WO2025250751A1

WO2025250751A1 - Circular rna compositions and methods

Info

Publication number: WO2025250751A1
Application number: PCT/US2025/031358
Authority: WO
Inventors: Akinola Emmanuel; Ganapathy Subramanian SANKARAN; Isin DALKILIC-LIDDLE; Karolina Anna KOSAKOWSKA; Muthusamy Jayaraman
Original assignee: Orna Therapeutics Inc; Renagade Therapeutics Management Inc
Current assignee: Orna Therapeutics Inc; Renagade Therapeutics Management Inc
Priority date: 2024-05-31
Filing date: 2025-05-29
Publication date: 2025-12-04
Anticipated expiration: 2026-11-30

Abstract

Circular RNA, along with related compositions, methods, and precursor RNA, are described herein. In some embodiments, the composition comprises a transfer vehicle and a circular RNA comprising a 3' self-spliced exon segment, a translation initiation element (TIE), an expression sequence encoding a chimeric antigen receptor (CAR), and a 5' self-spliced exon segment. In some embodiments, the transfer vehicle comprises an ionizable lipid disclosed herein. In some embodiments, the circular RNA and/or composition thereof has improved expression, functional stability, immunogenicity, ease of manufacturing, and/or half-life as compared to linear RNA. Also presented herein are methods and compositions for the manufacture and preparation of circularized RNAs, along with methods of administering said circular RNAs and related compositions to a subject in need thereof for treatment or prevention purposes.

Description

CIRCULAR RNA COMPOSITIONS AND METHODS

SEQUENCE LISTING

[1] The present application contains a Sequence Listing which has been submitted electronically in XML format. Said XML copy, created on August 24, 2024, is named “01318- 0015-00PCT_SL.xml” and is 32,780,395 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

[2] FIGS. 1 A-1D show % CD45, CD3, CD20, and CD19 cells following administration of a composition comprising LNP A or LNP B in combination with oRNA expressing a reporter (VHH) or a CD 19 CAR or a CD20 CAR, or a PBS control.

[3] FIGS. 2A-2C show hCD45+ cell frequency and cell count in the peripheral blood at 24 hours following administration of a composition comprising LNP A or LNP B in combination with linear mRNA expressing a reporter (VHH), oRNA expressing a reporter (VHH) or a CD 19 CAR or a CD20 CAR, or a PBS control.

[4] FIGS. 3A-3B show hCD45+ cell frequency and cell count in the peripheral blood at 7 days following administration of a composition comprising LNP A or LNP B in combination with oRNA expressing a reporter (VHH) or a CD 19 CAR or a CD20 CAR, or a PBS control.

[5] FIGS. 4A-4D show hCD45+, hCD20+, hCD19+, hCD3+ reporter (VHH) expression in the peripheral blood at 24 hours following administration of a composition comprising LNP A or LNP B in combination with oRNA expressing a reporter (VHH), or a PBS control.

[6] FIGS. 5A-5D show hCD45+, hCD20+, hCD19+, hCD3+ reporter (VHH) expression in the peripheral blood at 24 hours following administration of a composition comprising LNP A in combination with linear mRNA or oRNA expressing a reporter (VHH), or a PBS control.

[7] FIGS. 6A-6D show hCD45+, hCD20+, hCD19+, hCD3+ reporter (VHH) expression in the peripheral blood at 7 days following administration of a composition comprising LNP A or LNP B in combination with oRNA expressing a reporter (VHH), or a PBS control.

[8] FIGS. 7A-7D show hCD45+, hCD20+, hCD19+, hCD3+ reporter (VHH) expression in the spleen at 24 hours following administration of a composition comprising LNP A in combination with linear mRNA or oRNA expressing a reporter (VHH), or a PBS control.

[9] FIGS. 8A-8D show hCD45+, hCD20+, hCD19+, hCD3+ reporter (VHH) expression in the spleen at 7 days following administration of a composition comprising LNP A or LNP B in combination with oRNA expressing a reporter (VHH), or a PBS control.

[10] FIGS. 9A-9D show hCD45+, hCD20+, hCD19+, hCD3+ reporter (VHH) expression in bone marrow (lymphocyte and T cell) at 24 hours following administration of a composition comprising LNP A in combination with linear mRNA or oRNA expressing a reporter (VHH), or a PBS control.

[11] FIGS. 10A-10D show hCD45+, hCD20+, hCD19+, hCD3+ reporter (VHH) expression in bone marrow (lymphocyte and T cell) at 7 days following administration of a composition comprising LNP A or LNP B in combination with oRNA expressing a reporter (VHH), or a PBS control.

[12] FIGS. 11 A-l 1B show hCD45+ and hCD3+ CAR expression in peripheral blood at 24 hours following administration of a composition comprising LNP A or LNP B in combination with oRNA expressing CD 19 CAR or CD20 CAR, or a PBS control.

[13] FIGS. 12A-12B show hCD45+ and hCD3+ CAR expression in peripheral blood at 7 days following administration of a composition comprising LNP A or LNP B in combination with oRNA expressing CD 19 CAR or CD20 CAR, or a PBS control.

[14] FIGS. 13A-13B show hCD45+ and hCD3+ CAR expression in spleen at 24 hours following administration of a composition comprising LNP A in combination with oRNA expressing CD 19 CAR or CD20 CAR, or a PBS control.

[15] FIGS. 14A-14B show hCD45+ and hCD3+ CAR expression in spleen at 7 days following administration of a composition comprising LNP A or LNP B in combination with oRNA expressing CD 19 CAR or CD20 CAR, or a PBS control.

[16] FIGS. 15A-15B show hCD45+ and hCD3+ CAR expression in bone marrow at 24 hours following administration of a composition comprising LNP A combination with oRNA expressing CD 19 CAR or CD20 CAR, or a PBS control.

[17] FIGS. 16A-16B show hCD45+ and hCD3+ CAR expression in bone marrow at 7 days following administration of a composition comprising LNP A or LNP B in combination with oRNA expressing CD 19 CAR or CD20 CAR, or a PBS control.

[18] FIGS. 17A-17B show hCD20+ and hCD19+ cell frequency in peripheral blood (B cell depletion) at 24 hours following administration of a composition comprising LNP A or LNP B in combination with linear mRNA expressing reporter (VHH) or oRNA expressing reporter (VHH) or CD 19 CAR or CD20 CAR, or a PBS control.

[19] FIGS. 18A-18B show hCD20+ and hCD19+ cell frequency in peripheral blood (B cell depletion) at 7 days following administration of a composition comprising LNP A or LNP B in combination with oRNA expressing reporter (VHH) or CD 19 CAR or CD20 CAR, or a PBS control.

[20] FIGS. 19A-19C show hCD20+ and hCD19+ cell count in peripheral blood (B cell depletion) at 24 hours following administration of a composition comprising LNP A or LNP B in combination with linear mRNA expressing reporter (VHH) or oRNA expressing reporter (VHH) or CD 19 CAR or CD20 CAR, or a PBS control.

[21] FIGS. 20A-20C show hCD20+ and hCD19+ cell count in peripheral blood (B cell depletion) at 7 days following administration of a composition comprising LNP A or LNP B in combination with oRNA expressing reporter (VHH) or CD 19 CAR or CD20 CAR, or a PBS control.

[22] FIGS. 21A-21B show hCD20+ and hCD19+ cell frequency in spleen (B cell depletion) at 24 hours following administration of a composition comprising LNP A in combination with linear mRNA expressing reporter (VHH) or oRNA expressing reporter (VHH) or CD 19 CAR or CD20 CAR, or a PBS control.

[23] FIGS. 22A-22B show hCD20+ and hCD19+ cell frequency in spleen (B cell depletion) at 7 days following administration of a composition comprising LNP A or LNP B in combination with oRNA expressing reporter (VHH) or CD 19 CAR or CD20 CAR, or a PBS control.

[24] FIGS. 23A-23B show hCD20+ and hCD19+ cell frequency in bone marrow (B cell depletion) at 24 hours following administration of a composition comprising LNP A in combination with linear mRNA expressing reporter (VHH) or oRNA expressing reporter (VHH) or CD 19 CAR or CD20 CAR, or a PBS control.

[25] FIGS. 24A-24B show hCD20+ and hCD19+ cell frequency in bone marrow (B cell depletion) at 7 days following administration of a composition comprising LNP A or LNP B in combination with oRNA expressing reporter (VHH) or CD 19 CAR or CD20 CAR, or a PBS control.

[26] FIGS. 25A-25B show % CD19 CAR+ T cells using two cynomolgus donors following delivery of a composition comprising LNP A or LNP B in combination with oRNA expressing CD19 CAR, or mock control. FIGS. 25C-25D show % CD20 CAR+ T cells using the same two cynomolgus donors following delivery of a composition comprising LNP A or LNP B in combination with oRNA expressing CD20 CAR, or mock control.

[27] FIGS. 26A-26D show % CD19 CAR+ T cells using cynomolgus donor following delivery of a composition comprising LNP A or LNP B in combination with oRNA expressing CD 19 CAR, or mock control. FIGS. 26E-26H show % CD20 CAR+ T cells using human donor following delivery of a composition comprising LNP A or LNP B in combination with oRNA expressing CD20 CAR, or mock control.

[28] FIGS. 27A-27B show % CD19 CAR+ T cells using a human donor following delivery of a composition comprising LNP A or LNP B in combination with oRNA expressing CD 19 CAR, or mock control. FIGS. 27C-27D show % CD20 CAR+ T cells using a human donor following delivery of a composition comprising LNP A or LNP B in combination with oRNA expressing CD20 CAR, or mock control.

[29] FIGS. 28A-28B show % reporter (VHH)+ T cells using two cynomolgus donors following delivery of a composition comprising LNP A or LNP B in combination with linear mRNA or oRNA expressing reporter (VHH), or mock control.

[30] FIGS. 29A-29D show reporter (VHH) expression and gMFI in T cells using cynomolgus donor following delivery of a composition comprising LNP A or LNP B in combination with linear mRNA or oRNA expressing reporter (VHH), or mock control.

[31] FIGS. 30A-30B show reporter (VHH) expression and gMFI in T cells using a human donor following delivery of a composition comprising LNP A or LNP B in combination with linear mRNA or oRNA expressing reporter (VHH), or mock control.

DETAILED DESCRIPTION

[32] Circular RNA (circRNA or oRNA®) is a stable form of RNA that provides an advantage compared to linear RNA in structure and function, especially in the case of molecules that are prone to folding in an inactive conformation (Wang and Ruffner, 1998). 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 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. 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 protein replacement therapy and vaccination.

[33] 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 toxi cities. In addition to the toxi cities 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.

[34] In anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus, autologous T cells from five SLE patients “were transduced with a lentiviral anti-CD19 CAR vector, expanded and reinfused . . . into the patients after lymphodepletion with fludarabine and cyclophosphamide. CAR T cells expanded in vivo led to deep depletion of B cells, improvement of clinical symptoms and normalization of laboratory parameters including seroconversion of anti-double-stranded DNA antibodies. Remission of SLE according to DORIS criteria was achieved in all five patients after 3 months and the median (range) Systemic Lupus Erythematosus Disease Activity Index score after 3 months was 0 (2).” See Mackensen et al., Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus, Nature Medicine (2022); see also Nunez et al., Cytokine and reactivity profiles in SLE patients following anti-CD19 CART therapy, Molecular Therapy (2023).

[35] Because circRNAs are more stable and can be expressed in tissue-specific manner, and because using circRNAs can avoid the lymphodepletion step of traditional therapies, circRNAs provide an attractive alternative to traditional CAR therapies and other therapies. Accordingly, provided herein are circular RNA constructs that comprise an internal ribosome entry site (IRES) and at least one expression sequence encoding a binding molecule. In certain embodiments, the binding molecule encodes a CAR that targets a cancer antigen, for use in treating cancer. The circular RNA can be formulated with a transfer vehicle to facilitate and/or enhance the delivery and release of circRNA to one or more target cells. Accordingly, lipid nanoparticles (LNPs) or other transfer vehicles containing ionizable lipids may be used to deliver the circular RNA described herein, for example, to a patient in need of treatment.

[36] 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.

[37] 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.

[38] 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.

[39] 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.

[40] 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 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

[41] Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings:

[42] 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 moi eties 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.

[43] 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. [44] 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% 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.

[45] 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 selfspliced exon segment.

[46] 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.

[47] 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.

[48] 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 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.

[49] 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 Table

1 below.

Table 1 : Exemplary Splice Site Dinucleotides

[50] As used herein, the term “permutation site” refers to a site in an intron and/or exon (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.

[51] 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.

[52] 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.”

[53] As used herein, “accessory element” or “accessory sequences” refer 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. [54] 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 poly AC 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 bound and removed and wanted species, such as circular RNA, are eluted and separated from unwanted species.

[55] 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.

[56] 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. [57] 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, CHI, CH2 and CH3. Each light chain can comprise a light chain variable region (abbreviated herein as VL) and a light chain constant region. The 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 carboxyterminus 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 singlechain 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.

[58] 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 IgGl, IgG2, IgG3 and IgG4. “Isotype” refers to the Ab class or subclass (e.g., IgM or IgGl) 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 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.

[59] 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.

[60] 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.

[61] 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, 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.

[62] 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, 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, and thyroiditis, as well as in the generalized autoimmune diseases typified by human Lupus.

[63] “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.

[64] 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.

[65] “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 (K_D or K_d). Affinity may be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (K_D), and equilibrium association constant (K_A or K_a). The K_D is calculated from the quotient of k_off/k_on, whereas K_A is calculated from the quotient of k_on/k_off. kon refers to the association rate constant of, e.g., an antibody to an antigen, and koir refers to the dissociation of, e.g., an antibody to an antigen. The k_on and koir may be determined by techniques known to one of ordinary skill in the art, such as BIACORE® or KinExA.

[66] 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.

[67] 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.

[68] 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, nonHodgkin'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 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 carcinoma, 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, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, 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.

[69] 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.

[70] 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 linear starting material.

[71] 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 selfcleaving 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.

[72] 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.

[73] 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).

[74] 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.

[75] 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), 0X40 ligand, PD-L2, or programmed 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), 0X40, PD-1, or tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT).

[76] 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, CD 19a, 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), 0X40, PAG/Cbp, PD-1, PSGL1, SELPLG (CD 162), signaling lymphocytic activation molecule, SLAM (SLAMF1; CD 150; 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.

[77] 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 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).

[78] 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. Examples of acute phase-proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[79] 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.

[80] 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.

[81] 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 doublestranded. In most cases, genomic DNA is double-stranded.

[82] 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.

[83] 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 circular RNA polynucleotide).

[84] 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.

[85] As used herein, the term “heterologous” means from any source other than naturally occurring sequences.

[86] 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.

[87] An “eukaryotic initiation factor” or “elF” refers to a protein or protein complex used in assembling an initiator tRNA, 40S and 60S ribosomal subunits required for initiating eukaryotic translation.

[88] 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 Crystallogr 56(Pt 10): 1316-1323).

[89] 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.”

[90] 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.

[91] 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.

[92] 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.

[93] 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 predetermined 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 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.

[94] 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.

[95] 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.

[96] 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.

[97] 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.

[98] As used herein, a “terminal untranslated sequence” is a region of polynucleotide 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.

[99] 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.

[100] 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.

[101] 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.

[102] 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 selfcleave. 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 is capable of cleaving the splice site nucleotide of the terminal element.

[103] 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.

[104] 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.

[105] 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.

[106] “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 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. The “modified” nucleotide or nucleoside may be naturally occurring (e.g., pseudouridine) or synthetic. 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 modification, 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. 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 polynucleotide sequence contains at least one nucleotide or nucleoside having 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).

[107] 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.

[108] 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., 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-m ethyl deoxy guanosine, deaza- or aza-purines, deaza- or azapyrimidines, 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).

[109] 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.

[110] As used herein, “poly A” 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.

[ 111] 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).

[112] 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, Vai, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, 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.

[113] 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.

[114] 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.

[115] 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 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.

[116] 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.

[117] 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).

[118] 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.

[119] As used herein, “translation” refers to the formation of a polypeptide molecule by a ribosome based upon an RNA template.

[120] 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.

[121] 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.

[122] 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.

[123] 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 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.

[124] 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.

[125] 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

[126] 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.

[ 127] 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.

[128] 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.

[129] As used herein, the term “PEG” means any polyethylene glycol or other polyalkylene ether polymer.

[130] 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 on the lipid.

[131] 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.

[132] 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.

[133] 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.

[134] 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 C_6-20 alkyl) and at least one hydrophilic head-group (e.g., imidazole), each bound to a cleavable group (e.g., disulfide).

[135] 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.

[136] 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 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.

[137] 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.

[138] 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.

[139] 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.

[140] 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 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

[141] 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.

[142] 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.

[143] When a range of values is listed, it is intended to encompass each value and subrange within the range. For example, “C_1-6 alkyl” is intended to encompass, C₁, C₂, C₃, C₄, C₅, c₆, C_1-6, C_1-5, C_1-4, C_1-3, C_1-2, C_2-6, C_2-5, C_2-4, C_2-3, C_3-6, C_3-5, C_3-4, C_4-6, C_4-5, and C_{5 -6} alkyl.

[144] 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 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.

[145] As used herein, the term “alkyl” refers to both straight and branched chain C_1-40 hydrocarbons (e.g., C_6-20 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, “C_6-20” 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 (“C_1-10 alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C_1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C_1-8 alkyl”). In some embodiments, an alkyl group has

1 to 7 carbon atoms (“C_1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C_1-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C_1-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C_1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C_1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C_1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C₁ alkyl”). Examples of C_1-6 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, and the like.

[146] 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 carboncarbon triple bonds (e.g., 1, 2, 3, or 4 carbon-carbon triple bonds) (“C_2-20 alkenyl”). In certain embodiments, alkenyl does not contain any triple bonds. In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C_2-10 alkenyl”). In some embodiments, an alkenyl group has

2 to 9 carbon atoms (“C_2-9 alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C_2-8 alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C_2- ₇ alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C_2-6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C_2-5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C_2-4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C_2-3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C₂ alkenyl”). The one or more carboncarbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C_2-4 alkenyl groups include ethenyl (C₂), 1-propenyl (C₃), 2-propenyl (C₃), 1- butenyl (C₄), 2-butenyl (C₄), butadienyl (C₄), and the like. Examples of C_2-6 alkenyl groups include the aforementioned C_2-4 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.

[147] 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) (“C_2-20 alkynyl”). In certain embodiments, alkynyl does not contain any double bonds. In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C_2-10 alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C_2-9 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C_2-8 alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C_2- ₇ alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C_2-6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C_2-5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C_2-4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C_2-3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C₂ alkynyl”). The one or more carboncarbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C_2-4 alkynyl groups include, without limitation, ethynyl (C₂), 1-propynyl (C₃), 2- propynyl (C₃), 1-butynyl (C₄), 2-butynyl (C₄), and the like. Examples of C_2-6 alkenyl groups include the aforementioned C_2-4 alkynyl groups as well as pentynyl (C₅), hexynyl (C₆), and the like. Additional examples of alkynyl include heptynyl (C₇), octynyl (C₈), and the like.

[148] 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.

[149] 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.

[150] 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).

[151] 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 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:

[152] 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 "C_4-8 cycloalkyl," derived from a cycloalkane. Exemplary cycloalkyl groups include, but are not limited to, cyclohexanes, cyclopentanes, cyclobutanes and cyclopropanes.

[153] As used herein, “cyano” refers to -CN.

[154] 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 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).

[155] 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. [156] 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.

[157] As used herein, “oxo” refers to -C=O.

[158] 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 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.

[159] 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; — (C_1-4 straight or branched alkylene)O— N(R°)₂; or — (C_1-4 straight or branched alkylene)C(O)O— N(R°)₂, wherein each R° may be substituted as defined below and is independently hydrogen, C_1-6 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.

[160] 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^●, — (C_1-4 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 C_1-4 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.

[161] 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, C_1-6 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, C_1-6 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.

[162] 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, wherein each R^● is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently C_1-4 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.

[163] Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include wherein each is independently hydrogen, C_1-6 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 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.

[164] Suitable substituents on the aliphatic group of 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 C_1-4 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.

[165] 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.

[166] 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 the heteroatoms.

[167] 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.

[168] 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, carb oxami doaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy, aminocarboxamidoalkyl, cyano, alkoxyalkyl, perhaloalkyl, arylalkyloxyalkyl, and the like. In some embodiments, the substituent is selected from cyano, halogen, hydroxyl, and nitro. [ 169] 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, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 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⁺(C_1-4alkyl)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.

[170] 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. [171] 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, 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.

[172] 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.

[173] 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

[174] Provided herein are circular RNA constructs and related pharmaceutical compositions comprising transfer vehicles, wherein the circular RNA constructs are capable of in vivo delivery to immune cells for therapy or production of proteins. According to the present disclosure, the circular RNA provided herein can be injected into an animal (e.g., a human), such that a polypeptide encoded by the circular RNA molecule is expressed inside the animal, for example by immune cells and T cells.

[175] In some embodiments, provided herein are circular RNA polynucleotides comprising, optionally in the following order, a 3' self-spliced exon segment, optionally a first spacer, a translation initiation element (TIE) (e.g., comprising an Internal Ribosome Entry Site (IRES)), an expression sequence encoding a binding molecule (e.g., encoding a chimeric antigen receptor (CAR)), optionally a second spacer, and a 5' self-spliced exon segment.

[176] In certain embodiments, a circular RNA constructed is formulated into a pharmaceutical composition. In certain embodiments, the pharmaceutical composition comprises a transfer vehicle. In certain embodiments, a circular RNA construct comprising a TIE and at least one expression sequence encoding a binding molecule is formulated into a pharmaceutical composition comprising a transfer vehicle.

[177] In certain embodiments, pharmaceutical compositions comprising a circular RNA construct comprising a TIE and at least one expression sequence encoding a binding molecule, and a transfer vehicle are disclosed. In certain embodiments, the transfer vehicle facilitates and/or enhances the delivery and release of circular RNA to one or more target cells.

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

[179] In certain embodiments, the circular RNA constructs comprise a TIE and at least one expression sequence encoding a cytokine, immune checkpoint inhibitor, agonist, chimeric antigen receptor (CAR), inhibitory receptor agonist, one or more T-Cell Receptors, and/or B- cell Receptors.

[180] In some embodiments, a 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 circular RNA molecule is delivered in the transfer vehicle and each circular RNA encodes a separate subunit of the protein. Alternatively, a single circular RNA may be engineered to encode more than one subunit. In certain embodiments, separate circular RNA molecules encoding the individual subunits may be administered in separate transfer vehicles.

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

[182] The circular RNA is produced by transcription of a DNA template that results in formation of a precursor linear RNA polynucleotide capable of circularizing. Linear precursor RNA polynucleotides are provided for producing circular RNA constructs and related pharmaceutical compositions. The DNA template shares the same sequence as the precursor linear RNA polynucleotide prior to splicing of the precursor linear RNA polynucleotide. The DNA template shares the same sequence as the precursor linear RNA polynucleotide prior to splicing of the precursor linear RNA polynucleotide. In some embodiments, said linear precursor RNA polynucleotide undergoes splicing to remove a 3’ intron element and 5’ intron element during the process of circularization. In some embodiments, the resulting circular RNA polynucleotide lacks a 3’ intron element and a 5’ intron element, but maintains a 3’ exon element, an intervening region comprising a coding sequence, and a 5’ exon element. Circularization strategies are known in the art and described elsewhere herein. In certain embodiments, the resulting circular RNA can include a PIE (permuted intron-exon) region, a translation region (IRES and coding/noncoding elements), and a PIE region. The resulting permuted intron-exon (PIE) regions allow for 5’ and 3’ ends of the RNA to covalently link and form the circular RNA.

[183] In certain embodiments circular RNA provided herein is produced inside a 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.

[ 184] In certain embodiments, the circular RNA provided herein is inj ected into an animal

(e.g., a human), such that a polypeptide encoded by the circular RNA molecule is expressed inside the animal.

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

[186] In some embodiments, the circular RNA provided herein has higher functional stability than mRNA comprising the same expression sequence. In some embodiments, the circular RNA provided herein has higher functional stability than mRNA comprising the same expression sequence, modified nucleotides (e.g., 5moU modifications), an optimized UTR, a cap, and/or a poly A tail.

[187] In some embodiments, the circular RNA polynucleotide provided herein 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 provided herein has a functional half-life of 5-80, 10-70, 15-60, and/or 20-50 hours. In some embodiments, the circular RNA polynucleotide provided herein has a functional half-life greater than (e.g., at least 1.5-fold greater than, at least 2-fold greater than) that of an equivalent linear RNA polynucleotide encoding the same protein. In some embodiments, functional halflife can be assessed through the detection of functional protein synthesis.

[188] In some embodiments, the circular RNA polynucleotide provided herein has a halflife 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 provided herein has a half-life of 5-80, 10-70, 15-60, and/or 20-50 hours. In some embodiments, the circular RNA polynucleotide provided herein has a half-life greater than (e.g., at least 1.5-fold greater than, at least 2-fold greater than) that of an equivalent linear RNA polynucleotide encoding the same protein. In some embodiments, the circular RNA polynucleotide, or pharmaceutical composition thereof, has a functional half-life in 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 other 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.

[189] In some embodiments, the circular RNA provided herein may have a higher magnitude of expression than equivalent linear mRNA, e.g., a higher magnitude of expression 24 hours after administration of RNA to cells. In some embodiments, the circular RNA provided herein has a higher magnitude of expression than mRNA comprising the same expression sequence, 5moU modifications, an optimized UTR, a cap, and/or a polyA tail.

[ 190] In some embodiments, the circular RNA provided herein may be 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 provided herein 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 provided herein is associated with reduced production of IFN-β 1, 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 provided herein is associated with less IFN-β 1, 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 provided herein is less immunogenic than mRNA comprising the same expression sequence. In some embodiments, the circular RNA provided herein is less immunogenic than mRNA comprising the same expression sequence, modified nucleotides (e.g., 5moU modifications), an optimized UTR, a cap, and/or a polyA tail.

[191] In some embodiments, the circular RNA provided herein can be encapsulated by a transfer vehicle (e.g., LNPs), which can deliver the circular RNA constructs. Encapsulating the circular RNA in the transfer vehicle, for example can efficiently introduce the CAR genes to the T cells. The transfer vehicles can comprise, e.g., ionizable lipids, PEG-modified lipids, helper lipids, and/or structural lipids, that are capable of encapsulating the circular RNAs. Pharmaceutical compositions are provided for circular RNA constructs comprising an IRES, an expression sequence, and a transfer vehicle.

[192] In certain embodiments, the circular RNA constructs provided herein can be transfected into a cell as is or can be transfected in DNA vector form and transcribed in the cell. Transcription of circular RNA from a transfected DNA vector can be via added polymerases or polymerases encoded by nucleic acids transfected into the cell, or preferably via endogenous polymerases. Accordingly, also provided herein is a eukaryotic cell comprising a circular RNA polynucleotide provided herein. In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the eukaryotic cell is an immune cell. In some embodiments, the eukaryotic cell is a T cell, dendritic cell, macrophage, B cell, neutrophil, or basophil. Also provided herein is a prokaryotic cell comprising a circular RNA polynucleotide provided herein.

[193] In some embodiments, provided herein is a T cell, e.g., human T cell, comprising the circular RNA constructs provided herein. In some embodiments, provided herein is a helper T cell, e.g., human helper T cell, comprising the circular RNA constructs provided herein. In some embodiments, provided herein is a cytotoxic T cell, e.g., human cytotoxic T cell, comprising the circular RNA constructs provided herein. In some embodiments, provided herein is a NK cell, e.g., human NK cell, comprising the circular RNA constructs provided herein. In some embodiments, provided herein is a macrophage, e.g., human macrophage, comprising the circular RNA constructs provided herein. In some embodiments, provided herein is a monocyte, e.g., human monocyte, comprising the circular RNA constructs provided herein. In some embodiments, provided herein is a myeloid cell, human monocyte, comprising the circular RNA constructs provided herein. In some embodiments, these cells are present in the bone marrow. In some embodiments, these cells are present in the spleen. In some embodiments, these cells are present in the blood, e.g., peripheral blood.

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

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

[196] In certain embodiments, a provided polynucleotide (e.g., a DNA template, a precursor RNA polynucleotide, or a circular RNA polynucleotide) comprises modified nucleotides and/or modified nucleosides. 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); 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' -0- 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 ' -0- 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); 02yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7- cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G⁺ (archaeosine); D (dihydrouridine); m⁵Um (5,2' -O-dimethyluri dine); 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-methoxy carbonylmethyluridine); 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-sel enouridine); 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-m ethylinosine); 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); m^{2 7}G (N²,7-dimethyl guanosine); m^2,2,7G (N²,N²,7-trimethylguanosine); m³Um (3,2' -O-dimethyluri dine); m⁵D (5-methyldihydrouridine); CCm (5-formyl-2' -O- methylcytidine); m¹Gm (1,2' -O-dimethylguanosine); m¹Am (1,2' -O-dimethyladenosine); 5U (5-taurinomethyluridine); (5-taurinomethyl-2-thiouridine)); imG-14 (4- demethylwyosine); imG2 (isowyosine); or ac⁶A (N⁶-acetyladenosine).

[197] In some embodiments, the modified nucleoside may include a compound selected from the group of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2- thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl -uridine, 1-propynyl- pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2- thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4- thio-l-methyl-pseudouridine, 2 -thio- 1-methyl-pseudouridine, 1 -methyl- 1 -deazapseudouridine, 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-l-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, and N2,N2-dimethyl-6-thio- guanosine. In another embodiment, the modifications are independently selected from the group consisting of 5-methylcytosine, pseudouridine and 1 -methylpseudouridine.

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

[199] 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 ' selfspliced exon segment.

[200] 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.

[201] In some embodiments, the 5 ' combined accessory element comprises a 3’ selfspliced 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 3 ' nucleotide of a 3 ' splice site dinucleotide. In some embodiments, the 3’ selfspliced 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.

[202] 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 SEQ ID NOs: 2990-3668, 25573, and 25574. In some embodiments, the 3’ self-spliced exon segment is selected from an exon segment disclosed herein, e.g., in Table 2 or Table 3, or SEQ ID NOs: 2990-3668, 25573, and 25574. 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 2 or is e.g., 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides from the exonic sequences of Table 3. See also SEQ ID NOs: 2990-3668, 25573, and 25574.

[203] In some embodiments, the 3 ' combined accessory element comprises a 5’ selfspliced 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 5 ' nucleotide of a 5 ' splice site dinucleotide. In some embodiments, the 5’ selfspliced 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 II intron sequence.

[204] 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 SEQ ID NOs: 2990-3668, 25573, and 25574. In some embodiments, the 5’ self-spliced exon segment is selected from an exon segment disclosed herein, e.g., in Table 2 or Table 3, or SEQ ID NOs: 2990-3668, 25573, and 25574. 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 2 or is e.g., 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides from the exonic sequences of Table 3. See also SEQ ID NOs: 2990-3668, 25573, and 25574.

[205] 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 comprises a sequence selected from the sequences in Table 4 or a fragment thereof or a sequence selected from SEQ ID NOS: 1-2989 and 4045-25570. See, e.g., supra. In some embodiments the TIE comprises a coding sequence with which the TIE is not naturally associated.

[206] 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 3 ' Untranslated Region (UTR), a natural 5 ' Untranslated Region (UTR), a synthetic spacer sequence, an aptamer, and IncRNA, miRNA, and a miRNA sponge. In some embodiments, the noncoding element is or comprises the TIE.

[207] 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 2.

[208] In some embodiments, provided herein are circular RNA polynucleotides comprising, in the following order, i) a 5’ combined accessory element comprising a 3’ selfspliced 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 2 or Table 3, or SEQ ID NOs: 2990-3668, 25573, and 25574.

[209] In some embodiments, provided herein are circular RNA polynucleotides comprising, in the following order, i) a 5’ combined accessory element comprising a 3’ selfspliced 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 2 or Table 3, or SEQ ID NOs: 2990-3668, 25573, and 25574.

[210] In some embodiments, provided herein are circular RNA polynucleotides comprising, in the following order, i) a 5’ combined accessory element comprising a 3’ selfspliced 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’ selfspliced 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 2 or Table 3, or SEQ ID NOs: 2990-3668, 25573, and 25574.

[211] 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, wherein at least one of the 3’ or 5’ self-spliced exon segments is selected from an exon segment comprising a sequence selected from SEQ ID NOs: 2990-3668, 25573, and 25574.

[212] As a non-limiting example, a circular RNA polynucleotide comprises the following elements operably connected and arranged in the following sequence:

(a) a 3' exon segment comprising a Group I or Group II exon 3' nucleotide of a 3' splice site dinucleotide;

(b) an intervening region; and

(c) a 5' exon segment comprising a Group I or Group II exon 5' nucleotide of a 5' splice site dinucleotide.

[213] 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 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 SEQ ID NOs: 2990- 3187, 25573, and 25574.

[214] 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.

[215] 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 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.

[216] 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 3' exon segment comprising a 3' nucleotide of a 3' splice site dinucleotide,

(c) an intervening region,

(d) a 5' exon segment comprising a 5' nucleotide of a 5' 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.

[217] In some embodiments, element (d) comprises the first nucleotide of a 5 ' Group I or Group II splice site dinucleotide and a natural exon sequence. In some embodiments, element (b) comprises the second nucleotide of a 3 ' Group I or Group II exon splice site dinucleotide and a natural exon sequence.

[218] In some embodiments, in the circular RNA polynucleotide, the 5' exon element comprises the second nucleotide of a 3' Group I or Group II exon splice site dinucleotide and a natural exon sequence. In some embodiments, the 3' exon element fragment comprises the first nucleotide of a 5' Group I or Group II splice site dinucleotide and a natural exon sequence. In some embodiments, the 5' exon element comprises a 5' internal duplex; and the 3' exon element comprises a 3' internal duplex. In some embodiments, the 5' exon element comprises a 5' internal spacer. In some embodiments, the 3' exon element comprises a 3' internal spacer. [219] In some embodiments, the circular RNA polynucleotide comprises a 5’ internal duplex and a 3’ internal duplex. See, e.g., supra.

[220] In some embodiments, the circular RNA polynucleotide comprises a 5’ internal homology region and/or a 3’ internal homology region. See, e.g., supra.

[221] 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.

[222] 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.

[223] 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. 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). See, e.g., Figures 24B, 24C, and 24D. 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. [224] In some embodiments, the circular RNA polynucleotide is from about 50 nucleotides to about 15 kilobases in length.

[225] 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.

[226] 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. See, e.g., Example 8, demonstrating that self-splicing efficiency and/or circularization efficiency is linked to the structures herein, e.g., at Examples 1, 2, 8. 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 selfcircularization. See, e.g., id. 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. See, e.g., id. 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. See, e.g., id. 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. See, e.g., id. 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. See, e.g., id. 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-splicing. See, e.g., id. 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. See, e.g., id. 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. See, e.g., id. 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. See, e.g., id.

A. PRECURSORS

[227] Disclosed herein are precursor RNAs for producing circular RNAs. In some embodiments, the precursor RNA comprises both 5' intron and exon elements and 3' exon and intron elements or comprising only 3' exon and intron elements for producing circular RNAs with enhanced circularization efficiency.

[228] Accordingly, provided herein is a precursor RNA polynucleotide capable of producing a circular RNA polynucleotide after splicing, wherein the precursor RNA polynucleotide comprises both 5' 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 3' exon and intron elements.

[229] In some embodiments, a provided precursor RNA polynucleotide comprises (i) 3' permuted intron segment comprising a 5' nucleotide of a 3' splice site dinucleotide; and (ii) a 3' exon segment comprising a 3' nucleotide of a 3' splice site dinucleotide. Exemplary splice site dinucleotides are provided in the Table set forth herein. In some embodiments, a provided precursor RNA polynucleotide comprises (i) a 5' exon segment comprising a 5' nucleotide of a 5' splice site dinucleotide; and (ii) a 5' permuted intron segment comprising a 3' nucleotide of a 5' 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. [230] In some embodiments, a provided precursor RNA polynucleotide comprises (i) a 5' intron element comprising a 3' permuted intron segment comprising a 5' nucleotide of a 3' splice site dinucleotide; (ii) a 5' exon element comprising a 3' exon segment comprising a 3' nucleotide of a 3' splice site dinucleotide; (iii) a 3' exon element comprising a 5' exon segment comprising a 5' nucleotide of a 5' splice site; and (iv) a 3' intron element comprising a 5' permuted intron segment comprising a 3' nucleotide of a 5' 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 5' intron element comprising a 3' permuted intron segment comprising a 5' nucleotide of a 3' splice site dinucleotide; and (iii) a 5' exon element comprising a 3' exon segment comprising a 3' nucleotide of a 3' splice site dinucleotide.

[231] In some embodiments, a provided precursor RNA polynucleotide comprises a 5’ combined accessory element comprising (i) a 3' permuted intron segment comprising a 5' nucleotide of a 3' splice site dinucleotide; and (ii) a 3' exon segment comprising a 3' nucleotide of a 3' splice site dinucleotide. In some embodiments, element (ii) is located upstream to the intervening region. In some embodiments, the 5 ' combined accessory element comprises a 3 ' exon segment comprising a Group I or Group II exon 3 ' nucleotide of a 3 ' splice site dinucleotide.

[232] In some embodiments, a provided precursor RNA polynucleotide comprises a 3' combined accessory element comprising (i) a 5' exon segment comprising a 5' nucleotide of a 5' splice site dinucleotide; and (ii) a 5' permuted intron segment comprising a 3' nucleotide of a 5' splice site dinucleotide. In some embodiments, element (ii) is located downstream to the intervening region. In some embodiments, a 3 ' combined accessory element comprises a 5 ' exon segment comprising a Group I or Group II exon 5 ' nucleotide of a 5 ' splice site dinucleotide.

[233] In some embodiments, a provided precursor RNA polynucleotide comprises a 5’ combined accessory element, an intervening region, and a 3' combined accessory element. In some embodiments, (a) the 5’ combined accessory element comprises (i) a 3' permuted intron segment comprising a 5' nucleotide of a 3' splice site dinucleotide; and (ii) a 3' exon segment comprising a 3' nucleotide of a 3' splice site dinucleotide; and (b) the 3' combined accessory element comprising (i) a 5' exon segment comprising a 5' nucleotide of a 5' splice site dinucleotide; and (ii) a 5' permuted intron segment comprising a 3' nucleotide of a 5' splice site dinucleotide. In some embodiments, the 5' nucleotide of a 3' splice site dinucleotide, 3' nucleotide of a 3' splice site dinucleotide, 5' nucleotide of a 5' splice site dinucleotide and 3' nucleotide of a 5' splice site dinucleotide are optionally a combination of nucleotides or a portion of a sequence selected from SEQ ID NOS: 2990-3668.

[234] In some embodiments, the 5’ combined accessory element is located 5' to the intervening region; and the intervening region is located is 5' to the 3' combined accessory element.

[235] 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.

[236] In some embodiments, the precursor RNA polynucleotide is linear.

[237] In some embodiments, permuted intron-exon splicing results in circularization of 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 3' intron element and the 5' intron element. Accordingly, the circular RNA polynucleotide produced after splicing of the precursor RNA polynucleotide lacks the 3' intron segment and the 5' intron segment, but retains the 3' exon segment and the 5' exon segment.

[238] 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⁺).

[239] 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.

[240] 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 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.

[241] In various embodiments, provided herein is a circular RNA polynucleotide produced by circularization of a precursor RNA polynucleotide described herein.

[242] 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.

[243] 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.

[244] 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 may allow for the stabilization of RNA polynucleotides that generally suffer from short halflives and may improve the overall efficacy of exogenous mRNA in a variety of applications.

[245] 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).

[246] 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 poly A tail.

[247] 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.

[248] 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.

[249] 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.

[250] 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-β 1, 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.

[251] 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.

B. INTRON ELEMENTS, EXON ELEMENTS, TERMINAL ELEMENTS

[252] 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 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

[253] 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. [254] In some embodiments, an intron element comprises an intron derived from a transsplicing ribozyme. In some embodiments, the intron element comprises a Group I transsplicing 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.

[255] 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.

[256] 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 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.

[257] 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.

[258] In some embodiments, at least one of the exon segments is less than 15 nucleotides in length. In some embodiments, the 3' exon segment and/or 5' exon segment comprises a Group I exon segment or a Group II exon segment less than 15 nucleotides in length.

[259] 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 2 (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 2. 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 3 (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 3. See also SEQ ID NOs: 2990-3668, 25573, and 25574.

[260] 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.

[261] 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 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 2 or Table 3, below. b. TERMINAL ELEMENTS

[262] 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.

[263] 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.

[264] 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.

[265] 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.

[266] In some embodiments, the terminal element is capable of binding to a 3’ intron 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. MONOTRON

[267] 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.

[268] 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.

[269] 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, 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. [270] 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.

[271] 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 nucleophile can be a guanosine, e.g., a free guanosine that is introduced to the precursor RNA polynucleotide.

[272] 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.

[273] 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.

[274] 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 Table 2 or Table 3. 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 Table 2 or Table 3. 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 SEQ ID NOS: 2990-3668.

[275] In some embodiments, the terminal element is less than 500, less than 450, less than 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.

[276] In some embodiments, the terminal element is capable of directing or functionalizing the splicing activity of the monotron element.

[277] 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 postcircularization. In some embodiments, the terminal element is not excised upon cleavage and is retained post-cleavage. [278] 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 Table 2 or Table 3. 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 Table 2 or Table 3. 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 Table 2 or Table 3. 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 SEQ ID NOS: 2990-3668.

[279] In some embodiments, the Group I or Group II intron or introns, or portion thereof, are at least 10 nucleotides in length.

[280] 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- 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 3' exon segment and/or 5' 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 3' exon segments and/or 5' 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.

[281] In some embodiments, the monotron element is less than 500 nucleotides in length.

[282] 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.

[283] 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 5' affinity tag or a 3' affinity tag. In some embodiments, the monotron element comprises an affinity tag. In some embodiments, the monotron element comprises a 3' affinity tag or a 5' affinity tag.

[284] 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 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.

[285] 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 5' internal duplex sequence and a 3' internal duplex sequence. In embodiments where the terminal element is upstream of the monotron element, the 5' internal duplex sequence is positioned between the terminal element and the intervening region, and the 3' 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 5' internal duplex sequence is positioned between monotron and the intervening region, and the 3' internal duplex sequence is positioned between the intervening region and the terminal element. In some embodiments, the 5' internal duplex sequence and 3' 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 5' internal duplex sequence and/or 3' internal duplex sequence each have a GC content of at least 10%.

[286] 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 5' affinity tag is positioned adjacent to the 5' external spacer, and in certain embodiments is positioned 5' to the 5' external spacer. In some embodiments where the polynucleotide comprises a 3' affinity tag, the 3' affinity tag is positioned adjacent to the 3’ external spacer, and in certain embodiments is positioned 3' to the 3' external spacer.

[287] 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 5' internal duplex, the 5' internal duplex is positioned adjacent to the 5' internal spacer, and in certain embodiments is positioned 5' to the 5' internal spacer. In some embodiments where the polynucleotide comprises a 3' internal duplex, the 3' internal duplex is positioned adjacent to the 3' internal spacer, and in certain embodiments, the 3' internal duplex is positioned 3' to the 3' internal spacer.

[288] 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.

[289] 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 5' 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 3' 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.

[290] 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 5' Untranslated Region (UTR), a natural 3' Untranslated Region (UTR), a synthetic spacer sequence, an aptamer, TIE, a viral or eukaryotic IRES, or sequences selected from, e.g., IncRNA, miRNA, or a miRNA sponge.

[291] 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 sequence. In some embodiments, at least one of the exon segments is less than 15 nucleotides in length. In some embodiments, the 3' exon segment and/or 5' 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 3' exon segment, 5' exon segment, or a 3' and 5' permuted exon segment. In some embodiments, the exon sequence or fragment is in the terminal element.

[292] 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.

[293] 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 3' exon segment comprising a 3' nucleotide of a 3' splice site dinucleotide, (c) an intervening region, (d) a 5' exon segment comprising a 5' nucleotide of a 5' splice site dinucleotide, and (e) at least a portion of a monotron element. In some embodiments, (d) comprises the first nucleotide of a 5' Group I or Group II splice site dinucleotide and a natural exon sequence and (b) comprises the second nucleotide of a 3' 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 5' internal duplex and/or 3' internal duplex; a 5' internal spacer and/or 3' internal spacer. In some embodiments, the circular RNA polynucleotide is from about 50 nucleotides to about 15 kilobases in length.

[294] 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.

[295] 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 5' and 3' 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 5' and 3' Group I or Group II intron sequence comprises at least one deletion, insertion or substitution of at least one nucleotide. In some embodiments, the 5' or 3' Group I or Group II intronic sequences, or combinations thereof are sequenced.

[296] 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 5' and/or 3' internal and/or external spacer sequence of the DNA sequence encoding precursor RNA polynucleotide described above; (iii) altering the length of the 5' and/or 3' internal duplex sequence of the DNA sequence encoding the precursor RNA polynucleotide described above; (iv) altering the length of the 5' and/or 3' 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 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. d. EXEMPLARY ELEMENTS

[297] 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.

[298] 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.

[299] 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 Table 2 or Table 3.

[300] 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 tag, and a trailing untranslated sequence. In some embodiments, the affinity tag is a polyA affinity tag.

[301] In some embodiments, the 5' intron element is located 5' to the 5' exon element. In some embodiments, the 5' intron element is adjacent to the 5' exon element. In some embodiments, the 3' intron element is located 3' to the 3' exon element. In some embodiments, the 3' intron element is adjacent to the 3' exon element.

[302] In some embodiments, the 5' exon element comprises a 5' internal duplex sequence located 3' to the 3' exon segment. In some embodiments, the 3' exon element comprises a 3' internal duplex sequence located 5' to the 5' exon segment. In some embodiments, the 5' intron element comprises a 5' external duplex sequence located 5' to the 3' permuted intron segment. In some embodiments, the 3' intron element comprises a 3' external duplex sequence located 3' to the 5' permuted intron segment. In some embodiments, the 5' intron element is adjacent to the 5' exon element. In some embodiments, the 3' intron element is located 3' to the 3' exon element. In some embodiments, the 3' intron element is adjacent to the 3' exon element.

[303] In some embodiments, the 5' intron comprises a 5' affinity tag, a 5' external spacer, and the 3' permuted intron segment. In some embodiments, the 5' exon comprises the 3' exon segment, a 5' internal duplex sequence, and a 5' internal spacer. In some embodiments, the 5' affinity tag is adjacent to the 5' external spacer. In some embodiments, the 5' affinity tag is located 5' to the 5' external spacer. In some embodiments, the 5' internal duplex sequence is adjacent to the 5' internal spacer. In some embodiments, the 5' internal duplex sequence is located 5' to the 5' internal spacer. In some embodiments, the 3' exon comprises a 3' internal spacer, 3' internal duplex sequence, and the 5' exon segment. In some embodiments, the 3' intron comprises the 5' permuted intron segment, a 3' external spacer, and a 3' affinity tag. In some embodiments, the 3' affinity tag is adjacent to the 3' external spacer. In some embodiments, the 3' affinity tag is located 3' to the 3' external spacer. In some embodiments, the 3' internal duplex sequence is adjacent to the 3' internal spacer. In some embodiments, the 3' internal duplex sequence is located 3' to the 3' internal spacer. In some embodiments, the affinity tag is a polyA affinity tag.

[304] In some embodiments, the 5' exon comprises a 5' internal duplex sequence located between the 3' exon segment and the intervening region. In some embodiments, the 3' exon comprises a 3' internal duplex sequence positioned between the intervening region and the 5' exon segment. In some embodiments, the polynucleotide comprises a 5' internal duplex sequence and a 3' internal duplex sequence.

[305] In some embodiments, the 3' and 5' 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 3' permuted intron segment comprises a 3' Group I intron segment or a variant thereof. In some embodiments, the 5' permuted intron segment comprises a 5' Group I intron segment or a variant thereof. In some embodiments, the 3' permuted intron segment comprises a 3' Group II intron segment or a variant thereof. In some embodiments, the 5' permuted intron segment comprises a 5' Group II intron segment or a variant thereof. [306] In some embodiments, the 3' permuted intron segment or element, 5' permuted intron segment or element, or both the 3' and 5' 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 3' permuted intron element, 5' permuted intron element, or both the 3' and 5' permuted intron elements are at least 50 nucleotides in length. In some embodiments, the 3' permuted intron element, 5' permuted intron element, or both the 3' and 5' 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.

[307] In some embodiments, the 3' permuted intron element, 5' permuted intron element, or both the 3' and 5' permuted intron elements comprise a native Group I intron segment or Group II intron segment sequence. In some embodiments, the 3' permuted intron element, 5' permuted intron element, or both the 3' and 5' 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 3' permuted intron element, 5' permuted intron element, or both the 3' and 5' 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 3' permuted intron element, 5' permuted intron element, or both the 3' and 5' 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 3' permuted intron element, 5' permuted intron element, or both the 3' and 5' 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 3' Group I or Group II intron segment or the 5' 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 subtraction. In some embodiments, the mutations are deletions of two or more nucleotides of the 3' Group I or Group II intron segment or the 5' Group I or Group II intron segment, or combinations thereof. In some embodiments, the mutations are two or more deletions of the 5' Group I intron segment at the 3' end or two or more deletions of the 3' Group I intron segment at the 5' end.

[308] In some embodiments, the native Group I intron segment or Group II intron segment sequences are selected from a sequence in Table 2 or Table 3, below. In some embodiments, the 3' and/or 5' 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 2 or Table 3, below, or a fragment or segment thereof. In some embodiments, the 3' and/or 5' permuted intron element comprise a polynucleotide sequence selected from a sequence set forth in SEQ ID NOS: 2990-3668. In some embodiments, the 3' and/or 5' permuted intron element comprise a polynucleotide sequence selected from a sequence set forth in SEQ ID NOS: 3188-3668.

[309] In some embodiments, the 3' permuted intron segment comprises a 3' Group I or Group II intron segment derived from a gene selected from a genus and/or species selected from column 2 of Table 2 or Table 3; and/or the 5' permuted intron segment comprises a 5' Group I or Group II intron segment derived from a gene selected from a genus and/or species selected from column 2 of Table 2 or Table 3.

[310] In some embodiments, the 3' Group I or Group II intron segment or the 5' 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 2: Group I introns (flanked by 15nt exons)

Table 3: Group II introns (flanked by lOnt exons)

Ill

[311] In some embodiments, the 3' 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 therm ophila, 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, 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.

[312] 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 monotron element and 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. [313] 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 2 or Table 3. 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 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).

[314] 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).

[315] 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,

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,

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 therm ophila 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,

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.

[316] 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 translatable or biologically active inside a eukaryotic cell. In some embodiments, such a method comprises:

(i) inserting 5' and 3' 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.

[317] 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 5' and/or 3' Group I splice site dinucleotides. In some embodiments, the 5' or 3' 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.

[318] 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 5' intron element and/or 3' intron element of the DNA sequence encoding the precursor RNA polynucleotide described herein;

(ii) altering the length of the 5' and/or 3' internal and/or external spacer sequence of the DNA sequence encoding precursor RNA polynucleotide;

(iii) altering the length of the 5' and/or 3' internal duplex sequence of the DNA sequence encoding the precursor RNA polynucleotide;

(iv) altering the length of the 5' and/or 3' exon sequence of the DNA sequence encoding the precursor RNA polynucleotide; or

(v) 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 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. e. SPACER

[319] 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.

[320] 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”).

[321] 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 5' external spacer is located 5' to the 3' permuted intron segment. In some embodiments, the 5' internal spacer is located 3' to the 3' exon segment. In some embodiments, the 3' external spacer is located 3' to the 5' permuted intron segment. In some embodiments, the 3' external spacer is located 5' to the 5' exon segment.

[322] In certain embodiments, the polynucleotide comprises a 5' external spacer located between a leading untranslated sequence and the 5' or 3' intron element. In certain embodiments, the polynucleotide comprises a 3' external spacer located between the 3' or 5' intron element and a lagging untranslated sequence.

[323] In certain embodiments where the polynucleotide comprises a monotron, the 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.

[324] 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.

[325] In some embodiments, the polynucleotide further comprises an aptamer. In some embodiments, the aptamer is synthetic.

[326] 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.

[327] 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 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.

[328] 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.

[329] 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.

[330] 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 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. f. DUPLEX

[331] 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.

[332] 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 5' internal duplex sequence and a 3' internal duplex sequence. In some embodiments, the polynucleotide comprises a 5’ internal duplex located within the 3’ exon fragment. In some embodiments, the 5' internal duplex sequence is positioned between the 5' 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 3' internal duplex sequence is positioned between the intervening region and the 3' 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.

[333] In some embodiments, the polynucleotide comprises a monotron element, intervening region, and terminal element, and a 5' internal duplex sequence and a 3' internal duplex sequence. In some embodiments, if the terminal element is upstream of the monotron element, the 5' internal duplex sequence is positioned between the terminal element and the intervening region, and the 3' 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 5' internal duplex sequence is positioned between monotron and the intervening region, and the 3' 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.

[334] 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 5' internal duplex sequence and 3' 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.

[335] 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 5' internal duplex sequence and 3' 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.

[336] In some embodiments, the 5' internal duplex sequence and/or 3' internal duplex sequence each have a GC content of at least 10%.

[337] In other embodiments, the polynucleotide does not comprise of any duplex to optimize translation or circularization. g. AFFINITY SEQUENCES

[338] 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 5' affinity tag is located 5' to the 3' permuted intron segment. In some embodiments, the 3' affinity tag is located 3' to the 5' permuted intron segment.

[339] 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 5' affinity tag if the terminal element is located upstream of the monotron element, wherein the 5' affinity tag is located 5' to the terminal element; or (b) a 3' affinity tag if the monotron element is located upstream of the terminal element, wherein the 3' affinity tag is located 3' to the terminal element. In some embodiments, the monotron element comprises (a) a 3' affinity tag if the terminal element is located upstream of the monotron element, wherein the 3' affinity tag is located 3' to the monotron element; or (b) a 5' affinity tag if the monotron element is located upstream of the terminal element, wherein the 5' affinity tag is located 5' 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.

[340] 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.

[341] 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. 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.

[342] 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.

[343] 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.

[344] 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 KC1, 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 storage buffer. In some embodiments, the storage buffer comprises ImM sodium citrate, pH 6.5. h. LEADING SEQUENCES & LAGGING SEQUENCES

[345] 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.

[346] 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 3' external spacer located between the 3' intron element and a lagging untranslated sequence. In some embodiments, the polynucleotide a leading untranslated sequence at the 5' end. In some embodiments, the polynucleotide comprises a 5' external spacer located between a leading untranslated sequence and the 5' intron element.

[347] In some embodiments, the polynucleotide comprises a monotron element and a leading untranslated sequence. In some embodiments, the polynucleotide comprises a 5' 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.

[348] 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.

C. INTERVENING REGION

[349] 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.

[350] In some embodiments, the intervening region and/or core functional element comprises one or more 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 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 coding or non-coding region is a part of the core functional element or intervening region located between the 5’ end and 3’ end of the linear precursor RNA polynucleotide and resultant circular RNA.

[351] 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.

[352] 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.

[353] In some embodiments, the intervening region comprises one or more noncoding elements. In some embodiments, the noncoding element comprises an untranslated region (UTR) or fragment thereof. 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. In some embodiments, the noncoding element is or comprises a translation initiation element (TIE). In some embodiments, the noncoding element comprises a IncRNA, miRNA, or a miRNA sponge.

[354] 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.

[355] 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 wildtype viral IRES or eukaryotic IRES. See, e.g., PCT Application No. US2022/33091, which is incorporated herein by reference in its entirety.

[356] 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). a. TRANSLATION INITIATION ELEMENT

[357] 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.

[358] 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 or noncoding 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.

[359] 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. i. Natural TIES: viral & eukaryotic/cellular IRES

[360] 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) within the translation initiation element (TIE). In some embodiments, IRES specificity within a circular RNA can significantly enhance expression of specific proteins encoded within the coding element. In some embodiments, the IRES comprises a viral IRES or eukaryotic IRES.

[361] 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.

[362] 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 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.

[363] Different 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. In certain embodiments, potential IRES sequences can be bioinformatically identified based on sequence positions in viral sequences. However, the activity of such sequences has been previously uncharacterized. As demonstrated herein, such IRES sequences may have differing protein expression capability depending on cell type, for example in T cells, liver cells, or muscle cells. In some embodiments, the novel 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 previously described EMCV IRES sequences.

[364] 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 operably linked to a protein coding sequence. In some embodiments, the IRES comprises a sequence selected from the sequences in Table 4 or a fragment thereof or a sequence selected from SEQ ID NOS: 1-2989 and 4045-25570 (GIRES 0-10762), or a fragment thereof. In some embodiments, the IRES comprises a sequence 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 4 or a sequence selected from SEQ ID NOS: 1-2989 and 4045- 25570 (GIRES 0-10762), or a fragment thereof. See also, e.g., PCT Application No. US2022/33091 (WO202261490), which is incorporated herein by reference in its entirety.

[365] In some embodiments, the IRES 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, Pestivirus, or Flavivirus. In some embodiments herein, the IRES is selected from an Enterovirus, Kobuvirus, Parechovirus, Hunnivirus, Passerivirus, Mischivirus, and Cardiovirus.

[366] In some embodiments, the IRES 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 LEF 1 , Mouse HIF 1 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, Picobimavirus, HCV QC64, Human Cosavirus E/D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SHI, 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.

[367] In some embodiments, the IRES comprises in whole or in part a eukaryotic or cellular IRES. In certain embodiments, the IRES is an IRES 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, Clorf226, C21orf62, C2orfl5, C4BPB, C4orf22, C9orf84, CACNA1A, CALCOC02, 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, CHICI, CHMP2A, CHRNA2, CLCN3, CLEC12A, CLEC7A, CLECL1, CLRN1, CMSS1, CNIH1, CNR1, CNTN5, C0G4, C0MMD1, 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, FMRI, 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, LAMB3, LANCL1, LBX2, LCAT, LDHA, LDHAL6A, LEF1, LINC-PINT, LM03, 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, MY01A, MYT2, MZB1, NAP1L1, NAVI, NBAS, NCF2, NDRG1, NDST2, NDUFA7, NDUFB11, NDUFC1, NDUFS1, NEDD4L, NFAT5, NFE2L2, NFE2L2, NFIA, NHEJ1, NHP2, NITI, 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, PTCHI, 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, 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.

[368] In some embodiments, the cell is a myotube. In some embodiments, the IRES 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.

[369] In some embodiments, the cell is a hepatocyte. In some embodiments, the IRES 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.

[370] In some embodiments, the cell is a T cell. In some embodiments, the IRES 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.

[371] 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 comprises a sequence selected from SEQ ID NOS: 1-2989 and 4045-25570 (GIRES 0- 10762) or Table 4 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 IRES comprises a sequence 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 and 4045-25570 or Table 4 below, or a sequence selected from SEQ ID NOs: 1-2983 and 3282-3287 of PCT Application No. US2022/33091 (WO202261490). In some embodiments, the circular RNA disclosed herein comprises an IRES sequence 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 and 4045-25570 or Table 4 below, or a sequence selected from SEQ ID NOs: 1-2983 and 3282-3287 of PCT Application No. US2022/33091 (WO202261490). In some embodiments, the circular RNA disclosed herein comprises an IRES sequence selected from SEQ ID NOS: 1-2989 and 4045- 25570 or Table 4 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.

[372] Further exemplary IRES sequences are provided in Table 4. In some embodiments, the precursor RNA polynucleotide, circular RNA constructs and related pharmaceutical compositions disclosed herein comprise an IRES sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an IRES sequence in Table 4. In some embodiments, the precursor RNA polynucleotide, circular RNA constructs and related pharmaceutical compositions disclosed herein comprise an IRES sequence in Table 4.

Table 4: IRES Sequences

[373] Mutations of IRES and accessory sequences are encompassed herein to increase or reduce IRES activities, for example, by 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), modification of (e.g., mutations) alternative translation initiation sites, and creating chimeric/hybrid IRES sequences. In some embodiments, the IRES sequence in the polynucleotide disclosed herein comprises one or more of these modifications relative to a natural or native IRES. ii. Synthetic TIEs: aptamer complexes, modified nucleotides, IRES variants & other engineered TIEs

[374] In certain embodiments, a TIE provided herein is a synthetic TIE. In some embodiments, a synthetic TIE comprises aptamer complex, synthetic IRES, or other engineered TIE capable of initiating translation of a linear RNA or circular RNA polynucleotide.

[375] In some embodiments, 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 (elF) (e.g., certain aptamers disclosed in International Pat. Appl. No. PCTZEP2018/078794 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 (poly A binding protein), PTB, Argonaute protein family, HNRNPK (heterogeneous nuclear ribonucleoprotein K), or La protein. b. STOP CODON OR STOP CASSETTE

[376] 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.

D. ADDITIONAL ELEMENTS

[377] 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.

[378] 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.

[379] 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 (poly A 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 poly A, polyC, poly AC, 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.

[380] In certain embodiments, the polynucleotide, precursor RNA polynucleotide, or circular RNA comprises a IncRNA, 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 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.

[381] 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.

[382] In some embodiments, the miR-122 site can comprise the following sequence:

CAAACACCATTGTCACACTCCAA (SEQ ID NO: 4018).

E. MODIFICATIONS

[383] 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 insertions, deletions, addition, or subtraction of nucleotides, for example, the mutations in a permuted Group I and Group II intron segment.

[384] 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.

[385] 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.

[386] 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 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. [387] 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.

[388] 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.

[389] 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); 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); nflm (1,2' -O-dimethylinosine); m³C (3 -methylcytidine); Cm (2' -0- 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 ' -0- 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); 02yW (peroxywybutosine); oHyW (hydroxywybutosine); OhyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7- cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G⁺ (archaeosine); D (dihydrouridine); m⁵Um (5,2' -O-dimethyluri dine); 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-methoxy carbonylmethyluridine); 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-sel enouridine); 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-m ethylinosine); 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); m^2,7G (N²,7-dimethyl guanosine); m^2,2,7G (N²,N²,7-trimethylguanosine); m³Um (3,2' -O-dimethyluri dine); m⁵D (5-methyldihydrouridine); CCm (5-formyl-2' -O- methylcytidine); m¹Gm (1,2' -O-dimethylguanosine); m¹Am (1,2' -O-dimethyladenosine); 5U (5-taurinomethyluridine); (5-taurinomethyl-2-thiouridine)); imG-14 (4- demethylwyosine); imG2 (isowyosine); N1 -methylpseudouridine; or ac⁶A (N⁶- acetyladenosine). [390] In some embodiments, the modified nucleoside may include a compound selected from the group of 152yridine-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, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4- thio-l-methyl-pseudouridine, 2 -thio- 1-methyl-pseudouridine, 1 -methyl- 1 -deazapseudouridine, 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-l-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.

[391] In another embodiment, the modifications are independently selected from 5- methylcytosine, pseudouridine and 1 -methylpseudouridine.

[392] In some embodiments, the modified ribonucleosides include 5-methylcytidine, 5- methoxyuridine, 1-methyl-pseudouridine, N6-methyladenosine, and/or pseudouridine.

[393] In some embodiments, the modified nucleoside is N1 -methylpseudouridine.

[394] 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 alkyl carb ony 1 alky 1 ated nucl eoti des .

[395] 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.

[396] 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 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.

[397] 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.

[398] 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 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.

[399] 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 ' 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.

[400] 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.

[401] 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 maintain stability and resistance to immune activation as compared to a corresponding polynucleotide comprising no modified nucleotides and/or modified nucleosides.

[402] 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.

[403] 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 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.

[404] 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.

[405] 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 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.

[406] 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.

[407] 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.

[408] 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.

[409] 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 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.

[410] 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.

[411] 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.

[412] In some embodiments, the first and second precursor RNA polynucleotides further comprise spacers and/or homology arms.

[413] 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.

[414] 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 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 polynucleotide are modified.

[415] 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.

[416] 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.

[417] 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 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.

[418] 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.

[419] In some embodiments, in portions of the second 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 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.

[420] 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.

[421] 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.

[422] 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 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.

[423] 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.

[424] 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 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.

F. CODON OPTIMIZATION

[425] 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.

3. EXPRESSION SEQUENCES AND PAYLOADS

[426] In various embodiments, a provided polynucleotide (e.g., a DNA template, a linear precursor RNA polynucleotide, or a circular RNA polynucleotide) comprises at least one expression sequence encoding a therapeutic protein, e.g., a chimeric antigen receptor (CAR). In certain embodiments, the polynucleotide comprises at least one expression sequence encoding a binding molecule or portion thereof. In certain embodiments, the polynucleotide comprises the expression sequence and a TIE. In certain embodiments, the polynucleotide comprises the expression sequence and an IRES, wherein the IRES can facilitate expression of the protein when delivered in vivo. In some embodiments, the expression sequence 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 or resultant circular RNA.

[427] In some embodiments, the payload encoded by the provided polynucleotide (e.g., a DNA template, a linear precursor RNA polynucleotide, or a circular RNA polynucleotide) is a water channel or an aquaporin, e.g., a membrane protein that comprises transmembrane domains and is capable of forming a pore in the cell membrane. The aquaporin may be an animal aquaporin, a plant aquaporin, a bacterial aquaporin, a fungal aquaporin, a synthetic aquaporin, or derivative or functional fragment thereof. See, e.g., Verkman, Aquarporins, 2014; Adeoye et al., Structure and Function of Aquaporins, 2022. In some embodiments, the aquaporin is a mammalian aquaporin (AQPO to AQP12) or derivative or functional fragment thereof. In some embodiments, the aquaporin is an aquaglyceroporin and is capable of transporting glycerol.

[428] 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 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. In some embodiments, the payload of the provided polynucleotide is not a CAR.

[429] 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

[430] 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.

[431] 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 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.

[432] 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.

[433] In some embodiments, the CAR comprises an antigen binding domain specific for an antigen selected from the group CD 19, CD 123, 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 (GalNAca-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-l lRa), 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, member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), Poly sialic 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- la), 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 Bl, v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN), Ras Homolog Family Member C (RhoC), Tyrosinase-related protein 2 (TRP-2), Cytochrome P450 1B1 (CYP1B1), CCCTC-Binding Factor (Zinc Finger Protein)-Like, Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3), Paired box protein Pax-5 (PAX5), 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, αvβθ integrin, αvβ6 integrin, alphafetoprotein (AFP), B7-H6, ca-125, CA9, CD44, CD44v7/8, CD52, E-cadherin, EMA (epithelial membrane antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB4, epithelial tumor antigen (ETA), folate binding protein (FBP), kinase insert domain receptor (KDR), k-light chain, LI cell adhesion molecule, MUC18, NKG2D, oncofetal antigen (h5T4), tumor/testis-antigen 1B, GAGE, GAGE-1, BAGE, SCP-1, CTZ9, SAGE, CAGE, CT 10, 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.

[434] As a non-limiting example, in some embodiments, the circular RNA construct comprises an IRES and at least one expression sequence encoding a CAR targeting a cancer antigen. As a non-limiting example, in some embodiments, the circular RNA construct comprises an IRES and a CAR comprising an antigen binding domain specific for CD 19. In some embodiments, the circular RNA construct comprises an IRES and a CAR comprising an antigen binding domain specific for BCMA. In some embodiments, the circular RNA construct comprises an IRES and a CAR comprising an antigen binding domain specific for HER2. In some embodiments, the expression sequence is codon optimized.

[435] As a non-limiting example, in some embodiments, the circular RNA construct comprises a CAR comprising an antigen binding domain specific for CD 19 (B-lymphocyte antigen CD 19). CD 19 is a biomarker for normal and neoplastic B cells, as well as follicular dendritic cells. Diffuse large B cell lymphoma (DLBCL) is the most common lymphoma, accounting for about 25% to 30% of all the non-Hodgkin lymphomas, followed by FL. As CD 19 is expressed in over 95% of B-cell malignancies, it is an attractive target for immunotherapeutic approaches. One known example of a CAR T cell therapy targeting CD 19 is Yescarta® (Kite Pharma Inc., axicabtagene ciloleucel), an anti-CD19 28-ζ (28-zeta) CAR. Another known example of a CAR T cell therapy targeting CD 19 is Kymriah® (Novartis Pharmaceutical Corp., tisagenlecleucel), an anti-CD19 BB-ζ (BB-zeta) CAR. Accordingly, in some embodiments, the expression sequence of the circular RNA construct encodes a CAR, where the codon is directed to an anti-CD19 domain known in the art. In some embodiments, the CAR construct comprises an anti-CD19 binder. In some embodiments, the expression sequence is codon optimized.

[436] As a further non-limiting example, in some embodiments, the circular RNA construct comprises a CAR comprising an antigen binding domain specific for B-cell maturation antigen (BCMA). BCMA (also referred to as TNFRSF17 or CD269), is a member of the tumor necrosis factor receptor (TNFR) superfamily and is expressed by normal and malignant plasma cells and a small subset of B cells. BCMA a known biomarker for certain cancers, including multiple myeloma, and several BCMA-targeted CAR T therapies have been studied, where the constructs varied in their costimulatory domains, hinge regions, transmembrane domains, species used to generate the anti-BCMA scFVs, and the use of different modifications to address safety of the CAR-T therapy. See generally Shah et al., “B- cell maturation antigen (BCMA) in multiple myeloma: rationale for targeting and current therapeutic approaches,” Leukemia 34, 985-1005 (2020). Accordingly, in some embodiments, the expression sequence of the circular RNA construct encodes a CAR, where the codon is directed to an anti-BCMA domain known in the art. In some embodiments, the CAR construct comprises an anti-BCMA binder. In some embodiments, the expression sequence is codon optimized.

[437] As a further non-limiting example, in some embodiments, the circular RNA construct comprises a CAR comprising an antigen binding domain specific for Human Epidermal Growth Factor Receptor 2 (HER2). For example, the CAR can be directed to HER2- BB-ζ (BB-zeta) and/or HER2-28ζ (28-zeta). Accordingly, in some embodiments, the CAR construct comprises an anti-HER2 binder. In some embodiments, the expression sequence is codon optimized.

[438] In some embodiments, the circular RNA construct comprises a CAR comprising an antigen binding domain specific for CD20. CD20 is involved in “developing B-cells’ differentiation and development into plasma cells, which actively participate in B-cell activation and proliferation.” See, e.g., Yin et al., “The breakthrough and the future: CD20 chimeric antigen receptor T-cell therapy for hematologic malignancies” (2022). Engineered CD20 CAR T cells have been used ex vivo for B-cell malignancies and hematologic malignancies, e.g., B-cell hematologic malignancies, e.g., non-Hodgkin lymphoma. See, e.g., id.; see also Chen et al., “CAR-T: What Is Next” (2023). In some embodiments, the circular RNA construct comprises a CAR comprising an antigen binding domain specific for CD20, wherein the anti-CD20 binder is selected from rituximab, ibritumomab, obinutuzumab, otuzumab, ocrelizumab, odronextamab, plamotamab, paramotumab, veltuzumab, ofatumumab, ublituximab, epcoritamab, glofitamab, tositumomab, ripertamab, repatumab, mosunetuzumab, zebituzumab, divozilimab, MB-CART2019.1 (Miltenyi), BVX20-CD20 (Biocon), CPO-107, Leu 16, MRG001, MT-3724, CMG1A46, IMM-0306, JMT-601, ACE- 1831, BAT-4406F, C-CAR039, or a functional fragment thereof. In some embodiments, the expression sequence is codon optimized.

Table 5: Exemplary Constructs (DNA Templates)

[439] In some embodiments, the circular RNA constructs and related pharmaceutical compositions comprise the expression sequences described in Table 6, Table 7, Table 9, Table 10 below. 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 6, Table 7, Table 9, Table 10, wherein the codon sequence produces a protein having the desired sequence.

[440] The exemplary anti-CD19 binder sequences in Table 6 are codon-optimized and correspond to an anti-CD19 28-ζ (28 zeta) CAR. The amino acid sequence corresponding to the nucleotide sequences in Table 6 is set forth below. In some embodiments, the circular RNA constructs and related pharmaceutical compositions disclosed herein comprise a CAR sequence encoding a polypeptide that comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to said sequence or binding fragment thereof.

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSIEVMYPPPYLDNEKSNGTIIHVKG KHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNM TPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 25580)

[441] In some embodiments, the circular RNA constructs and related pharmaceutical compositions disclosed herein comprise an IRES sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an IRES from Table 4, an IRES from a construct of Table 5, and a CAR sequence encoding a polypeptide comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence above or binding fragment thereof. In some embodiments, said circular RNA further comprises a CD28z costimulatory domain as described herein and optionally exhibits increased activity compared to a suitable control having an alternate costimulatory domain. In some embodiments, said circular RNA further comprises a 4-1BB costimulatory domain as described herein and optionally exhibits increased activity compared to a suitable control having an alternate costimulatory domain.

Table 6: Codon Optimized Sequences (anti-CD19 28-ζ)

[442] Table 7 sets forth nucleotide and amino acid sequences for additional exemplary anti-CD19 binder sequences that are not codon-optimized. The sequences are directed to an anti-CD 1928- (28 zeta) CAR. In some embodiments, the circular RNA constructs and related pharmaceutical compositions disclosed herein comprise a CAR sequence encoding a polypeptide that comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the CAR sequences of Table 7 or binding fragments thereof.

[443] In some embodiments, the circular RNA constructs and related pharmaceutical compositions disclosed herein comprise an IRES sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an IRES from Table 4, an IRES from a construct of Table 5, and a CAR sequence encoding a polypeptide comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the CAR sequences of Table 6 or Table 7 or binding fragments thereof. In some embodiments, said circular RNA further comprises a CD28z costimulatory domain as described herein and optionally exhibits increased activity compared to a suitable control having an alternate costimulatory domain. In some embodiments, said circular RNA further comprises a 4- 1BB costimulatory domain as described herein and optionally exhibits increased activity compared to a suitable control having an alternate costimulatory domain.

Table 7: Additional Codon Amino Acid and Nucleotide Sequences (anti-CD 19 28-ζ)

[444] Table 9 sets forth nucleotide and amino acid sequences for additional exemplary binder sequences that are not codon-optimized, including a mouse anti-CD19 binder, anti- BCMA binders, and anti-HER2 binders. In some embodiments, the circular RNA constructs and related pharmaceutical compositions disclosed herein comprise a CAR sequence encoding a polypeptide that comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the CAR sequences of Table 9 or binding fragments thereof.

[445] Table 10 sets forth amino acid sequences for exemplary anti-CD20 binders. In some embodiments, the circular RNA constructs and related pharmaceutical compositions disclosed herein comprise a CAR sequence encoding a polypeptide that comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the CAR sequences of Table 10 or binding fragments thereof.

[446] In some embodiments, the circular RNA constructs and related pharmaceutical compositions disclosed herein comprise an IRES sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an IRES from Table 4, an IRES from a construct of Table 5, and a CAR sequence encoding a polypeptide comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the CAR sequences of Table 9 or Table 10 or binding fragments thereof. In some embodiments, said circular RNA further comprises a CD28z costimulatory domain as described herein and optionally exhibits increased activity compared to a suitable control having an alternate costimulatory domain. In some embodiments, said circular RNA further comprises a 4- 1BB costimulatory domain as described herein and optionally exhibits increased activity compared to a suitable control having an alternate costimulatory domain.

[447] As a non-limiting example, in some embodiments, the circular RNA constructs and related pharmaceutical compositions disclosed herein comprise a CAR sequence encoding a polypeptide that comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to CAR sequence 10J of Table 10 or binding fragments thereof, and an IRES sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an IRES from Table 4.

Table 9: Additional Codon Amino Acid and Nucleotide Sequences Table 10: Exemplary Binders

[448] 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.

[449] In some embodiments, the scFv, heavy variable domain, light variable domain, heavy CDR sequences, and/or light CDR sequences of the proteins listed in the tables herein may be used.

[450] In some embodiments, the therapeutic protein is selected from, e.g, 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 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, e.g., SEQ ID NOS: 4019-4040, or a fragment thereof.

[451] In some embodiments, the expression sequence encodes a therapeutic protein. In some embodiments, the expression sequence encodes, e.g., 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, e.g., an immune checkpoint inhibitor. In some embodiments, the expression sequence encodes, e.g., an agonist (e.g., a TNFR family member such as CD137L, OX40L, ICOSL, LIGHT, or CD70). In some embodiments, the expression sequence encodes, e.g., a chimeric antigen receptor. In some embodiments, the expression sequence encodes, e.g., 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, e.g, an inhibitory receptor antagonist. In some embodiments, the expression sequence encodes, e.g, one or more TCR chains (alpha and beta chains or gamma and delta chains). In some embodiments, the expression sequence encodes, e.g, 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, CD 19, CD20, or BCMA etc.). In some embodiments, the expression sequence encodes, e.g, 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, e.g, a GvHD (e.g, anti-HLA-A2 CAR-Tregs).

[452] 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. ii. Hinge / spacer domain

[453] 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 CD1 la (IT GAL), CD1 lb (IT GAM), CD1 1c (ITGAX), CD1 Id (IT GAD), CD 18 (ITGB2), CD 19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B-cell antigen receptor complex-associated alpha chain), CD79B (B-cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD 100 (SEMA4D), CD 103 (ITGAE), CD 134 (0X40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD 160 (BY55), CD 162 (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 (CD1 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.

[454] 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. iii. Transmembrane domain

[455] 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.

[456] 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 1c, 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; CD 150; 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.

[457] 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, 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.

[458] 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 Al (Eph Al), EPH receptor A2 (EphA2), (EPH receptor A3) Eph A3, 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 Bl (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

[459] 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 (Q. 4-1BB, CD28, CD3 zeta may comprise less than the whole 4-1BB, CD28 or CD3 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).

[460] In some embodiments, a costimulatory domain comprises the amino acid sequence of SEQ ID NO: 4041 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL) or 4042

(QVQLVQSGAEVEKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINP NSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCASGWDFDYWGQG TLVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQSPSSLSASVGDRVTITCRASQSIRY YLSWYQQKPGKAPKLLIYTASILQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCL QTYTTPDFGPGTKVEIK). See, e.g., PCT Application No. US2022/33091 (WO202261490), which is incorporated herein by reference in its entirety. v. Intracellular signaling domain

[461] 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.

[462] 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, CD 100 (SEMA4D), CD 103, CD 160 (BY55), CD 18, CD 19, 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 1c, CD1 Id, 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 (CD 162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMF1; CD 150; 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.

[463] 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 of SEQ ID NO: 4043 (R VKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR). See, e.g., PCT Application No. US2022/33091, which is incorporated herein by reference in its entirety.

4. PRODUCTION OF POLYNUCLEOTIDES

A. Precursor RNA preparation

[464] 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.

[465] 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) 259:631 1.

[466] 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 (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.

[467] 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 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.

[468] 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.

[469] 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).

[470] 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 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.

[471] 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.

B. Circular RNA preparation

[472] 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 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+).

[473] 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 aMg2+ 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.

[474] 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 DNA.

[475] 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.

[476] 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).

[477] 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.

[478] 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 ' fragment of the sequence of interest.

[479] 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). [480] 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.

[481] 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 1 A), and

(c) the 3 ’ fragment of the sequence of interest.

[482] 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.

[483] In some embodiments, a first precursor comprises an optional first external homology region (Arm 1 A), 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., IRES), the 5' fragment of the sequence of interest (e.g., coding region), a second exon fragment (Exon 2A), a second intron fragment (5 ' 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).

[484] In some embodiments, either the first precursor or the second precursor comprises a monotron. [485] In some embodiments, the first precursor comprises an optional first external homology region (Arm 1 A), 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., 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).

[486] 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., 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 sequence, and an optional second external homology region (Arm 1B).

[487] 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., 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).

[488] 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.

[489] 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.

[490] 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 embodiments, purification comprises one or more of the following steps: phosphatase treatment, HPLC size exclusion purification, and RNase R digestion. In some embodiments, purification comprises the following steps in order: RNase R digestion, phosphatase treatment, and HPLC size exclusion purification. In some embodiments, purification comprises reverse phase HPLC. In some embodiments, the purified composition contains less double stranded RNA, DNA splints, triphosphorylated RNA, phosphatase proteins, protein ligases, capping enzymes and/or nicked RNA than a control unpurified composition. 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.

[491] In some embodiments, provided herein is a method of measuring RNA species and/or RNA integrity. In some embodiments, the method measures an amount of a desired circular RNA and/or an amount of an undesired RNA, e.g., a linear precursor RNA and/or a nicked RNA, within a purified composition. In some embodiments, the amount is the % of an RNA species from the total RNA species. In some embodiments, provided herein is a method of measuring RNA species comprising the steps of: (1) obtaining a sample comprising a circular RNA and a precursor RNA, wherein the precursor RNA comprises a self-splicing intron fragment, and wherein the circular RNA is produced by (a) self-circularizing the precursor RNA to produce the circular RNA as described herein, and (b) purifying the circular RNA; (2) subjecting the sample to gel electrophoresis, e.g., optionally capillary gel electrophoresis, and separating the precursor RNA and the circular RNA; (3) measuring the precursor RNA and measuring the circular RNA. In some embodiments, the method comprises: (1) obtaining a sample comprising a circular RNA and a precursor RNA, (2) subjecting the sample to gel electrophoresis and separating the precursor RNA and the circular RNA; and (3) measuring the levels of precursor RNA and the circular RNA in the sample. Kits and assays thereof are contemplated herein.

[492] In some embodiments, the purified composition is less immunogenic and/or less reactogenic than a control unpurified composition. In some embodiments, cells exposed to the purified composition in vitro and/or in vivo produce less of an immunogenic and/or reactogenic molecule, e.g., cytokine, TNFα, RIG-I, IL-2, IL-6, interferon (INF), e.g., IFN-α, IFN-β, IFN- β1, IFNγ, INFλ, than cells exposed to a control unpurified composition. In some embodiments, the cells are cells that respond to dsRNA. In some embodiments, the cells are cells that express toll-like receptors (TLRs), e.g., epithelial cells, fibroblast cells, immune cells. See, e.g., Kawasaki and Kawai, Toll-like receptor signaling pathways, 2014; Farina et al., dsRNA activation of endothelin-1 and markers of vascular activation in endothelial cells and fibroblasts, 2011. In some embodiments, the cells are immune cells, e.g., B cells, dendritic cells, macrophages. In some embodiments, provided herein is a method of measuring a cellular response following exposure to the purified composition. In some embodiments, the cellular response is a measurement of the amount of one or more immunogenic and/or reactogenic molecules, e.g., cytokine, TNFα, RIG-I, IL-2, IL-6, interferon (INF), e.g., IFN-α, IFN-β, IFN- β 1, IFNγ, INFλ. In some embodiments, the cells are exposed to the purified composition in vitro, and the cells or cellular supernatant may be assessed for the immunogenic and/or reactogenic molecule, e.g., using an antibody -based detection method known in the art, e.g., ELISA, MSD, Western blot. In some embodiments, the purified composition is administered to a subject in vivo, and a sample is obtained from the subject, e.g., a blood sample, a serum sample, a cell sample, and/or a tissue sample, may be assessed for the immunogenic and/or reactogenic molecule, e.g., using an antibody -based detection method known in the art, e.g., ELISA, MSD, Western blot. In some embodiments, provided herein is a method of measuring an immunogenic and/or reactogenic molecule, e.g., cytokine, TNFα, RIG-I, IL-2, IL-6, interferon (INF), e.g., IFN-α, IFN-β, IFN-β 1, IFNγ, INFλ .

[493] In some embodiments, provided herein is a method of measuring the levels of an immunogenic and/or reactogenic molecule, comprising the steps of: (1) contacting cells in vitro or in vivo with a composition comprising a desired circular RNA and an undesired RNA (e.g., linear precursor RNA and/or nicked RNA), wherein the circular RNA is produced by: (a) selfcircularizing a precursor RNA comprising a self-splicing intron fragment to produce the circular RNA as described herein, and (b) purifying the circular RNA; (2) obtaining a first sample comprising the cells and/or a supernatant of the cells; and (3) detecting the level of at least one immunogenic and/or reactogenic molecule in said sample. In some embodiments, the method further comprises (4) contacting cells in vitro or in vivo with a control composition that omits step (b) purifying the circular RNA; (5) obtaining a second sample comprising the cells and/or a supernatant of the cells; (6) detecting the level of at least one immunogenic and/or reactogenic molecule in the second sample (baseline), and (7) comparing the level of the at least one molecule detected in the first sample to the level detected in the second sample. In some embodiments, the method comprises the steps of: (1) contacting a first sample of cells with a composition comprising a circular RNA and a precursor RNA; and (2) detecting the level of at least one immunogenic and/or reactogenic molecule in said sample; optionally wherein the molecule is selected from the group consisting of a cytokine, TNFα, RIG-I, IL-2, IL-6, interferon, IFN-α, IFN-β, IFN-β 1, IFNγ, and INFλ . In some embodiments, the method further comprises comparing the level of the at least one immunogenic and/or reactogenic molecule in said sample to the levels of a control sample or a baseline value. In some embodiments, the molecule is RIG-I. In some embodiments, the molecule is TNFα. In some embodiments, the molecule is IFN. In some embodiments, the molecule is IFN-β. Kits and assays thereof are contemplated herein.

[494] 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 is 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.

[495] 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 l. lkb 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 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, 3’ PIE splice site, and/or 5’ splice site.

[496] Further methods for preparing circular RNA are 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

[497] 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.

[498] 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

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- l-yl)tetracosa- 15,18-dien- 1 -amine (HGT5000), (15Z,18Z)— N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-l- yl)tetracosa-4,15,18-trien-l-amine (HGT5001), and (15Z,18Z) — N,N-dimethyl-6-((9Z,12Z)- octadeca-9,12-dien-l-yl)tetracosa-5,15,18-trien-l-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.

[499] In some embodiments, the cationic lipid N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride or “DOTMA” is used. (Feigner 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 noncationic 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 -di oleyloxy -N- [2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-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)-l-(cis,cis-9,12- octadecadienoxy)propane or “CLinDMA,” 2-[5’-(cholest-5-en-3-beta-oxy)-3’-oxapentoxy)-3- dimethy 1 - l-(cis,cis-9’ , 1-2’-octadecadienoxy)propane or “CpLinDMA,” N,N-dimethyl-3,4- dioleyloxybenzylamine or “DMOBA,” 1,2-N,N’ -di oleylcarbamyl-3 -dimethylaminopropane or “DOcarbDAP,” 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or “DLinDAP,” 1,2-N,N’- Dilinoleylcarbamyl-3-dimethylaminopropane or “DLincarbDAP,” 1,2-Dilinoleoylcarbamyl-3- dimethylaminopropane or “DLinCDAP,” 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]- dioxolane or “DLin-K-DMA,” 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane or “DLin- K-XTC2-DMA,” and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-l-yl)-l,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).

[500] 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), l,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. [501] 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.

[502] 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.

[503] 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:

[504] 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 1-1 through 1-41, 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 11-46, 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-l 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-l 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 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.

[505] 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.

[506] 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.

[507] 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.

[508] 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.

[509] 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.

[510] 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.

[511] 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” 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-lH-cyclopenta[a]phenanthren-3-yl 3-(lH-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-lH-cyclopenta[a]phenanthren-3-yl 3-(lH-imidazol-4-yl)propanoate, as represented by structure (XIII). In some embodiments, an ionizable lipid is described by US patent publication number 20190314284.

[512] 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.

[513] 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.

[514] 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 lOa-lOf, 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.

[515] In some embodiments, the one or more of the cationic or ionizable lipids are represented by 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.

[516] In some embodiments, R_a is hydrogen. In some embodiments, R_a is hydroxyl.

[517] In some embodiments, the ionizable lipid is represented by Formula (la -1), Formula (la-

2), or Formula (la-3):

[518] In some embodiments, the ionizable lipid is represented by Formula (Ib-1), Formula (Ib- 2), or Formula (Ib-3):

[519] 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):

[520] 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:

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

[522] In various embodiments, the one or more of the cationic or ionizable lipids are represented by Formula (I*): wherein: n* is an integer between 1 to 7,

R^a is hydrogen or hydroxyl,

R^b 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.

[523] In some embodiments, the one or more of the cationic or ionizable lipids are represented by 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, hydroxy carbonyl, alkyloxy carbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl,

(alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkyl sulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkyl sulfonealkyl; and R₂ is selected from a group consisting of:

[524] 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:

[525] In some embodiments, R₁ and R₃ are the same. In some embodiments, R₁ and R₃ are different.

[526] In some embodiments, the one or more of the cationic or ionizable lipids are represented by Formula (II- 1) or Formula (II-2):

[527] In some embodiments, the ionizable lipid is selected from an ionizable lipid of WO2015/095340 (lipid number 123 of Table 3). In some embodiments, the ionizable lipid is selected from an ionizable lipid ofWO2021/021634, WO2020/237227, or WO2019/236673 (lipid numbers 124- 127 of Table 3). In some embodiments, the ionizable lipid is selected from an ionizable lipid of WO2021226597 and WO2021113777 (lipid numbers 128-131 of Table 3).

[528] In some embodiments, the transfer vehicle comprises an ionizable lipid selected from an ionizable lipid represented in Table 3. 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 3, 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 3, below.

[529] In some embodiments, the one or more of the cationic or ionizable lipids are represented by 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, orN(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.

[530] In some embodiments, the one or more of the cationic or ionizable lipids are represented by 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, orN(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 8: Exemplary Ionizable Lipid Structures

[531] In some embodiments, an ionizable lipid is a compound of Formula (15): or is a pharmaceutically acceptable salt thereof, wherein: n* is an integer from 1 to 7;

R^a is hydrogen or hydroxyl;

R^h is hydrogen or C₁-C₆ alkyl; R¹ is C₁-C₃₀ alkyl or R^1*;

R² is C₁-C₃₀ alkyl orR^2*;

R^1* and R^2* 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 R^1*, (ii) R² is R^2*, or (iii) R¹ is R^1* and R² is R^2*.

[532] In some embodiments of Formula (15), R^a is hydrogen and the ionizable lipid is of

Formula (16): or is a pharmaceutically acceptable salt thereof, wherein: n* is an integer from 1 to 7.

[533] In some embodiments of Formula (16), the ionizable lipid is of 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;

Z^A and Z^B are each independently selected from and -OC(O)O-; where denotes the attachment point to -(CH₂)_q- or -(CH₂)_q'-; and

R^9A, R^9B, R^10A and R^10B are each independently C₁-C₂₀ alkyl or C₂-C₂₀ alkenyl.

[534] In some embodiments of Formula (17), Z^A and Z^B are and the ionizable lipid is of Formula (17a-1)

[535] In some embodiments of Formula (17), Z^A and Z^B are and the ionizable lipid is of Formula (17a-2)

[536] In some embodiments of Formula (17), Z^A and Z^B are -O(C)(O)O-, and the ionizable lipid is represented by Formula (17a-3):

[537] In some embodiments of Formula (15), R^a is hydroxyl and the ionizable lipid is of Formula (18): or is a pharmaceutically acceptable salt thereof, wherein: n* is an integer from 1 to 7;

R^h is hydrogen or C₁-C₆ alkyl;

R¹ is C₁-C₃₀ alkyl or R^1*

R² is C₁-C₃₀ alkyl or R^2*;

R^1* and R^2* are independently selected from:

-(CH₂)_qC(O)O(CH₂)_rC(R⁸)(R⁹)(R¹⁰),

-(CH₂)_qOC(O)(CH₂)_rC(R⁸)(R⁹)(R¹⁰), and

R⁸ is hydrogen or R¹¹;

R⁹, R¹⁰, and R¹¹ are each independently C₁-C₂₀ alkyl or C₂-C₂₀-alkenyl; wherein (i) R¹ is R^1*, (ii) R² is R^2*, or (iii) R¹ is R^1* and R² is R^2*; and wherein, for (iii), (a) R^1* and R^2* are different or (b) R⁹ and R¹⁰ have different numbers of carbon atoms for at least one of R^1* and R^2*.

[538] In some embodiments of Formula (18), the ionizable lipid of is of 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;

Z^A and Z^B are each independently selected from and -OC(O)O-; where denotes the attachment point to -(CH₂)_q- or -(CH₂)_q ;-and R^9A, R^9B, R^1OA and R^10B are each independently C₁-C₂₀ alkyl or C₂-C₂₀ alkenyl.

[539] In some embodiments of Formula (19), Z^A and Z^B are and the ionizable lipid is of Formula (19a-1):

[540] In some embodiments of Formula (19), Z^A and Z^B are and the ionizable lipid is of Formula (19a-2):

[541] In some embodiments of Formula (19), Z^A and Z^B are -O(C)(O)O-, and the ionizable lipid is represented by Formula (19a-3):

[542] In some embodiments of Formula (15), R¹ is C₁-C₃₀ alkyl, and the ionizable lipid is of Formula (20): or is a pharmaceutically acceptable salt thereof, wherein: Z^A is selected from and -OC(O)O-; where denotes the attachment point to -(CH₂)_q-;

R^9A and R^1OA 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.

[543] In some embodiments of Formula (20), Z^A is and the ionizable lipid is of Formula (20a- 1):

[544] In some embodiments of Formula (20), Z^A is and the ionizable lipid is of Formula (20a-2):

[545] In some embodiments of Formula (20), Z^A is -OC(O)O-, and the ionizable lipid is of Formula (20a-3):

[546] In some embodiments of Formula (15), R² is C₁-C₃₀ alkyl, and the ionizable lipid is of

Formula (21): or is a pharmaceutically acceptable salt thereof, wherein:

Z^B is selected from and -OC(O)O-; where denotes the attachment point to -(CH₂)_q -;

R^9B and R^10B 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.

[547] In some embodiments of Formula (21), Z^B is and the ionizable lipid is of

Formula (21a-1):

[548] In some embodiments of Formula (21), Z^B is and the ionizable lipid is of

Formula (2 la-2):

[549] In some embodiments of Formula (21), Z^B is -OC(O)O-, and the ionizable lipid is of Formula (2 la-3):

[550] In some embodiments, an ionizable lipid is selected from the table below:

[551] In some embodiments, an ionizable lipid of the present disclosure is represented by

Formula (22): or is a pharmaceutically acceptable salt thereof, wherein:

R^a is hydrogen or hydroxyl;

R¹ is C₁-C₃₀ alkyl or R^1*;

R² is C₁-C₃₀ alkyl or R^2*;

R^1* and R^2* 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 R^1*, (ii) R² is R^2*, or (iii) R¹ is R^1* and R² is R^2*; and 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) R^A, R^B, or R^c, wherein

R^A is selected from:

R^C is selected from:

with the proviso that the ionizable lipid is not:

[552] In some embodiments of Formula (22), R³ is selected from:

[553] In some embodiments of Formula (22), R¹ is R^1*, R² is R^2*, and the ionizable lipid is of

Formula (23): wherein: 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;

Z^A and Z^B are each independently selected from and -OC(O)O-; where denotes the attachment point to -(CH₂)_q- or -(CH₂)_q -; and

R^5A, R^5B, R^6A and R^6B are each independently C₁-C₂₀ alkyl or C₂-C₂₀ alkenyl.

[554] In some embodiments of Formula (23), Z^A and Z^B are and the ionizable lipid is of Formula (23a- 1):

[555] In some embodiments of Formula (23), Z^A and Z^B are and the ionizable lipid is of Formula (23a-2)

[556] In some embodiments of Formula (23), Z^A and Z^B are -O(C)(O)O-, and the ionizable lipid is represented by Formula (23a-3):

[557] In some embodiments of Formula (22), R² is C₁-C₃₀ alkyl, and the ionizable lipid is of

Formula (25): or is a pharmaceutically acceptable salt thereof, wherein:

R^5B and R^6B 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.

[558] In some embodiments of Formula (25), Z^B is and the ionizable lipid is of Formula (25a-1):

[559] In some embodiments of Formula (25), Z^B is and the ionizable lipid is of Formula (25a-2):

[560] In some embodiments of Formula (25), Z^B is -OC(O)O-, and the ionizable lipid is of Formula (25a-3):

[561] In some embodiments, an ionizable lipid is selected from the table below:

[562] In some embodiments, an ionizable lipid is selected from the table below:

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.

[564] 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.

[565] 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.

[566] 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.

[567] In some embodiments, an LNP described herein comprises an ionizable lipid of Table Z:

Table Z - Exemplary Ionizable Lipids

[568] In some embodiments, the ionizable lipid is MC3.

Series “A”

[569] 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)

[570] 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;

R^xx 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 each R^ww 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 R^ww is linear C₄-C₁₂ alkyl.

[571] In some embodiments, ionizable lipids ofthe present disclosure have a structure ofFormula (X), wherein R^xx is H. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein R^xx is optionally substituted C₁-C₆ alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein R^xx is C₁ alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure ofFormula (X), wherein R^xx is C₂ alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein R^xx is C₃ alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein R^xx is C₄ alkyl. In some embodiments, ionizable lipids ofthe present disclosure have a structure of Formula (X), wherein R^xx is C₅ alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein R^xx is C₆ alkyl.

[572] In some embodiments, ionizable lipids of the present disclosure have a structure ofFormula (X), wherein each R^ww 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 R^ww 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 R^ww 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 R^ww is branched C₄-C₁₂ alkenyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each R^ww 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 R^ww is C₉-C₁₂ alkenyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each R^ww is linear C₄-C₁₂ alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each R^ww 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 R^ww is C₆-C₁₄ alkyl, 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 R^ww 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.,

[573] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each R^ww 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 R^ww is C₉-C₁₂ alkenyl.

[574] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each R^ww 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.

[575] 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).

[576] 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).

[577] 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).

[578] 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 Cl 1 alkyl. In some embodiments, ionizable lipids of the present disclosure have 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.

[579] 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 Cl 1 alkenyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein each Rww is C12 alkenyl.

[580] 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.

[581] 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.

[582] 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 (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.

[583] 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.

[584] 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.

[585] 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. [586] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula

(X), wherein ee is 1.

[587] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X), wherein ee is 0.

Formula (X-A)

[588] 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;

R^xx is selected from hydrogen and optionally substituted C₁-C₆ alkyl; and each R^ww is independently selected from the group consisting of C₄-C₁₄ alkyl or (linear or branched C₃-C₅ alkylenyl)-(branched C₅-C₇alkcnyl).

[589] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein R^xx is hydrogen. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein R^xx is C₁ alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein R^xx is C₂ alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein R^xx is C₃ alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein R^xx is C₄ alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein R^xx is C₅ alkyl. In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (X-A), wherein R^xx is C₆ alkyl.

[590] 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.

[591] In some embodiments, ionizable lipids ofthe present disclosure have a structure ofFormula (X-A), wherein each dd is 1 or 3. In some embodiments, ionizable lipids ofthe present disclosure have a structure ofFormula (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.

[592] In some embodiments, ionizable lipids of the present disclosure have a structure ofFormula (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 ofFormula (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 ofthe 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.

[593] In some embodiments, ionizable lipids of the present disclosure have a structure ofFormula (X-A), wherein each R^ww is (linear or branched C₃-C₅ alkylenyl) -(branched C₅-C₇alkenyl), e.g., (branched C₅ alkylenyl)-(branched C₅alkenyl), e.g.,

[594] 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:

Table (LA). Non-Limiting Examples of Ionizable Lipids with an Acyclic Core

Series “CY”

[595] 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)

[596] In some embodiments, an LNP disclosed herein comprises an ionizable lipid of Formula (CY) or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from the group consisting of -OH, -OAc, R^1a,

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-;

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 wherein the bond marked with an is attached to X⁴ or X⁵; 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); R^1a is:

R^2a, R^2b, and R^2c are independently hydrogen and C₁-C₆ alkyl;

R^3a, R^3b, and R^3c are independently hydrogen and C₁-C₆ alkyl;

R^4a, R^4b, and R^4c are independently hydrogen and C₁-C₆ alkyl;

R^5a, R^5b, and R^5c 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)

[597] In some embodiments, the present disclosure comprises a compound of any of the below Formulae:

[598] In some embodiments, the present disclosure includes a compound of Formula (CY -IV-d), (CY-IV-e), or (CY-IV-f)

Formula (CY-IV’)

[599] 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’)

[600] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CY-VI' ): 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').

[601] 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.

[602] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (CYVI' ), or a pharmaceutically acceptable salt thereof, wherein XI is C2-C6 alkylenyl.

[603] 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-.

[604] 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.

[605] 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.

[606] 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:

[607] 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:

[608] 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.

[609] 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-.

[610] 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.

[611] 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.

[612] 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.

[613] 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.

[614] 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.

[615] 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.

[616] 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.

[617] 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.

[618] 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.

[619] In some embodiments, Lipids of the Present Disclosure are selected from any lipid in Table (I-B) below or a pharmaceutically acceptable salt thereof:

Table (I-B). Non-Limiting Examples of Ionizable Lipids with a Cyclic Core

Series “C”

[620] 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.

[621 ] In one embodiment, the disclosure provides a compound of Formula IA: or a pharmaceutically acceptable salt or solvate thereof, wherein:

A is selected from the group consisting of -N(R^1a)- and -C(R')-OC(=O)(R^8a)-;

R^1a is -L¹-R¹;

L¹ is C₂-C₆ alkylenyl or -(CH₂)_2-6-OC(=O)-;

R¹ is selected from the group consisting of -OH,

R^2a, R^2b, and R^2c are independently selected from the group consisting of hydrogen and C₁-C₆ alkyl;

R^3a, R^3b, and R^3c are independently selected from the group consisting of hydrogen and C₁-C₆ alkyl;

R^4a, R^4b, and R^4c are independently selected from the group consisting of hydrogen and C₁-C₆ alkyl;

R^5a, R^5b, and R^5c are independently selected from the group consisting of hydrogen and C₁-C₆ alkyl;

R^6a, R^6b, and R^6c are independently selected from the group consisting of hydrogen and C₁-C₆ alkyl; or

R^6a and R^6b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; and R^6c is selected from the group consisting of hydrogen and C₁C₆ alkyl;

R^7a, R^7b. and R^7c are independently selected from the group consisting of hydrogen and C₁-C₆ alkyl; or R^7a and R^7b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; and R^7c is selected from the group consisting of hydrogen and C₁C₆ alkyl;

R' is selected from the group consisting of hydrogen and C₁-C₆ alkyl;

R^8a is - L²-R⁸;

L² is C₂-C₆ alkylenyl; R⁸ is selected from the group consisting of -NR^9aR^9b,

R^9a and R^9b are independently selected from the group consisting of hydrogen and C₁-C₆ alkyl; or

R^9a and R^9b 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(R^12a)-, -N(R^12a)C(=O)-, - OC(=O)N(R^12a)-, - N(R^12a)C(=O)O-, and -OC(=O)O-;

R^12a 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(R^12b)-, -OC(=O)N(R^12b)-, and - OC(=O)O-;

R^12b 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, 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.

[622] 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(R^1a)- and -C(R')-OC(=O)(R^8a)-; R^1a is -L¹-R¹;

L¹ is C₂-C₆ alkylenyl or -(CH₂)_2-6-OC(=O)-;

R¹ is selected from the group consisting of -OH,

R^6a, R^6b, and R^6c are independently selected from the group consisting of hydrogen and C₁-C₆ alkyl; or R^6a and R^6b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; and R^6c is selected from the group consisting of hydrogen and C₁C₆ alkyl; R^7a, R^7b. and R^7c are independently selected from the group consisting of hydrogen and C₁-C₆ alkyl; or R^7a and R^7b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; and R^7c is selected from the group consisting of hydrogen and C₁C₆ alkyl;

R' is selected from the group consisting of hydrogen and C₁-C₆ alkyl;

R^8a is - L²-R⁸;

Q¹ is C₁-C₂₀ alkylenyl;

R^12a is selected from the group consisting of hydrogen and C₁-C₆ alkyl;

X¹ is optionally substituted C₁-C₁₅ alkylenyl; or

X¹ is a bond;

Z¹ is selected from the group consisting of optionally substituted C₅-C₁₂ bridged cycloalkylenyl,

Q² is C₁-C₂₀ alkylenyl;

R^12b is selected from the group consisting of hydrogen and C₁-C₆ alkyl; X² is optionally substituted C₁-C₁₅ alkylenyl; or

X² is a bond;

[623] In one embodiment, the disclosure provides a compound of Formula IC: or a pharmaceutically acceptable salt or solvate thereof, wherein:

L¹ is C₂-C₆ alkylenyl or -(CH₂)_2-6-OC(=O)-;

R¹ is selected from the group consisting of -OH,

R^3a, R^3b, and R^3c are independently selected from the group consisting of hydrogen and C₁-C₆ alkyl; R^4a, R^4b, and R^4c are independently selected from the group consisting of hydrogen and C₁-C₆ alkyl;

R^7a, R^7b, and R^7c are independently selected from the group consisting of hydrogen and C₁-C₆ alkyl; or R^7a and R^7b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; and R^7c is selected from the group consisting of hydrogen and C₁C₆ alkyl;

R' is selected from the group consisting of hydrogen and C₁-C₆ alkyl;

R^8a is - L²-R⁸;

Q¹ is C₁-C₂₀ alkylenyl;

R^12a 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;

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;

R^12b is selected from the group consisting of hydrogen and C₁-C₆ alkyl;

X² is optionally substituted C₁-C₁₅ alkylenyl; or

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.

[624] 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.

[625] 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.

[626] 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(R^1a)- and -C(R')-OC(=O)(R^8a)-; R^1a is - L¹-R¹;

L¹ is C₂-C₆ alkylenyl or -(CH₂)_2-6-OC(=O)-;

R¹ is selected from the group consisting of -OH,

R^2a, R^2b, and R^2c are independently selected from the group consisting of hydrogen and C₁-C₆ alkyl; R^3a R^3b, and R^3c are independently selected from the group consisting of hydrogen and C₁-C₆ alkyl; R^4a, R^4b, and R^4c are independently selected from the group consisting of hydrogen and C₁-C₆ alkyl;

R^6a, R^6b, and R^6c are independently selected from the group consisting of hydrogen and C₁-C₆ alkyl; or R^6a and R^6b taken together with the nitrogen atom to which they are attached form a 4-to 8-membered heterocyclo; and R^6c is selected from the group consisting of hydrogen and C₁C₆ alkyl;

R' is selected from the group consisting of hydrogen and C₁-C₆ alkyl;

R^8a is - L²-R⁸;

L² is C₂-C₆ alkylenyl;

R⁸ is selected from the group consisting of -NR^9aR^9b,

Q¹ is C₁-C₂₀ alkylenyl;

R^12a 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;

Z¹ is optionally substituted C₅-C₁₂ bridged cycloalkylenyl;

Q² is C₁-C₂₀ alkylenyl;

R^12b 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.

[627] In some embodiments, the disclosure provides a compound of Formula ID or a pharmaceutically acceptable salt or solvate thereof, wherein Z1 is not adamantyl.

[628] In one embodiment, the disclosure provides a compound of Formula I: or a pharmaceutically acceptable salt or solvate thereof, wherein:

L¹ is C₂-C₆ alkylenyl;

R¹ is selected from the group consisting of -OH,

R' is selected from the group consisting of hydrogen and C₁-C₆ alkyl;

R^8a is - L²-R⁸;

L² is C₂-C₆ alkylenyl;

R⁸ is -NR^9aR^9b;

Q¹ is C₁-C₂₀ alkylenyl;

R^12a is selected from the group consisting of hydrogen and C₁-C₆ alkyl;

X¹ is C₁-C₁₅ alkylenyl; or

X¹ is a bond;

Z¹ is selected from the group consisting of C₄-C₁₂ cycloalkylenyl,

Q² is C₁-C₂₀ alkylenyl;

R^12b is selected from the group consisting of hydrogen and C₁-C₆ alkyl;

X² is C₁-C₁₅ alkylenyl; or

X² is a bond;

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.

[629] 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: Table (I-C). Non-Limiting Examples of Ionizable Lipids with a Constrained Arm

Series “CX”

[630] 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.

[631] 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. [632] In some embodiments, a compound of the present disclosure is represented by Formula (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(R^a)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-; R^2' 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 R^a is independently optionally substituted C₁-C₆ alkyl; or two R^a 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.

[633] In some embodiments, a compound of the present disclosure is represented by Formula

(CX-i): or a pharmaceutically acceptable salt thereof, wherein

R¹ is -(CH₂)_1-6N(R^a)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 R^a is independently optionally substituted C₁-C₆ alkyl; or two R^a 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. [634] 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

[635] 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”

[636] 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.

[637] In some embodiments, a compound of the present disclosure is represented by Formula (CZ-I) or a pharmaceutically acceptable salt thereof, wherein

R¹ is -(CH₂)_1-6N(R^a)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 R^a is independently optionally substituted C₁-C₆ alkyl; or two R^a 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.

[638] In some embodiments, the present disclosure comprises a compound selected from any lipid in Table (I-E) below or a pharmaceutically acceptable salt thereof: Table (I-E). Non-Limiting Examples of Ionizable Lipids

Series “S”

[639] 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.

Formula (S-I)

[640] 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; R¹ is OH, N(R³)₂, each R is independently -H or C₁-C₆ aliphatic; each R² is independently selected from optionally substituted C_2-14alkyl and C_2-14alkenyl, wherein any -(CH₂)₂- of the C₂-C₁₄ alkyl can be optionally replaced with C₃-C₆ cycloalkylenyl; each R³ independently selected from is H and C_1-6alkyl; n is selected from 1 to 6; and each p is independently selected from 1 to 6.

Formula (S-M)

[641] 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, , 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; R¹ is OH, N(R³)₂, each R is independently -H or C₁-C₆ aliphatic; each R³ independently selected from is H and C_1-6alkyl;

R⁴ is -CH(SR⁶)(SR⁷); 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.

[642] 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.

[643 ] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula

(S-Mb) or a pharmaceutically acceptable salt thereof, wherein: each R³ independently selected from is H and C_1-6 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.

[644] 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

Series “AT”

[645] 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.

[646] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (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: ii) A is CH; wherein the bond 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;

R^Z 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 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), -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-;

R⁸ and R⁹ are each independently optionally substituted G-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’)

[647] 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' ): or a pharmaceutically acceptable salt thereof, wherein R¹, R, X¹, Z, X², X³, X⁴, R^Z, 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’”)

[648] 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' " ):

[649] or a pharmaceutically acceptable salt thereof, wherein R¹, R, X¹, Z, X², X³, X⁴, R^Z, R², R³, R⁶, R⁷, R⁸, and R⁹ are as described in Formula (AT) or as otherwise described in any embodiments below.

Formula (AT-M)

[650] 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): or a pharmaceutically acceptable salt thereof, wherein R¹, R, X², X³, X⁴, R^Z, 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.

Formula (AT-N’)

[651] 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' ):

[652] or a pharmaceutically acceptable salt thereof, wherein R¹, R, X¹, X², X³, X⁴, R^Z, 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’)

[653] 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' ): or a pharmaceutically acceptable salt thereof, wherein R¹, R, X¹, X², X³, Y¹, Y², X⁴, R^Z, R², R³, R⁶, R⁷, R⁸, and R⁹ are as described in Formula (AT) or as otherwise described in any embodiments below.

Formula (AT-P’”)

[654] 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- P' ' ' ):

or a pharmaceutically acceptable salt thereof, wherein R¹, R, X¹, X², X³, R², R³, X⁴, R^Z, R⁶, R⁷, R⁸, and R⁹ are as described in Formula (AT) or as otherwise described in any embodiments below.

[655] 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

Series “AC”

[656] 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.

[657] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula (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 ;

X⁴ is a bond or C₂-C₆ 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 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

[658] 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):

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. [659] 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

Series “CO”

[660] 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.

[661] The present disclosure provides compound of Formula (CO) : 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 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 an 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-;

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-.

[662] In certain embodiments, the compound of Formula (CO) is a compound of any of the below Formulae:

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.

[663] 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 Series “CC”

[664] 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.

[665] The present disclosure provides compound of Formula (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-;

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 an "*" 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 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. [666] In certain embodiments, the compound of Formula (CC) is a compound of any one of the Formulae below:

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.

[667] 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

Series “CT”

[668] Described below are a number of exemplary ionizable lipids of the present disclosure.

[669] In some embodiments, ionizable lipids of the present disclosure have a structure of Formula

(CT) or a pharmaceutically acceptable salt thereof, wherein: i) A is N; Z is a bond, 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, ii) A is CH; marked with an "*" is attached to X¹;

X¹ is a bond or optionally substituted C₁-C₆ aliphatic;

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

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

[670] 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:

or a pharmaceutically acceptable salt thereof, wherein R¹, R, X¹, Z, X², X³, X⁴, R^Z, 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.

[671 ] 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

Series “AX”

[672] Described below are a number of exemplary ionizable lipids of the present disclosure.

Formula (AX)

[673] 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 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)R^H, each R is independently -H or C₁-C₆ aliphatic; each R^H is C₁-C₆ aliphatic-OH; each X¹ and X^A is independently a bond or optionally substituted C₁-C₆, aliphatic; each Y¹ is independently selected from and a bond; wherein tire 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. wherein the bond marked with an "*" is attached to X⁴ or X⁵; each X⁶ and X⁷is 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 -( 1(0)0-;

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 R⁶ 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 bicy clic or multicyclic C₄-C₁₄ cy cloalkyl or optionally substituted bridged bicyclic or multicyclic 4-14 membered heterocyclyl; wherein one or more of X¹, X^A, 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.

[674] The present disclosure, in some embodiments, provides compounds of Formula (AX) 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;

X¹ and X^A 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-; 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-;

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.

[675] The present disclosure, in some embodiments, provides compounds of Formula (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 tire group consisting of a bond, wherein tire bond marked with an "*" is atached to X or X ; each X⁶ and X⁷is 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). Additional Formulae

[676] The present disclosure, in some embodiments, provides compounds of any one of the Formulae below:

or a pharmaceutically acceptable salt thereof, wherein R¹, R, X¹, X^A, 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

[677] As disclosed in Formula (AX), in certain embodiments, A is In certain embodiments, A is In certain embodiments, A is a multivalent adamantyl core, . In certain embodiments, A is In certain embodiments, A is In certain embodiments, A is In certain embodiments, A is a multivalent (lS,5S,7S)-2,4,10-trioxaadamantane core, In certain embodiments, A is In certain embodiments, A is [678] In certain embodiments, A is wherein * is attached to X¹. In certain embodiments, A is In certain certain embodiments, A is In certain embodiments, A is wherein * is attached to X¹. In certain embodiments, A is wherein * is attached to X¹.

[679] In certain embodiments, A is selected from:

n and m

[680] 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 is 1. In certain embodiments, m is 1 and n is 1. In certain embodiments, m is 1 and n is 0. Y¹

[681] 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, Y is a bond. X¹

[682] 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 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-.

X^A

[683] As disclosed in Formula (AX), in certain embodiments X^A is a bond or optionally substituted C₁-C₆ aliphatic. In certain embodiments, X^A is a bond. In certain embodiments, X^A is a bond or unsubstituted C₁-C₆ aliphatic. In certain embodiments, X^A is unsubstituted C₁-C₆ aliphatic. In certain embodiments, X^A is a bond or optionally substituted C₁-C₆ alkylene. In certain embodiments, X^A is optionally substituted C₁-C₆ alkylene. In certain embodiments, X^A is a bond or unsubstituted C₁-C₆ alkylene. In certain embodiments, X^A is unsubstituted C₁-C₆ alkylene. In certain embodiments, X^A is a bond or unsubstituted C₁-C₄ alkylene. In certain embodiments, X^A is unsubstituted C₁-C₄ alkylene. In certain embodiments, X^A is a bond or unsubstituted C₁-C₂ alkylene. In certain embodiments, X^A is unsubstituted C₁-C₂ alkylene. In certain embodiments, X^A is unsubstituted C₂-C₆ alkylene. In certain embodiments, X^A is optionally substituted methylene. In certain embodiments, R² is optionally substituted C₂ alkylene. In certain embodiments, X^A is optionally substituted C₃ alkylene. In certain embodiments, X^A is optionally substituted C₄ alkylene. In certain embodiments, X^A is optionally substituted C₅ alkylene. In certain embodiments, X^A is optionally substituted C₆ alkylene. In certain embodiments, X^A is -(CH₂)-. In certain embodiments, X^A is -(CH₂)₂-. In certain embodiments, X^A is -(CH₂)₃-. In certain embodiments, X^A is -(CH₂)₄-. In certain embodiments, X^A is -(CH₂)₅-. In certain embodiments, X^A is -(CH₂)₆-. R¹

[684] As disclosed in Formula (AX), in certain embodiments R¹ is selected from -OH, -OAc, - [685] In certain embodiments, R¹ is OH, -NR₂, or In certain embodiments, R¹ is -NMe₂, -NEt₂, 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, R¹ is In certain embodiments, R¹ is

[686] In certain embodiments, -X¹-Y¹-X^A-R¹ is

R

[687] 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

[688] 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.

[689] 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 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-.

[690] 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 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-.

[691] 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.

[692] 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.

[693] 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

[694] 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.

[695] 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 and Y³ are each independently certain embodiments, Y² is In certain embodiments, Y² is In certain embodiments, Y² is In certain embodiments, Y² is . In certain 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, Y³ is In certain embodiments, Y³ is In certain embodiments, Y³ is . In certain embodiments, Y³ is In certain embodiments, Y³ is In certain emb ³ odiments, Y 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 In certain embodiments, Y³ and Y² are both

X⁴ and X⁵ [696] 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.

[697] 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-.

[698] 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-.

[699] 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 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.

[700] 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.

[701] 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-.

[702] 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 a-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 a-position and β-position from Y² or Y³, respectively.

[703] 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

[704] As disclosed in Formula (AX””), in certain embodiments, Y⁴ and Y⁵ are each 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.

[705] In certain embodiments, Y⁴ and Y⁵ are each independently . In certain embodiments, Y⁴ and Y⁵ are each independently In certain embodiments, In certain embodiments, Y⁴ is In certain embodiments, Y⁵ is 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

[706] 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

[707] 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. [708] In certain embodiments, R² is selected from

R³

[709] 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. [710] In certain embodiments, R3 is selected from

[711] In certain embodiments, R² and R³ are the same. In certain embodiments, R² and R³ are different.

R⁶ and R⁷

[712] 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-.

[713] In certain embodiments, R⁶ and R⁷ are the same. In certain embodiments, R⁶ and R⁷ are different.

[714] 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.

[715] In certain embodiments, R⁷ is optionally substituted C₁-C₁₄ aliphatic. In certain 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, R⁷is 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, R⁷is optionally substituted -(CH₂)₅CH₃. In certain embodiments, R⁷is 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.

[716] In certain embodiments, each R⁶ and R⁷ are each independently selected from

R⁸ and R⁹

[717] 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-.

[718] In certain embodiments, R⁸ and R⁹ are the same. In certain embodiments, R⁸ and R⁹ are different.

[719] 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 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.

[720] 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, R⁹is 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, R⁹is 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.

[721] In certain embodiments, each R⁸ and R⁹ are each independently selected from

R¹⁰ and R¹¹

[722] 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 multicyclic 4-14 membered heterocyclyl.

[723] 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.

[724] 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 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.

[725] 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

 [726] 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

Series “TL ”

[727] Described below are a number of exemplary ionizable lipids of the present disclosure.

Formula (TL)

[728] The present disclosure, in some embodiments, provides compounds of Formula

(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 -( (O)O-.

Z¹ is selected from the group consisting of: 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,

523 each R is independently -H or C₁-C₆ aliphatic;

X^Z is a bond or optionally substituted C₁-C₆ aliphatic;

R^Z is NR₂ or OH;

Y¹ is selected from the group consi sting 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 R 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-; R¹² is -CH(OR¹³)(OR¹⁴), -CH(SR¹³)(SR¹⁴), -CH(R¹³)(R¹⁴), -R¹³, optionally substituted C₅-C₁₈ aliphatic, or optionally substituted C₁-C₁₄ aliphatic-R¹³, 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-Cg 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 R⁵ 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-R¹³; wherein one or more methylene linkages of R¹³ and R¹⁴ are each optionally and independently replaced with an optionally substituted C₃-Cg cycloalkylenyl, phenyl, - O-, -NH-, -S-, -SS-, -C(O)-, -OC(O)O-, -OC(O)-, -NHC(O)-, or -C(O)O-; each of R¹⁰, R¹¹, and R¹³ 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, R ¹¹ or R¹³ 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

[729] The present disclosure, in some embodiments, provides compounds of any one of the Formulae below:

Wherein R¹, R, X¹, L¹, L², Z¹, X², X³, X⁴, X⁵, Y², Y³, Y⁴, R², R³, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, and R¹⁵ are as described in Formula (TL”).

[730] 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:

[731] 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.

[732] 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.

[733] 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-l-yl)tetracosa-15,18-dien-l-amine (HGT5000), (15Z,18Z)-N,N-dimethyl- 6-((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)tetracosa-4, 15,18-trien- 1 -amine (HGT 5001), 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-l-yl)-l,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLinKC2-DMA)) (See, WO 2010/042877; Semple el 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-lH-cyclopenta[a]phenanthren-3-yl 3-(lH-imidazol-4- yl)propanoate (ICE), (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,l 2-dien- l-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-l-amine (HGT5001), (15Z,18 Z)-N,N-dimethyl-6-((9Z,12Z)- octadeca-9,12-dien-l-yl)tetracosa-5,15,18-trien-l-amine (HGT5002), 5- carboxyspermylglycine-dioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-l-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-hydroxy ethyl ammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3- beta-oxybutan-4-oxy)-l-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5 ' -(cholest- 5-en-3-beta-oxy)-3 ' -oxapentoxy)-3-dimethyl-l-(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-[l,3]-di oxolane (DLin-K-DMA), 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane (DLin-K-XTC2-DMA) or GL67, or mixtures thereof.

[734] 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-lH-cyclopenta[a]phenanthren-3-yl 3-(lH-imidazol-4- yl)propanoate (ICE), as disclosed in International Application No. PCT/US2010/058457, incorporated herein by reference.

[735] Also contemplated are ionizable lipids such as the dialkylamino-based, imidazole- based, and guanidinium-based lipids.

[736] In some embodiments, an ionizable lipid is described by US patent publication number 20190314284.

[737] 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.

[738] In some embodiments, an ionizable lipid is as described in international patent application PCT/US2019/015913.

[739] Preparation methods for the above compounds and compositions are described herein below and/or known in the art.

[740] 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-butyldiphenyl silyl or trimethyl silyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino, and guanidino include, e.g., t-butoxy carbonyl, 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.

[741] 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.

[742] 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.

[743] 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

[744] In certain embodiments, transfer vehicle compositions for the delivery of circular RNA comprise an amine lipid. In certain embodiments, an ionizable lipid is an amine lipid.

[745] 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-di enoate. 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.

[746] 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.

[747] Biodegradable lipids include, for example, the biodegradable lipids of WO2017/173054, WO2015/095340, and WO2014/136086.

[748] Lipid clearance may be measured by methods known by persons of skill in the art. See, for example, Maier, M.A., etal. Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics. Mol. Ther. 2013, 21(8), 1570-78.

[749] 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.

[750] 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 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.

[751] 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

[752] In some embodiments, the ionizable lipid is described in US patent 9,708,628.

[753] 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

R^3a— (Y^a— R^2a)n^a— X^a— R^{1 a}— SH, and

R^3b— (Y^b— R^2b)n^b— X^b— R^1b- 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.

[754] 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.

[755] 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

[756] In some embodiments, an ionizable lipid is described in US patent 9,765,022.

[757] 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. [758] 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 nonaromatic 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.

[759] 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.

[760] 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

[761] 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 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.

[762] 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 nitrogenbased dendritic structures.

[763] 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, WO2017/004 143, WO2017/075531, WO2017/117528, WO2011/022460, WO2013/148541, WO2013/116126, WO2011/153120, WO2012/044638, WO2012/054365, WO2011/090965, WO2013/016058, WO2012/162210, WO2008/042973, WO2010/129709, WO2010/144740, WO20 12/099755, WO2013/049328, WO2013/086322, WO2013/086373, WO2011/071860, WO2009/132131, WO2010/048536, WO2010/088537, WO2010/054401, WO2010/054406, WO20 10/054405, WO2010/054384, WO2012/016184, WO2009/086558, WO2010/042877, WO20 11/000106, WO2011/000107, WO2005/120152, WO2011/141705, WO2013/126803, WO2006/007712, WO2011/038160, WO2005/121348, WO2011/066651, WO2009/127060, WO201 1/141704, 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)

[764] 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.

[765] The use and inclusion of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-Octanoyl- Sphingosine-l-[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 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.

[766] 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

[767] 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.

[768] 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.

[769] 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-1, 2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). [770] 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.

[771] In some embodiments the PEG-modified lipids are a modified form of PEG-DMG.

PEG-DMG has the following structure:

[772] In some embodiments, the PEG lipid is a compound of Formula (Pl): 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.

[773] For example, R is C17 alkyl. For example, the PEG lipid is a compound of Formula (Pl-a): or a salt or isomer thereof, wherein r is an integer between 1 and 100.

[774] In some embodiments the PEG-modified lipids are a modified form of PEG-C18, or PEG-1. PEG-1 has the following structure: [775] 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.

[776] In some embodiments, an LNP comprises one, two or more PEGylated lipid or PEG-modified lipid. A PEGylated lipid may be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG- modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG- modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c- DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[777] In some embodiments, the PEGylated lipid is selected from (R)-2,3- bis(octadecyloxy)propyl-l-(methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG-S- DSG, PEG-S-DMG, PEG-PE, PEG-PAA, PEG-OH DSPE C18, PEG-DSPE, PEG-DSG, PEG- DPG, PEG-DOMG, PEG-DMPE Na, PEG-DMPE, PEG-DMG2000, PEG-DMG C14, PEG- DMG 2000, PEG-DMG, PEG-DMA, PEG-Ceramide C16, PEG-C-DOMG, PEG-c-DMOG, PEG-c-DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, PEG2k-C11, PEG2000-PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000-DMG, PEG2000- C-DMA, PEG2000, PEG200, PEG(2k)-DMG, PEG DSPE C18, PEG DMPE C14, PEG DLPE C12, PEG Click DMG C14, PEG Click C12, PEG Click C10, N(Carbonyl- methoxypolyethylenglycol-2000)-1,2-distearoyl-sn-glycero3-phosphoethanolamine, Myrj52, mPEG-PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG-2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE, mPEG2000-DMPE, mPEG2000-DMG, mDPPE-PEG2000, 1,2-distearoyl- sn-glycero-3-phosphoethanolamine-PEG2000, HPEG-2K-LIPD, Folate PEG-DSPE, DSPE- PEGMA 500, DSPE-PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2k, DSPE-PEG2000maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE-mPEG, DPG- PEGMA, DOPE-PEG2000, DMPE-PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE- mPEG2000, DMG-PEGMA, DMG-PEG2000, DMG-PEG, distearoyl-glycerol- polyethyleneglycol, C18PEG750, C18PEG5000, C18PEG3000, C18PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 Ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14-PEG 2000, C14-PEG, C14PEG, 14:0-PEG2KPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, (R)- 2,3-bis(octadecyloxy)propyl-l-(methoxypoly(ethyleneglycol)2000)propylcarbamate, (PEG)- C-DOMG, PEG-C-DMA, and DSPE-PEG-X.

[778] In some embodiments, the LNP comprises a PEGylated lipid disclosed in one of 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 2019/232095 Al; WO 2021/077067; WO 2019/152557; US 2015/0203446; US 2017/0210697; US 2014/0200257; or WO 2019/089828 A1, each of which is incorporated by reference herein in their entirety.

[779] In some embodiments, the LNP comprises a PEGylated lipid substitute in place of the PEGylated lipid. All embodiments disclosed herein that contemplate a PEGylated lipid should be understood to also apply to PEGylated lipid substitutes. In some embodiments, the LNP comprises a polysarcosine-lipid conjugate, such as those disclosed in US 2022/0001025 A1, which is incorporated by reference herein in its entirety. In some embodiments the LNP comprises a polyoxazoline-lipid conjugate, such as those disclosed in US 2022/0249695 A1, which is incorporated by reference herein in its entirety.

Series “PL ”

[780] In some embodiments, the LNP comprises a PEGylated lipid disclosed and described in PCT Application WO 2024/044728 A1, which is incorporated by reference herein, in its entirety. In certain embodiments, the PEGylated lipid is a lipid of any one of formulas PL-I' , PL-I' ' , PL-I, PL-Ia, PL-Ib, PL-Iaa, PL-Iab, PL-Iac, PL-Iad, PL-Iae, PL-Iaf, PL-Iag, PL-Iah, PL-Iba, PL-Ibb, PL-Ibc, PL-Ibd, PL-Ibe, PL-Ibf, PL-Ibg, PL-Ibh, PL-Ica, PL-Icb, PL- Icc, PL-Icd, PL-Id PL-Ie, PL-If, PL-Ig, PL-Ih, PL-Ii, PL-Iha, PL-Ihb, PL-Ihc, PL-Ihd, PL-Iia, PL-lib, PL-Iic, PL-Iid, PL-Ij, PL-Ik, L-Il, PL-Im, PL-In, PL-Io, PL-Ip, PL-Iq, PL-Ioa, PL-Iob, PL-Ioc, PL-Iod, PL-Ioe, PL-Iof, PL-Iog, PL-Ioh, PL-Ipa, PL-Ipb, PL-Ipc, PL-Ipd, PL-Ipe, PL- Ipf, PL-Ipg, PL-Iph, PL-Iqa, PL-Iqb, PL-Iqc, PL-Iqd, PL-Ir, PL-Is, PL-It, PL-Iu, PL-Iv, PL- Iw, PL-Iva, PL-Ivb, PL-Ivc, PL-Ivd, PL-Iwa, PL-Iwb, PL-Iwc, PL-Iwd, PL-Ix, PL-Ixx, PL-Iy, PL-Iyy, PL-Iyyy, PL-Iz, PL-Izz, PL-Izzz, PL-II' , PL-II' ' , PL-II, PL-IIc, PL-IId, PL-IIe, PL- Ilf, PL-IIg, PL-IIh, PL-IIa, PL-IIb, PL-IIk, PL-IIm or PL-IIn.

[781] In some embodiments, the PEGylated lipid is a compound of formula PL-I' : or a pharmaceutically acceptable salt thereof, wherein:

A¹ is a saturated 5-6 membered carbocyclic ring or a saturated 5-6 membered heterocyclic ring containing 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the carbocyclic ring and heterocyclic ring are substituted with t occurrences of R⁴;

X¹ is -N(H)-, -N(C_1-6 alkyl)-, -C_1-6 aliphatic-N(H)-, -C_1-6 aliphatic-N(C_1-6 alkyl)-, -O- or -C_1-6 aliphatic-O-;

L¹ is -C(O)(C_1-6 aliphatic)C(O)-N(R)-, -C(O)(C_1-6 aliphatic)-N(R)C(O)-, -C(O)(C_1-6 aliphatic)C(O)O-, -C(O)(C_1-6 aliphatic)C(O)-, -C(O)(C_1-6 aliphatic)C(O)OCH₂-, -C(O)(C_1-6 aliphatic)-, -C(O)(C_1-6 aliphatic)-N(R)-, or -C(O)-;

L² and L³ are independently a covalent bond or C_1-6 alkylene wherein one methylene unit of the C_1-6 alkylene is optionally replaced with -O-, -NR-, -S-, -S-S-, -S(O)-, -S(O)₂-, -C(O)-, - C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, -N(R)C(O)-, - N(R)C(O)N(R)-, -C(R⁵)=N-, or -C(R⁵)=N-O-;

R¹ is H, C_1-6 alkyl, -(C_1-6 alkyl)-N₃, -(C_1-6 alkyl)-SH, or C_3-8 alkynyl;

R² and R³ are independently a straight or branched C_{6 -30} alkyl, straight or branched C_{6 -30} alkenyl, or straight or branched C_{6 -30} alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C_3-6 carbocyclic ring or phenylene; wherein the alkyl, alkenyl, and alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x;

R⁴ is C_1-4 alkyl;

R⁵ is C_1-6 alkyl or C_2-14 alkenyl; each R is independently hydrogen or an optionally substituted group selected from C_1-6 aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each R^x is independently halogen, -CN, -OR, -SR, -C(O)R, -C(O)OR, or -OC(O)OR; n is an integer from 10-75, inclusive; m is 0, 1, 2, 3, or 4; and t is 0, 1, or 2.

[782] In some embodiments, the PEGylated lipid is a compound of formula PL-II' : or a pharmaceutically acceptable salt thereof, wherein:

L¹ is -C(O)(C_1-6 aliphatic)C(O)-, -C(O)(C_1-6 aliphatic)-, or -C(O)-;

L² and L³ are a covalent bond or C_1-6 alkylene wherein one methylene unit of the C_1-6 alkylene is optionally replaced with -O-, -NR-, -S-, -S-S-, -S(O)-, -S(O)₂-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R)-, -N(R)C(O)O-, -C(O)N(R)-, -N(R)C(O)-, -N(R)C(O)N(R)-, -C(R⁶)=N-, or -C(R⁶)=N- O-;

R² and R³ are independently straight or branched C_6-30 alkyl, straight or branched C_6-30 alkenyl, or straight or branched C_6-30 alkynyl; wherein 1, 2, or 3 methylene units are independently and optionally replaced by a saturated or partially unsaturated C_3-6 carbocyclic ring or phenylene; wherein the alkyl, alkenyl, and alkynyl and any carbocyclic ring or phenylene is substituted with m instances of R^x;

R⁶ is C_1-6 alkyl or C_2-14 alkenyl; each R is independently hydrogen or an optionally substituted group selected from C_1-6 aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each R^x is independently halogen, -CN, -OR, -SR, -C(O)R, -C(O)OR, or OC(O)OR; n is an integer from 10-75, inclusive; and m is 0, 1, 2, 3, or 4.

[783] In some embodiments, the PEGylated lipid compound is one of those shown in Table (I-X), or a pharmaceutically acceptable salt thereof.

Table (I-X). Exemplary PEGylated Compounds

Series “FS”

[784] In some embodiments, the LNP comprises a PEGylated lipid disclosed and described in PCT Application PCT/US2025/014133, which is incorporated by reference herein.

[785] In some embodiments, the PEG lipid is a compound of Formula (VI): wherein:

P is a PEG moiety;

Y^B is an optional linking moiety selected from -OC(O)-, -C(O)O-, -OC(O)O-, - OC(O)NH-, -NHC(O)O-, and -NHC(O)NH-; L is an optional linker selected from optionally substituted C1-C10 alkylene, optionally substituted C1-C10 heteroalkylene, optionally substituted C2-C10 alkenylene, and optionally substituted C2-C10 alkynylene;

Y^A is selected from -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NH-, -NHC(O)O-, and - NHC(O)NH-; m is an integer selected from 1 to 10; and

R² and R³ are each independently optionally substituted C1-C15 alkyl or optionally substituted C2-C15 -alkenyl.

In some embodiments, the PEG lipid is a compound of Formula (VI): wherein:

P is a PEG moiety of formula z is an integer selected from 1 to 200;

R is hydrogen or methyl;

Y^B is an optional linking moiety selected from -OC(O)-, -C(O)O-, -OC(O)O-, - OC(O)NH-, -NHC(O)O-, and -NHC(O)NH-;

L is an optional linker selected from optionally substituted C1-C10 alkylene, optionally substituted C1-C10 heteroalkylene, optionally substituted C2-C10 alkenylene, and optionally substituted C2-C10 alkynylene;

R² and R³ are each independently optionally substituted C1-C15 aliphatic.

[786] In some embodiments, the PEGylated lipid compound is one of those shown in Table (I-Z), or a pharmaceutically acceptable salt thereof.

Table (I-Z). Exemplary PEGylated Compounds

[787] In some embodiments of the compounds of Table (I-Z), z is selected from 1 to 200. In some embodiments, z is an integer selected from 30 to 75. In some embodiments, z is an integer selected from 35 and 50. In some embodiments, z is 45. In some embodiments, z is 44. In some embodiments, z is 43. In some embodiments, z is 42. In some embodiments, z is 41. In some embodiments, z is 40. In some embodiments, z is 46. In some embodiments, z is 47. All individual species of a compound of Table 2B having any above described value of “z” are to be understood as specifically contemplated herein as an exemplary compound of the present disclosure (E.g. Compound P131, wherein z = 45; P131, wherein z = 44; P131, wherein z = 43;. ..etc.).

C. HELPER LIPIDS

[788] 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.

[789] 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.

[790] 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.

[791] 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.

[792] 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.

[793] 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.

[794] 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-l,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), l-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), l-paimitoyl-2-myristoyl phosphatidylcholine (PMPC), l-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 phosphatidyl ethanol 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.

[795] In certain embodiments, the helper lipid is a phospholipid selected from the nonlimiting group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1.2-dioleoyl- sn-glycero-3 -phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocho line (POPC), 1,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuc cinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1- hexadecyl-sn-glycero-3 -phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1,2-diarachidonoyl-sn-glycero-3 -phosphocholine, 1,2-didocosahexaenoyl- sn-glycero-3 -phosphocholine, 1,2-diphytanoylsn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2- diarachidonoyl-sn-glycero-3 -phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG), sodium (S)-2-ammonio-3-((((R)-2-(oleoyloxy)-3-

(stearoyloxy)propoxy)oxidophosphoryl)oxy)propanoate (L-α-phosphatidylserine; Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl-phosphatidylethanolamine4-(N- maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), di oleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidyl ethanolamine (DPPE), 1,2-Dielaidoyl-sn- phosphatidylethanolamine (DEPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl- phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2- dioleoyl-sn-glycero-3 -phosphate (18: 1 PA; DOPA), ammonium bis((S)-2-hydroxy-3- (oleoyloxy)propyl) phosphate (18: 1 DMP; LBPA), 1,2-dioleoyl-sn-glycero-3-phospho-(l ' - myo-inositol) (DOPI; 18:1 PI), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), 1,2- dilinoleoyl-sn-glycero-3-phospho-L-serine (18:2 PS), l-palmitoyl-2-oleoyl-sn-glycero-3- phospho-L-serine (16:0-18: 1 PS; POPS), l-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (18:0-18: 1 PS), l-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (18:0-18:2 PS), 1- oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18: 1 Lyso PS), l-stearoyl-2-hydroxy-sn- glycero-3-phospho-L-serine (18:0 Lyso PS), and sphingomyelin. In some embodiments, an LNP comprises DSPC. In certain embodiments, an LNP comprises DOPE. In some embodiments, an LNP comprises both DSPC and DOPE.

[796] In some embodiments, an LNP comprises a phospholipid selected from 1- pentadecanoyl-2-oleoyl-sn-glycero-3-phosphocholine, l-myristoyl-2-palmitoyl-sn-glycero-3- phosphocholine, l-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine, l-palmitoyl-2- myristoyl-sn-glycero-3-phosphocholine, l-palmitoyl-2-stearoyl-sn-glycero-3- phosphocholine, 1 -palmitoyl-2-oleoyl-glycero-3 -phosphocholine, 1 -palmitoyl-2-linoleoyl-sn- glycero-3 -phosphocholine, l-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, 1- palmitoyl-2-docosahexaenoyl-sn-glycero-3 -phosphocholine, l-stearoyl-2-myristoyl-sn- glycero-3 -phosphocholine, l-stearoyl-2-palmitoyl-sn-glycero-3 -phosphocholine, l-stearoyl-2- oleoyl-sn-glycero-3 -phosphocholine, 1 -stearoyl-2-linoleoyl-sn-glycero-3 -phosphocholine, 1 - stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, l-stearoyl-2-docosahexaenoyl-sn- glycero-3 -phosphocholine, 1 -oleoyl-2-myristoyl-sn-glycero-3 -phosphocholine, 1 -oleoyl-2- palmitoyl-sn-glycero-3 -phosphocholine, 1 -oleoyl-2-stearoyl-sn-glycero-3 -phosphocholine, 1 - palmitoyl-2-acetyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phospho-(l ' - myo-inositol-3 ' ,4' -bisphosphate), 1,2-dioleoyl-sn-glycero-3-phospho-(l ' -myo-inositol- 3 ' ,5 ' -bisphosphate), 1,2-dioleoyl-sn-glycero-3-phospho-(l ' -myo-inositol -4' ,5 ' - bisphosphate), 1,2-dioleoyl-sn-glycero-3-phospho-(r-myo-inositol-3',4',5'-trisphosphate), 1,2-dioleoyl-sn-glycero-3-phospho-(l ' -myo-inositol-3 ' -phosphate), 1,2-dioleoyl-sn-glycero- 3-phospho-(l ' -myo-inositol-4 ' -phosphate), 1,2-dioleoyl-sn-glycero-3-phospho-(l'-myo- inositol-5'-phosphate), 1,2-dioleoyl-sn-glycero-3-phospho-(l ' -myo-inositol), 1,2-dioleoyl-sn- glycero-3-phospho-L-serine, and l-(8Z-octadecenoyl)-2-palmitoyl-sn-glycero-3- phosphocholine. In some embodiments, the LNP comprises a phospholipid selected from DSPS (Distearoylphosphatidylserine), DSPG (1,2-distearoyl-sn-glycero-3-phospho-(l'-rac- glycerol)), DSPA (1,2-Distearoyl-sn-glycero-3-phosphate), diPhyPC (1,2-diphytanoyl-sn- glycero-3 -phosphocholine), diPhy-diether-PC (1,2-di-O-phytanyl-sn-glycero-3- phosphocholine), diPhy PE (1,2-diphytanoyl-sn-glycero-3 -phosphoethanolamine), diPhy - diether-PE (1,2-di-O-phytanyl-sn-glycero-3 -phosphoethanolamine), diPhyPS (1,2- diphytanoyl-sn-glycero-3 -phospho-L-serine), diPhyPG ( 1 ,2-diphytanoyl-sn-glycero-3 - phospho-(l'-rac-glycerol)), diPhyP A (1,2-diphytanoyl-sn-glycero-3 -phosphate), Egg PA (L-α- phosphatidic acid), and Soy PA (L-α-phosphatidic acid).

[797] In some embodiments, the LNP comprises a phospholipid selected from 18: 1 (A9- Cis) PE (DOPE), 18:0-18: 1 PE (SOPE), CI6-18:1 PE, 16:0-18: 1 PE (POPE), 18: 1 BMP (S,R), 18:0-18: 1 PC (SOPC), 16:0-18: 1 PC (POPC), 4ME 16:0 Diether PE (4Me), 18: 1 (A9-Trans) PE (DEPE), 16: 1 PE (DPPE), and CL. In certain embodiments, the LNP comprises a phospholipid described or disclosed in Alvarez-Benedicto, et al. (Biomater. Sci., 2022, 10, 549) and Li, et al. (Asian Journal of Pharmaceutical Sciences, 2015, 10, 81-98).

[798] In certain embodiments, the phospholipid is a sphingoid lipid or sphingolipid, such as, but not limited to sphingomyelin. As used herein, the terms "sphingoid lipid" and "sphingolipid" are meant to refer to a class of lipids containing a backbone comprising a sphingoid base. An exemplary sphingoid base is sphingosine. In certain embodiments, the LNP comprises a sphingolipid selected from Egg Sphingomyelin (Egg SM / ESM / (2S,3R,E)- 3-hydroxy-2-palmitamidooctadec-4-en-l-yl (2-(trimethylammonio)ethyl) phosphate), Brain or Porcine Sphingomyelin (Brain SM / (2S,3R,E)-3-hydroxy-2-stearamidooctadec-4-en-l-yl (2- (trimethylammonio)ethyl) phosphate), Milk or Bovine Sphingomyelin (Milk SM / (2S,3R,E)- 3-hydroxy-2-tricosanamidooctadec-4-en-l-yl (2-(trimethylammonio)ethyl) phosphate), 28:0 SM (N-octacosanoyl-D-erythro-sphingosylphosphorylcholine), 14:0 SM (N-myristoyl-D- erythro-sphingosylphosphorylcholine), 16: 1 SM (N-palmitoleoyl-D-erythro- sphingosylphosphorylcholine), 12:0 Dihydro SM (N-lauroyl-D-erythro- sphinganylphosphorylcholine), Lyso SM (Sphingosylphosphorylcholine), Lyso SM

(Sphingosylphosphorylcholine), Lyso SM (dihydro) (Sphinganine Phosphorylcholine), 24: 1

SM (N-nervonoyl-D-erythro-sphingosylphosphorylcholine), 24:0 SM (N-lignoceroyl-D- erythro-sphingosylphosphorylcholine), 18: 1 SM (N-oleoyl-D-erythro- sphingosylphosphorylcholine), 18:0 SM (N-stearoyl-D-erythro- sphingosylphosphorylcholine), 17:0 SM (N-heptadecanoyl-D-erythro- sphingosylphosphorylcholine), 16:0 SM (N-palmitoyl-D-erythro- sphingosylphosphorylcholine), 12:0 SM (N-lauroyl-D-erythro-sphingosylphosphorylcholine), 06:0 SM (N-hexanoyl-D-erythro-sphingosylphosphorylcholine), 02:0 SM (N-acetyl-D- erythro-sphingosylphosphorylcholine), 3-O-methyl Lyso SM (3-O-methyl- spingosylphosphorylcholine), 3-O-methyl-N-methyl Lyso SM (3-O-methyl-N-methyl- spingosylphosphorylcholine), and 3-N-methyl Lyso SM (3-N-methyl- spingosylphosphorylcholine).

[799] In some embodiments, the LNP comprises a phospholipid comprising at least one constrained tail, such as those described by Gan, et al. (Bioeng Transl Med. 2020 Sep; 5(3): elO16L). In certain embodiments, the phospholipid is one selected from:

[800] In some embodiments, the LNP comprises a phospholipid comprising a ceramide analogue having a triazole linkage, such as those described by Kim et al., Bioorg. Med. Chem. Lett., 17(16), 2007, 4584-4587.

[801] In some embodiments, the LNP comprises a phospholipid disclosed in WO

2023/141470, which is incorporated by reference herein, in its entirety. In certain embodiments, the phospholipid is

[802] In some embodiments, the LNP comprises a phospholipid disclosed in WO 2022/040641, which is incorporated by reference herein, in its entirety.

[803] In some embodiments, a phospholipid tail may be modified in order to promote endosomal escape as described in U.S. Application Publication 2021/0121411, which is incorporated herein by reference.

[804] In some embodiments, the LNP comprises a phospholipid disclosed in one of 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 2019/232095 Al; WO 2021/077067; WO 2019/152557; US 2017/0210697; or WO 2019/089828 A1, each of which is incorporated by reference herein in their entirety.

[805] In some embodiments, the LNP comprises a phospholipid disclosed in PCT Publication WO2023141470A2, which is incorporated by reference herein in its entirety. In certain embodiments, the LNP comprises a phospholipid of Formula (I) of PCT Publication WO2023141470A2, including but not limited to 2-ammonioethyl ((S)-3-(((S)-12- methyltetradecanoyl)oxy)-2-((13-methyltetradecanoyl)oxy)propyl) phosphate.

D. STRUCTURAL LIPIDS

[806] 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, 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.

[807] In an embodiment, a structural lipid is described in international patent application [808] 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. In certain embodiments, the structural lipid is selected from the group consisting of, cholesterol, fecosterol, fucosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, cholic acid, sitostanol, litocholic acid, tomatine, ursolic acid, alpha-tocopherol, Vitamin D3, Vitamin D2, Calcipotriol, botulin, lupeol, oleanolic acid, beta-sitosterol-acetate and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid is a cholesterol analogue disclosed by Patel, et al., Nat Commun., 11, 983 (2020), which is incorporated herein by reference in its entirety. In some embodiments, the structural lipid comprises cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or any combinations thereof. In some embodiments, a structural lipid is described in international patent application WO2019152557A1, which is incorporated herein by reference in its entirety.

[809] 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.

[810] 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.

[811] In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol.

[812] 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

[813] 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.

[814] 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.

[815] 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., FEB S 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.

[816] 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.

[817] 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.

[818] 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.).

[819] 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. [820] 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 polynucleotide(s), particularly with respect to the environments into which such polynucleotides will be exposed.

[821] 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.

[822] 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).

[823] 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.

[824] In some embodiments, the lipid nanoparticle comprises one or more cationic lipids, ionizable lipids, or poly β-amino esters. In some embodiments, the nanoparticle comprises one or more non-cationic lipids. In some embodiments, the 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. Accordingly, in some embodiments, a lipid nanoparticle of a composition comprises one or more ionizable lipids, one or more phospholipids, one or more structural lipids, and one or more PEG lipids. In some embodiments, the lipid nanoparticle comprises a targeting moiety, wherein the targeting moiety mediates receptor-mediated endocytosis selectively into cells of a selected cell population in the absence of cell selection or purification. In some embodiments, the lipid nanoparticle comprises more than one circular RNA construct.

[825] 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.

[826] 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.

[827] 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 embodiment.

[828] 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.

[829] 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.

[830] 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

90 nm and/or from about 70 to about 90 nm. Each possibility represents a separate embodiment.

[831] A nanoparticle composition may be relatively homogenous. A poly dispersity 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 poly dispersity 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, 0.1 1, 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 poly dispersity index of a nanoparticle composition may be from about 0.10 to about 0.20. Each possibility represents a separate embodiment.

[832] 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.

[833] 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 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%.

[834] In some embodiments, the lipid nanoparticle has a poly dispersity 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.

[835] 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.

[836] In some embodiments, an LNP has a diameter of at least about 20nm, 30 nm, 40nm, 50nm, 60nm, 70nm, 80nm, or 90nm. In some embodiments, an LNP has a diameter of less than about 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, or 160nm. In some embodiments, an LNP has a diameter of less than about 120 nm. In some embodiments, an LNP has a diameter of less than about 100nm. In some embodiments, an LNP has a diameter of less than about 90nm. In some embodiments, an LNP has a diameter of less than about 80nm. In some embodiments, an LNP has a diameter of about 60- 100nm. In some embodiments, an LNP has a diameter of about 50-120nm. In some embodiments, an LNP has a diameter of about 75- 80nm.

[837] In some embodiments, the lipid nanoparticle compositions of the present disclosure are described according to the respective molar ratios of the component lipids in the formulation. As a non-limiting example, the mol-% of the ionizable lipid may be from about 10 mol-% to about 80 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 20 mol-% to about 70 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 30 mol-% to about 60 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 35 mol-% to about 55 mol-%. As a nonlimiting example, the mol-% of the ionizable lipid may be from about 40 mol-% to about 50 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 30 mol-% to about 40 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 28 mol-% to about 40 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 25 mol-% to about 45 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 25 mol-% to about 35 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 10 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 15 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 20 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 25 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 30 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 33 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 35 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 40 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 45 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 55 mol-%. In some embodiments, the mol-% of the ionizable lipid is about 60 mol-%. [838] In some embodiments, the mol-% of the phospholipid may be from about 1 mol-% to about 50 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 2 mol-% to about 45 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 3 mol-% to about 40 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 4 mol-% to about 35 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 5 mol-% to about 30 mol-%. In some embodiments, the mol- % of the phospholipid may be from about 10 mol-% to about 20 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 5 mol-% to about 20 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 25 mol-% to about 40 mol- %. In some embodiments, the mol-% of the phospholipid may be from about 35 mol-% to about 40 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 20 mol- % to about 60 mol-%. In some embodiments, the mol-% of the phospholipid is from about 30 mol-% to about 60 mol-%. In some embodiments, the mol-% of the phospholipid is from about 35 mol-% to about 55 mol-%. In some embodiments, the mol-% of the phospholipid is from about 35 mol-% to about 45 mol-%. In some embodiments, the mol-% of the phospholipid is about 10 mol-%. In some embodiments, the mol-% of the phospholipid is about 15 mol-%. In some embodiments, the mol-% of the phospholipid is about 20 mol-%. In some embodiments, the mol-% of the phospholipid is about 25 mol-%. In some embodiments, the mol-% of the phospholipid is about 30 mol-%. In some embodiments, the mol-% of the phospholipid is about 35 mol-%. In some embodiments, the mol-% of the phospholipid is about 40 mol-%. In some embodiments, the mol-% of the phospholipid is about 45 mol-%. In some embodiments, the mol-% of the phospholipid is about 55 mol-%. In some embodiments, the mol-% of the phospholipid is about 60 mol-%.

[839] In some embodiments, the mol-% of the phospholipid as described above comprises two or more phospholipids at an individual mol-% that totals to an aforementioned amount. In certain embodiments, the mol-% of the phospholipid is about 20 mol-% each of two phospholipids. In certain embodiments, the mol-% of the phospholipid is about 15 mol-% each of two phospholipids. In certain embodiments, the mol-% of the phospholipid is about 25 mol- % each of two phospholipids. In certain embodiments, the mol-% of the phospholipid is about 30 mol-% each of two phospholipids. In certain embodiments, the mol-% of the phospholipid is about 15 mol-% of a first phospholipid and about 20 mol-% of a second phospholipid. In certain embodiments, the mol-% of the phospholipid is about 30 mol-% of a first phospholipid and about 10 mol-% of a second phospholipid. In certain embodiments, the mol-% of the phospholipid is about 25 mol-% of a first phospholipid and about 10 mol-% of a second phospholipid. In certain embodiments, the mol-% of the phospholipid is about 25 mol-% of a first phospholipid and about 20 mol-% of a second phospholipid. In certain embodiments, the mol-% of the phospholipid is about 15 mol-% of a first phospholipid and about 20 mol-% of a second phospholipid.

[840] In some embodiments, the mol-% of the structural lipid may be from about 10 mol-% to about 80 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 15 mol-% to about 35 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 20 mol-% to about 70 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 30 mol-% to about 60 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 35 mol-% to about 55 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 20 mol-% to about 40 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 20 mol-% to about 30 mol- %. In some embodiments, the mol-% of the structural lipid may be from about 40 mol-% to about 50 mol-%.

[841] In some embodiments, the mol-% of the PEG lipid may be from about 0.1 mol-% to about 10 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 0.2 mol-% to about 5 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 0.5 mol-% to about 3 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 1 mol-% to about 2 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 1.5 mol-% to about 2.5 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 1 mol-% to about 3 mol-%. In some embodiments, the mol-% of the PEG lipid may be about 1.5 mol-%. In some embodiments, the mol-% of the PEG lipid may be about 2.5 mol-%.

[842] In some embodiments, (a) the PEG lipid is PEG2k-DMG or PEG2k-DSPE or a mixture thereof; (b) the structural lipid is cholesterol; and (c) the phospholipid, non-ionizable lipid or zwitterionic lipid is a sphingolipid or DSPC or a mixture thereof.

[843] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 0 mol% to about 10 mol% of PEG lipid; (b) about 0 mol% to about 30 mol% structural lipid; (c) about 20 mol% to about 45 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 30 mol% to about 60 mol% of a Lipid of the Disclosure.

[844] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 1 mol% to about 2 mol% of PEG lipid; (b) about 25 mol% to about 40 mol% structural lipid; (c) about 20 mol% to about 45 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 30 mol% to about 60 mol% of a Lipid of the Disclosure.

[845] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 2 mol% of PEG lipid; (b) about 25 mol% structural lipid; (c) about 40 mol% phospholipid, non- ionizable lipid or zwitterionic lipid; and (d) about 33 mol% of a Lipid of the Disclosure.

[846] In some embodiments, the lipid component of the nanoparticle comprises: (a) about

2.5 mol% of PEG lipid; (b) about 39 mol% structural lipid; (c) about 10 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 48.5 mol% of a Lipid of the Disclosure.

[847] In some embodiments, the lipid component of the nanoparticle comprises: (a) about

1.5 mol% of PEG lipid; (b) about 40 mol% structural lipid; (c) about 10 mol% phospholipid, non-ionizable lipid or zwitterionic lipid; and (d) about 48.5 mol% of a Lipid of the Disclosure.

[848] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 1 mol% to about 3 mol% of the one or more PEG lipids; (b) about 15 mol% to about 35 mol% of the one or more structural lipids; (c) about 30 mol% to about 60 mol% of the one or more phospholipids; and (d) about 25 mol% to about 45 mol% of the one or more ionizable lipids.

[849] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 1.5 mol% to about 2.5 mol% of the one or more PEG lipids; (b) about 20 mol% to about 30 mol% of the one or more structural lipids; (c) about 35 mol% to about 45 mol% of the one or more phospholipids; and (d) about 28 mol% to about 40 mol% of the one or more ionizable lipids.

[850] In some embodiments, the lipid component of the nanoparticle comprises: (a) about 1.5 mol% to about 2.5 mol% of the one or more PEG lipids; (b) about 20 mol% to about 40 mol% of the one or more structural lipids; (c) about 25 mol% to about 40 mol% of the one or more phospholipids; and (d) about 28 mol% to about 40 mol% of the one or more ionizable lipids.

[851] In certain embodiments, the lipid component of the nanoparticle composition comprises about 30 mol % to about 60 mol % ionizable lipid, about 0 mol % to about 30 mol % phospholipid, about 18.5 mol % to about 48.5 mol % structural lipid, and about 0 mol% to about 10 mol% of PEG lipid, provided that the total mol % does not exceed 100%. In certain embodiments, the lipid component of the nanoparticle composition comprises about 20 mol % to about 45 mol % ionizable lipid, about 30 mol % to about 60 mol % phospholipid, about 10 mol % to about 30 mol % structural lipid, and about 0 mol% to about 10 mol% of PEG lipid, provided that the total mol % does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition comprises about 35 mol % to about 55 mol % ionizable lipid, about 5 mol % to about 25 mol % phospholipid, about 30 mol % to about 40 mol % structural lipid, and about 0 mol % to about 10 mol % of PEG lipid, provided that the total mol % does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition comprises about 30 mol % to about 40 mol % ionizable lipid, about 35 mol % to about 45 mol % phospholipid, about 20 mol % to about 30 mol % structural lipid, and about 0.5 mol % to about 5 mol % of PEG lipid, provided that the total mol % does not exceed 100%. In certain embodiments, the lipid component of the nanoparticle composition comprises about 25 mol % to about 45 mol % ionizable lipid, about 35 mol % to about 50 mol % phospholipid, about 10 mol % to about 25 mol % structural lipid, and about 1 mol% to about 5 mol% of PEG lipid, provided that the total mol % does not exceed 100%. In a particular embodiment, the lipid component comprises about 50 mol % ionizable lipid, about 10 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol% of PEG lipid. In another particular embodiment, the lipid component comprises about 40 mol % ionizable lipid, about 20 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular embodiment, the lipid component comprises about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 40 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular embodiment, the lipid component comprises about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 39 mol % structural lipid, and about 2.5 mol % of PEG lipid. In another particular embodiment, the lipid component comprises about 33 mol % ionizable lipid, about 40 mol % phospholipid, about 25 mol % structural lipid, and about 2 mol % of PEG lipid. In some embodiments, the phospholipid is DOPE or DSPC. In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is a sphingolipid. In some embodiments, the phospholipid is a sphingomyelin. In other embodiments, the PEG lipid is PEG-DMG (eg. PEG2K-DMG). In other embodiments, the PEG lipid is PEG-DSPE (eg. PEG2K-DSPE). In other embodiments, the PEG lipid is PEG-DMPE (eg. PEG2K-DMPE). In other embodiments, the structural lipid is cholesterol. In other embodiments, the PEG lipid is PEG-DMG and/or the structural lipid is cholesterol. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DSPC. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is sphingomyelin. In some embodiments, the PEG lipids is PEG-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In certain embodiments, the LNP comprises about 33mol% ionizable lipid (eg. at least one ionizable lipid of a Formula described herein), about 40mol% of a sphingolipid, about 25mol% cholesterol and about 2mol% PEG2K-DMG. In some embodiments, the PEG lipids is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is DSPC. In some embodiments, the PEG lipids is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is sphingomyelin. In some embodiments, the PEG lipids is PEG-DSPE, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DOPE. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DOPC. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DLPC. In some embodiments, the PEG lipids is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is DOPS. In some embodiments, the PEG lipids is PEG-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a phosphatidylcholine lipid and a sphingolipid. In some embodiments, the PEG lipids is PEG- DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a phosphatidylcholine lipid and phosphatidylserine lipid. In some embodiments, the PEG lipids is PEG-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a phosphatidylcholine lipid and a phosphoethanolamine lipid. In some embodiments, the PEG lipids is PEG-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a sphingolipid and phosphatidylserine lipid. In some embodiments, the PEG lipids is PEG-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of a sphingolipid and a phosphoethanolamine lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 20mol% of a sphingolipid, about 20mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 10mol% of a sphingolipid, about 30mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 30mol% of a sphingolipid, about 10mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 20mol% sphingomyelin, about 20mol% of a DSPC, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 10mol% sphingomyelin, about 30mol% of a DSPC, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 30mol% sphingomyelin, about 10mol% of a DSPC, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 25mol% cholesterol, about 2mol% of a PEGylated lipid, and about 40% of a mixture of phosphatidylcholine, phosphatidyl serine, phosphoethanolamine, and sphingoid lipids. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 25mol% cholesterol, about 2mol% of a PEGylated lipid, and about 40% of a mixture of phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids, wherein each of the phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids is present in an amount less than 30 mol% of the total lipid component of the LNP. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 25mol% cholesterol, about 2mol% of a PEGylated lipid, and about 40% of a mixture of phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids, wherein each of the phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids is present in an amount less than 25 mol% of the total lipid component of the LNP. In certain embodiments, LNP is any one of the aforementioned in this paragraph wherein the PEG lipid is PEG2k-DMG. In certain embodiments, LNP is any one of the aforementioned in this paragraph wherein the PEG lipid is PEG2k-DSPE.

[852] In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % DSPC, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % sphingomyelin, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % DOPE, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % DOPC, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % DLPC, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % DOPS, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 40 mol % phospholipid, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In another particular embodiment, LNP comprises about 33 mol % ionizable lipid, about 20 mol % sphingomyelin, about 20 mol% DSPC, about 25 mol % cholesterol, and about 2 mol % of PEG lipid. In certain embodiments, LNP is any one of the aforementioned in this paragraph wherein the PEG lipid is PEG2k-DMG. In certain embodiments, LNP is any one of the aforementioned in this paragraph wherein the PEG lipid is PEG2k-DSPE.

[853] In certain embodiments, the LNP comprises about 43mol% ionizable lipid, about 15mol% of a sphingolipid, about 15mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 25mol% of a sphingolipid, about 15mol% of a non- sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In certain embodiments, the LNP comprises about 33mol% ionizable lipid, about 15mol% of a sphingolipid, about 25mol% of a non-sphingolipid phospholipid, about 25mol% cholesterol and about 2mol% of a PEGylated lipid. In some embodiments, the PEG lipid is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In some embodiments, the PEG lipid is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DSPE. [854] In another particular embodiment, the lipid component includes about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 38.5 mol % structural lipid, and about 3 mol % of PEG lipid. In another particular embodiment, the lipid component includes about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 38 mol % structural lipid, and about 3.5 mol % of PEG lipid. In some embodiments, the PEG lipid is PEG2K-DPPE, the structural lipid is cholesterol, and the phospholipid is a DSPC or a mixture of DSPC and sphingomyelin. In some embodiments, the PEG lipid is PEG2K-DPPE, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DPPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39.5 mol% cholesterol and about 2 mol% PEG2K- DPPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DPPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38.5 mol% cholesterol and about 3 mol% PEG2K-DPPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38 mol% cholesterol and about 3.5mol% PEG2K-DPPE. In some embodiments, the PEG lipid is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is a DSPC or a mixture of DSPC and sphingomyelin. In some embodiments, the PEG lipid is PEG2K-DMG, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39.5 mol% cholesterol and about 2 mol% PEG2K-DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38.5 mol% cholesterol and about 3 mol% PEG2K-DMG. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38 mol% cholesterol and about 3.5mol% PEG2K-DMG. In some embodiments, the PEG lipid is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is a DSPC or a mixture of DSPC and sphingomyelin. In some embodiments, the PEG lipid is PEG2K-DSPE, the structural lipid is cholesterol, and the phospholipid is a mixture of DSPC and sphingomyelin. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 40mol% cholesterol and about 1.5mol% PEG2K-DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39.5 mol% cholesterol and about 2 mol% PEG2K-DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 39mol% cholesterol and about 2.5mol% PEG2K-DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38.5 mol% cholesterol and about 3 mol% PEG2K-DSPE. In certain embodiments, the LNP comprises about 48.5mol% ionizable lipid, about 10mol% of a phospholipid (such as DSPC), about 38 mol% cholesterol and about 3.5mol% PEG2K-DSPE.

[855] In some embodiments, the LNP further comprises a targeting moiety. In some embodiments, the targeting moiety is an antibody or a fragment thereof.

[856] The amount of active agent in a nanoparticle composition may depend on the size, composition, desired target and/or application, or other properties of the nanoparticle composition as well as on the properties of the active agent. For example, the amount of active agent useful in a nanoparticle composition may depend on the size, sequence, and other characteristics of the active agent. The relative amounts of active agent and other elements (e.g., lipids) in a nanoparticle composition may also vary. In some embodiments, the wt/wt ratio of the lipid component to payload in a nanoparticle composition is from about 5 : 1 to about 60: 1, such as 5: 1, 6: 1, 7:1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, and 60: 1. The amount of a payload in a nanoparticle composition may, for example, be measured using absorption spectroscopy (e.g., ultraviolet- visible spectroscopy).

[857] In some embodiments, a nanoparticle composition of the present disclosure is formulated to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in an RNA active agent (e.g., a linear or circular mRNA payload). In general, a lower N:P ratio is preferred. The one or more enzymes, lipids, and amounts thereof is selected to provide an N:P ratio from about 2: l to about 30: 1, such as 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 12: 1, 14: 1, 16: 1, 18: 1, 20: 1, 22: 1, 24: 1, 26: 1, 28: 1, or 30: 1. In certain embodiments, the N:P ratio is from about 2: 1 to about 8: 1. In other embodiments, the N:P ratio is from about 5: 1 to about 8: 1. For example, the N:P ratio is about 5.0: 1, about 5.5: 1, about 5.67: 1, about 6.0: 1, about 6.5: 1, or about 7.0: 1.

F. METHODS FOR PREPARING LIPID NANOPARTICLES (LNP) FORMULATIONS

[858] 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.

[859] 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).

[860] For certain cationic lipid nanoparticle formulations of RNA, in order to achieve 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.

[861] 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.

[862] 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.

[863] 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 to 170 mM, 50 mM to 160 mM, 50 mM to 150 mM, or 50 mM to 100 mM.

[864] 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.

[865] 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.

[866] 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.

[867] 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.

[868] 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.

[869] 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.

[870] 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 3. Ethanol phase contained ionizable Lipid 128 or Lipid 129 from Table 3, 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 IL 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

[871] 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).

[872] 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).

[873] 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 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).

[874] 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.

[875] 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).

[876] 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 lipid component includes a combination of cholesterol and cholesterol oleate.

[877] 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).

[878] 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.

[879] 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.

[880] 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 (IncRNA), 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).

[881] 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.

[882] 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.

[883] 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

[884] 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).

[885] 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 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.

[886] 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 a chain antibodies, T-cell β chain antibodies, T-cell y chain antibodies, T-cell 5 chain antibodies, CCR7 antibodies, CD3 antibodies, CD4 antibodies, CD5 antibodies, CD7 antibodies, CD8 antibodies, CD11 b antibodies, CD11 c antibodies, CD16 antibodies, CD 19 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 antibodies, CD80 antibodies, CD95 antibodies, CD117 antibodies, CD127 antibodies, CD133 antibodies, CD137 (4-1BB) antibodies, CD163 antibodies, F4/80 antibodies, IL-4Ra 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.

[887] 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.

[888] 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.

[889] 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 protein or enzyme.

[890] 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

[891] 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).

[892] In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier or excipient. 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.

[893] 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.

[894] In some embodiments, the pharmaceutical composition comprises a targeting moiety. The targeting moiety mediates receptor-mediated endocytosis, endosome fusion, or 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.

[895] 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.

[896] With respect to pharmaceutical compositions, the pharmaceutically acceptable carrier can be any of those conventionally used and is limited only by chemi co-phy si cal 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.

[897] 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.

[898] 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.

[899] 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.

[900] In certain embodiments, the therapeutic agents provided herein can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or LNPs or liposomes. [901] 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.

[902] 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.

[903] 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 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.

[904] 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.

[905] 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).

[906] 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.

[907] 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.

[908] 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 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).

[909] 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

[910] 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.

[911] 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.

[912] 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.

[913] 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). 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.

[914] 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. [915] 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. 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, lymphocytic choriomeningitis virus, parvovirus Bl 9, 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, Mycoplasma species, Borrelia burgdorferi, Legionalla pneumophila, Colstridium botulinum, Cory neb acterium 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, 0mpl6, 0mpl9, 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-PDl, anti- 41BB, PD-L1, Tim-3, Lag-3, TIGIT, GITR, and anti-CD3.

[916] 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.

[917] 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 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., CD 19 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. [918] In some embodiments, the subject has an autoimmune disease or disorder.

[919] 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.

[920] In some embodiments, the subject has a cancer selected from the group consisting of acute myeloid leukemia (AML); alveolar rhabdomyosarcoma; B cell malignancies; bladder 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.

[921] 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.

[922] In the field of ex vivo CAR-T cell therapeutics, “T cell expansion is limited by low rates, and T-cell products of limited functionality” (see, e.g., Cheung et al., Nature Biotechnology, 2018) and the efficacy “is limited by the poor proliferation and persistence of the engineered T cells” (see, e.g., Zhang et al., Nature Biomedical Engineering, 2024) (the contents of both of which are hereby incorporated by reference). In some embodiments, the method of treating the subject comprises administering a bioscaffold in addition to administering the circular RNA encoding the CAR or composition thereof.

[923] In some embodiments, the bioscaffold is an injectable biomaterial. In some embodiments, the bioscaffold is a biomaterial-derived injectable. In some embodiments, the bioscaffold comprises or is derived from mesoporous silica rods. In some embodiments, the bioscaffold is loaded with interleukin-2. In some embodiments, the bioscaffold comprises a lipid bilayer, e.g., prepared with anti-CD3 and anti-CD28. Bioscaffolds are known and have been generated in the art. See, e.g., Kim et al., Nature Biotechnology, 2014; Cheung 2018 supra, Li et al., Nature Materials, 2018; Dellacherie et al., Advanced Functional Materials, 2020; Zhang et al., Nature Protocols, 2020; Zhang 2024 supra (the contents of each of which are hereby incorporated by reference).

[924] In some embodiments, administering the circular RNA encoding the CAR in combination with administration of the bioscaffold is capable of ameliorating disease in the subject as compared to control absent bioscaffold administration. In some embodiments, administering the circular RNA encoding the CAR in combination with administration of the bioscaffold is capable of improving activation, proliferation, stimulation, restimulation, and/or persistence of immune cells (e.g., T cells, NK cells, etc.) in the subject as compared to a control absent bioscaffold administration.

[925] In some embodiments, the bioscaffold is administered to a subject having cancer. In some embodiments, the bioscaffold is injected into the subject. In some embodiments, the bioscaffold is injected subcutaneously into the subject. In some embodiments, the bioscaffold is administered to a subject having a solid tumor. The bioscaffold may be delivered into or adjacent to the solid tumor. In some embodiments, the bioscaffold is injected into or adjacent to the site of an oral cancer. In some embodiments, the bioscaffold is injected into or adjacent to the site of cervical cancer.

[926] In some embodiments, the bioscaffold is administered prior to administration of the circular RNA encoding the CAR or composition thereof. In these embodiments, the bioscaffold may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more days prior to administration of the circular RNA encoding the CAR or composition thereof, which may allow for vascularization of the administered bioscaffold material.

[927] In some embodiments, the bioscaffold is administered concurrently with administration of the circular RNA encoding the CAR or composition thereof. In some embodiments, the bioscaffold is administered after administration of the circular RNA encoding the CAR or composition thereof.

EXAMPLES

[928] 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.

[929] 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. EXAMPLE 1: Synthesis of Exemplary Lipids

[930] Ionizable lipids and PEG lipids of Series "A", "CY", "C", "CX", "CZ", "PL", "S", "AT", "AC", "CO", and "CC" were prepared as reported in PCT Publications WO2023044343A1, WO2023044333A1, WO2023122752A1, WO2024044728A1 and WO2023196931A1 and PCT Application PCT/US2024/019990 using appropriate starting materials and procedures as would be apparent to a person of ordinary skill in the art. Additional lipids of the present disclosure were made by methods and procedures as would be apparent to a person of ordinary skill in the art, as described in one or more publications or patent applications disclosed herein. Exemplary additional synthetic procedures and schema are included below.

Synthesis of selected intermediates

Synthesis of 4,4-bis(3,7-dimethyloctyl)oxy)butane nitrile (L4L-2) [Procedure A]

[931] To a 100 mL round bottom flask, 4,4-dimethoxybutanenitrile (3.0 g, 23.2 mmol), 3,7- dimethyloctan-l-ol (11.0 g, 69.7 mmol) and pyridinium p-toluenesulfonate (0.29 g 1.2 mmol) were added. The resulting mixture was stirred at 120 ° C for 4h and cooled to room temperature. EtOAc (50 mL) and H₂O (20 mL) were added in, and the resulting phases were separated. The aqueous phase was extracted with EtOAc (50 mL). Combined organic extracts were washed with H₂O (20 mL) and dried over anhydrous MgSO₄. Filtration and concentration provided crude material which was purified by flash column chromatography (SiO₂: 0 to 10% ethyl acetate in hexanes gradient) to yield L4L-2 as colorless oil (6.6 g, 74%); ¹HNMR (CDCI₃) δ 4.50-4.53 (t, 1H), 3.58-3.60 (m, 2H), 3.41 - 3.49 (m, 2H), 2.39 - 2.44 (t, 2H), 1.92-1.94 (q, 2H), 1.50-1.55 (m, 6H), 1.38-1.42 (m, 2H), 1.11 - 1.14 (m, 14H) 0.88-0.84 (t, 18H); CIMS m/z [M+H]⁺ 381.

Synthesis of 4,4-bis((3,7-dimethyloctyl) oxy) butanoic acid (L4L-3) [Procedure B]

[932] To a 100 mL round bottom flask containing a solution of L4L-2 (8.2 g, 21 mmol) in ethanol

(50 mL) was added a solution of KOH (3.6 g, 64 mmol) in water (50 mL). After completion of addition, the mixture was stirred at 120 º C for 20h. The volatiles were removed, and the reaction pH was adjusted to 5. EtOAc (150 mL) and H₂O (60 mL) were added, and the resulting phases were separated. The aqueous phase was extracted with EtOAc (50 mL). Combined organic extracts were washed with H₂O (60 mL x 2) and dried over anhydrous MgSO₄. Filtration and concentration provided L4L-3 (6.4 g, 74%) which was used for the next step without further purification. ¹HNMR (CDCl₃) δ 4.54 (t, 1H), 3.60-3.65 (m, 2H), 3.45- 3.49 (m, 2H), 2.39 - 2.44 (t, 2H), 1.92 - 1.94 (m, 2H), 1.50 - 1.95 (m, 6H),

1.26 - 1.55 (m, 8H), 1.11 - 1.14 (m, 6H). 0.84 - 0.88 (d, 18H); CIMS m/z [M-H]' 399.

Synthesis of 4,4-bis(octyloxy)butanoic acid (L4L-4)

[933] Prepared following Procedures A & B described in Compound L4L-3 synthesis, replacing 3,7-dimethyloctan-l-ol with octan-l-ol. Compound L4L-4 was isolated as light-yellow oil in a yield of 11.8 g (98%). ¹HNMR (CDCl₃) δ: 4.53-4.56 (t, 1H), 3.57-3.60 (m, 2H), 3.40-3.43 (m, 2H), 2.39- 2.41 (t, 2H), 1.90-1.95 (m, 2H), 1.54-1.56 (M, 4H), 1.26 (bs, 28H), 0.85-0.87 (t, 6H); CIMS m/z [M- H] 371.

Synthetic Scheme for Compound AX-3

Synthesis of (1R,3R,5S)-3,5-dihydroxyadamantan-l-yl 2-chloroacetate (L122A-2)

[934] A solution of L122A-1 (5.0 g, 27.14 mmol) in DMF (100 mL) was heated to 45° C under nitrogen atmosphere. Triethylamine (11.0 mL, 81.4 mmol) and chloroacetyl chloride (5.4 mL, 67.9 mmol) were added portion wise to the above solution and the reaction was monitored by TLC. Upon consumption of the starting material, the reaction mixture was concentrated under reduced pressure and the crude was purified by flash chromatography (SiO₂: 0-100% ethyl acetate in hexanes gradient and then 0-5% methanol in dichloromethane gradient) to yield L122A-2 as beige solid (3.01 g, 43%); ¹HNMR (CDCl₃) δ 3.98 (s, 2H), 2.42 (p, J= 3.2 Hz, 1H), 2.09 (s, 4H), 1.98 (d, J= 3.1 Hz, 2H), 1.82 - 1.69 (m, 2H), 1.68 - 1.59 (m, 4H).

Synthesis of (lR,3R,5S)-3,5-dihydroxyadamantan-l-yl diethylglycinate (L122A-3)

[935] A mixture of L122A-2 (3.00 g, 11.51 mmol) and diethylamine (12.0 mL, 115.1 mmol) in DCM (50 mL) was stirred at room temperature for 48 h. The mixture was concentrated under reduced pressure and the crude was purified by flash chromatography (SiO₂ : 0-5% methanol in dichloromethane gradient) to afford L122A-3 as brownish solid (2.47 g, 72%); ¹HNMR (CDCl₃) δ 3.24 (s, 2H), 2.64 (q, J= 6.5 Hz, 4H), 2.47 - 2.32 (m, 1H), 2.10 (s, 4H), 1.98 (s, 2H), 1.81 - 1.66 (m, 6H), 1.06 (t, J= 7.1 Hz, 6H); CIMS m/z [M+H] 298.2.

Synthesis of (lR,3S,5S)-5-((diethylglycyl)oxy)adamantane-l,3-diyl bis(4,4-bis(octyloxy) butanoate) (Compound AX- 3)

[936] A mixture of L122A-3 (0.70 g, 2.35 mmol), 4,4-bis(octyloxy)butanoic acid (2.43 g, 7.06 mmol), DCC (1.94 g, 7.06 mmol) and DMAP (288 mg, 2.35 mmol) in DCM (50 mL) was stirred at room temperature for 24 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by flash chromatography (SiO₂: 0-20 % ethyl acetate in hexane) to afford Compound L122A as yellow oil (1.0 g, 45%); ¹H NMR (CDCl₃) ) δ 4.47 (t, J = 5.6 Hz, 2H), 3.64 - 3.50 (m, 4H), 3.47 - 3.33 (m, 4H), 3.23 (s, 2H), 2.64 (q, J = 7.2 Hz, 4H), 2.47 (p, J= 12.0 Hz, 7H), 2.30 (t, J= 7.6 Hz, 4H), 2.09 - 1.94 (m, 6H), 1.87 (q, J = 7.4 Hz, 4H), 1.60 - 1.50 (m, 8H), 1.40 - 1.18 (m, 40H), 1.05 (t, J= 7.2 Hz, 6H), 0.88 (t, J= 6.8 Hz, 12H); CIMS m/z [M+H] 950.8; Analytical HPLC column: Agilent Zorbax SB-C18, 5 μm, 4.6x 150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 70% to 100% in 10 min then 100 % for 5 min, flow rate: ImL/min, column temperature: 20±2 ° C, detector: ELSD, t_R= 7.7 min, purity: > 99%; UPLC column: Waters Aquity UPLC® CSHTM, C18, 1.7 μm, 3.0x 150 mm, (Part No. 186005302), mobile phase A: water with 0.1% trifluoroacetic acid, mobile phase B: acetonitrile with 0.1% trifluoroacetic acid, use gradient: A in B 60% to 100% in 10 min, flow rate: ImL/min, column temperature: 55±2 ° C, detector: CAD, t_R= 15.2 min, purity: 99 %.

Synthetic Scheme for Compound AX-5

Synthesis of (lS,3R,5S, 7S)-3,5-dihydroxyadamantan-l-yl 4-bromobutanoate (LI 23-1)

[937] The starting material L122A-1 (3 g, 16.28 mmol) and pyridine (2 mL, 24.42 mmol) were dissolved in DMF (42 mL). 4-Bromobutanoyl chloride (1.98 mL, 16.28 mmol) was added slowly to the above solution through a syringe over 30 min at room temperature. The reaction mixture was then stirred at room temperature for 20 h. The solvent was evaporated under reduced pressure and the crude product was subjected to silica gel column using 0 - 10% MeOH in DCM as eluent to afford L123-1 (1.2 g, 22%) as yellow oil; ¹H-NMR (400 MHz, CDCl₃) δ 3.58 (t, 2H), 2.43 (t, 2H), 2.44-2.36 (m, 1H),

2.14-2.04 (m, 2H), 1.82-1.66 (m, 3H), 1.62 (bs, 5H), 1.56 (bs, 4H).

Synthesis of (lR,3S,5S, 7S)-3-(2-(bicyclo[2.2.2]octan-l-yl)acetoxy)-5-hydroxyadamantan-l-yl 4- bromobutanoate (LI 23-2) [938] 2-(Bicyclo[2.2.2]octan-l-yl)acetic acid (606 mg, 3.6 mmol) was dissolved in DCM (6 mL) at 0 ° C. After adding DMF (12 uL), a solution of oxalyl chloride (468 uL, 5.4 mmol) in DCM (6 ml) was dropped in through syringe. After addition finished, the reaction mixture was allowed to warm to room temperature and stirred for 2 h. The solvent was evaporated under reduced pressure and the crude 2-(bicyclo[2.2.2]octan-l-yl)acetyl chloride was used directly for the next step without further purification.

[939] The above crude acid chloride was dissolved in DCM (3 mL) and added slowly to a stirring solution of L123-1 (1.2 g, 3.6 mmol) and pyridine (438 uL, 5.4 mmol) in DCM (15 mL) over a period of one hour. After the addition finished, the reaction mixture was stirred at room temperature for 20 h. The solvent was evaporated to give the crude product which was subjected to silica gel column using 0 - 5% MeOH in DCM as eluent to afford L123-2 (0.9 g, 52%) as yellow oil; ¹H-NMR (400 MHz, CDCl₃) δ 3.58 (t, 2H), 2.50-2.34 (m, 5H), 2.18-2.04 (m, 2H), 1.72-1.34 (m, 25H).

Synthesis of (lR,3S,5S, 7S)-3-(2-(bicyclo[2.2.2]octan-l-yl)acetoxy)-5-hydroxyadamantan-l-yl 4- (diethylamino)butanoate (L123-3)

[940] The starting material L123-2 (650 mg, 1.34 mmol) was dissolved in diethylamine (15 mL) at room temperature. An excess amount of potassium carbonate and potassium iodide were then added to the reaction mixture. The reaction was sealed and stirred at 90 ° C for 20 h. Diethylamine was evaporated to provide the crude product which was purified by silica gel column using 0 - 20% MeOH in DCM as eluent to afford L123-3 (350 mg, 55%) as light-yellow oil; ¹H-NMR (400 MHz, CDCl₃) δ 2.50 (q, 4H), 2.44-2.28 (m, 4H), 2.23 (t, 2H), 2.18-2.02 (m, 5H), 2.00-1.82 (m, 6H), 1.72-1.58 (m, 4H), 1.58-1.28 (m, 13H), 0.99 (t, 6H); CIMS m/z [M+H]⁺ 476.3.

Synthesis of (1S,3S,5R, 7S)-3-(2-(bicyclo[2.2.2]octan-l-yl)acetoxy)-5-((4-(diethylamino) butanoyl) oxy)adamantan-l-yl (9Z,12Z)-octadeca-9,12-dienoate (Compound AX-5)

[941] A mixture of linoleic acid (295 mg, 1.05 mmol), DCC (260 mg, 1.26 mmol) and 1,4- dimethylpyridinium 4-methylbenzenesulfonate (180 mg, 0.61 mmol) in DCM (15 mL) was stirred at room temperature for 5 min to give a clear solution. The starting material L123-3 (250 mg, 0.53 mmol) was then added to the above solution and the resulting mixture was stirred at room temperature for 3 days. The solvent was evaporated to give the crude product which was subjected to silica gel column using 0 - 20% MeOH in DCM with 1% NH₄OH as eluent to afford a mixture of product and impurities. This mixture was dissolved in ethyl acetate (2 mL), washed by aq. HC1 (0.2 N, 2 mL x3), sat. aq. sodium bicarbonate (2 mL x2) and brine (2 mL). The organic phase was dried over anhydrous Na₂SO₄. Filtration and concentration provided Compound L123 (101 mg, 26%) as yellow oil; ¹H-NMR (400 MHz, CDCl₃) δ 5.42-5.27 (m, 4H), 2.76 (t, 2H), 2.54-2.36 (m, 13H), 2.27-2.14 (m, 4H), 2.09-1.96 (m,

10H), 1.93 (s, 2H), 1.73-1.66 (m, 2H), 1.66-1.58 (m, 2H), 1.58-1.48 (m, 7H), 1.48-1.38 (m, 6H), 1.38- 1.22 (m, 14H), 0.99 (t, 6H), 0.88 (t, 3H); CIMS m/z [M+H]⁺ 738.5; Analytical HPLC column: Agilent Zorbax SB-C18, 5 μm, 4.6x 150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 70% to 100% in 5 min, then 100% for 10 min. Flow rate: 1 mL/min, column temperature: 20±2 ° C, detector: ELSD, 7.05 min, purity: >99.9%; UPLC column: Thermo Scientific Hypersil GOLD C18, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 5% to 100% in 5 min, then 100% for 15 min. Flow rate: 1 mL/min, column temperature: 20±2 ° C, detector: CAD, t_R= 8.02 min, purity: 89.7%.

Synthetic Scheme for Compound AX-1

Synthesis of (1R, 3S, 5R)-5-((diethylglycyl)oxy)adamantane-l, 3-diyl (9Z, 9 'Z, 12Z, 12'Z)-bis(octadeca-

9,12-dienoate) (Compound AX- 1)

[942] To an ice bath cooled solution of linoleic acid (566 mg, 2.02 mmol) in DCM (5 mL) under nitrogen atmosphere was added oxalyl chloride (230 μl, 2.69 mmol) and DMF (50 μl) by syringe. The mixture was stirred at room temperature for 2 h and concentrated. The residue was co-evaporated with toluene (10 mL X 3). The crude acid chloride was then dissolved in DCM (5 mL) and dropped into a solution of L122A-3 (100 mg, 0.34 mmol) and pyridine (160 μl, 2.02 mmol) in DCM (5 mL) at 45 °C over a period of 15 min. The resulting mixture was stirred at 45 °C for 2h and at room temperature for 18h. The reaction mixture was concentrated under reduced pressure and the crude was purified using flash chromatography (SiO₂: first 0-20% ethyl acetate in hexane gradient, then 0-4% methanol in dichloromethane gradient) to afford Compound AX-1 as yellow oil (69 mg, 25%); ¹HNMR (CDCl₃) δ 5.38-5.36 (m, 8H), 3.26 (s, 2H), 2.78 (t, J = 5.9 Hz, 4H), 2.66 (q, J = 7.2 Hz, 4H), 2.55-2.42 (m, 7H), 2.22 (t, J = 7.6 Hz, 4H), 2.09-2.04 (m, 14H), 1.61-1.52 (m, 4H), 1.37-1.30 (m, 28H), 1.07 (t, J = 7.2 Hz, 6H), 0.90 (t, J = 6.7 Hz, 6H); CIMS m/z [M+H] 822.7; Analytical HPLC column: Agilent Zorbax SB- C18, 5 μm, 4.6x 150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 70% to 100% in 10 min then 100 % for 5 min, flow rate: ImL/min, column temperature: 20±2 ° C, detector: ELSD, t_R= 7.8 min, purity: > 99%;

UPLC column: Waters Aquity UPLC® CSHTM, C18, 1.7 μm, 3.0x 150 mm, (Part No. 186005302), mobile phase A: water with 0.1% trifluoroacetic acid, mobile phase B: acetonitrile with 0.1% trifluoroacetic acid, use gradient: A in B 60% to 100% in 10 min, flow rate: ImL/min, column temperature: 55±2 ° C, detector: CAD, t_R= 13.9 min, purity: 93.13 %.

Synthetic Scheme for Compound AX-2

Synthesis of (lR,3R,5S)-3-((diethylglycyl)oxy)-5-hydroxyadamantan-l-yl (9Z,12Z)-octadeca-9,12- dienoate (L123C-1)

[943] To an ice bath cooled solution of linoleic acid (373 mg, 1.33 mmol) in DCM (5 mL) under nitrogen atmosphere was added oxalyl chloride (155 μl, 1.82 mmol) and DMF (50 μl) by syringe. The mixture was stirred at room temperature for 2 h and concentrated. The residue was co-evaporated with toluene (10 mL X 3). The crude acid chloride was then dissolved in DCM (5 mL) and dropped into a solution of L122A-3 (180 mg, 0.61 mmol) and pyridine (120 μl, 1.51 mmol) in DCM (5 mL) at 45 °C over a period of 15 min. The resulting mixture was stirred at 45 °C for 2h and at room temperature for 18h. The reaction mixture was concentrated under reduced pressure and the crude was purified using flash chromatography (SiO₂: 0-2% methanol in dichloromethane gradient) to afford L123C-1 as yellowish oil (144 mg, 43%); ¹HNMR (CDCl₃) δ 4.82-5.62 (4H), 3.24 (s, 2H), 2.77 (t, J = 5.6 Hz, 2H),

2.68-2.62 (q, J = 7.2 Hz, 4H), 2.47-2.39 (m, 3H), 2.23-2.17 (m, 2H), 2.13-1.98 (m, 10H), 1.64-1.52 (m, 6H), 1.37-1.20 (m, 14H), 1.05 (t, J = 7.2 Hz, 6H), 0.88 (t, J = 6.9 Hz, 3H); CIMS m/z [M+H] 560.4.

Synthesis of (lR,3S,5S)-3-(2-(hicyclo[2.2.2]octan-l-yl)acetoxy)-5-((diethylglycyl)oxy) adamantan-1- yl ( 9Z, 12Z)-octadeca-9, 12-dienoate ( Compound AX-2)

[944] To an ice bath cooled solution of 2-(bicyclo[2.2.2]octan-l-yl)acetic acid (195 mg, 1.16 mmol) in DCM (5 mL) under nitrogen atmosphere was added oxalyl chloride (208 μl, 2.32 mmol) and DMF (50 μl) by syringe. The mixture was stirred at room temperature for 2 h and concentrated. The residue was co-evaporated with toluene (10 mL X 3). The crude acid chloride was then dissolved in DCM (5 mL) and dropped into a solution of L123C-1 (130 mg, 0.23 mmol) and pyridine (184 μl, 2.32 mmol) in DCM (5 mL) at 45 °C over a period of 15 min. The resulting mixture was stirred at 45 °C for 2h and at room temperature for 18h. The reaction mixture was concentrated under reduced pressure and the crude was purified using flash chromatography (SiO₂: 0-2% methanol in dichloromethane gradient) to afford Compound AX-2 as yellow oil (63 mg, 38%); ¹HNMR (CDCl₃) δ 5.39-5.30 (m, 4H), 3.24 (s, 2H), 2.77 (t, J = 6.1 Hz, 2H), 2.64 (q, J = 7.2 Hz, 4H), 2.54-2.41 (m, 7H), 2.20 (t, J = 7.4 Hz, 2H), 2.08- 2.03 (m, 10H), 1.94 (s, 2H), 1.57-1.25 (m, 29H), 1.05 (t, J = 7.2 Hz, 6H), 0.88 (t, J = 6.9 Hz, 3H); CIMS m/z [M+H] 710.4; Analytical HPLC column: Agilent Zorbax SB-C18, 5 μm, 4.6x 150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 70% to 100% in 10 min then 100 % for 5 min, flow rate: 1 mL/min, column temperature: 20±2 ° C, detector: ELSD, 9.7 min, purity: > 99%; UPLC column: Waters Aquity UPLC® CSHTM, C18, 1.7 μm, 3.0 x 150 mm, (PartNo. 186005302), mobile phase A: water with 0.1% trifluoroacetic acid, mobile phase B: acetonitrile with 0.1% trifluoroacetic acid, use gradient: A in B 60% to 100% in 10 min, flow rate: ImL/min, column temperature: 55±2 ° C, detector: CAD, 11.4 min, purity: 94.22 %.

Synthetic Scheme for Compound AX-8

Synthesis of (lS,3S,5R)-3-(2-(bicyclo[2.2.2]octan-l-yl)acetoxy)-5-hydroxyadamantan-l-yl diethylglycinate (L125A-1)

[945] A mixture of L122A-3 (300 mg, 1.01 mmol), 2-(bicyclo[2.2.2]octan-l-yl)acetic acid (170 mg, 1.01 mmol), DCC (416 mg, 2.02 mmol) and DMAP (123 mg, 1.01 mmol) in DCM (10 mL) was stirred at room temperature for 24 h. The reaction mixture was fdtered over Celite. The fdtrate was concentrated under reduced pressure and the residue was purified using flash chromatography ( SiO₂ : 0- 5 % methanol in dichloromethane) to give impure product which was repurified using flash chromatography (SiO₂, 0-100% ethyl acetate in hexanes gradient) to afford L125A-1 as yellow oil (140 mg, 31%); ¹HNMR (CDCl₃) δ 3.22 (s, 2H), 2.63 (q, J = 7.2 Hz, 4H), 2.42-2.38 (m, 3H), 2.15-2.09 (m, 4H), 2.03-1.97 (m, 4H), 1.94 (s, 2H), 1.64 (d, J = 2.7 Hz, 2H), 1.56-1.49 (m, 10H), 1.45-1.41 (m, 4H), 1.04 (t, J = 7.1 Hz, 6H); CIMS m/z [M+H] 448.3.

Synthesis of (lR,3S,5R)-3-(2-(hicyclo[2.2.2]octan-l-yl)acetoxy)-5-((diethylglycyl)oxy) adamantan-1- yl 4,4-his(octyloxy)hutanoate) (Compound AX-8)

[946] A mixture of L125A-1 (140 mg, 0.31 mmol), 4,4-bis(octyloxy)butanoic acid (323 mg, 0.94 mmol), DCC (387 mg, 1.88 mmol) and DMAP (38 mg, 0.31 mmol) in DCM (10 mL) was stirred at room temperature for 24 h. The reaction mixture was filtered through Celite. The filtrate was concentrated under reduced pressure and the residue was purified using flash chromatography (SiO₂: 0- 50 % ethyl acetate in hexanes gradient) to afford Compound AX-8 as yellow oil (145 mg, 58%); ¹HNMR (CDCl₃) δ 4.47 (t, J = 5.5 Hz, 1H), 3.56 (dd, J = 15.7, 7.0 Hz, 2H), 3.40 (dd, J = 15.6, 6.9 Hz,

2H), 3.23 (s, 2H), 2.64 (q, J = 7.1 Hz, 4H), 2.54-2.41 (m, 7H), 2.31 (t, J = 7.4 Hz, 2H), 2.06- 2.02 (m, 6H), 1.95 (s, 2H), 1.87 (dd, J = 13.3, 7.1 Hz, 2H), 1.61- 1.50 (m, 14H)1.45 (d, J = 9.2 Hz, 4H), 1.28 (s, 20H), 1.05 (t, J = 7.1 Hz, 6H), 0.90-0.87 (m, 6H); CIMS m/z [M+H] 774.6; Analytical HPLC column: Agilent Zorbax SB-C18, 5 μm, 4.6* 150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 70% to 100% in 10 min then 100 % for 5 min, flow rate: ImL/min, column temperature: 20±2 ° C, detector: ELSD, t_R= 7.2 min, purity: 99.8 %; UPLC column: Waters Aquity UPLC® CSHTM, C18, 1.7 μm, 3.0 x 150 mm, (Part No. 186005302), mobile phase A: water with 0.1% trifluoroacetic acid, mobile phase B: acetonitrile with 0.1% trifluoroacetic acid, use gradient: A in B 60% to 100% in 10 min, flow rate: ImL/min, column temperature: 55±2 ° C, detector: CAD, t_R= 11.8 min, purity: > 96.55 %.

Synthetic Scheme for Compound AX-4

Synthesis of (lR,3S,5R)-5-((diethylglycyl)oxy)adamantane-l,3-diyl bis(2-(bicyclo[2.2.2] octan-1- yl)acetate) (L125A-2)

[947] To an ice bath cooled solution of 2-(bicyclo[2.2.2]octan-l-yl)acetic acid acid (300 mg, 1.78 mmol) in DCM (5 mL) under nitrogen atmosphere was added oxalyl chloride (623 μl, 7.26 mmol) and DMF (50 μl) by syringe. The mixture was stirred at room temperature for 2 h and concentrated. The residue was co-evaporated with toluene (10 mL X 3). The crude acid chloride was then dissolved in DCM (5 mL) and dropped into a solution of L122A-3 (360 mg, 1.21 mmol) and pyridine (586 μl, 7.26 mmol) in DCM (5 mL) at 45 °C over a period of 15 min. The resulting mixture was stirred at 45 °C for 2h and at room temperature for 18h. The reaction mixture was concentrated under reduced pressure and the crude was purified using flash chromatography (SiO₂: 0-30 % ethyl acetate in hexanes gradient) to afford AX-4 as yellow oil (64 mg, 9%); ¹HNMR (CDCl₃) δ 3.22 (s, 2H), 2.63 (q, J = 7.0 Hz, 4H), 2.53-2.39 (m, 7H), 2.09-2.03 (m, 6H), 1.93 (s, 4H), 1.44 (s, 26H), 1.04 (t, J = 7.1 Hz, 6H); CIMS m/z [M+H] 598.4; Analytical HPLC column: Agilent Zorbax SB-C18, 5 μm, 4.6x 150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 70% to 100% in 10 min then 100 % for 5 min, flow rate: ImL/min, column temperature: 20±2 °C, detector: ELSD, 3.9 min, purity: > 99%; UPLC column: Waters Aquity UPLC® CSHTM, C18, 1.7 μm, 3.0x 150 mm, (Part No. 186005302), mobile phase A: water with 0.1% trifluoroacetic acid, mobile phase B: acetonitrile with 0.1% trifluoroacetic acid, use gradient: A in B 60% to 100% in 10 min, flow rate: ImL/min, column temperature: 55±2 ° C, detector: CAD, t_R= 6.2 min, purity: 93.09 %.

Synthetic Scheme for Compound AX-6

Synthesis of ((1S,3R,5S, 7S)-3,5-bis(hydroxymethyl)adamantan-l-yl)methyl 4-(diethylamino) butcmoate (L150-2)

[948] A solution of 4-(diethylamino)butanoic acid hydrochloride (0.64 g, 3.3 mmol), EDC (5.0 g, 26.5 mmol), DIPEA (0.5 mL) and DMAP (813 mg, 6.6 mmol) in DCM (20 mL) was stirred at room temperature for 30 min. The starting alcohol L150-1 (1.5 g, 6.6 mmol) was added in. The reaction mixture was stirred at room temperature for one hour and concentrated under reduced pressure. The residue was purified by column chromatography (SiO₂, solvent 0-10% MeOH/DCM) to provide L150- 2 (1.3 g, 53%) as colorless liquid; ¹H NMR(CDCl₃): 0.98 (t, 6H), 1.38-1.34 (m, 6H), 1.45-1.40 (m, 6H), 1.78-1.73 (m, 2H), 2.18- 2.16 (m, 3H), 2.51-246 (m, 4H), 2.40 (t, 2H), 2.31 (t, 2H), 3.26-3.23 (m, 4H), 3.74-3.72 (m, 2H); CIMS m/z [M+H]⁺ 368.1. Synthesis of ((1R,3S,5R, 7R)-5-(((4-(diethylamino)butanoyl)oxy)methyl)adamantane-l , 3- diyl)bis (methylene) bis(4, 4-bis(octyloxy)butanoate) (Compound AX-6)

[949] A solution of 4,4-bis(octyloxy)butanoic acid (1.8 g, 5.2 mmol), EDC (1.6 g, 8.7 mmol) and DMAP (267 mg, 2.1 mmol) in DCM (15 mL) was stirred at room temperature for 30 min. The starting alcohol L150-2 (800 mg, 2.1 mmol) was added in. The reaction mixture was stirred at room temperature for 12 h and concentrated under reduced pressure. The residue was purified by column chromatography (SiCE, solvent 0-10% MeOH/DCM) to provide Compound AX-6 (1.2 g, 55%) as colorless liquid; ¹H NMR (CDCl₃): 0.85 (t, 12H), 1.11 (t, 6H), 1.4-1.25 (m, 46H), 1.45- 1.44 (m, 6H), 1.61-1.50 (m, 8H), 1.93-1.86 (m, 6H), 2.22- 2.10 (s, 1H), 2.40-2.35 (m, 6H), 2.69-2.58 (m, 6H), 3.41- 3.33 (m, 4H), 3.57-3.51 (m, 4H), 3.72 (s, 6H), 4.47 (t, 2H); CIMS m/z [M+H] ⁺ 1020.8. Analytical HPLC column: Agilent Zorbax SB-C18, 5 μm, 4.6x 150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 5% to 100% in 17 min, flow rate: ImL/min, column temperature: 20±2 ° C, detector: ELSD, t_R= 7.93 min, purity: > 99%; UPLC column: Waters Aquity UPLC® CSHTM, C18, 1.7 μm, 3.0x 150 mm, (Part No. 186005302), mobile phase A: water with 0.1% trifluoroacetic acid, mobile phase B: acetonitrile with 0.1% trifluoroacetic acid, use gradient: B in A 60% to 100% in 17 min, flow rate: 0.5 mL/min, column temperature: 20±2 °C, detector: CAD, t_R= 13.50 min, purity: > 99%.

Synthetic Scheme for Compound AX-7 Synthesis of ((1R,3S,5R, 7R)-5-(((4-(diethylamino)butanoyl)oxy)methyl)adamantane-l , 3- diyl)bis (methylene) bis(4, 4-bis(octylthio)butanoate) (Compound AX-7)

[950] A solution of 4,4-bis(octylthio)butanoic acid (184 mg, 0.49 mmol), EDC (156 mg, 0.81 mmol) and DMAP (25 mg, 0.2 mmol) in DCM (5 mL) was stirred at room temperature for 20 min. The starting alcohol L150-2 (75 mg, 0.2 mmol) was added in. The reaction mixture was stirred at room temperature for 12 h and concentrated under reduced pressure. The residue was purified by column chromatography (SiCE, solvent 0-10% MeOH/DCM) to provide Compound AX-7 (125 mg, 56%) as colorless liquid; ¹H NMR(CDCl₃): 0.85 (t, 12H), 1.16 (t, 6H), 1.36-1.20 (m, 46H), 1.43- 1.42 (m, 6H), 1.57-1.53 (m, 6H), 1.92-1.88 (m, 2H), 2.10- 2.05 (m, 4H), 2.16-2.19 (m, 1H), 2.39 (t, 2H), 2.52-2.63 (m, 14H), 2.66-2.88 (m, 6H), 3.74 (s, 6H), 3.79 (t, 2H); CIMS m/z [M+H]⁺ 1084.7; Analytical HPLC column: Agilent Zorbax SB-C18, 5 μm, 4.6x 150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 5% to 100% in 17 min, flow rate: ImL/min, column temperature: 20±2 ° C, detector: ELSD, t_R= 8.35 min, purity: > 99%; UPLC column: Waters Aquity UPLC® CSHTM, C18, 1.7 μm, 3.0 x 150 mm, (Part No. 186005302), mobile phase A: water with 0.1% trifluoroacetic acid, mobile phase B: acetonitrile with 0.1% trifluoroacetic acid, use gradient: B in A 60% to 100% in 17 min, flow rate: 0.5 mL/min, column temperature: 20±2 ° C, detector: CAD, t_R= 13.58 min, purity: 85.57%.

Synthetic Scheme for Compound AX-9

Synthesis of ((1R,3S,5R, 7R)-5-(((4-(diethylamino)butanoyl)oxy)methyl)adamantane-l , 3- diyl)bis (methylene) bis(3-pentyloctanoate)) (Compound AX-9)

[951] A solution of 3-pentyloctanoic acid (105 mg, 0.49 mmol), EDC (156 mg, 0.81 mmol) and

DMAP (25 mg, 0.2 mmol) in DCM (5 mL) was stirred at room temperature for 30 min. The starting alcohol L150-2 (75 mg, 0.2 mmol) was added in. The reaction mixture was stirred at room temperature for 12 h and concentrated under reduced pressure. The residue was purified by column chromatography (SiO₂, solvent 0-10% MeOH/DCM) to provide Compound AX-9 (88 mg, 66%) as colorless liquid; ¹H NMR(CDCl₃): 0.85 (t, 12H), 1.33-1.27 (m, 44H), 1.48-1.42 (m, 6H), 1.86- 1.79 (m, 2H), 2.07-1.97 (m, 2H), 2.18-2.14 (m, 1H), 2.24- 2.22 (m, 4H), 2.42 (t, 2H), 2.98-2.86 (m, 6H), 3.73-3.71 (m, 6H); CIMS m/z [M+H]⁺ 760.6; Analytical HPLC column: Agilent Zorbax SB-C18, 5 μm, 4.6x 150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 5% to 100% in 17 min, flow rate: ImL/min, column temperature: 20±2 ° C, detector: ELSD, t_R= 6.59 min, purity: > 99%; UPLC column: Waters Aquity UPLC® CSHTM, C18, 1.7 μm, 3.0x 150 mm, (Part No. 186005302), mobile phase A: water with 0.1% trifluoroacetic acid, mobile phase B: acetonitrile with 0.1% trifluoroacetic acid, use gradient: B in A 60% to 100% in 17 min, flow rate: 0.5 mL/min, column temperature: 20±2 ° C, detector: CAD, t_R= 11.88 min, purity: 92.45%.

Synthetic Scheme for Compound AX-10

Synthesis of ((1R,3S,5R, 7R)-5-(hydroxymethyl)adamantane-l,3-diyl)bis(methylene) his(3- pentyloctcmoate) (L156-1)

[952] A solution of 3-pentyloctanoic acid (428 mg, 2.0 mmol), EDC (768 mg, 4.0 mmol) and DMAP (122 mg, 1.0 mmol) in DCM (6 mL) was stirred at room temperature for 30 min, and then added dropwise into a solution of the starting alcohol L150-1 (226 mg, 1.0 mmol) in DMF (4 mL) over a period of 30 min. The resulting reaction mixture was stirred at room temperature for 20 h and concentrated under reduced pressure. The residue was purified by column chromatography (SiCE, 0- 100% EtOAc in hexane) to provide L156-1 (253 mg, 41%) as colorless liquid; ¹H-NMR (300 MHz, CDCl₃) δ 3.72 (s, 4H), 3.28 (s, 2H), 2.23 (d, J= 6.8 Hz, 4H), 2.19 (m, 1H), 1.91-1.75 (m, 2H), 1.41- 1.30 (m, 6H), 1.29-1.12 (m, 38H), 0.87 (t, J =7.2 Hz. 12H).

Synthesis of ((1R,3S,5S, 7S)-5-(((quinuclidine-4-carbonyl)oxy)methyl)adamantane-l,3- diyl)bis (methylene) bis(3-pentyloctanoate) (Compound AX- 10)

[953] A mixture of quinuclidine-4-carboxylic acid hydrochloride (115 mg, 0.6 mmol), DCC (205 mg, 0.8 mmol), 1,4-dimethylpyridinium 4-methylbenzenesulfonate (120 mg, 0.44 mmol) and DIPEA (0.5 mL) in DCM (10 mL) was stirred at room temperature for 15 min. A solution of L156-1 (250 mg, 0.4 mmol) in DCM (2 mL) was then added in and the resulting mixture was stirred at room temperature for 3 days. The solvent was evaporated to give the crude product which was subjected to silica gel column using 0 - 20% MeOH in DCM with 1% NH₄OH as eluent to afford Compound AX-10 (60 mg, 20%) as slightly yellow oil; ¹H-NMR (300 MHz, CDCl₃) δ 3.80 (s, 2H), 3.74 (s, 4H), 3.36-3.32 (m, 6H), 2.23 (d, J= 6.8 Hz, 4H), 2.21 (m, 1H), 2.18-2.05 (m, 6H), 1.90-1.76 (m, 2H), 1.51-1.40 (m, 6H), 1.32-1.15 (m, 38H), 0.87 (t, J=7.2 Hz, 12H); APCI-MS: m/z [M+H]⁺ 756.6. Analytical HPLC column: Agela Durashell C18, 3 μm (Catalog No. DC930505-0), 4.6x 150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 70% to 100% in 5 min, then 100% for 10 min. Plow rate: 1 mL/min, column temperature: 20±2 º C, detector: t_R= 7.82 min, purity: 96.24 %; UPLC column: Waters Aquity UPLC® CSHTM, C18, 1.7 μm, 3.0x 150 mm, (Part No. 186005302), mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 40% to 100% in 5 min, then 100% for 15 min. Flow rate: 0.5 mL/min, column temperature: 20±2 °C, detector: CAD, t_R= 12.38 min, purity: 92.85%.

Synthetic Scheme for Compound AX-11

Synthesis of ((1R,3S,5R, 7R)-3-(((4-(diethylamino)butanoyl)oxy)methyl)-5-(hydroxymethyl) adamantan-l-y I) methyl 3 -pentyloctanoate (L158-1)

[954] A solution of 3-pentyloctanoic acid (121 mg, 0.56 mmol), EDC (542 mg, 2.8 mmol) and DMAP (86 mg, 0.70 mmol) in DCM (5 mL) was stirred at room temperature for 20 min. The starting alcohol L150-2 (260 mg, 0.70 mmol) was added in. The reaction mixture was stirred at room temperature for 12 h and concentrated under reduced pressure. The residue was purified by column chromatography (SiO₂, solvent 0-10% MeOH/DCM) to provide L158-1 (85 mg, 22%) as colorless liquid; ¹H NMR(CDCl₃): 0.86, (t, 6H), 1.16 (t, 6H), 1.26-1.24 (m, 21H), 1.44-1.35 (m, 7H), 1.92-1.81 (m, 3H), 2.24- 2.22 (m, 3H), 2.38-2.35 (m, 2H), 2.75-2.67 (m, 5H), 3.01 (s, 2H), 3.27 (s, 2H), 3.74-3.72 (m, 4H); CIMS m/z [M+H]⁺ 564.5.

Synthesis of ((1S,3R,5R, 7R)-3-((2-(bicyclo[2.2.2]octan-l-yl)acetoxy)methyl)-5-(((4-

(diethylamino)hutanoyl)oxy)methyl)adamantan-l-yl)methyl 3 -pentyloctanoate (Compound AX-11)

[955] A solution of 2-(bicyclo [2.2.2] octan-l-yl)acetic acid (38 mg, 0.22 mmol), EDC (115 mg, 0.60 mmol) and DMAP (18 mg, 0.15 mmol) in DCM (5 mL) was stirred at room temperature for 20 min. The starting alcohol L158-1 (85 mg, 0.15 mmol) was added in. The reaction mixture was stirred at room temperature for 12 h and concentrated under reduced pressure. The residue was purified by column chromatography (SiCE, solvent 0-10% MeOH/DCM) to provide Compound AX-11 (78 mg, 74%) as colorless liquid; ¹HNMR(CDCl₃): 0.85 (t, 6H), 1.28- 1.19 (m, 27H), 1.53-1.43 (m, 20H), 1.85- 1.78 (m, 1H), 2.03-1.97 (m, 4H), 2.23-2.19 (m, 3H), 2.42 (t, 2H), 2.97-2.85 (m, 6H), 3.72-3.67 (m, 6H); QMS m/z [M+H]⁺ 714.6. Analytical HPLC column: Agilent Zorbax SB-C18, 5 μm, 4.6x 150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 5% to 100% in 17 min, flow rate: ImL/min, column temperature: 20±2 ° C, detector: ELSD, 6.70 min, purity: > 99%; UPLC column: Waters Aquity UPLC® CSHTM, C18, 1.7 μm, 3.0 x 150 mm, (Part No. 186005302), mobile phase A: water with 0.1% trifluoroacetic acid, mobile phase B: acetonitrile with 0.1% trifluoroacetic acid, use gradient: B in A 60% to 100% in 17 min, flow rate: 0.5 mL/min, column temperature: 20±2 ° C, detector: CAD, t_R= 10.10 min, purity: 94.04%.

Synthetic Scheme for Compound AX-12

Synthesis of ((1S,3R,5S, 7S)-3,5-bis(hydroxymethyl)adamantan-l-yl)methyl 3-pentyloctanoate (L160- 1)

[956] A solution of 3-pentyloctanoic acid (428 mg, 2.0 mmol), EDC (768 mg, 4.0 mmol) and DMAP (122 mg, 1.0 mmol) in DCM (12 mL) was stirred at room temperature for 30 min, and then added dropwise into a solution of the starting alcohol L150-1 (452 mg, 2.0 mmol) in DMF (6 mL) over a period of 30 min. The resulting reaction mixture was stirred at room temperature for 18 h and concentrated under reduced pressure. The residue was purified by column chromatography (SiCE, 0- 100% EtOAc in hexane) to provide L160-1 (330 mg, 70%) as colorless liquid; ¹H-NMR (300 MHz, CDCl₃) δ 3.74 (s, 4H), 3.29-3.28 (m, 2H), 2.23 (d, J= 6.8 Hz, 2H), 2.22-2.18 (m, 1H), 1.91-1.76 (m, 1H), 1.49-1.40 (m, 6H), 1.35-1.15 (m, 22H), 0.87 (t, J=7.2 Hz, 6H). Synthesis of ((1S,3R,5R, 7R)-3-((2-(bicyclo[2.2.2]octan-l-yl)acetoxy)methyl)-5- (hydroxymethyl)adamantan-l-yl)methyl 3 -pentyloctanoate (LI 60-2)

[957] A solution of 2-(bicyclo[2.2.2]octan-l-yl)acetic acid (125 mg, 0.74 mmol), EDC (320mg, 1.66 mmol) and DMAP (52 mg, 0.41 mmol) in DCM (12 mL) was stirred at room temperature for 30 min, and then added dropwise into a solution of the starting alcohol L160-1 (350 mg, 0.83 mmol) in DMF (4 mL) over a period of 30 min. The reaction mixture was stirred at room temperature for 20 h and concentrated under reduced pressure. The residue was purified by column chromatography (SiCE, 0-100% EtOAc in hexane) to provide L160-2 (181 mg, 50%) as colorless liquid; ¹H-NMR (300 MHz, CDCl₃) δ 3.73 (s, 2H), 3.69 (s, 2H), 3.31-3.28 (m, 2H), 2.23 (d, J= 6.8 Hz, 2H), 2.22-2.18 (m, 1H), 2.05 (s, 2H), 1.88-1.76 (m, 1H), 1.60-1.50 (m, 13H), 1.49-1.39 (m, 6H), 1.38-1.15 (m, 22H), 0.87 (t, J =7.2 Hz, 6H).

Synthesis of ((1R,3S,5R, 7R)-3-((2-(bicyclo[2.2.2]octan-l-yl)acetoxy)methyl)-5-(((3- pentyloctanoyl)oxy)methyl)adamantan-l-yl)methyl quinuclidine-4-carhoxylate (Compound AX-12)

[958] A mixture of quinuclidine-4-carboxylic acid hydrochloride (130 mg, 0.67 mmol), DCC (232 mg, 0.9 mmol), 1,4-dimethylpyridinium 4-methylbenzenesulfonate (135 mg, 0.49 mmol) and DIPEA (0.5 mL) in DCM (10 mL) was stirred at room temperature for 15 min. A solution of L160-2 (260 mg, 0.45 mmol) in DCM (2 mL) was then added and the resulting mixture was stirred at room temperature for 3 days. The solvent was evaporated to give the crude product which was subjected to silica gel column using 0 - 20% MeOH in DCM with 1% NH₄OH as eluent to afford Compound AX- 12 (156 mg, 48%) as slightly yellow oil; ¹H-NMR (300 MHz, CDCl₃) δ 3.73 (s, 4H), 3.69 (s, 2H), 2.92- 2.87 (m, 6H), 2.23 (d, J= 6.8 Hz, 2H), 2.21-2.18 (m, 1H), 2.03 (s, 2H), 1.85-1.76 (m, 1H), 1.73-1.65 (m, 6H), 1.60-1.49 (m, 13H), 1.50-1.39 (m, 6H), 1.38-1.16 (m, 22H), 0.85 (t, 7 =7.0 Hz, 6H); APCI- MS: m/z [M+H]⁺ 710.5; Analytical HPLC column: Agela Durashell C18, 3 μm (Catalog No. DC930505-0), 4.6 * 150 mm, mobile phase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 70% to 100% in 5 min, then 100% for 10 min. Flow rate: 1 mL/min, column temperature: 20±2 ° C, detector: t_R= 6.76 min, purity: > 99%; UPLC column: Waters Aquity UPLC® CSHTM, C18, 1.7 μm, 3.0x 150 mm, (Part No. 186005302)), mobilephase A: acetonitrile with 0.1% trifluoroacetic acid, mobile phase B: water with 0.1% trifluoroacetic acid, use gradient: A in B 40% to 100% in 5 min, then 100% for 15 min. Flow rate: 0.5 mL/min, column temperature: 20±2 ° C, detector: CAD, t_R= 8.30 min, purity: 96.29%.

[959] In some embodiments, the below synthetic scheme can be used for the synthesis of exemplary LNPs disclosed herein, including but not limited to those in series "CT". EXAMPLE 2A: LNP Formulations - oRNA expression

[960] Ionizable lipids, phospholipid, cholesterol, and PEG lipid were dissolved in pure ethanol at the specified mol% ratios with atotal lipid concentration of 15.9 mM. A 0.075 mg/mL mRNA solution was prepared using acidic buffer (pH 4.0-5.0) containing linear mRNA or circular RNA encoding VHH or a CAR protein. The nucleotide and lipid solutions were mixed at a 3 : 1 volume ratio using the Knauer mixing system at a 30 mL/min total flow rate resulting in rapid mixing and self-assembly of LNPs. Formulations were further dialyzed against cryopreservation buffer using a Repligen TFF system and concentrated prior to sterile filtration (0.2 μm pore size). The particle size and polydispersity index (PDI) of formulations was measured by dynamic light scattering (DLS) using a Zetasizer Ultra (Malvem Panalytical). RNA encapsulation efficiency (EE%) was determined by Ribogreen assay.

Table XA: In Vivo VHH and oRNA CAR Formulations

EXAMPLE 2B: LNP Formulations F - VHH Formulations

[961] Ionizable lipids, phospholipid, cholesterol, and a PEG lipid were dissolved in pure ethanol at the specified mol% ratios with a total lipid concentration of 10.8 mM. A 0.10 mg/mL mRNA solution was prepared using acidic buffer (pH 4.0-5.0) containing mRNA encoding VHH. The nucleotide and lipid solutions were mixed at a 3 : 1 volume ratio using the NanoAssemblr microfluidic system at a 12 mL/min total flow rate resulting in rapid mixing and self-assembly of LNPs. Formulations were further dialyzed against PBS (pH 7.4) overnight at 4 ° C, concentrated using centrifugal filtration and filtered (0.2 μm pore size). The particle size and polydispersity index (PDI) of formulations was measured by dynamic light scattering (DLS) using a Zetasizer Ultra (Malvem Panalytical). RNA encapsulation efficiency (EE%) was determined by Ribogreen assay. Table XB: In Vivo VHH Formulation

VHH m RNA

GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCG

CCACCAUGGGAGUGAAGGUGCUGUUCGCUCUCAUCUGCAUUGCUGUGGCUGAG

GCCCAGGUGCAGCUGCAAGAGUCUGGCGGCGGACUGGUGCAACCUGGCGGCAG

CUUGAGACUGAGCUGUGCUGCCAGCGGCUUCACGCUGGAUUACUACUACAUCG

GGUGGUUUAGACAGGCCCCCGGCAAGGAGAGGGAAGCCGUCUCCUGCAUUAGC

GGCUCAUCCGGGAGCACCUACUACCCGGAUAGCGUCAAGGGCAGAUUCACCAU CUCCCGGGACAACGCCAAGAACACGGUGUACCUGCAGAUGAACAGCCUGAAGC CCGAGGACACGGCUGUGUACUACUGCGCUACCAUCAGGAGCAGCAGCUGGGGC GGCUGCGUCCAUUACGGAAUGGACUACUGGGGCAAAGGCACCCAGGUGACCGU GUCCAGCGGCACCACCAGACUGCUGUCUGGACACACCUGUUUCACACUGACAG GGCUGCUGGGCACCCUGGUGACCAUGGGGCUGCUUACAUAAUGAAUUAAUAG UGUAGUGACUAGUGACUGACUAGGAUCUGGUUACCACUAAACCAGCCUCAAG AACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUACAAAAU GUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUU UCUUCACAUGCGGCCG (SEQ ID NO: 25620)

EXAMPLE 3: Assessment of Constructs Comprising IRESs and oCARs

[962] CD3+ T cells from two healthy donors (609C, first row; 4003, second row) were activated with αCD3/CD28 tetrameric complexes for 3 days and then electroporated with CD19 CAR oRNA sequences at a 10ng/100K cells oRNA dose and evaluated. 74 total sequences were evaluated, comprising a combination of CD 19 CAR sequences comprising the codon optimized sequences of Table 6 and 16 IRES sequences of Table 4, and an IRES corresponding to a base CD19 control. Starting at 24 hours post-electroporation up to 120 hours, CD19 CAR expression (T cell MFI), i.e., the level of expression per cell over time, was evaluated via flow cytometry using the anti -idiotypic antibody FMC63. Starting at 24 hours post-electroporation and up to 120 hours, frequency of CD19 CAR expressing cells (% CAR positive cells by IRES over time, i.e., the percent of cells or average signal of the cells expressing over time) was evaluated via flow cytometry using the anti -idiotypic antibody FMC63. See also WO2024102677.

[963] CD3+ T cells from two healthy donors (4003, first row; 609C, second row) were activated with αCD3/CD28 tetrameric complexes for 3 days and then electroporated with CD 19 CAR oRNA sequences. 74 total sequences were evaluated, comprising a combination of CD 19 CAR sequences comprising the codon optimized sequences of Table 6 and 18 IRES sequences of Table 4. Frequency of CD19 CAR expressing cells was analyzed via flow cytometry at 24- 120hr timepoints post-electroporation. Frequency of CD19 CAR expression was rank ordered for each timepoint pursuant to the tables below for each of Donor 609C and Donor 4003 as compared to a base control CD 19 CAR and a combination HER2 standard control sequence. See also id. Donor 609C oCAR+ cells (% T cells post- electroporation)

Donor 4003 oCAR+ cells (% T cells post- electroporation) [964] oRNA constructs comprising a base CD 19 CAR construct in combination with different IRESs described in Table 4 and Table 5, were assessed for % Nalm6 lysis in two donors (Donor 4003 and Donor 609C). CD3+ T cells from two healthy donors (609C; 4003) were activated with αCD3/CD28 tetrameric complexes for 3 days and then electroporated with the CD 19 CAR oRNA sequences. Nalm6 killing was assessed at 24 and 48 hours for each CD 19 CAR base oRNA construct along with a mock negative control, Nalm6 alone, and the base alone.

[965] Coculture supernatants were collect and analyzed using Meso Scale Discovery (MSD) (Agilent Technologies, Santa Clara, CA) for IFNγ and IL-2 expression. CD3+ T cells from two healthy donors (609C, first row; 4003, second row) were activated with αCD3/CD28 tetrameric complexes for 3 days and then electroporated with CD 19 CAR oRNA sequences. 74 total sequences were evaluated, comprising a combination of 5 CD19 CAR sequences comprising the codon optimized sequences of Table 6 and 18 IRES sequences of Table 4. 24 hours post-electroporation, transfected T cells were co-cultured with Nalm6 cells for up to 48 hours. At 24-hour and 48-hour timepoints, supernatants were collected and IL-2 and IFN-γ amounts were quantified via MSD.

EXAMPLE 4: In Vivo Tumor Efficacy

[966] NSG mice were engrafted with Nalm6-Luciferase (Luc) tumor cells and 4 days later were engrafted with human PBMCs. Starting on Day 5, the mice were treated 4 times every other day with vehicle (PBS), control, or anti-CD19 LNP-oCAR compounds at doses of 1.0 mg/kg, 0.3 mg/kg, and 0.1 mg/kg. PBS control is representative of mice with engrafted Nalm6- luc tumor cells only and treated with vehicle. LNP used herein comprised PEG-ionizable lipid 86 of Table 8. Animals were then whole-body imaged via IVIS to monitor luciferase expression from Nalm6 cells. Nalm6 tumor burden is plotted as Total Flux (photons/second over a region of interest) of the liver, spleen, kidney, lung, and heart of luciferase expression at each imaging timepoint.

[967] Total Flux (photons/second) was observed over time for oRNA constructs with expression sequences directed to HER2 and CD 19 and to assess the effect of anti -turn or efficacy after 4 doses using different lipids of LNP-circRNAs, where the effects of PEG lipids in the transfer vehicle was also assessed. NSG mice were engrafted with Nalm6-Luciferase (Luc) tumor cells and 3 days later were engrafted with human PBMCs. Starting on Day 1, the mice were treated 4 times every other day with vehicle (PBS) or anti-CD19 LNP-oCAR compounds. LNPs delivering the oCAR constructs comprised of PEG-modified lipids of control lipids (11 and 62) and lipids of Formula I, including lipids 16, 45, and 86 of Table 8) at an ionizable lipid to phosphate ratio (IL:P) of 5.7. The ionizable lipid: helper lipid: cholesterol: PEG-lipid molar ratio of these LNPs was 50: 10:38.5: 1.5. The formulations of the LNPs are further detailed below. PBS control is representative of mice with engrafted Nalm6- luc tumor cells only and treated with vehicle. Animals were then whole-body imaged via IVIS to monitor luciferase expression from Nalm6 cells.

[968] The anti-CD19 oRNA-LNP construct described above was assessed in vivo in mice engrafted with Nalm6 tumor cells and dosing schedules (every-other-day dosing post- engraftment, weekly dosing post-engraftment, and every-other-week dosing post-engraftment) were compared. Dose timing groups were evaluated at doses of 1.0 mg/kg, 0.3 mg/kg, and 0.1 mg/kg across four PBMC donors (two studies, two donors per study, A and B). The experimental protocol is shown in the table below.

[969] NSG MHC I/II double knockout mice were engrafted with Nalm6-Luciferase (Luc) tumor cells and 4 days later were engrafted with human PBMCs. Starting on Day 5 post-Nalm6 engraftment, the mice were treated 4 times (as shown in the table below in the dosing days column) with the anti-CD19 oRNA-LNP, PBS control, or HER2 control as shown herein. Total flux (photons/second) was observed over time for the CD 19 oRNA construct. Animals were then whole-body imaged twice weekly via IVIS to monitor luciferase expression from Nalm6 cells.

[970] The CD 19 oRNA-LNP treated animals showed tumor control and improved survival compared to controls treated with the HER2 oRNA construct described herein.

EXAMPLE 5: BCMA oCAR, CD19 oCAR, HER2 oCAR

[971] Expression of circular RNAs encoding anti -BCMA CAR was studied in vitro. T cells from a single donor were activated for 3 days with anti-CD3 and CD28 beads and allowed to rest for 24 hours. Concurrently, engineered circular RNA constructs were designed to encode a BCMA-41BB ζ chimeric antigen receptor (CAR). For comparison purposes, "mock" or control circular RNAs encoded a HER2 or CD19 CAR. The circular RNAs were developed from an in vitro translation (IVT) reaction of DNA comprising a T7 polymerase promoter, permuted Anabaena intron exon segments, internal ribosome entry site (IRES) from Caprine Kobuvirus, Hunnivirus, Apodemus Picomavirus, or Picornavirales internal spacers, optionally internal homology arms, and a XI ab restriction site. DNA templates comprised of sequences from the table below. The donor T cells were electroporated with the circular RNAs at either 10 ng, 30ng, or 100 ng per 0.1x10⁶ T cell to form CAR-T cells. As a control, "Mock" T cells not electroporated with circular RNAs were analyzed. T cells were then allowed to rest for 24 hours after electroporation. 24 hours after electroporation, the CAR-T cells were counted and assessed for BCMA CAR expression. Resulting circular RNA with anti-BCMA CAR encoding regions were given a commercially available soluble BCMA detection reagent containing R- phycoerythrin (PE) fluorophore (e.g., from AcroBiosy stems, Delaware), anti -Whitlow PE linker antibody detection reagent, or an anti-G4S linker PE detection reagent (e.g., G4S- AF647). Anti-BCMA, CD 19, or HER2 expression was assessed using fluorescence activated cell sorting (FACS) and gating flow cytometry methods known in the art at one or more timepoints from 24 to 72 hours post electroporation.

[972] Expression of circular RNAs encoding anti-BCMA CAR co-cultured with MM1S was studied. As a preliminary testing of various cell lines used, target protein expression on multiple myeloma positive cells (e.g., MM1S, NCI-H929, and RPMI-8226) and negative target cell line (e.g., Nalm6 target cell line) was analyzed using methods known in the art. Donor T cells were thawed and activated with anti-CD3/CD28 solutions. Three days after activation, the cells were washed. On the following day, the cells were electroporated with a range of 50 ng dosages of circular RNA encoding a BCMA-41BB ζ or BCMA-CD28 ζ CAR or CD19- CD28 ζ CAR per 0.1 X10⁶ T cells to form engineered CAR-T cells (oCAR-T cells). Selected oCAR-T cells were analyzed for percent live cells for 6 circular RNA constructs encoding BCMA CARs and compared to the circular RNA construct comprising a CD 19 28 CAR and a control comprising an EP Buffer only. Circular RNAs were formed from DNA templates present in Table 5. The cells were given a commercially available soluble BCMA detection reagent containing R-phycoerythrin (PE) fluorophore (e.g., from AcroBiosystems, Delaware), anti-Whitlow PE detection reagent, or anti-G4S linker detection reagent (e.g., G4S-AF647). 24 hours post electroporation of the circular RNA into the T cells, oCAR-T cells were subgated on live T cells based on FACS results and amount of detectable reagent was collected. [973] The selected oCAR-T cells were co-cultured with targeted multiple myeloma cells (e.g., MM1S), NCI-H929, Nalm6 or K562.CD19 cells at an E:T ratio of 1 : 1 on the day following electroporation of the circular RNAs to the donor T cells. The oCAR-T cells were given soluble BCMA-PE or anti-Whitlow.PE detection reagent.

[974] BCMA CAR expression and CD 19 CAR expression were studied in multiple myeloma positive and negative target cell lines. Donor T cells were thawed and activated with anti-CD3/CD28 solutions. Three days after activation, the cells were washed. On the following day, the cells were electroporated with a range of 10-30 ng dosages of circular RNA encoding a BCMA-41BB ζ CAR or CD19CD28 ζ CAR per 0.1 X10⁶ T cells to form engineered CAR-T cells (oCAR-T cells). Circular RNAs comprised in the following order: a ' 3 Anabaena exon, an internal ribosome entry site (IRES), a coding region encoding a BCMA CAR or CD 19 CAR, and a 5 ' Anabaena exon. Circular RNAs were formed from an IVT reaction of DNA templates from Table 5. As a control, Mock T cells were used, wherein the Mock T cells were not electroporated with any circular RNAs. A day following electroporation of the circular RNAs to the T cells, the oCAR-T cells were co-cultured with target multiple myeloma (MM) positive or negative T cells at a 1 : 1 E:T ratio. MM positive T cells comprised of MM1 S, NCI- 14929 or RPM 1-8226. MM negative T cells comprised of a CD 19 positive T cell line (i.e., Nalm6 cell line). As a control, the target tumor cell without co-culturing with the T cells was analyzed for comparison purposes. The circular RNAs were given a commercially available soluble BCMA detection reagent containing R-phycoerythrin (PE) fluorophore or soluble CD 19 detection reagent containing quantum dot (qdot) fluorophore (e.g., from AcroBiosystems, Delaware). The oCAR-T cells were also placed into a commercially available live-cell analysis portfolio system (e.g., an IncuCyte) and analyzed 0-72 or 0-96 hours post co-culture for cytotoxicity.

[975] Cytotoxicity of BCMA targeted killing on MM1S was studied via FACS. BMCA positive cells were acquired and prepped. The cells were electroporated with 30 ng per 0.1X10⁶ cells of circular RNA comprising a 3 ' Anabaena exon, an internal ribosome entry site (IRES), a BCMA-41 BB ζ CAR, and a 5 ' Anabaena exon. Circular RNAs were formed from an IVT reaction of DNA templates from Table 5. On the following day, the cells were then co-cultured with MM1S cells and dyed with FSC-A, CD3-Brilliant Violet 650-A, LD-fixable Near IR-A for fluorescence activated cell sorting analysis (FACS).

[976] As a follow up, FACS cytotoxicity assays were performed for circular RNAs encoding BCMA-41BB ζ, CD19-CD28 ζ or HER2-CD28 ζ CAR and an IRES at either 10, 30, 50 or 100 ng per 0.1X10⁶ T cells. Circular RNAs were formed from an IVT reaction of DNA templates from Table 5. The T cells containing circular RNAs (oCAR-T) were co-cultured with one of four target cell - either MM1S (a BCMA positive cell line), NCI-H929 (a BCMA positive cell line) Nalm6 cells (a CD 19 positive cell line), a CD 19 T stable cell line (e.g., K562.CD19) - at an E:T ratio of 1 : 1. 24 hours post co-culture of the oCAR-T cells with the MM1S, Nalm6, NCI-H929, or CD19 T stable cells. MM1S +Mock (i.e., MM1S tumor cells cocultured with T cells not electroporated with circular RNAs), MM1 S (i.e., MM1 S tumor cells not cocultured with T cells), Nalm6 + Mock (i.e., Nalm6 tumor cells cocultured with T cells not electroporated with circular RNAs), Nalm6 (i.e., Nalm6 tumor cells not co-cultured with T cells), Mock + NCI-H929 (i.e., NCI-H929 tumor cells cocultured with T cells not electroporated with circular RNAs), and Mock + K562.CD19 (i.e., K562.CD19 tumor cells cocultured with T cells not electroporated with circular RNAs) were used as controls. Resulting FACS imaging for each of the cell types post 24 hours after co-culturing the T cells with the target cells was calculated.

[977] The circular RNA constructs encoding BCMA CAR were tested in vivo. NSG mice were prepared at Day -1. 5M U266 cells were injected at Day 0. 10M PBMC cells (n=2 donors) were injected at Day 4. The circular RNA constructs were injected either at EOD (2 mpk), i.e., Day 8, Day 10, Day 13, and Day 15, or QW (2 mpk), i.e., Day 8, Day 15, and Day 22. Controls were HER2 oCAR-treated mice and untreated mice. Twice weekly, tumor burden was quantified via IVIS as described herein. EOD dosing demonstrated BCMA oCAR-dependent tumor control. QW dosing demonstrated BCMA oCAR-dependent tumor control.

[978] Circular RNAs encoding HER2 CARs induced cytotoxicity in vitro. Plates were coated with 0.01% poly-L-ornithine solution or 5 μL/mL fibronectin diluted in 0.1% BSA. The dilute apoptosis reagent (e.g., Annexin V) was prepared in a medium and cell treatments are prepared. Cell treatments comprised of circular RNAs comprising a 3 ' exon segment, a Caprine Kobuvirus or a Hunnivirus internal ribosome entry site (IRES), a coding region encoding HER2, and a 5 ' exon segment with an E:T ratio of 1 : lor 1 :2. Circular RNAs were derived from DNA Templates from Table 5 that underwent IVT reactions 100 μL/well of 25,000-50,000 of HER2 positive BT474 or SKBR2 target cells were placed into the coated 96- well plates and allowed to adhere overnight. After about 24 hours, the BT474/SKBR3 cells were adhered and the cell treatment at 0 and 100 ng dosages containing the circular RNAs and Annexin V were added to the BT474 or SKBR2 cells. As a control, some BT474 /SKBR3 cells were not given any circular RNAs but were given Annexin V. All the plates containing the target cells were analyzed in a live-cell analysis portfolio system (e.g., an IncuCyte) which captured images every 2-3 hours. In the imaging alive Nalm6 cells retained a green fluorescence, and Annexin V apoptotic cells had a red luminescence. % apoptotic cells were then calculated by measuring the amount of ((green area + red area)/green area).

[979] Further, activated PBMC T cells from a single donor were prepared. Separately, circular RNAs encoding HER2-CD28 ζ, HER2-41BB ζ or CD19-CD28 ζ CAR prepared from an IVT reaction of DNA templates comprising a 3 ' intron segment, a 3 ' exon segment, an internal ribosome entry site (IRES), a coding region encoding either a HER2.28 ζ, HER2.BB ζ or CD19-28 ζ CAR, a 5 ' exon segment, and a 5' intron segment. The circular RNAs were transfected onto the activated PBMC T cells using frozen or fresh lipid nanoparticles comprising an ionizable lipid from Table 8 at a concentration listed in the table below. For comparison purposes, activated PBMC T cells were given no circular RNAs as a control. On the day of post-delivery of the circular RNAs to activated PBMC T cells, the circular RNA- activated PBMC T cells were co-cultured with one of three HER2 positive cell lines (i.e., BT474, SKBR3, JIMT1). The resulting co-cultured cells given Annexin V and were analyzed in live-cell analysis portfolio system (e.g., an IncuCyte).

[980] Female NSG immune deficient mice (aged 8 to 12 weeks) with 1X10⁷ JIMT-1 or BT-474 tumor cells (in subcutaneous flank engraftment) were prepared to have an average tumor size of 50-100 mm³. IX 10⁷ human PBMC T cells from two different donors were activated with anti-CD3 and anti-CD8 for 72 hours, then activation was removed and cells were prepared in PBS for injection. On the fifth day after human PBMC thawed, 200 μL of 50X10⁶/mL of the stimulated human PBMC T cells were injected into the tail vein of mice intravenously. A day after the activated human PBMC T cells were injected, the mice were dosed with either docetaxel (control, no PBMC) at 10 mL/kg, PBS (control), or LNP described herein comprising circular RNAs encoding HER2.28 ζ, HER2.BB ζ or CD19.28 ζ CAR at 3mg/kg intravenously. Circular RNAs further comprised internal ribosome entry sites (IRESes) derived from Caprine Kobuvirus or Hunnivirus. The mice were dosed every other day (within a four-hour timeframe) for a total of 4 dosages.

[981] Frozen human T cells were thawed and activated with Stemcell CD3/CD28 antibody cocktail for 72 hours. Post activation T cells were electroporated with circular RNA as described herein comprising different IRES sequences and the HER2 CAR sequences as shown in Construct N and Construct O above, with either 28z or BBz domains. T cells that were electroporated (EP) but had no circular RNA (mock) or had a circular RNA encoding CD 19 CAR served as controls. After EP, cells were allowed to recover for 24 hours, following which they were assessed for expression by FACS at 24, 48 and 72 hours post transfection. HER2 CAR was detected using soluble HER2 fluorokines (soluble proteins conjugated to fluorophores).

[982] Incucyte assays were performed. 24 hours after EP, cells expressing the circular RNA encoding HER2 CAR were plated on Incucyte plates at an E:T of 1 : 1 with HER2 positive, GFP positive BT474 target cells in T cell growth media without IL2. Annexin V, which stains dead cells, was added to the co-culture to track the accumulation of dead cells. The plate was read on the Incucyte instrument for 5 days. Target cell death was assessed by analyzing the percent of green target cells that were stained with Annexin V and comparing to the total GFP positive population in the well as well as to the mock control. Expression kinetics were assessed for circular RNAs encoding HER2 CAR and comprising different IRES sequences over 72 hours. Cytotoxicity function of BT474 target cells by Incucyte was assessed for circular RNAs encoding HER2 CAR and comprising different IRES sequences.

[983] RAJI control in natural killer (NK) cells using circular RNA encoding CD19 CAR in NOG-IL15 mice was assess. See, e.g., Katano et al., Long-term maintenance of peripheral blood derived human NK cells in a novel human IL- 15- transgenic NOG mouse, Sci Rep (2017), the contents of which are hereby incorporated by reference herein ("mouse strain expressing transgenic human interleukin- 15 (IL- 15) using the severe immunodeficient NOD/Shi-scid-IL-2R. 7 null (NOG) mouse genetic background (NOG-IL-15 Tg). Human natural killer (NK) cells, purified from the peripheral blood (hu-PB-NK) of normal healthy donors, proliferated when transferred into NOG-IL-15 Tg mice").

[984] NOG-IL15 mice were engrafted with a CD19+ Raji-luciferase cell line at Day 0. On Day 3, primary human NK cells purified from peripheral blood were engrafted into recipient animals. On Day 8, mice were left untreated, or treated i.v. with vehicle, circular RNA encoding mOX40L CAR encapsulated in LNP (1 mg/kg), or circular RNA encoding CD19 CAR encapsulated in LNP (1 mg/kg). Mice were treated every two days for 10 doses. Tumor burden was assessed using IVIS imaging as described herein. Tumor clearance was observed in this mouse model mouse model. Mice treated with circular RNA encoding CD19 CAR encapsulated in LNP exhibited tumor control until the study endpoint at Day 24.

[985] NSG-QUAD mice were engrafted with human CD34+ cord blood. At 10 weeks post-engraftment, animals were left untreated (control) or treated with circular RNA comprising a sequence for mOX40L encapsulated in LNP. Animals were sacrificed after 24, 48, or 72 hours. mOX40L expression was analyzed by flow cytometry on CD33+, CD33+ CD14+ or CD33+CD64+ myeloid cells in the blood, bone marrow and spleen. mOX40L was detected on the surface of CD33+ myeloid cells in the bone marrow and spleen at each timepoint, with peak expression observed at 72 hours LNP-circRNA treatment.

EXAMPLE 6: Use of circular RNA encoding CD19 CAR in autoimmune disease

[986] The ability of circular RNA encoding CD 19 CAR to deplete human B cells in a CD34+ engrafted humanize mouse model was assessed using a CD34+ NOD.Cg-PrkdcscidlL- 2rgeml/Smoc strain at 19 weeks. The CD34+ Humanized Mice (HiMice) were generated by Invivocue. Five- to six- week-old, female NOD-PrkdcscidIL2rgeml/Smoc mice, at approximately 16-22g, were sub-lethally irradiated and engrafted with human CD34+ hematopoietic stem cells via i.v. During the humanization process, mice were monitored over the course of 12 to 14 weeks post engraftment to ensure immune lineage differentiation & maturation. HiMice with minimal 10% of human CD45+ reconstitution at week 12- or 14 were used in this experiment. HiMice were also supplemented with cytokines GM-CSF, IL-3, and IL-4 to enhance myeloid lineage, B cell differentiation, and preserve T cell development. Once engraftment was confirmed, a total of 5 doses of either mWasabi oRNA encapsulated in an LNP disclosed herein or CD 19 CAR oRNA encapsulated in the same LNP were administered

1.v. weekly. Peripheral blood was collected post 3 days of each LNP-oRNA injection and analyzed for immune subsets. Spleens were harvested and processed 3 days after the 5th LNP- oRNA injection for immune profiling.

[987] Cells from blood or spleen were first stained with 50 μl of live/dead solution (1 :400 dilution in PBS) for 10 minutes at room temperature. The cells were then pelleted by centrifugation and resuspended with mouse and human Fc receptor blocking reagent (in 25 μ l of FACS buffer) for 10 minutes at room temperature to prevent non-specific binding. Subsequently, fluorescent-labeled surface markers (in 25 μl of FACS buffer) were added to the cells. After approximately 30 minutes of incubation in the dark, the cells were washed before acquiring the FACS data using a Fortessa™ X-20 flow cytometer (BD Biosciences) with FACSDiva software. The FACS data were then analyzed with FlowJo software (Tree Star Inc).

[988] The circular RNA encoding CD 19 CAR mediated B-cell specific depletion in CD34+ humanized mice. B cell-mediated killing in groups treated with CD 19 CAR oRNA encapsulated in LNP was maintained upon 5 doses within the peripheral blood and spleen. Minimal LNP effect was observed on total CD20+ B cell frequency and count in the peripheral blood.

EXAMPLE 7: LNP oCAR

[989] Exemplary ionizable lipids, LNPs and formulations thereof (see, e.g., Examples 1-

2, Table 8) were combined with linear mRNA or circular RNA expressing reporter (VHH) or CD19 CAR or CD20 CAR (see, e.g., Examples 3-6, Table 5, Table 10).

[990] Immunodeficient NOD SCID gamma (NSG) mice with transgenic expression of human IL3, GM-CSF (CSF2) and SCF (KITLG) cytokines (NSG-SGM3) mice were engrafted with human cord blood-derived CD34+ hematopoietic stem cells (hCD34+ NSG-SGM3), 22 weeks old, were pre-bled at Day -7 and administered (1) IX PBS (control), (2) a composition comprising lipid nanoparticle A (LNP A) or lipid nanoparticle B (LNP B) and linear mRNA expressing a VHH reporter at 1.0 mg/kg, (3) a composition comprising LNP A or LNP B and oRNA expressing the VHH reporter at 1.0 mg/kg, (4) a composition comprising LNP A or LNP B and oRNA expressing CD 19 CAR (oCAR) comprising the binding and IRES sequences of construct M in Table 5, at 1.0 mg/kg, or (5) a composition comprising LNP A and oRNA expressing CD20 CAR (oCAR) comprising binding sequences 10J in Table 10 and IRES sequence from Table 4, at 1.0 mg/kg. At 24 hours following administration, reporter or CAR expression was measured and B cell depletion was measured in the blood, spleen, and bone narrow. At 7 days following administration, reporter or CAR expression was measured and B cell depletion was measured in the blood, spleen, and bone narrow. See, e.g., Figs. 1 A-24B.

[991] Each of LNP A and LNP B in combination with oRNA expressing reporter demonstrated reporter (VHH) expression and delivery to peripheral blood lymphocytes 24 hours following administration. LNP A in combination with linear mRNA expressing reporter demonstrated reporter (VHH) expression and delivery to peripheral blood, splenic, and bone marrow cells 24 hours following administration.

[992] Each of LNP A and LNP B in combination with oRNA expressing CD19 CAR demonstrated CD 19 CAR expression and delivery to peripheral blood and splenic T cells 24 hours following administration.

[993] Each of LNP A and LNP B in combination with oRNA expressing CD19 CAR demonstrated peripheral blood B cell depletion. LNP B in combination with oRNA expressing CD 19 CAR demonstrated splenic B cell depletion 7 days following administration. Each of LNP A and LNP B in combination with oRNA expressing CD 19 CAR demonstrated bone marrow B cell depletion 7 days following administration.

[994] LNP A in combination with oRNA expressing CD20 CAR demonstrated expression and delivery to peripheral blood 24 hours following administration. LNP A in combination with oRNA expressing CD20 CAR demonstrated expression and delivery to peripheral blood 7 days following administration. LNP A in combination with oRNA expressing CD20 CAR demonstrated expression and delivery to splenic T cells 24 hours following administration. [995] LNP A in combination with oRNA expressing CD20 CAR demonstrated peripheral blood B cell depletion. LNP A in combination with oRNA expressing CD20 CAR demonstrated splenic B cell depletion 24 hours following administration.

EXAMPLE 8: Cynomolgus and Human Donor T Cells

[996] As described in Example 7, exemplary ionizable lipids, LNPs and formulations thereof (see, e.g., Examples 1-2, Table 8) were combined with linear mRNA or circular RNA expressing reporter (VHH) or CD19 CAR or CD20 CAR (see, e.g., Examples 3-6, Table 5, Table 10).

[997] LNP A was combined with oRNA expressing reporter (VHH), oRNA expressing CD 19 CAR, oRNA expressing CD20 CAR, or linear mRNA expressing reporter (VHH). T cells were obtained from two cynomolgus donors and one human donor. Cynomolgus T cells were transfected at a dose of 50 ng, 200 ng, or 500 ng per 100,000 cells. Human T cells were transfected at a dose of 200 ng per 100,000 cells. Human T cells were thawed and activated after 24 hours with an anti-CD3/CD28 cocktail for 3 days. T cell media was changed and cells were rested for 24 hours prior to treatment with LNP A in combination with the reporter (VHH) or CD 19 CAR constructs. Cynomolgus T cells were thawed and immediately activated with an anti-CD2/CD3/CD28 cocktail for 3 days. T cell media was changed and cells were rested for 48 hours prior to treatment with LNP A in combination with the reporter (VHH) or CD 19 CAR constructs. FACS was conducted using the panel below.

[998] LNP A in combination with oRNA expressing CD 19 CAR delivered and expressed

CD 19 CAR in cynomolgus and human T cells. LNP A in combination with oRNA or linear mRNA expressing reporter (VHH) delivered and expressed reporter in cynomolgus and human T cells. Lipid A in combination with oRNA expressing CD20 CAR delivered and expressed CD20 CAR in cynomolgus and human T cells. See, e.g., Figs. 25A-30B. EXAMPLE 9A: LNP Delivery to T and NK cells - Non-Human Primates

[999] LNP formulations were prepared as described in Example 2B. Each formulation was administered to 3 Cynomolgus monkeys on Day 1 via 60-min intravenous infusion into an appropriate peripheral vein using an infusion pump at a dose level of 2.0 mg/kg mRNA (dose volume of 5 mL/kg; concentration 0.4 mg/mL). Blood samples were collected at 6 and 24 hours after dosing for peripheral blood mononuclear cells, and at 24 hours the test subjects were terminated. Peripheral blood mononuclear cell were purified from blood and cryopreserved in Cryostor CS10 at a density of approximately 5e6 PBMC per mL. On day of analysis, cryopreserved PBMC were thawed into pre-warmed complete RPMI containing RPMI 1640 (GIBCO, 72400-047) with 10% heat inactivated FBS (GIBCO, A38400-01) and 1% Pen/Strep (GIBCO, 15-140-122). Cells were spun down and resuspended in RPMI 1640 supplemented with 50U/mL benzonase (EMD, 70664- 10KUN) and incubated for 10 minutes at 37°C. Cells were then spun down and resuspended in complete RPMI and counted (Cellaca MX, PerkinElmer MX-AOPI). Cells were diluted, plated (5,000,000 per well) in a 96-well round bottom plate (Costar 3799), and stained for flow cytometry. Test subject femurs were flushed for collection of bone marrow and spleens and lungs were collected for analysis by flow cytometry. Up to 1 gram of harvested spleen per animal was dissociated into single cell suspension of splenocytes using the gentleMACS Octo Dissociator with Heaters (Miltenyi 130- 096-427) with the Multi Tissue Dissociation Kit I (Miltenyi 130-110-201) per manufacturer’s instructions. Dissociated splenocytes were then passed through a 70μm filter (Miltenyi 130- 098-462) and washed with 1x PBS (ThermoFisher 10010049) containing 2mM EDTA (ThermoFisher 15575-020), 0.5% BSA (Miltenyi 130-091-376) and 1x + 1x Antimycotic/ Antibiotic (ThermoFisher 15240-062). Red blood cells were lysed using ACK Lysing Buffer (Thermo Fisher A1049201) and washed twice with 1x PBS + 2mM EDTA + 0.5% BSA, passing the cell suspension through an additional 70μm filter prior to the last wash. Following final wash, cells were resuspended in 1x PBS + 2mM EDTA + 0.5% BSA + 1x Antimycotic/ Antibiotic and counted (Cellaca MX, PerkinElmer MX-AOPI). Cells were diluted, plated (5,000,000 per well) in a 96-well round bottom plate (Costar 3799), and stained for flow cytometry. Up to 3 grams of harvested lung per animal was dissociated into single cell suspension of cells using the gentleMACS Octo Dissociator with Heaters (Miltenyi 130-096- 427) with the Multi Tissue Dissociation Kit I (Miltenyi 130-110-201) per manufacturer’s instructions. Dissociated lung cells were then passed through a 70μm filter (Miltenyi 130-098- 462) and washed with 1x PBS + 2mM EDTA + 0.5% BSA + 1x Antimycotic/ Antibiotic. Red blood cells were lysed using ACK Lysing Buffer (Thermo Fisher Al 049201) and washed twice with 1x PBS + 2mM EDTA + 0.5% BSA + 1x Antimycotic/ Antibiotic, passing the cell suspension through an additional 70μm filter prior to the last wash. Following final wash, cells were resuspended in 1x PBS + 2mM EDTA + 0.5% BSA + 1x Antimycotic/ Antibiotic and counted (Cellaca MX, PerkinElmer MX-AOPI). Cells were diluted, plated (2,000,000 per well) in a 96-well round bottom plate (Costar 3799), and stained for flow cytometry. Flushed bone marrow was centrifuged and cells were collected, passed through a 70μm filter, and washed with 1x PBS + 2mM EDTA + 0.5% BSA+ 1x Antimycotic/ Antibiotic. Red blood cells were lysed using ACK Lysing Buffer and washed twice with 1x PBS + 2mM EDTA + 0.5% BSA+ Ix Antimycotic/ Antibiotic, passing the cell suspension through an additional 70μm filter prior to the last wash. Following final wash, cells were resuspended in 1x PBS + 2mM EDTA + 0.5% BSA+ 1x Antimycotic/ Antibiotic and counted. Cells were diluted, plated (15 million per well) in a 96-well bottom plate, and stained for flow cytometry. Cells were stained in 1x PBS with Live/Dead Fixable Aqua (Invitrogen L34966) at 1 : 1000 for 20min at room temperature. Cells were then washed twice with Cell Staining Buffer (BioLegend 420201) and incubated with Fc block for 5min at 4°C and surface antibody stains either in full or FMO (fluorescence minus one) master mixes (panels and dilutions shown below in Tables 5A-5D) added on top of the Fc Block for an additional 30min at 4°C. Cells were then washed three times with Cell Staining Buffer and fixed with Cytofix (BD 554655) at 4°C for 20min. Cells were washed twice with 1x PBS and filtered through a 30-40 μm filter (Pall 8027) and acquired on cytometer(Sony ID7000 Cell Analyzer: Cat # LE-ID7000C with UV/V/B/YG/R lasers). Single stain controls were made using UltraComp eBeads (ThermoFisher 01-3333-41) and ArC Amine Reactive Compensation Bead Kit (ThermoFisher Al 0346). Analysis was performed using Flowjo (BD V10.8.1). Cells were identified with markers in Tables 5E-5H and VHH reporter+ gates placed so that the negative control would be ≤0.5%⁺.

[1000] The percentage of cells of the noted varieties expressing VHH mRNA is reported below in Table 51 (as the mean of 3 animals). Robust VHH expression was detected in many organs, including splenic T cells and NK cells. Formulation F-32 performed especially well for delivery to T cells, higher than any other comparative LNP formulation known in the art. Additionally, Formulation F-32 demonstrated remarkable delivery to NK cells and bulk HSPCs in bone marrow. Table 5A: Splenocyte surface antibody stains used in Example 9A

Table 5B: PBMC surface antibody stains used in Example 9A

Table 5C: NHP Bone Marrow antibodies used in Example 9A

Table 5D: Lung Antibodies used in Example 9A

Table 5E: Definition of Cell Subsets in NHP Spleen by Flow Cytometry used in Example 9A

Table 5F: Definition of Cell Subsets in NHP PBMC by Flow Cytometry used in Example 9

Table 5G: Definition of Cell Subsets in NHP BM by Flow Cytometry used in Example 9 *Lineage is defined as including CD3, CD4, CD8, CD14, CD16, CD20, CD11b, and CD11c.

Table 5H: Definition of Cell Subsets in NHP Lung by Flow Cytometry used in Example 9A

Table 51: Percentage of cells of various types expressing VHH mRNA

EXAMPLE 9B: LNP Delivery to T and NK cells over a timecourse in a non-terminal study - Non-Human Primates

[1001] LNP formulations were prepared as described in Example 2B. Each formulation was administered to 3 Cynomolgus monkeys on Day 1 via 60-min intravenous infusion into an appropriate peripheral vein using an infusion pump at a dose level of 2.0 mg/kg mRNA (dose volume of 5 mL/kg; concentration 0.4 mg/mL). Blood samples were collected at 6, 24 and 48 hours after dosing for peripheral blood mononuclear cells, and bone marrow aspirates were collected at 24 hours after dosing. Animals were not terminated at the end of the study and no adverse events were reported. Peripheral blood mononuclear cell were purified from blood and cryopreserved in Cryostor CS10 at a density of approximately 5e6 PBMC per mL. On day of analysis, cryopreserved PBMC were thawed into pre-warmed complete RPMI containing RPMI 1640 (GIBCO, 72400-047) with 10% heat inactivated FBS (GIBCO, A38400-01) and 1% Pen/Strep (GIBCO, 15-140-122). Cells were spun down and resuspended in RPMI 1640 supplemented with 50U/mL benzonase (EMD, 70664- 10KUN) and incubated for 10 minutes at 37°C. Cells were then spun down and resuspended in complete RPMI and counted (Cellaca MX, PerkinElmer MX-AOPI). Cells were diluted, plated (3,000,000 per well) in a 96-well round bottom plate (Costar 3799), and stained for flow cytometry. Bone marrow aspirate was centrifuged, and cells were collected, passed through a 70μm filter, and washed with 1x PBS + 2mM EDTA + 0.5% BSA+ 1x Antimycotic/ Antibiotic. Red blood cells were lysed using ACK Lysing Buffer and washed twice with 1x PBS + 2mM EDTA + 0.5% BSA+ 1x Antimycotic/ Antibiotic, passing the cell suspension through an additional 70μm filter prior to the last wash. Following final wash, cells were resuspended in 1x PBS + 2mM EDTA + 0.5% BSA+ 1x Antimycotic/ Antibiotic and counted. Cells were diluted, plated (15 million per well) in a 96-well bottom plate, and stained for flow cytometry. Cells were stained in 1x PBS with Live/Dead Fixable Aqua (Invitrogen L34966) at 1 : 1000 for 20min at room temperature. Cells were then washed twice with Cell Staining Buffer (BioLegend 420201) and incubated with Fc block for 5min at 4°C and surface antibody stains either in full or FMO (fluorescence minus one) master mixes (panels and dilutions being the same as those reported in Example 9, Tables 5B and 5C) added on top of the Fc Block for an additional 30min at 4°C. Cells were then washed three times with Cell Staining Buffer and fixed with Cytofix (BD 554655) at 4°C for 20min. Cells were washed twice with 1x PBS and filtered through a 30-40 μm filter (Pall 8027) and acquired on cytometer(Sony ID7000 Cell Analyzer: Cat # LE-ID7000C with UV/V/B/YG/R lasers). Single stain controls were made using UltraComp eBeads (ThermoFisher 01-3333-41) and ArC Amine Reactive Compensation Bead Kit (ThermoFisher A10346). Analysis was performed using Flowjo (BD V10.8.1). Cells were identified with markers in Example 9, Tables 5F and 5G, and VHH reporter+ gates placed so that the negative control would be ≤0.5%⁺.

[1002] The percentage of cells of the noted varieties expressing VHH mRNA is reported below in Table 5 J (as the mean of 3 animals). Robust VHH expression was detected in PBMCs at both the 24hr and 48hr time points, as well as in bone marrow aspirates at 24 hr. Formulations F-32c and F-33c performed especially well for delivery to T cells, confirming the results observed in Example 9A.

Table 5 J: Percentage of cells of various types expressing VHH mRNA

EXAMPLE 10: LNP Delivery to T and NK cells -Humanized CD34+ NSG mice

[1003] LNP/mRNA formulations were prepared as described in Example 2B. CD34+ Humanized Mice (Hu-NSG, Jackson laboratory), were injected via tail vein with 1.0 mg/kg VHH mRNA formulated in LNP formulations F-32a and F-33a in a total volume of 5mL/kg. Each formulation was dosed in 3 mice and an additional 1 mouse was dosed with PBS and an additional 2 mice were dosed with an fLuc mRNA LNP to serve as a control. 6 h post injection whole blood was collected with EDTA anticoagulant for flow cytometry analysis, and 24 h post injection animals were euthanized by CO₂ inhalation, and spleens, femurs, and tibiae/fibulae and whole blood were harvested. Harvested spleens were dissociated into single cell suspension of splenocytes using the gentleMACS Octo Dissociator with Heaters (Miltenyi 130-096-427) with the Mouse Spleen Dissociation Kit (Miltenyi 130-095-926) per manufacturer’s instructions. Dissociated splenocytes were then passed through a 70μm filter (Miltenyi 130-098-462) and washed with 1x PBS (ThermoFisher 10010049) containing 2mM EDTA (ThermoFisher 15575-020) and 0.5% BSA (Miltenyi 130-091-376). Red blood cells were lysed using ACK Lysing Buffer (Thermo Fisher A1049201) and washed twice with 1x PBS + 2mM EDTA + 0.5% BSA, passing the cell suspension through an additional 70μm filter prior to the last wash. Following final wash, cells were resuspended in 1x PBS + 2mM EDTA + 0.5% BSA and counted (Cellaca MX, PerkinElmer MX-AOPI). Cells were diluted, plated (5,000,000 per well) in a 96-well round bottom plate (Costar 3799), and stained for flow cytometry. Bone marrow (BM) was harvested from the bones by using a 25G needle (BD 305122) to flush the marrow cavity with 1x PBS + 2mM EDTA + 0.5% BSA. Cells were collected, passed through a 70μm filter, and washed with 1x PBS + 2mM EDTA + 0.5% BSA. Red blood cells were lysed using ACK Lysing Buffer and washed twice with 1x PBS + 2mM EDTA + 0.5% BSA, passing the cell suspension through an additional 70μm filter prior to the last wash. Following final wash, cells were resuspended in 1x PBS + 2mM EDTA + 0.5% BSA and counted. Cells were diluted, plated (10 million per well) in a 96-well bottom plate, and stained for flow cytometry. Whole blood was collected into EDTA (Corning, 46-034-CI) as an anti-coagulant, and processed by pipetting 50 μL of blood into 150 μL of PBS into a 96-well round bottom plate. Cells were spun at 300xg for 5 minutes, supernatants were discarded and cell pellet was resuspended in 250 μL of ACK lysis buffer and incubated for 5 minutes at room temperature. Cells then had 50 μL of wash buffer added on top of the ACK lysis buffer and were spun at 300xg for 5 minutes. Cells were then treated with one more ACK lysis procedure, washed with PBS and then proceeded to flow cytometry staining. Cells were stained in 1x PBS with Live/Dead Fixable Aqua (Invitrogen L34966) at 1 : 1000 for 20min at room temperature. Cells were then washed twice with Cell Staining Buffer (BioLegend 420201) and incubated with Fc block for 5min at 4°C and surface antibody stains either in full or FMO master mixes (panel and dilutions shown below in Tables 6A-6C) added on top of the Fc Block for an additional 30min at 4°C. Cells were then washed three times with Cell Staining Buffer and fixed with Cytofix (BD 554655) at 4°C for 30min. Cells were washed twice with 1x PBS and filtered through a 30-40 μm filter (Pall 8027) and acquired on a cytometer (ThermoFisher Attune NXT with a laser configuration of Blue(3)/Red(3)/Violet(4)/Yellow(4)equipped with a high-throughput autosampler (ThermoFisher CytKick) or (Sony ID7000 Cell Analyzer: Cat # LE-ID7000C with UV/V/B/YG/R lasers). Compensation was performed using UltraComp eBeads and ArC Amine Reactive Compensation Bead Kit (ThermoFisher Al 0346). Analysis was performed using Flowjo (BD V10.8.1). Cells were identified with markers in Tables 6D, 6E and 6F and VHH reporter+ gates placed so that the negative control would be ≤0.5%⁺.

[1004] The percentage of cells of the noted varieties expressing VHH mRNA is reported below in Table 6G (as the mean of 3 animals). Remarkably high immune cell delivery, including delivery to splenic T cells and whole blood T cells, was observed. Additionally, saturation level delivery was observed for F-32a and F-33a in both HSCs and T cells in the collected bone marrow samples.

Table 6A: Splenocyte surface antibody stains used in Example 10

Table 6B: Whole Blood surface antibody stains used in Example 10

Table 6C: Bone Marrow surface antibody stains used in Example 10

Table 6D: Definition of Cell Subsets in humanized CD34+ NSG mice Spleen by Flow Cytometry used in Example 10

Table 6E: Definition of Cell Subsets in humanized CD34+ NSG mice whole blood by Flow

Cytometry used in Example 10

Table 6F: Definition of Cell Subsets in humanized CD34+ NSG mice Bone Marrow by Flow

Cytometry used in Example 10

Table 6G: Percentage of cells of various types expressing VHH mRNA

EXAMPLE 11: LNP Delivery to T and NK cells -WT C57B1/6 mice

[1005] LNP/mRNA formulations were prepared as described in Example 2B. Wildtype C57bl/6 mice (C57BL/6J (Jackson Laboratory 000664)), were injected via tail vein with 1.0 mg/kg VHH mRNA formulated in LNP formulations F-32b, F-33b and F-34 in a total volume of 5mL/kg. Each formulation was dosed in 3 mice and an additional 1 mouse was dosed with PBS and an additional 2 mice were dosed with an fLuc mRNA LNP to serve as a control. 16 h post injection, animals were euthanized by CO₂ inhalation, and spleens, femurs, and tibiae/fibulae and whole blood were harvested. Harvested spleens were dissociated into single cell suspension of splenocytes using the gentleMACS Octo Dissociator with Heaters (Miltenyi 130-096-427) with the Mouse Spleen Dissociation Kit (Miltenyi 130-095-926) per manufacturer’s instructions. Dissociated splenocytes were then passed through a 70μm filter (Miltenyi 130-098-462) and washed with 1x PBS (ThermoFisher 10010049) containing 2mM EDTA (ThermoFisher 15575-020) and 0.5% BSA (Miltenyi 130-091-376). Red blood cells were lysed using ACK Lysing Buffer (Thermo Fisher A1049201) and washed twice with 1x PBS + 2mM EDTA + 0.5% BSA, passing the cell suspension through an additional 70μm filter prior to the last wash. Following final wash, cells were resuspended in 1x PBS + 2mM EDTA + 0.5% BSA and counted (Cellaca MX, PerkinElmer MX-AOPI). Cells were diluted, plated (5,000,000 per well) in a 96-well round bottom plate (Costar 3799), and stained for flow cytometry. Bone marrow (BM) was harvested from the bones by using a 25G needle (BD 305122) to flush the marrow cavity with 1x PBS + 2mM EDTA + 0.5% BSA. Cells were collected, passed through a 70μm filter, and washed with 1x PBS + 2mM EDTA + 0.5% BSA. Red blood cells were lysed using ACK Lysing Buffer and washed twice with 1x PBS + 2mM EDTA + 0.5% BSA, passing the cell suspension through an additional 70μm filter prior to the last wash. Following final wash, cells were resuspended in 1x PBS + 2mM EDTA + 0.5% BSA and counted. Cells were diluted, plated (10 million per well) in a 96-well bottom plate, and stained for flow cytometry. Whole blood was collected into EDTA (Corning, 46-034-CI) as an anti-coagulant, and processed by pipetting 50 μL of blood into 150 μL of PBS into a 96-well round bottom plate. Cells were spun at 300xg for 5 minutes, superanatants were discarded and cell pellet was resuspended in 250 μL of ACK lysis buffer and incubated for 5 minutes at room temperature. Cells then had 50 μL of wash buffer added on top of the ACK lysis buffer and were spun at 300xg for 5 minutes. Cells were then treated with one more ACK lysis procedure, washed with PBS and then proceeded to flow cytometry staining. Cells were stained in 1x PBS with Live/Dead Fixable Aqua (Invitrogen L34966) at 1 : 1000 for 20min at room temperature. Cells were then washed twice with Cell Staining Buffer (BioLegend 420201) and incubated with Fc block (splenocytes and whole blood) or labeled CD 16/32 antibody (bone marrow) for 5min at 4°C and surface antibody stains either in full or FMO master mixes (panel and dilutions shown below in Tables 7A-7C) added on top of the Fc Block for an additional 30min at 4°C. Cells were then washed three times with Cell Staining Buffer and fixed with Cytofix (BD 554655) at 4°C for 30min. Cells were washed twice with 1x PBS and filtered through a 30-40 μm filter (Pall 8027) and acquired on cytometer (Sony ID7000 Cell Analyzer: Cat # LE- ID7000C with UV/V/B/YG/R lasers). Compensation was performed using UltraComp eBeads and ArC Amine Reactive Compensation Bead Kit (ThermoFisher Al 0346). Analysis was performed using Flowjo (BD V10.8.1). Cells were identified with markers in Tables 7D-7F and VHH reporter+ gates placed so that the negative control would be ≤0.5%⁺.

[1006] The percentage of cells of the noted varieties expressing VHH mRNA is reported below in Table 7G (as the mean of 3 animals). VHH expression was detected in many of the same cells and organs detected for NHPs and humanized mice (Examples 9 and 10 respectively). Without intending to be limited to any particular theory, it was predicted that wild type mice would demonstrate overall lower expression levels than the other two model organisms, illustrating the difficulty in translatability and precise predictability between different species in the art of the present disclosure.

Table 7A: Splenocyte surface antibody stains used in Example 11

Table 7B: Whole Blood surface antibody stains used in Example 11

Table 7C: Bone Marrow surface antibody stains used in Example 11

Table 7D: Definition of Cell Subsets in WT mice Spleen by Flow Cytometry used in Example 11

Table 7E: Definition of Cell Subsets in WT mice whole blood by Flow Cytometry used in

Example 11

Table 7F: Definition of Cell Subsets in WT mice Bone Marrow by Flow Cytometry used in

Example 11

Table 7G: Percentage of cells of various types expressing VHH mRNA

EXAMPLE 12: LNP Delivery of oRNA CAR Payload to T and NK cells - Non-Human Primates [1007] LNP formulations were prepared as described in Example 2 A. Each formulation was administered to Cynomolgus monkeys on Day 1 via 60-min intravenous infusion into an appropriate peripheral vein using an infusion pump at a dose level of 2.0 mg/kg mRNA of oRNA (dose volume of 5 mL/kg; concentration 0.4 mg/mL). Each LNP formulation and control was dosed in the following number of animals: PBS control - 2; F-1 - 2; F-2 - 3; F-3 - 5; F-4 - 3. Blood samples were collected at baseline, 6hr, 24hr, 48hr, 72hr and 168hr post dosing, processed to isolate Peripheral Blood Mononuclear Cells (PBMC) and cryopreserved for later flow cytometry analysis. Bone marrow aspirates were collected at 1 Day and 7 Days post dosing for analysis by flow cytometry. Cryopreserved PBMC were thawed and washed with complete RPMI supplemented with 10% heat-inactivated FBS (GIBCO 72400-047 and A38400-01) before incubating with plain RPM with 50 U/mL Benzonase (EMD 7066-10KUN) at 37°C for 15 minutes. Cells were then centrifuged, resuspended in cRPMI and counted (Cellaca MX, PerkinElmer MX-AOPI). Cells were plated at 5 million cells per well in a 96- well round bottom plate (Costar 3799) and stained for flow cytometry. Bone marrow aspirate was centrifuged, and cells were resuspended in 1x PBS + 2mM EDTA + 0.5% BSA+ 1x Antimycotic/Antibiotic wash buffer, passed through a 70μm filter, and washed with wash buffer. Red blood cells were lysed using ACK Lysing Buffer (Thermo Fisher A1049201) and washed twice with wash buffer, passing the cell suspension through an additional 70μm filter prior to the last wash. Following final wash, cells were resuspended in wash buffer and counted. Cells were diluted, plated (5 million per well) in a 96-well bottom plate, and stained for flow cytometry. Briefly, cells were stained in 1x PBS with Live/Dead Fixable Aqua (Invitrogen L34966) at 1 : 1000 for 20min at room temperature. Cells were then washed twice with Cell Staining Buffer (BioLegend 420201) and incubated with Fc block for 5min at 4°C and surface antibody stains either in full or FMO (fluorescence minus one) master mixes (panels and dilutions shown below in Tables 8A-8B) added on top of the Fc Block for an additional 30min at 4°C. Cells were then washed and stained with Streptavidin-BV785 (BioLegend 405249) at 4°C for 20min. Cells were then washed three times with Cell Staining Buffer, resuspended in 1x PBS and filtered through a 30-40 μm filter (Pall 8027) and acquired on cytometer (Sony ID7000 Cell Analyzer: Cat # LE-ID7000C with UV/V/B/YG/R lasers). Single stain controls were made using UltraComp eBeads (ThermoFisher 01-3333-41) and ArC Amine Reactive Compensation Bead Kit (ThermoFisher Al 0346). Analysis was performed using Flowjo (BD V10.8.1). Cells were identified with markers in Tables 8C-8D. VHH reporter+ gates, antiCD 19-CAR (FMC63)+ gates and anti-CD20-CAR (Linker)+ gates were placed so that the negative control (PBS dosed subjects) would be ≤0.5%⁺.

Table 8A: PBMC surface antibody stains used in Example 12

Table 8B: NHP Bone Marrow antibodies used in Example 12

Table 8C: Definition of Cell Subsets in NHP PBMC by Flow Cytometry used in Example 12

Table 8D: Definition of Cell Subsets in NHP BM by Flow Cytometry used in Example 12

Table 8E: Expression of VHH mRNA or oRNA cargo in various cell types over time, reported as % of cells VHH+ (mean of n=2 animals for F-l, mean of n=3 animals for F-2)

Table 8F: Expression of CD19-CAR oRNA cargo in various cell types over time, reported as % of cells CD19+ (mean of n=3 animals)

Table 8G: Expression of CD20-CAR oRNA cargo in various cell types over time, reported as % of cells CD20+ (mean of n=5 animals)

Table 8H: B cell depletion in PBMC over time

Table 8I: B cell Absolute Counts in Bone Marrow

INCORPORATION BY REFERENCE

[1008] 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.

[1009] This description and exemplary embodiments should not be taken as limiting. For 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. EMBODIMENTS

Embodiment 1. A pharmaceutical composition comprising: a. a circular RNA polynucleotide comprising, in the following order: i. a 3' self-spliced exon segment; ii. a translation initiation element; iii. an expression sequence encoding a chimeric antigen receptor (CAR); iv. a 5' self-spliced exon segment; and b. a lipid nanoparticle comprising: i. one or more ionizable lipids; ii. one or more phospholipids, in an amount of about 20 mol% to about 60 mol% of the total lipid content of the lipid nanoparticle; iii. one or more structural lipids; and iv. one or more PEG lipids.

Embodiment 2. The pharmaceutical composition of embodiment 1, wherein the 3’ selfspliced exon segment is a Group I or Group II self-spliced exon segment, and/or wherein the 5’ self-spliced exon segment is a Group I or Group II self-spliced exon segment.

Embodiment 3. The pharmaceutical composition of embodiment 1 or 2, comprising a 5’ internal duplex and/or a 3’ internal duplex.

Embodiment 4. The pharmaceutical composition of any one of embodiments 1-3, comprising a 5’ internal spacer and/or a 3’ internal spacer.

Embodiment 5. The pharmaceutical composition of any one of embodiments 1-4, wherein the translation initiation element is an Internal Ribosome Entry Site (IRES).

Embodiment 6. The pharmaceutical composition of any one of embodiments 1-5, wherein the circular RNA polynucleotide is from about 50 nucleotides to about 15 kilobases in length.

Embodiment 7. The pharmaceutical composition of any one of embodiments 1-6, wherein the circular RNA polynucleotide: a. has an in vivo duration of therapeutic effect in a subject of at least about 10 hours; b. has a functional half-life of at least about 10 hours; c. 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; d. 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; e. 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; and/or f. has an in vivo functional half-life in a subject greater than that of an equivalent linear RNA polynucleotide having the same expression sequence.

Embodiment 8. The pharmaceutical composition of any one of embodiments 1-7, wherein the circular RNA polynucleotide comprises at least one modified A, C, G, or U nucleotide or nucleoside.

Embodiment 9. The pharmaceutical composition of embodiment 8, wherein the modified nucleotide or nucleoside is: a) one or more of m⁵U (5-methyluridine); m⁶A (N⁶ -methyladenosine); s²U (2- thiouridine); Ψ (pseudouridine); Um (2'-O-methyluridine); m¹A (1 -methyladenosine); m²A (2 -methyladenosine); Am (2’-O-methyladenosine); ms²m⁶A (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); 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 (l- methylguanosine); m²G (N² -methylguanosine); m⁷G (7-m ethylguanosine); Gm (2'-0- methylguanosine); m²2G (N²,N²-dimethylguanosine); m²Gm (N²,2’-O- dimethylguanosine); m²2Gm (N²,N²,2’-O-trimethylguanosine); Gr(p) (2’-O- ribosylguanosine(phosphate)); yW (wybutosine); 02yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl- queuosine); manQ (mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G⁺ (archaeosine); D (dihydrouridine); m⁵Um (5,2’ -O-dimethyluri dine); 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-methoxy carbonylmethyl -

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); m⁶2A (N⁶,N⁶-dimethyladenosine); Im (2’ -O-m ethylinosine); m⁴C (N⁴-methylcytidine); m⁴Cm (N⁴,2’-O-dimethylcytidine); hm⁵C (5-hydroxymethylcytidine); m³U (3 -methyluridine); cm⁵U (5- carboxymethyluridine); m⁶Am (N⁶,2’-O-dimethyladenosine); m⁶2Am (N⁶,N⁶,O-2’- trimethyladenosine); m^2,7G (N²,7-dimethylguanosine); m^2,2,7G (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); (5-taurinomethyluridine); taurinomethyl-2-thiouridine)); imG-14 (4-demethylwyosine); imG2 (isowyosine); or ac⁶A (N⁶-acetyladenosine); pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza- uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,

3 -methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5- propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1- taurinomethyl-pseudouridine, 5-taurinomethyl-2 -thio-uridine, 1 -taurinomethyl-4-thio- uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l-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-pseudoisocyti dine, 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-m ethoxy-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-m ethylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2- methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo- guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, Nl- methylpseudouridine, and N2,N2-dimethyl-6-thio-guanosine; or b) 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, N1 -methylpseudouridine, and alkylcarbonylalkylated nucleotides; or c) is selected from one or more of 5-methylcytidine, 5-methoxyuridine, 1-methyl- pseudouridine, N6-methyladenosine, and/or pseudouridine.

Embodiment 10. The pharmaceutical composition of any one of embodiments 1-9, wherein the CAR comprises an anti-BCMA antigen binding molecule, optionally selected from a nucleotide or amino acid sequence in Table 5 or Table 9 or functional fragment thereof, e.g., CDR sequences.

Embodiment 11. The pharmaceutical composition of any one of embodiments 1-9, wherein the CAR comprises an anti-CD19 antigen binding molecule, optionally selected from a nucleotide or amino acid sequence in Table 5, Table 6, Table 7, or Table 9 or functional fragment thereof, e.g., CDR sequences.

Embodiment 12. The pharmaceutical composition of any one of embodiments 1-9, wherein the CAR comprises an anti-CD20 antigen binding molecule, optionally selected from an amino acid sequence in Table 10 or functional fragment thereof, e.g., CDR sequences.

Embodiment 13. The pharmaceutical composition of any one of embodiments 1-9, wherein the CAR comprises an anti-HER2 antigen binding molecule, optionally selected from a nucleotide or amino acid sequence in Table 5 or Table 9 or functional fragment thereof, e.g., CDR sequences. Embodiment 14. The pharmaceutical composition of any one of embodiments 1-13, wherein the lipid nanoparticle comprises about 25 mol% to about 45 mol% of the one or more ionizable lipids, as a proportion of the total lipid content of the lipid nanoparticle.

Embodiment 15. The pharmaceutical composition of any one of embodiments 1-14, wherein the lipid nanoparticle comprises about 15 mol% to about 35 mol% of the one or more structural lipids, as a proportion of the total lipid content of the lipid nanoparticle.

Embodiment 16. The pharmaceutical composition of any one of embodiments 1-15, wherein the lipid nanoparticle comprises about 1 mol% to about 3 mol% of the one or more PEG lipids, as a proportion of the total lipid content of the lipid nanoparticle.

Embodiment 17. The pharmaceutical composition of any one of embodiments 1-16, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of the one or more PEG lipids; (b) about 15 mol% to about 35 mol% of the one or more structural lipids; (c) about 30 mol% to about 60 mol% of the one or more phospholipids; and (d) about 25 mol% to about 45 mol% of the one or more ionizable lipids.

Embodiment 18. The pharmaceutical composition of any one of embodiments 1-16, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of the one or more PEG lipids; (b) about 20 mol% to about 30 mol% of the one or more structural lipids; (c) about 35 mol% to about 45 mol% of the one or more phospholipids; and (d) about 28 mol% to about 40 mol% of the one or more ionizable lipids.

Embodiment 19. The pharmaceutical composition of any one of embodiments 1-18, wherein the one or more ionizable lipids comprises a compound selected from those depicted in any one of Tables (I-A), (I-B), (I-C), (I-D), (I-E), (I-F), (I-G), (I-H), (I-I), (I- J), (I-K), or (I- L), an enantiomer thereof, or any mixture of enantiomers thereof, or a pharmaceutically acceptable salt of any such compound.

Embodiment 20. The pharmaceutical composition of any one of embodiments 1-18, wherein the one or more ionizable lipids comprises a compound of Formula (AX) or a pharmaceutically acceptable salt thereof, wherein:

Y¹ is selected from the group consisting of and bond; wherein the bond marked with an "*" is attached to X ; 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-; 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.

Embodiment 21. The pharmaceutical composition of any one of embodiments 1-20, wherein the one or more phospholipids are selected from 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero- phosphocholine (DMPC), 1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho line (POPC), 1,2- di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuc cinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl -sn- glycero-3 -phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine,

1.2-diarachidonoyl-sn-glycero-3 -phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, 1,2-diphytanoylsn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3 -phosphoethanolamine, 1,2- diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), sodium (S)-2-ammonio-3-((((R)-2-(oleoyloxy)-3- (stearoyloxy)propoxy)oxidophosphoryl)oxy)propanoate (L-α-phosphatidylserine; Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl-phosphatidylethanolamine4-(N- maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), di oleoylphosphatidyl glycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Dielaidoyl-sn- phosphatidylethanolamine (DEPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3- phosphate (18: 1 PA; DOPA), ammonium bis((S)-2-hydroxy-3-(oleoyloxy)propyl) phosphate (18: 1 DMP; LBPA), 1,2-dioleoyl-sn-glycero-3-phospho-(l’ -myo-inositol) (DOPI; 18: 1 PI),

1.2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), 1,2-dilinoleoyl-sn-glycero-3- phospho-L-serine (18:2 PS), l-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (16:0-18: 1 PS; POPS), l-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (18:0-18: 1 PS), l-stearoyl-2- linoleoyl-sn-glycero-3-phospho-L-serine (18:0-18:2 PS), l-oleoyl-2-hydroxy-sn-glycero-3- phospho-L-serine (18: 1 Lyso PS), l-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:0 Lyso PS), and sphingomyelin, or combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the above phospholipids.

Embodiment 22. The pharmaceutical composition of any one of embodiments 1-20, wherein the one or more phospholipids are selected from DSPC and egg sphingomyelin, or a combination thereof.

Embodiment 23. The pharmaceutical composition of any one of embodiments 1-22, wherein the one or more structural lipids are selected from cholesterol, fecosterol, fucosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, cholic acid, sitostanol, litocholic acid, tomatine, ursolic acid, alpha-tocopherol, Vitamin D3, Vitamin D2, Calcipotriol, botulin, lupeol, oleanolic acid, beta-sitosterol-acetate and any combinations thereof.

Embodiment 24. The pharmaceutical composition of any one of embodiments 1-23, wherein the one or more PEG lipids is selected from DMG-PEG2k, DSPE-PEG2k, DSG- PEG2k, DMPE-PEG2k, DPPE-PEG2k and mixtures thereof

Embodiment 25. The pharmaceutical composition of any one of embodiments 1-24, wherein the one or more phospholipids comprises one or more selected from phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids or a combination thereof.

Embodiment 26. The pharmaceutical composition of any one of embodiments 1-24, wherein the one or more phospholipids comprises 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), sphingomyelin or a combination thereof.

Embodiment 27. The pharmaceutical composition of any one of embodiments 1-26, wherein the one or more phospholipids comprises two or more phospholipids, such that no single phospholipid makes up more than 30 mol% of the total lipid content of the nanoparticle.

Embodiment 28. The pharmaceutical composition of any one of embodiments 1-27, wherein the lipid nanoparticle comprises about 40 mol% 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC).

Embodiment 29. The pharmaceutical composition of any one of embodiments 1-27, wherein the lipid nanoparticle comprises about 40 mol% sphingomyelin.

Embodiment 30. The pharmaceutical composition of any one of embodiments 1-29, wherein the phospholipid is sphingomyelin, the structural lipid is cholesterol, and the PEG lipid is DMG-PEG2k.

Embodiment 31. The pharmaceutical composition of any one of embodiments 1-30, wherein the ionizable lipid is a compound of Formula (AX), the phospholipid is sphingomyelin, the structural lipid is cholesterol, and the PEG lipid is DMG-PEG2k. Embodiment 32. The pharmaceutical composition of any one of embodiments 1-24, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of DMG-PEG2k; (b) about 15 mol% to about 35 mol% cholesterol; (c) about 30 mol% to about 60 mol% sphingomyelin; and (d) about 25 mol% to about 45 mol% of the one or more ionizable lipids.

Embodiment 33. The pharmaceutical composition of any one of embodiments 1-24, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of DMG-PEG2k; (b) about 15 mol% to about 35 mol% cholesterol; (c) about 30 mol% to about 60 mol% sphingomyelin; and (d) about 25 mol% to about 45 mol% of an ionizable lipid of Formula (AX).

Embodiment 34. The pharmaceutical composition of any one of embodiments 1-24, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of DMG- PEG2k; (b) about 20 mol% to about 30 mol% cholesterol; (c) about 35 mol% to about 45 mol% sphingomyelin; and (d) about 28 mol% to about 40 mol% of the one or more ionizable lipids.

Embodiment 35. The pharmaceutical composition of any one of embodiments 1-24, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of DMG- PEG2k; (b) about 20 mol% to about 30 mol% cholesterol; (c) about 35 mol% to about 45 mol% sphingomyelin; and (d) about 28 mol% to about 40 mol% of an ionizable lipid of Formula (AX).

Embodiment 36. The pharmaceutical composition of any one of embodiments 1-24, wherein the lipid nanoparticle comprises (a) about 2 mol% of DMG-PEG2k; (b) about 25 mol% cholesterol; (c) about 40 mol% sphingomyelin; and (d) about 33 mol% of an ionizable lipid of Formula (AX).

Embodiment 37. The pharmaceutical composition of any one of embodiments 1-24, wherein the lipid nanoparticle comprises (a) about 2 mol% of DMG-PEG2k; (b) about 25 mol% cholesterol; (c) about 40 mol% sphingomyelin; and (d) about 33 mol% of the one or more ionizable lipids.

Embodiment 38. The pharmaceutical composition of any one of embodiments 1-24, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of DMG-PEG2k; (b) about 15 mol% to about 35 mol% cholesterol; (c) about 30 mol% to about 60 mol% DSPC; and (d) about 25 mol% to about 45 mol% of the one or more ionizable lipids.

Embodiment 39. The pharmaceutical composition of any one of embodiments 1-24, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of DMG-PEG2k; (b) about 15 mol% to about 35 mol% cholesterol; (c) about 30 mol% to about 60 mol% DSPC; and (d) about 25 mol% to about 45 mol% of an ionizable lipid of Formula (AX).

Embodiment 40. The pharmaceutical composition of any one of embodiments 1-24, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of DMG- PEG2k; (b) about 20 mol% to about 30 mol% cholesterol; (c) about 35 mol% to about 45 mol% DSPC; and (d) about 28 mol% to about 40 mol% of the one or more ionizable lipids.

Embodiment 41. The pharmaceutical composition of any one of embodiments 1-24, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of DMG- PEG2k; (b) about 20 mol% to about 30 mol% cholesterol; (c) about 35 mol% to about 45 mol% DSPC; and (d) about 28 mol% to about 40 mol% of an ionizable lipid of Formula (AX).

Embodiment 42. The pharmaceutical composition of any one of embodiments 1-24, wherein the lipid nanoparticle comprises (a) about 2 mol% of DMG-PEG2k; (b) about 25 mol% cholesterol; (c) about 40 mol% DSPC; and (d) about 33 mol% of an ionizable lipid of Formula (AX).

Embodiment 43. The pharmaceutical composition of any one of embodiments 1-24, wherein the one or more phospholipids comprises a mixture of sphingomyelin and DSPC, the structural lipid is cholesterol, and the PEG lipid is DMG -PEG2k.

Embodiment 44. The pharmaceutical composition of any one of embodiments 1-24, wherein the ionizable lipid of Formula (AX), the one or more phospholipids comprise a mixture of sphingomyelin and DSPC, the structural lipid is cholesterol, and the PEG lipid is DMG-PEG2k.

Embodiment 45. The pharmaceutical composition of any one of embodiments 1-24, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of DMG-PEG2k; (b) about 15 mol% to about 35 mol% cholesterol; (c) about 30 mol% to about 60 mol% of a combination of sphingomyelin and DSPC; and (d) about 25 mol% to about 45 mol% of the one or more ionizable lipids.

Embodiment 46. The pharmaceutical composition of any one of embodiments 1-24, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of DMG-PEG2k; (b) about 15 mol% to about 35 mol% cholesterol; (c) about 30 mol% to about 60 mol% of a combination of sphingomyelin and DSPC; and (d) about 25 mol% to about 45 mol% of an ionizable lipid of Formula (AX).

Embodiment 47. The pharmaceutical composition of any one of embodiments 1-24, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of DMG- PEG2k; (b) about 20 mol% to about 30 mol% cholesterol; (c) about 35 mol% to about 45 mol% of a combination of sphingomyelin and DSPC; and (d) about 28 mol% to about 40 mol% of the one or more ionizable lipids.

Embodiment 48. The pharmaceutical composition of any one of embodiments 1-24, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of DMG- PEG2k; (b) about 20 mol% to about 30 mol% cholesterol; (c) about 35 mol% to about 45 mol% of a combination of sphingomyelin and DSPC; and (d) about 28 mol% to about 40 mol% of an ionizable lipid of Formula (AX).

Embodiment 49. The pharmaceutical composition of any one of embodiments 1-24, wherein the lipid nanoparticle comprises (a) about 2 mol% of DMG-PEG2k; (b) about 25 mol% cholesterol; (c) about 40 mol% of a combination of sphingomyelin and DSPC; and (d) about 33 mol% of an ionizable lipid of Formula (AX).

Embodiment 50. The pharmaceutical composition of any one of embodiments 1-24, wherein the lipid nanoparticle comprises (a) about 2 mol% of DMG-PEG2k; (b) about 25 mol% cholesterol; (c) about 40 mol% of a combination of sphingomyelin and DSPC; and (d) about 33 mol% of the one or more ionizable lipids.

Embodiment 51. The pharmaceutical composition of any one of embodiments 1-24, wherein the lipid nanoparticle comprises about 20 mol% 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) and about 20 mol% sphingomyelin. Embodiment 52. The pharmaceutical composition of any one of embodiments 1-51, wherein the one or more ionizable lipids is compound AX-6.

Embodiment 53. A method of expressing CAR in an immune cell by administering the pharmaceutical composition of any one of embodiments 1-52.

Embodiment 54. A method of expressing CAR in a T cell by administering the pharmaceutical composition of any one of embodiments 1-52.

Embodiment 55. A method of expressing CAR in blood, optionally selected from whole blood or peripheral blood, by administering the pharmaceutical composition of any one of embodiments 1-52.

Embodiment 56. A method of expressing CAR in spleen by administering the pharmaceutical composition of any one of embodiments 1-52.

Embodiment 57. A method of expressing CAR in bone marrow by administering the pharmaceutical composition of any one of embodiments 1-52.

Embodiment 58. A method of treating a subject in need thereof, comprising administering to the subject the pharmaceutical composition of any one of embodiments 1- 52.

Embodiment 59. The pharmaceutical composition of any one of embodiments 1-52 for use as a medicament.

Embodiment 60. The method or use of embodiment 58 or embodiment 59, wherein the subject’s B cell count is decreased post-treatment as compared to pre-treatment.

Claims

1. A pharmaceutical composition comprising: a. an RNA polynucleotide comprising an expression sequence selected from: i. an expression sequence encoding an anti-CD19 antigen binding molecule comprising:

1. a variable heavy (VH) region having at least 95% identity to a VH of SEQ ID NO: 25580 or a binding fragment thereof; and a variable light (VL) region having at least 95% identity to a VL of SEQ ID NO: 25580 or a binding fragment thereof; or

2. a VH region having at least 95% identity to a VH of Table 5, Table 6, Table 7, or Table 9 or a binding fragment thereof; and a VL region having at least 95% identity to a VL of Table 5, Table 6, Table 7, or Table 9 or a binding fragment thereof; ii. an expression sequence encoding an anti-BCMA antigen binding molecule comprising a VH region having at least 95% identity to a VH of Table 5 or Table 9 or a binding fragment thereof; and a VL region having at least 95% identity to a VL of Table 5 or Table 9 or a binding fragment thereof; iii. an expression sequence encoding an anti-CD20 antigen binding molecule comprising a VH region having at least 95% identity to a VH of Table 10 or a binding fragment thereof; and a VL region having at least 95% identity to a VL of Table 10 or a binding fragment thereof; or iv. an expression sequence encoding an anti-HER2 antigen binding molecule comprising a VH region having at least 95% identity to a VH of Table 5 or Table 9 or a binding fragment thereof; and a VL region having at least 95% identity to a VL of Table 5 or Table 9 or a binding fragment thereof; and b. a lipid nanoparticle comprising: i. one or more ionizable lipids; ii. one or more phospholipids, in an amount of about 20 mol% to about 60 mol% of the total lipid content of the lipid nanoparticle; iii. one or more structural lipids; and iv. one or more PEG lipids; wherein the one or more ionizable lipids comprises a compound of Formula AX”” : 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)R^H, each R is independently -H or C₁-C₆ aliphatic; each R^H is C₁-C₆ aliphatic-OH; each X¹ and X^A 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 and 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, and wherein the bond marked with an is attached to X⁴ or X⁵; each X⁶ and X⁷is 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-;

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, X^A, 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.

2. The pharmaceutical composition of claim 1, wherein the RNA polynucleotide is a circular RNA polynucleotide.

3. The pharmaceutical composition of claims 1 or 2, wherein the circular RNA polynucleotide comprises, in the following order: a. a 3’ self-spliced exon segment; b. a translation initiation element; c. the expression sequence, wherein the expression sequence encodes an antiCD 19 antigen binding molecule; and d. a 5’ self-spliced exon segment.

4. The pharmaceutical composition of claim 3, wherein the 3’ self-spliced exon segment is a Group I or Group II self-spliced exon segment, and/or wherein the 5’ self-spliced exon segment is a Group I or Group II self-spliced exon segment.

5. The pharmaceutical composition of claims 3 or 4, comprising a 5’ internal duplex and/or a 3’ internal duplex.

6. The pharmaceutical composition of any one of claims 3-5, comprising a 5’ internal spacer and/or a 3’ internal spacer.

7. The pharmaceutical composition of any one of claims 3-6, wherein the translation initiation element is an Internal Ribosome Entry Site (IRES).

8. The pharmaceutical composition of any one of claims 2-7, wherein the circular RNA polynucleotide is from about 50 nucleotides to about 15 kilobases in length.

9. The pharmaceutical composition of any one of claims 2-7, wherein the circular RNA polynucleotide comprises at least one modified A, C, G, or U nucleotide or nucleoside.

10. The pharmaceutical composition of claim 9, wherein the modified nucleotide or nucleoside is: d) one or more of m⁵U (5-methyluridine); m⁶A (N⁶ -methyladenosine); s²U (2- thiouridine); Ψ (pseudouridine); Um (2'-O-methyluridine); m¹A (1 -methyladenosine); m²A (2 -methyladenosine); Am (2’-O-methyladenosine); ms²m⁶A (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); 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); m^m (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 (l- methylguanosine); m²G (N² -methylguanosine); m⁷G (7-m ethylguanosine); 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); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G⁺ (archaeosine); D (dihydrouridine); m⁵Um (5,2’ -O-dimethyluri dine); 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-methoxy carbonylmethyl - 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); cmnmVU (5- carboxymethylaminomethyl-2-thiouridine); m⁶ 2A (N⁶,N⁶-dimethyladenosine); Im (2’ -0-m ethylinosine); 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); m^2,7G (N²,7-dimethylguanosine); m^2,2,7G (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); (5-taurinomethyluridine); (5- taurinomethyl-2-thiouridine)); imG-14 (4-demethylwyosine); imG2 (isowyosine); or ac⁶A (N⁶-acetyladenosine); pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza- uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,

3 -methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5- propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1- taurinomethyl-pseudouridine, 5-taurinomethyl-2 -thio-uridine, 1 -taurinomethyl-4-thio- uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l-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-pseudoisocyti dine, 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-m ethoxy-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-m ethylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2- methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo- guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N1- methylpseudouridine, and N2,N2-dimethyl-6-thio-guanosine; or e) 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, N1 -methylpseudouridine, and alkylcarbonylalkylated nucleotides; or f) is selected from one or more of: 5-methylcytidine, 5-methoxyuridine, 1-methyl- pseudouridine, N6-methyladenosine, and/or pseudouridine.

11. The pharmaceutical composition of any one of claims 1-10, wherein the VH region comprises three complementarity determining regions (HCDR1, HCDR2, HCDR3) of SEQ ID NO: 25580 and the VL region comprises three CDRs (LCDR1, LCDR2, LCDR3) of SEQ ID NO: 25580.

12. The pharmaceutical composition of any one of claims 1-11, wherein the lipid nanoparticle comprises about 25 mol% to about 45 mol% of the one or more ionizable lipids, as a proportion of the total lipid content of the lipid nanoparticle.

13. The pharmaceutical composition of any one of claims 1-12, wherein the lipid nanoparticle comprises about 15 mol% to about 35 mol% of the one or more structural lipids, as a proportion of the total lipid content of the lipid nanoparticle.

14. The pharmaceutical composition of any one of claims 1-13, wherein the lipid nanoparticle comprises about 1 mol% to about 3 mol% of the one or more PEG lipids, as a proportion of the total lipid content of the lipid nanoparticle.

15. The pharmaceutical composition of any one of claims 1-14, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of the one or more PEG lipids; (b) about 15 mol% to about 35 mol% of the one or more structural lipids; (c) about 30 mol% to about 60 mol% of the one or more phospholipids; and (d) about 25 mol% to about 45 mol% of the one or more ionizable lipids.

16. The pharmaceutical composition of any one of claims 1-15, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of the one or more PEG lipids; (b) about 20 mol% to about 30 mol% of the one or more structural lipids; (c) about 35 mol% to about 45 mol% of the one or more phospholipids; and (d) about 28 mol% to about 40 mol% of the one or more ionizable lipids.

17. The pharmaceutical composition of any one of claims 1-16, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of the one or more PEG lipids; (b) about 20 mol% to about 40 mol% of the one or more structural lipids; (c) about 25 mol% to about 40 mol% of the one or more phospholipids; and (d) about 28 mol% to about 40 mol% of the one or more ionizable lipids.

18. The pharmaceutical composition of any one of claims 1-17, wherein the one or more ionizable lipids comprises a compound of Formula (AX) or a pharmaceutically acceptable salt thereof, wherein:

Y¹ is selected from the group consisting of and bond; wherein the bond marked with an is attached to X ; 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;

19. The pharmaceutical composition of any one of claims 1-18, wherein the one or more ionizable lipids comprises a compound of Formula AX-T’: or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from -OH, -OAc, -NR₂, each R is independently -H or C₁-C₆ aliphatic;

X² and X³ are each independently a bond or optionally substituted C₁-C₁₂ aliphatic;

X⁴ and X⁵ are each independently optionally substituted C₁-C₆ aliphatic;

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.

20. The pharmaceutical composition of any one of claims 1-19, wherein the ionizable lipid is selected from:

21. The pharmaceutical composition of any one of claims 1-20, wherein the one or more phospholipids are selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1.2-dioleoyl- sn-glycero-3 -phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn- glycero-3-phosphocho line (POPC), 1,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuc cinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3 -phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn- glycero-3 -phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoylsn-glycero-3- phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3 -phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3 -phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3 -phosphorac^ 1 -glycerol) sodium salt (DOPG), sodium (S)-2-ammonio-3-((((R)-2-(oleoyloxy)-3- (stearoyloxy)propoxy)oxidophosphoryl)oxy)propanoate (L-α-phosphatidylserine; Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl-phosphatidylethanolamine4-(N- maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), di oleoylphosphatidyl glycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Dielaidoyl-sn- phosphatidylethanolamine (DEPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3- phosphate (18: 1 PA; DOPA), ammonium bis((S)-2-hydroxy-3-(oleoyloxy)propyl) phosphate (18: 1 DMP; LBPA), 1,2-dioleoyl-sn-glycero-3-phospho-(l’ -myo-inositol) (DOPI; 18: 1 PI), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), 1,2-dilinoleoyl-sn-glycero-3- phospho-L-serine (18:2 PS), l-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (16:0-18: 1 PS; POPS), l-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (18:0-18: 1 PS), l-stearoyl-2- linoleoyl-sn-glycero-3-phospho-L-serine (18:0-18:2 PS), l-oleoyl-2-hydroxy-sn-glycero-3- phospho-L-serine (18: 1 Lyso PS), l-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:0 Lyso PS), and sphingomyelin, or combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the above phospholipids.

22. The pharmaceutical composition of any one of claims 1-20, wherein the one or more phospholipids are selected from DSPC and egg sphingomyelin, or a combination thereof.

23. The pharmaceutical composition of any one of claims 1-22, wherein the one or more structural lipids are selected from cholesterol, fecosterol, fucosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, cholic acid, sitostanol, litocholic acid, tomatine, ursolic acid, alpha-tocopherol, Vitamin D3, Vitamin D2, Calcipotriol, botulin, lupeol, oleanolic acid, beta-sitosterol-acetate and any combinations thereof.

24. The pharmaceutical composition of any one of claims 1-23, wherein the one or more PEG lipids is selected from DMG-PEG2k, DSPE-PEG2k, DSG-PEG2k, DMPE-PEG2k, DPPE-PEG2k and mixtures thereof.

25. The pharmaceutical composition of any one of claims 1-24, wherein the one or more phospholipids comprises one or more selected from phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids or a combination thereof.

26. The pharmaceutical composition of any one of claims 1-24, wherein the one or more phospholipids comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), sphingomyelin or a combination thereof.

27. The pharmaceutical composition of any one of claims 1-26, wherein the one or more phospholipids comprises two or more phospholipids, such that no single phospholipid makes up more than 30 mol% of the total lipid content of the nanoparticle.

28. The pharmaceutical composition of any one of claims 1-27, wherein the lipid nanoparticle comprises about 40 mol% 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

29. The pharmaceutical composition of any one of claims 1-27, wherein the lipid nanoparticle comprises about 40 mol% sphingomyelin.

30. The pharmaceutical composition of any one of claims 1-29, wherein the phospholipid is sphingomyelin, the structural lipid is cholesterol, and the PEG lipid is DMG-PEG2k.

31. The pharmaceutical composition of any one of claims 1-30, wherein the ionizable lipid is a compound of Formula (AX), the phospholipid is sphingomyelin, the structural lipid is cholesterol, and the PEG lipid is DMG-PEG2k.

32. The pharmaceutical composition of any one of claims 1-24, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of DMG-PEG2k; (b) about 15 mol% to about 35 mol% cholesterol; (c) about 30 mol% to about 60 mol% sphingomyelin; and (d) about 25 mol% to about 45 mol% of the one or more ionizable lipids.

33. The pharmaceutical composition of any one of claims 1-24, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of DMG-PEG2k; (b) about 15 mol% to about 35 mol% cholesterol; (c) about 30 mol% to about 60 mol% sphingomyelin; and (d) about 25 mol% to about 45 mol% of an ionizable lipid of Formula (AX).

34. The pharmaceutical composition of any one of claims 1-24, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of DMG-PEG2k; (b) about 20 mol% to about 30 mol% cholesterol; (c) about 35 mol% to about 45 mol% sphingomyelin; and (d) about 28 mol% to about 40 mol% of the one or more ionizable lipids.

35. The pharmaceutical composition of any one of claims 1-24, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of DMG-PEG2k; (b) about 20 mol% to about 30 mol% cholesterol; (c) about 35 mol% to about 45 mol% sphingomyelin; and (d) about 28 mol% to about 40 mol% of an ionizable lipid of Formula (AX).

36. The pharmaceutical composition of any one of claims 1-24, wherein the lipid nanoparticle comprises (a) about 2 mol% of DMG-PEG2k; (b) about 25 mol% cholesterol;

(c) about 40 mol% sphingomyelin; and (d) about 33 mol% of an ionizable lipid of Formula (AX).

37. The pharmaceutical composition of any one of claims 1-24, wherein the lipid nanoparticle comprises (a) about 2 mol% of DMG-PEG2k; (b) about 25 mol% cholesterol; (c) about 40 mol% sphingomyelin; and (d) about 33 mol% of the one or more ionizable lipids.

38. The pharmaceutical composition of any one of claims 1-24, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of DMG-PEG2k; (b) about 15 mol% to about 35 mol% cholesterol; (c) about 30 mol% to about 60 mol% DSPC; and (d) about 25 mol% to about 45 mol% of the one or more ionizable lipids.

39. The pharmaceutical composition of any one of claims 1-24, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of DMG-PEG2k; (b) about 15 mol% to about 35 mol% cholesterol; (c) about 30 mol% to about 60 mol% DSPC; and (d) about 25 mol% to about 45 mol% of an ionizable lipid of Formula (AX).

40. The pharmaceutical composition of any one of claims 1-24, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of DMG-PEG2k; (b) about 20 mol% to about 30 mol% cholesterol; (c) about 35 mol% to about 45 mol% DSPC; and (d) about 28 mol% to about 40 mol% of the one or more ionizable lipids.

41. The pharmaceutical composition of any one of claims 1-24, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of DMG-PEG2k; (b) about 20 mol% to about 30 mol% cholesterol; (c) about 35 mol% to about 45 mol% DSPC; and (d) about 28 mol% to about 40 mol% of an ionizable lipid of Formula (AX).

42. The pharmaceutical composition of any one of claims 1-24, wherein the lipid nanoparticle comprises (a) about 2 mol% of DMG-PEG2k; (b) about 25 mol% cholesterol; (c) about 40 mol% DSPC; and (d) about 33 mol% of an ionizable lipid of Formula (AX).

43. The pharmaceutical composition of any one of claims 1-24, wherein the one or more phospholipids comprises a mixture of sphingomyelin and DSPC, the structural lipid is cholesterol, and the PEG lipid is DMG-PEG2k.

44. The pharmaceutical composition of any one of claims 1-24, wherein the ionizable lipid of Formula (AX), the one or more phospholipids comprise a mixture of sphingomyelin and DSPC, the structural lipid is cholesterol, and the PEG lipid is DMG-PEG2k.

45. The pharmaceutical composition of any one of claims 1-24, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of DMG-PEG2k; (b) about 15 mol% to about 35 mol% cholesterol; (c) about 30 mol% to about 60 mol% of a combination of sphingomyelin and DSPC; and (d) about 25 mol% to about 45 mol% of the one or more ionizable lipids.

46. The pharmaceutical composition of any one of claims 1-24, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of DMG-PEG2k; (b) about 15 mol% to about 35 mol% cholesterol; (c) about 30 mol% to about 60 mol% of a combination of sphingomyelin and DSPC; and (d) about 25 mol% to about 45 mol% of an ionizable lipid of Formula (AX).

47. The pharmaceutical composition of any one of claims 1-24, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of DMG-PEG2k; (b) about 20 mol% to about 30 mol% cholesterol; (c) about 35 mol% to about 45 mol% of a combination of sphingomyelin and DSPC; and (d) about 28 mol% to about 40 mol% of the one or more ionizable lipids.

48. The pharmaceutical composition of any one of claims 1-24, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of DMG-PEG2k; (b) about 20 mol% to about 30 mol% cholesterol; (c) about 35 mol% to about 45 mol% of a combination of sphingomyelin and DSPC; and (d) about 28 mol% to about 40 mol% of an ionizable lipid of Formula (AX).

49. The pharmaceutical composition of any one of claims 1-24, wherein the lipid nanoparticle comprises (a) about 2 mol% of DMG-PEG2k; (b) about 25 mol% cholesterol;

(c) about 40 mol% of a combination of sphingomyelin and DSPC; and (d) about 33 mol% of an ionizable lipid of Formula (AX).

50. The pharmaceutical composition of any one of claims 1-24, wherein the lipid nanoparticle comprises (a) about 2 mol% of DMG-PEG2k; (b) about 25 mol% cholesterol;

(c) about 40 mol% of a combination of sphingomyelin and DSPC; and (d) about 33 mol% of the one or more ionizable lipids.

51. The pharmaceutical composition of any one of claims 1-24, wherein the lipid nanoparticle comprises about 20 mol% 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and about 20 mol% sphingomyelin.

52. The pharmaceutical composition of any one of claims 1-51, wherein the one or more ionizable lipids is compound AX-6.

53. A method of expressing CAR in an immune cell by administering the pharmaceutical composition of any one of claims 1-52.

54. A method of expressing CAR in a T cell by administering the pharmaceutical composition of any one of claims 1-52.

55. A method of expressing CAR in blood, optionally selected from whole blood or peripheral blood, by administering the pharmaceutical composition of any one of claims 1-52.

56. A method of expressing CAR in spleen by administering the pharmaceutical composition of any one of claims 1-52.

57. A method of expressing CAR in bone marrow by administering the pharmaceutical composition of any one of claims 1-52.

58. A method of treating a subject in need thereof, comprising administering to the subject the pharmaceutical composition of any one of claims 1-52.

59. The pharmaceutical composition of any one of claims 1-52 for use as a medicament.

60. The method or use of claim 58 or claim 59, wherein the subject’s B cell count is decreased post-treatment as compared to pre-treatment.

61. A method of measuring RNA species in a composition, comprising:

(1) obtaining a sample comprising a circular RNA and a precursor RNA,

(2) subjecting the sample to gel electrophoresis and separating the precursor RNA and the circular RNA; and

(3) measuring the levels of precursor RNA and the circular RNA in the sample.

62. A method of measuring a level of an immunogenic and/or reactogenic molecule, comprising:

(1) contacting a first sample of cells with a composition comprising a circular RNA and a precursor RNA; and

(2) detecting the level of at least one immunogenic and/or reactogenic molecule in said sample; optionally wherein the molecule is selected from the group consisting of a cytokine, TNFα, RIG-I, IL-2, IL-6, interferon, IFN-α, IFN-β, IFN-β1, IFNγ, and INFλ.