity of the translated protein or enzyme. 6. PHARMACEUTICAL COMPOSITIONS [863] In certain embodiments, provided herein are compositions (e.g., pharmaceutical compositions) comprising a therapeutic agent provided herein. In some embodiments, the therapeutic agent is a circular RNA polynucleotide provided herein. In some embodiments the therapeutic agent is a vector provided herein. In some embodiments, the therapeutic agent is a cell comprising a circular RNA, a precursor polynucleotide, or vector provided herein (e.g., a human cell, such as a human T cell). [864] In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, the composition comprises a circular RNA polynucleotide provided herein and a pharmaceutically acceptable salt, buffer, diluent, or combination, and optionally a transfer vehicle. In some embodiments, and as described elsewhere herein, the pharmaceutical composition comprises at least one circular RNA polynucleotide and a transfer vehicle. In some embodiments, the transfer vehicle comprises at least one of (i) an ionizable lipid, (ii) a structural lipid, and (iii) a PEG-modified lipid. [865] In some embodiments, the transfer vehicle is a nanoparticle or lipid nanoparticle. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP), a core-shell nanoparticle, a biodegradable nanoparticle, a biodegradable lipid nanoparticle, a polymer nanoparticle, a polyplex or a biodegradable polymer nanoparticle. [866] In some embodiments, the pharmaceutical composition comprises a targeting moiety. The targeting moiety mediates receptor-mediated endocytosis, endosome fusion, or 642
Attorney Docket No. 01318-0014-00PCT OR-043WO direct fusion into selected cells of a selected cell population or tissue in the absence of cell isolation or purification. In some embodiments, the pharmaceutical composition comprises a targeting moiety operably connected to the nanoparticle. In some embodiments, the targeting moiety is a small molecule, scFv, nanobody, peptide, cyclic peptide, di or tri cyclic peptide, minibody, polynucleotide aptamer, engineered scaffold protein, heavy chain variable region, light chain variable region, or a fragment thereof. In some embodiments, less than 1%, by weight, of the polynucleotides in the composition are double stranded RNA, DNA splints, DNA template, or triphosphorylated RNA. In some embodiments, less than 1%, by weight, of the polynucleotides and proteins in the pharmaceutical composition are double stranded RNA, DNA splints, DNA template, triphosphorylated RNA, phosphatase proteins, protein ligases, RNA polymerases, and capping enzymes. [867] In some embodiments, the compositions provided herein comprise a therapeutic agent provided herein in combination with other pharmaceutically active agents or drugs, such as anti-inflammatory drugs or antibodies capable of targeting B cell antigens, e.g., anti-CD20 antibodies, e.g., rituximab. [868] With respect to pharmaceutical compositions, the pharmaceutically acceptable carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active agent(s), and by the route of administration. The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the therapeutic agent(s) and one which has no detrimental side effects or toxicity under the conditions of use. [869] The choice of carrier will be determined in part by the particular therapeutic agent, as well as by the particular method used to administer the therapeutic agent. Accordingly, there are a variety of suitable formulations of the pharmaceutical compositions provided herein. In certain embodiments, the pharmaceutical composition comprises a preservative. In some embodiments, the pharmaceutical composition comprises a buffering agent. [870] In some embodiments, the concentration of therapeutic agent in the pharmaceutical composition can vary, e.g., less than about 1%, or at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or about 50% or more by weight, and can be selected primarily by fluid volumes, and viscosities, in accordance with the particular mode of administration selected. 643
Attorney Docket No. 01318-0014-00PCT OR-043WO [871] Formulations for oral, aerosol, parenteral (e.g., subcutaneous, intravenous, intraarterial, intramuscular, intradermal, intraperitoneal, and intrathecal), and topical administration are known in the art. More than one route can be used to administer the therapeutic agents provided herein, and in certain instances, a particular route can provide a more immediate and more effective response than another route. [872] In certain embodiments, the therapeutic agents provided herein can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or LNPs or liposomes. [873] In some embodiments, the composition comprises a precursor RNA polynucleotide described herein, a polynucleotide described herein, a circular RNA polynucleotide described herein, or combinations thereof; and a transfer vehicle described herein. In some embodiments, the transfer vehicle comprises at least one of (i) an ionizable lipid, (ii) a structural lipid, and (iii) a PEG-modified lipid. In some embodiments, the composition further comprises a targeting moiety. [874] In some embodiments, the therapeutic agents provided herein are formulated in time-released, delayed release, or sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to, cause sensitization of the site to be treated. Such systems can avoid repeated administrations of the therapeutic agent, thereby increasing convenience to the subject and the physician, and may be particularly suitable for certain composition embodiments provided herein. In one embodiment, the compositions of the present disclosure are formulated such that they are suitable for extended-release of the circRNA contained therein. Such extended-release compositions may be conveniently administered to a subject at extended dosing intervals. For example, in one embodiment, the compositions of the present disclosure are administered to a subject twice a day, daily or every other day. In an embodiment, the compositions of the present disclosure are administered to a subject twice a week, once a week, every ten days, every two weeks, every three weeks, every four weeks, once a month, every six weeks, every eight weeks, every three months, every four months, every six months, every eight months, every nine months or annually. [875] In some embodiments, a protein encoded by a polynucleotide is produced by a target cell for sustained amounts of time. For example, the protein may be produced for more than one hour, more than four, more than six, more than 12, more than 24, more than 48 hours, or more than 72 hours after administration. In some embodiments the polypeptide is expressed at a peak level about six hours after administration. In some embodiments the expression of the polypeptide is sustained at least at a therapeutic level. In some embodiments, the polypeptide 644
Attorney Docket No. 01318-0014-00PCT OR-043WO is expressed at least at a therapeutic level for more than one, more than four, more than six, more than 12, more than 24, more than 48, or more than 72 hours after administration. In some embodiments, the polypeptide is detectable at a therapeutic level in patient tissue (e.g., liver or lung). In some embodiments, the level of detectable polypeptide is from continuous expression from the circRNA composition over periods of time of more than one, more than four, more than six, more than 12, more than 24, more than 48, or more than 72 hours after administration. [876] In certain embodiments, a protein encoded by a polynucleotide is produced at levels above normal physiological levels. The level of protein may be increased as compared to a control. In some embodiments, the control is the baseline physiological level of the polypeptide in a normal individual or in a population of normal individuals. In other embodiments, the control is the baseline physiological level of the polypeptide in an individual having a deficiency in the relevant protein or polypeptide or in a population of individuals having a deficiency in the relevant protein or polypeptide. In some embodiments, the control can be the normal level of the relevant protein or polypeptide in the individual to whom the composition is administered. In other embodiments, the control is the expression level of the polypeptide upon other therapeutic intervention, e.g., upon direct injection of the corresponding polypeptide, at one or more comparable time points. [877] In certain embodiments, the levels of a protein encoded by a polynucleotide are detectable at 3 days, 4 days, 5 days, or 1 week or more after administration. Increased levels of protein may be observed in a tissue (e.g., liver or lung). [878] In some embodiments, the method yields a sustained circulation half-life of a protein encoded by a polynucleotide. For example, the protein may be detected for hours or days longer than the half-life observed via subcutaneous injection of the protein or mRNA encoding the protein. In some embodiments, the half-life of the protein is 1 day, 2 days, 3 days, 4 days, 5 days, or 1 week or more. [879] In some embodiments, the modified polynucleotide described herein is a circular RNA that affects net charge, and therefore may be suitable for use with a delivery or transfer vehicle comprising an ionizable lipid. [880] Different types of release delivery systems are available and known to those of ordinary skill in the art. See, e.g., U.S. Patent 5,075,109, U.S. Patents 4,452,775, 4,667,014, 4,748,034, and 5,239,660, U.S. Patents 3,832,253 and 3,854,480. In some embodiments, the therapeutic agent can be conjugated either directly or indirectly through a linking moiety to a targeting moiety. Methods for conjugating therapeutic agents to targeting moieties is known in 645
Attorney Docket No. 01318-0014-00PCT OR-043WO the art. See, e.g., Wadwa et al., J, Drug Targeting 3:111 (1995) and U.S. Patent 5,087,616. In some embodiments, the therapeutic agents provided herein are formulated into a depot form, such that the manner in which the therapeutic agent is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent 4,450,150). [881] In certain embodiments, the compositions may be loaded with diagnostic radionuclide, fluorescent materials or other materials that are detectable in both in vitro and in vivo applications. For example, suitable diagnostic materials may include Rhodamine- dioleoylphosphatidylethanolamine (Rh-PE), Green Fluorescent Protein circRNA (GFP circRNA), Renilla Luciferase circRNA and Firefly Luciferase circRNA. 7. THERAPEUTIC METHODS [882] Provided herein are methods of treating a subject in need thereof comprising administering a therapeutically effective amount of the circular RNA provided herein and/or a composition comprising the circular RNA provided herein. Provided herein are also methods of preventing a disease or disorder in a subject in need thereof comprising a therapeutically effective amount of circular RNA provided herein and/or a composition comprising the circular RNA provided herein. In some embodiments, in addition to the circular RNA, a delivery vehicle, and optionally, a targeting moiety operably connected to the delivery vehicle is administered. [883] In certain aspects, provided herein is a method of producing a protein of interest in a subject in need thereof by introducing or administering a pharmaceutical composition comprising a circular RNA, described herein. [884] In certain embodiments, the therapeutic agents provided herein (e.g., circular RNA and/or composition comprising the circular RNA) are co-administered with one or more additional therapeutic agents (e.g., in the same pharmaceutical composition or in separate pharmaceutical compositions). In some embodiments, the therapeutic agent provided herein can be administered first and the one or more additional therapeutic agents can be administered second, or vice versa. Alternatively, the therapeutic agent provided herein and the one or more additional therapeutic agents can be administered simultaneously. [885] In some embodiments, the subject is a mammal. In some embodiments, the mammal referred to herein can be any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, or mammals of the order Logomorpha, such as rabbits. The mammals may be from the order Carnivora, including Felines (cats) and Canines (dogs). 646
Attorney Docket No. 01318-0014-00PCT OR-043WO The mammals may be from the order Artiodactyla, including Bovines (cows) and Swines (pigs), or of the order Perssodactyla, including Equines (horses). The mammals may be of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). Preferably, the mammal is a human. [886] In some embodiments, provided herein is a method of treating an autoimmune disorder in a subject by introducing or administering the circular RNA construct provided herein and/or a composition comprising the circular RNA construct provided herein. [887] In some embodiments, provided herein is a method of vaccinating a subject by introducing or administering the circular RNA construct provided herein and/or a composition comprising the circular RNA construct provided herein. [888] In these embodiments, a circular RNA vaccine comprises one or more circular RNA polynucleotides, which encode one or more wild type or engineered proteins, peptides or polypeptides (e.g., antigens, adjuvant, or adjuvant-like proteins). In some embodiments, the one or more circular RNA polynucleotide encodes an antigen or adjuvant derived from an infectious agent. In some embodiments the infectious agent from which the antigen or adjuvant is derived or engineered includes, but is not limited to a virus, bacterium, fungus, protozoan, and/or parasite. In some embodiments, the antigen is a viral antigen. In an embodiment, the antigen is a SARS-CoV-2 antigen. In an embodiment, the antigen is SARS-CoV-2 spike protein. In an embodiment, the antigen is selected from or derived from the group consisting of rotavirus, foot and mouth disease virus, influenza A virus, influenza B virus, influenza C virus, H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, human parainfluenza type 2, herpes simplex virus, Epstein-Barr virus, varicella virus, porcine herpesvirus 1, cytomegalovirus, lyssavirus, Bacillus anthracis, anthrax PA and derivatives, poliovirus, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis E, distemper virus, venezuelan equine encephalomyelitis, feline leukemia virus, reovirus, respiratory syncytial virus, Lassa fever virus, polyoma tumor virus, canine parvovirus, papilloma virus, tick borne encephalitis virus, rinderpest virus, human rhinovirus species, Enterovirus species, Mengovirus, paramyxovirus, avian infectious bronchitis virus, human T-cell leukemia-lymphoma virus 1, human immunodeficiency virus-1, human immunodeficiency virus-2, norovirus, lymphocytic choriomeningitis virus, parvovirus B19, adenovirus, rubella virus, yellow fever virus, dengue virus, bovine respiratory syncitial virus, corona virus, Bordetella pertussis, Bordetella bronchiseptica, Bordetella parapertussis, Brucella abortis, Brucella melitensis, Brucella suis, Brucella ovis, Brucella species, Escherichia coli, Salmonella species, Salmonella typhi, 647
Attorney Docket No. 01318-0014-00PCT OR-043WO Streptococci, Vibrio cholera, Vibrio parahaemolyticus, Shigella, Pseudomonas, tuberculosis, avium, Bacille Calmette Guerin, Mycobacterium leprae, Pneumococci, Staphlylococci, Enterobacter species, Rochalimaia henselae, Pasteurella haemolytica, Pasteurella multocida, Chlamydia trachomatis, Chlamydia psittaci, Lymphogranuloma venereum, Treponema pallidum, Haemophilus species, Mycoplasma bovigenitalium, Mycoplasma pulmonis, Mycoplasma species, Borrelia burgdorferi, Legionalla pneumophila, Colstridium botulinum, Corynebacterium diphtheriae, Yersinia entercolitica, Rickettsia rickettsii, Rickettsia typhi, Rickettsia prowsaekii, Ehrlichia chaffeensis, Anaplasma phagocytophilum, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Schistosomes, trypanosomes, Leishmania species, Filarial nematodes, trichomoniasis, sarcosporidiasis, Taenia saginata, Taenia solium, Leishmania, Toxoplasma gondii, Trichinella spiralis, coccidiosis, Eimeria tenella, Cryptococcus neoformans, Candida albican, Aspergillus fumigatus, coccidioidomycosis, Neisseria gonorrhoeae, malaria circumsporozoite protein, malaria merozoite protein, trypanosome surface antigen protein, pertussis, alphaviruses, adenovirus, diphtheria toxoid, tetanus toxoid, meningococcal outer membrane protein, streptococcal M protein, influenza hemagglutinin, cancer antigen, tumor antigens, toxins, clostridium perfringens epsilon toxin, ricin toxin, pseudomonas exotoxin, exotoxins, neurotoxins, cytokines, cytokine receptors, monokines, monokine receptors, plant pollens, animal dander, and dust mites. In some embodiments, the adjuvant is selected from or derived from the group consisting of BCSP31, MOMP, FomA, MymA, ESAT6, PorB, PVL, Porin, OmpA, PepO, OmpU, Lumazine synthase, Omp16, Omp19, CobT, RpfE, Rv0652, HBHA, NhhA, DnaJ, Pneumolysin, Falgellin, IFN-alpha, IFN-gamma, IL-2, IL-12, IL-15, IL-18, IL-21, GM-CSF, IL-1b, IL-6, TNF-a, IL-7, IL-17, IL-1Beta, anti-CTLA4, anti-PD1, anti-41BB, PD-L1, Tim-3, Lag-3, TIGIT, GITR, and anti-CD3. [889] In some embodiments, provided herein is a method of treating cancer in a subject by introducing or administering the circular RNA construct provided herein and/or a composition comprising the circular RNA construct provided herein. [890] In some embodiments, the circular RNA construct encodes a CAR, the CARs have biological activity, e.g., ability to recognize an antigen, e.g., CD19, HER2, or BCMA, such that the CAR, when expressed by a cell, is able to mediate an immune response against the cell expressing the antigen, e.g., CD19, HER2, or BCMA, for which the CAR is specific. Thus, in certain embodiments, provided herein are methods of treating and/or preventing a disease in a subject (e.g., mammalian subject, such as a human subject). Without being bound to a particular 648
Attorney Docket No. 01318-0014-00PCT OR-043WO theory or mechanism, where the circular RNA encodes a CAR, the CARs have biological activity, e.g., ability to recognize an antigen, e.g., CD19 or BCMA, such that the CAR, when expressed by a cell, is able to mediate an immune response against the cell expressing the antigen, e.g., CD19 or BCMA, for which the CAR is specific. In this regard, an embodiment provided herein provides a method of treating or preventing an autoimmune disease in a subject, comprising administering to the subject the circular RNA therapeutic agents, and/or the pharmaceutical compositions provided herein in an amount effective to treat or prevent autoimmune disease in the subject. [891] In some embodiments, the subject has an autoimmune disease or disorder. [892] Adoptive T-cell immunotherapy is a rapidly growing field, in particular in cancer treatments. In general, chimeric antigen receptor (CAR) T cell or “CAR-T” engagement of CD19-expressing cancer cells results in T-cell activation, proliferation and secretion of inflammatory cytokines and chemokines resulting in tumor cell lysis. However, while CAR-T therapies have become an important tool in cancer treatments, they have toxic side effects and involve complex procedures. Treatment with CAR-T can lead to a large and rapid release of cytokines into the blood and can cause cytokine release syndrome (CRS) or CAR-T cell-related encephalopathy syndrome (CRES), also referred to as neurotoxicity associated with CAR-T. CRS is the most common and well-described toxicity associated with CAR-T therapy, occurring in over 90% of patients at any grade and is characterized by high fever, hypotension, hypoxia and/ or multiple organ toxicity and can lead to death. Neurotoxicity is characterized by damage to nervous tissue that can cause tremors, encephalopathy, dizziness or seizures. Additionally, prior to infusion, the patients generally undergo lymphodepletion. Lymphodepletion is known to increase CAR-T cell expansion and enhanced efficacy of infused CAR-T cells by, for example, altering the tumor phenotype and microenvironment. However, lymphodepletion agents often cause side effects to the patients. For example, lymphodepletion can cause neutropenia, anemia, thrombocytopenia, and immunosuppression, leading to a greater risk of infection, along with other toxicities. In addition to the toxicities associated with targeted CAR-T therapies, there are procedures, specialized equipment, and costs involved in producing the modified lymphocytes. CAR-T therapies require an assortment of protocols to isolate, genetically modify, and selectively expand the redirected cells before infusing them back into the patient. [893] In some embodiments, the subject has a cancer selected from the group consisting of: acute myeloid leukemia (AML); alveolar rhabdomyosarcoma; B cell malignancies; bladder 649
Attorney Docket No. 01318-0014-00PCT OR-043WO cancer (e.g., bladder carcinoma); bone cancer; brain cancer (e.g., medulloblastoma and glioblastoma multiforme); breast cancer; cancer of the anus, anal canal, or anorectum; cancer of the eye; cancer of the intrahepatic bile duct; cancer of the joints; cancer of the neck; gallbladder cancer; cancer of the pleura; cancer of the nose, nasal cavity, or middle ear; cancer of the oral cavity; cancer of the vulva; chronic lymphocytic leukemia; chronic myeloid cancer; colon cancer; esophageal cancer, cervical cancer; fibrosarcoma; gastrointestinal carcinoid tumor; head and neck cancer (e.g., head and neck squamous cell carcinoma); Hodgkin lymphoma; hypopharynx cancer; kidney cancer; larynx cancer; leukemia; liquid tumors; lipoma; liver cancer; lung cancer (e.g., non-small cell lung carcinoma, lung adenocarcinoma, and small cell lung carcinoma); lymphoma; mesothelioma; mastocytoma; melanoma; multiple myeloma; nasopharynx cancer; non-Hodgkin lymphoma; B-chronic lymphocytic leukemia; hairy cell leukemia; Burkitt's lymphoma; ovarian cancer; pancreatic cancer; cancer of the peritoneum; cancer of the omentum; mesentery cancer; pharynx cancer; prostate cancer; rectal cancer; renal cancer; skin cancer; small intestine cancer; soft tissue cancer; solid tumors; synovial sarcoma; gastric cancer; teratoma; testicular cancer; thyroid cancer; and ureter cancer. [894] In some embodiments, the subject has an autoimmune disorder selected from scleroderma, Grave's disease, Crohn's disease, Sjogren's disease, multiple sclerosis, Hashimoto's disease, psoriasis, myasthenia gravis, autoimmune polyendocrinopathy syndromes, Type I diabetes mellitus (TIDM), autoimmune gastritis, autoimmune uveoretinitis, polymyositis, colitis, thyroiditis, and the generalized autoimmune diseases typified by human Lupus. EXAMPLES [895] Wesselhoeft et al., (2019) RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In vivo. Molecular Cell. 74(3), 508-520; Wesselhoeft et al., (2018) Engineering circular RNA for Potent and Stable Translation in Eukaryotic Cells. Nature Communications. 9, 2629; and M. Puttaraju et al, (1992) Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons. Nucleic Acids Research. 20(20): 5357-5364 are incorporated by reference in their entirety. [896] The disclosure is further described in detail by reference to the following examples but are not intended to be limited to the following examples. These examples encompass any and all variations of the illustrations with the intention of providing those of ordinary skill in the art with complete disclosure and description of how to make and use the subject disclosure and are not intended to limit the scope of what is regarded as the disclosure. 650
Attorney Docket No. 01318-0014-00PCT OR-043WO EXAMPLE 1: Synthetic Internal Ribosome Entry Site (IRES) Design and Engineering [897] 2,838 naturally occurring internal ribosome entry sites (IRES) sequences were collected and/or derived from publicly available viral genomic databases. The IRES sequences were inserted into circular RNAs encoding a luciferase reporter protein (e.g., Gaussia Luciferase); the resulting circular RNAs were transfected into primary human hepatocytes (PHH), T cell lymphoma (TCL), and/or myotubes and analyzed for luciferase expression levels as compared to comparative IRES 1. IRESs were characterized as low expressing based on the fold change calculated as compared to comparative IRES 1 (i.e., if the circular RNA comprising the tested IRES had a lower or similar value of relative light units (fluorescent activity) as compared to a circular RNA comprising the comparative IRES 1, it was characterized as low expressing). IRES constructs with a fold change value of less than 1.0 were characterized as low expressing. IRESs were characterized as high expressing based on a fold change value of 1.0 or greater as compared to comparative IRES 1 (i.e., if the circular RNA comprising the tested IRES had a higher value of relative light units as compared to a circular RNA comprising the comparative IRES 1, it was characterized as high expressing). [898] For both low and high expressing IRES sequences, sequence structure clustering was performed. The resulting clustering is shown in FIG. 1. [899] The IRES sequences were analyzed as compared to an exemplary Type I IRES (e.g., CVB3 IRES). Folding constraints were generated for one or more IRES sequences to aid with alignment of the tested IRES to the exemplary IRES. Sequences between domains (e.g., linker spaces) were defined and the domains themselves were folded between the linker spaces in a commercially or publicly available folding software (e.g., RNAFOLD, Vienna). Domains for one or more IRES were identified based on sequence homology to the exemplary IRES (e.g., CVB3 IRES). For example, tested IRES sequences belonging to Type I IRES were analyzed for homology of domains and annotated for similarities and differences in the sequences as compared to an annotated CVB3 IRES (annotated CVB3 IRES is illustrated in FIG. 2). The IRES group I intron sequences were aligned to correspond with one or more domains of the CVB3 IRES. [900] The naturally occurring IRES was subject to engineering using one or more deletions, substitutions, and/or additions at the primary structure, including the inclusion or deletion of structural motifs present in high expressing naturally occurring IRES to form a 651
Attorney Docket No. 01318-0014-00PCT OR-043WO synthetic set of IRES. Following the engineering, the synthetic IRES or one or more of its domains was assessed using the folding software, reviewed for its initial folding conformation (i.e., the structure shown by folding software prior to using folding constraints), and given folding constraints and refolded using the folding software (i.e., constrained folded IRES). The initial folding conformation and constrained folded IRES variations were compared to other IRES (e.g., CVB3) to select for and/or generate: a desired shift in subdomain or domain structure (for IRES variations having additions and/or deletions (e.g., for C-loop variants or cryptic codons); locations of predicted favorable truncations; optimal locations for aptamer additions; or other alterations to form variations of synthetic IRES. [901] Certain synthetic IRESs were engineered to comprise specific point mutations along a naturally occurring IRES (e.g., sequences 1-1 through 1-34 of Table 1). [902] Certain synthetic IRESs were engineered to comprise cryptic codons mutations, wherein either whole cryptic codons were deleted or one or two nucleotides or nucleosides (i.e., altering the cryptic codon into a noncryptic codon) were deleted from the naturally occurring IRES (e.g., sequences 1-35 through 1-47 of Table 1). [903] Certain synthetic IRESs were engineered to add a Kozak sequence, a AUN-AUN- AUN-AUN-AUN sequence (i.e., N= “A” or “U”), a polyA sequence, or an aptamer, e.g., EIF4E aptamer sequence (e.g., at the end of Domain IV) to a naturally occurring IRES (e.g., sequences 1-48 through 1-58 of Table 1). [904] Certain synthetic IRESs were engineered to comprise an additional domain(s) or part(s) of domain(s) as compared to a naturally occurring IRES. Certain synthetic IRESs were engineered such that whole domain(s) or part(s) of domain(s) of a first IRES was replaced by that of a second IRES (e.g., sequences 1-59 through 1-63, 1-140 through 1-189, 1-225 through 1-232, 1-239 through 1-295 of Table 1). For example, a J/K-L loop of a first IRES was replaced by a domain, e.g., Domain V, of a second IRES (sequences 1-99 through 1-101 of Table 1). In the alternative, the Domain V of a first IRES was replaced by a J/K-L loop of a second IRES (sequences 1-102 through 1-104 of Table 1). [905] Certain synthetic IRESs were engineered to delete a SL2 region compared to a naturally occurring IRES. Certain synthetic IRESs were engineered to delete a SL3 region compared to a naturally occurring IRES. Certain synthetic IRESs were engineered to delete Domain II compared to a naturally occurring IRES. Certain synthetic IRESs were engineered to delete Domain III compared to a naturally occurring IRES. (See, e.g., sequences 1-198 through 1-203 of Table 1.) 652
Attorney Docket No. 01318-0014-00PCT OR-043WO [906] Certain synthetic IRESs were engineered to replace a SL1 region with an EIF4E aptamer sequence (e.g., Aptamer EIF4E sequence 1 or 2). Certain synthetic IRESs were engineered to replace a SL2 region with an EIF4E aptamer sequence (e.g., Aptamer EIF4E sequence 1 or 2). Certain synthetic IRESs were engineered to replace a SL3 region with an EIF4E aptamer sequence (e.g., Aptamer EIF4E sequence 1 or 2). Certain synthetic IRESs were engineered to replace a stem loop region with an EIF4E aptamer sequence (e.g., Aptamer EIF4E sequence 1 or 2). [907] Certain synthetic IRESs were engineered by adding a EIF4E aptamer sequence to the 3’ end of a domain, e.g., Domain IV, of a naturally occurring IRES, without deleting any nucleotides of the naturally occurring IRES (e.g., sequences 1-203 through 1-220, 1-221 through 1-224, and 1-233 through 1-235 of Table 1). [908] Certain synthetic IRESs were engineered by mutating Domain IV of naturally occurring Type I IRES in the C-loop region or in other stem loop region(s) (e.g., sequences 1- 64 through 1-99 of Table 1). [909] Certain synthetic IRESs were engineered to add a PTB or PPT sequence to a naturally occurring IRES that does not naturally comprise the PTB or PPT sequence. Certain synthetic IRESs were engineered to mutate a PTB or PPT sequence within a naturally occurring IRES. Certain synthetic IRESs were engineered such that a PTB or PPT sequence of a first IRES was replaced by that of a second IRES. (See, e.g., sequences 1-105 through 1-139 of Table 1). [910] Certain synthetic IRESs were engineered to delete a negative ITAF sequence(s) of a naturally occurring IRES. Certain synthetic IRESs were engineered to mutate a negative ITAF sequence(s) of a naturally occurring IRES. (See, e.g., sequences 1-190 through 1-197 of Table 1.) [911] Certain synthetic IRESs were engineered to add an AUG sequence to the 3’ end of a Kozak sequence from the naturally occurring IRES (e.g., sequences 1-236 through 1-238 of Table 1). [912] Certain additional synthetic IRESs were engineered (e.g., sequences 1-307 through 1-314 of Table 1). [913] In the experiments described herein, sequences 1-296 through 1-306 of Table 1 were used as exemplary controls. EXAMPLE 2: 653
Attorney Docket No. 01318-0014-00PCT OR-043WO Synthesis, Formulation, and Transfection of Synthetic IRES [914] Synthetic IRESs from Example 1 were inserted into circular RNAs. The circular RNAs were engineered from a DNA template comprising intron sequences, exon sequences, a synthetic IRES, an expression sequence encoding a Gaussia luciferase, and XbaI site. DNA templates were randomized onto a plate prior to synthesis. Each plate contained 2 µL of 1µg/µL plamid and 8 µL of pure water. The DNA templates were linearized using XBaI and then allowed to undergo an in vitro synthesis (IVT) reaction. The IVT product was purified to remove unspliced RNA and introns using oligo dT purification methods. Buffer exchange was performed on the IVT products using SPRI beads (via RNAXP clean) to yield purified circular RNA. The purified circular RNA solutions were normalized to 90 ng/µL and quality controlled on a commercially available fragment analyzer. [915] 50 ng of the engineered circular RNAs were formulated into lipid nanoparticles (LNP) comprising Lipid 86 from Table 4. The LNPs formulated with circular RNAs were transfected in vitro into activated primary human T-cells or cynomolgus T-cells. After 24 hours, luciferase expression was determined. [916] The resulting luciferase expression was provided in relative light units (RLU), fold change, and calculated Z score values relative to comparative IRES 1 were provided in Table X (for primary human T cells) and Table Y (cynomolgus T cells below) for IRESs comprising sequences 1-1 through 1-306 of Table 1. Mutating Domain I was shown to improve expression as compared to control (IRES with unmutated Domain I). Retaining Domain II of a naturally occurring IRES was shown to improve expression as compared to control (synthetic IRES comprising one or more mutations in the domain). Retaining Domain III of a naturally occurring IRES was shown to improve expression as compared to control (synthetic IRES comprising one or more mutations in the domain). Retaining Domain IV of a naturally occurring IRES was shown to improve expression as compared to control (synthetic IRES comprising one or more mutations in the domain). Retaining Domain V of a naturally occurring IRES was shown to improve expression as compared to control (synthetic IRES comprising one or more mutations in the domain). [917] Replacing Domain IV from a first IRES having lower expression, as determined by the assessment described above, with that of a second IRES having higher expression was shown to improve expression as compared to control (un-engineered naturally occurring first IRES). As shown in the table below, adding a EIF4 aptamer sequence to the 5’ end of a naturally occurring IRES can improve expression as compared to control (IRES without added 654
Attorney Docket No. 01318-0014-00PCT OR-043WO EIF4 aptamer). As shown in the table below, adding a EIF4G/EIF3 aptamer sequence to Domain IV, e.g., to the two- or three-nt bulges of Domain IV, of a naturally occurring IRES can improve expression as compared to control (IRES without added EIF4G/EIF3 aptamer). As shown in the table below, adding a PPT sequence in Domain IV to a naturally occurring IRES was shown to improve expression as compared to control (IRES without having a PPT sequence). As shown in the table below, retaining a PPT sequence in Domain IV of a naturally occurring IRES was shown to improve expression as compared to control (synthetic IRES comprising one or more mutations in PPT sequence). Mutating GNRA tetra loops in Domain IV was shown to improve expression as compared to control (IRES with unmutated GNRA tetra loops). Mutating or deleting a negative ITAF sequence was shown to improve expression as compared to control (IRES with retained negative ITAF sequence). Replacing the post- Domain VII terminal stem of a naturally occurring IRES with the AUN repeat described above, a Kozak sequence, or a 11-nucleotide consensus sequence of a Type I IRES (i.e., AACACAACAAA) was shown to improve expression as compared to control (IRES without terminal stem replacement). Adding a cryptic codon to a naturally occurring IRES was shown to improve expression as compared to control (IRES without added cryptic codon). Table X: Primary Human T Cells
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660
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661
Attorney Docket No. 01318-0014-00PCT OR-043WO
Table Y: Cynomolgus T cells
662
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663
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664
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665
Attorney Docket No. 01318-0014-00PCT OR-043WO
666
Attorney Docket No. 01318-0014-00PCT OR-043WO
667
Attorney Docket No. 01318-0014-00PCT OR-043WO
668
Attorney Docket No. 01318-0014-00PCT OR-043WO
INCORPORATION BY REFERENCE [918] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated as being incorporated by reference herein. [919] This description and exemplary embodiments should not be taken as limiting. For 669
Attorney Docket No. 01318-0014-00PCT OR-043WO the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages, or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about,” to the extent they are not already so modified. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. 670