Constrained ionizable cationic lipids and lipid nanoparticles

Abstract

Ionizable cationic lipids, methods for synthesizing the same, intermediates useful in synthesis of the ionizable cationic lipids, and methods of synthesizing the intermediates are disclosed. The ionizable cationic lipids are useful as a component of lipid nanoparticles (LNP), which in turn can be used for delivering nucleic acids into cells in vivo or ex vivo. LNP compositions are also disclosed, including LNPs comprising a functionalized lipid to enable conjugation of a binding moiety, and targeted LNPs (tLNPs), that is, LNPs in which a binding moiety has been conjugated to the functionalized lipid and can serve as a targeting moiety to direct the tLNPs to a desired tissue or cell type.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority U.S. provisional application number 63 / 632,944, filed April 11, 2024, U.S. provisional application number 63 / 654,720, filed May 31, 2024, U.S. provisional application number 63 / 654,930, filed May 31, 2024, U.S. provisional application number 63 / 654,928, filed May 31,2024, U.S. provisional application number 63 / 708,461, filed October 17, 2024, and U.S. provisional application number 63 / 708,529, filed October 17, 2024; the disclosures of which are expressly incorporated by reference herein. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] This application contains a Sequence Listing that has been submitted electronically and is hereby incorporated by reference in its entirety. The Sequence Listing was created on March 12, 2025, is named “CTX-019PR3_24-0507-US-PR03_Sequence Listing.xml”, and is 3,691 bytes in size. BACKGROUND

[0003] Lipid formulations have been used in the laboratory for delivering nucleic acids into cells. Early formulations that were based on the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and the ionizable, fusogenic lipid dioleoylphosphatidyl ethanolamine (DOPE) had a large particle size and were problematic when used in vivo, exhibiting clearance that was too rapid, tropism for the lung, and toxicity. Lipid nanoparticles (LNPs) comprising ionizable cationic lipids have been developed to address these issues to the extent that RNA-based products, such as the siRNA ONPATTRO® and two mRNA-based SARS-CoV-2 vaccines, have received regulatory approval and entered the marketplace.

[0004] However, there is limited ability to control which tissues or cells take up the LNP once administered. LNP administered intravenously are taken up primarily in the liver, lung, or spleen depending to a significant degree on net charge and particle size. It is possible to direct >90% of LNP to the liver by a combination of formulation and intravenous administration, for example. Intramuscular administration can provide a clinically useful level of local delivery and expression. LNP can be redirected to other tissues or cell types by conjugating to the LNP a binding moiety with specificity for the target tissue or cell type, for example, conjugating an antibody to an LNP (see, e.g., Endsley and Ho, J. Acquir. Immune Defic. Syndr. 61:417, 2012; Ramishetti et al., ACS Nano 9:6706, 2015; Veiga et al., Nat. Comms. 9:4493, 2018; US Patent No. 10,920,246). Nonetheless, avoiding uptake by the liver remains a challenge. Moreover, with current systems only a minor portion of the encapsulated nucleic acid is successfully delivered to the cells of interest and into the cytoplasm. Current formulations may release only 2-5% of the administered RNA into the cytoplasm (see, for example, Gilleron et al., 2013, Nat. Biotechnol. 31:638-646 and Munson et al., 2021, Commun. Biol. 4:211-224). There are remaining issues of off-target delivery, poor efficiency of release of nucleic acid into the cytoplasm, and toxicity associated with accumulation of the component lipids.

[0005] Thus, there exists a need to address issues of off-target delivery, poor efficiency of release of therapeutic agents into the cytoplasm, and toxicity associated with accumulation of the component lipids. SUMMARY

[0006] This disclosure fulfills the needs for addressing issues of off-target delivery, poor efficiency of release of therapeutic agents and provides further related advantages.

[0007] In certain aspects, this disclosure provides constrained ionizable cationic lipids having a structure of formula M6 set forth herein.

[0008] In certain aspects, this disclosure provides constrained ionizable cationic lipids having a structure of formula M3 set forth herein.

[0009] In some embodiments, this disclosure provides ionizable cationic lipids CICL-251, CICL-252, CICL-253, CICL-254, CICL-255, CICL-256, CICL-256, CICL-257, CICL-258, CICL-259, CICL-260, CICI-261, CICL-262, CICL-263, CICL-264, CICL-265, CICL-266, CICL-267, CICL-268, CICL-269, CICL-270, CICL-271, CICL-272, CICL-273, CICL-274, CICL-275, CICL-276, CICL-277, CICL-278, CICL-279, CICL-280, CICL-281, CICL-282, CICL-283, CICL-284, CICL-285, CICL-286, CICL-287, CICL-288, CICL-289, CICL-290, CICL-304, CICL-305, CICL-306, CICL-307, and CICL-308 set forth herein.

[0010] In some embodiments, this disclosure provides ionizable cationic lipids CICL-251-154, CICL-251-155, CILC-251-157, CICL-251-159, CICL-251-160, CICL-251-167, CICL-253-154, CICL-253-155, CILC-253-157, CICL-253-159, CICL-253-160, CICL-253-167, CICL-255-154, CICL-255-155, CICL-255-157, CICL-255-159, CICL-255-160, CICL-255-167, CICL-258-154, CICL-258-155, CICL-258-157, CICL-258-159, CICL-258-160, CICL-258-167, CICL-260-154, CICL-260-155, CICL-260-157, CICL-260-159, CICL-260-160, CICL-260-167, CICL-261-154, CICL-261-155, CICL-261-157, CICL-261-159, CICL-261-160, CICL-261-167, CICL-264-154, CICL-264-166, CICL-264-157, CICL-264-159, CICL-264-160, CICL-264-167, CICL-266-154, CICL-266-155, CICL-266-157, CICL-266-159, CICL-266-160, CICL-266-167, CICL-268-154, CICL-268-155, CICL-268-157, CICL-268-159, CICL-268-160, CICL-268-167, CICL-270-154, CICL-270-155, CICL-270-157, CICL-270-159, CICL-270-160, CICL-270-167, CICL-272-154, CICL-272-155, CICL-272-157, CICL-272-159, CICL-272-160, CICL-272-167, CICL-273-154, CICL-273-155, CICL-273-157, CICL-273-159, CICL-273-160, CICL-273-167, CICL-275- 154, CICL-275-155, CICL-275-157, CICL-275-159, CICL-275-160, CICL-275-167, CICL-278-154, CICL-278-155, CICL-278-157, CICL-278-159, CICL-278-160, CICL-278-167, CICL-280-154, CICL-280-155, CICL-280-157, CICL-280-159, CICL-280-160, CICL-280-167, CICL-281-154, CICL-281-155, CICL-281-157, CICL-281-159, CICL-281-160, CICL-281-167, CICL-284-154, CICL-284-155, CICL-284-157, CICL-284-159, CICL-284-160, CICL-284-167, CICL-286-154, CICL-286-155, CICL-286-157, CICL-286-159, CICL-286-160, CICL-286-167, CICL-288-154, CICL-288-155, CICL-288-157, CICL-288-159, CICL-288-160, CICL-288-167, CICL-290-154, CICL-290-155, CICL-290-157, CICL-290-159, CICL-290-160, CICL-290-167, CICL-304-154, CICL-304-155, CICL-304-157, CICL-304-159, CICL-304-160, CICL-304-167, CICL-305-154, CICL-305-155, CICL-305-157, CICL-305-159, CICL-305-160, CICL-305-167, CICL-306-154, CICL-306-155, CICL-306-157, CICL-306-159, CICL-306-160, CICL-306-167, CICL-307-154, CICL-307-155, CICL-307-157, CICL-307-159, CICL-307-160, CICL-308-167, CICL-308-154, CICL-308-155, CICL-308-157, CICL-308-159, CICL-308-160, and CICL-308-167 set forth herein.

[0011] In some embodiments, this disclosure provides ionizable cationic lipids CICL-252-164, CICL-254-164, CICL-256-164, CICL-257-164, CICL-259-164, CICL-262-164, CICL-263-164, CICL-265-164, CICL-267-164, CICL-269-164, CICL-271-164, CICL-274-164, CICL-276-164, CICL-277-164, CICL-279-164, CICL-282-164, CICL-283-164, CICL-285-164, CICL-287-164, and CICL-289-164 set forth herein.

[0012] In certain aspects, this disclosure provides methods of synthesizing ionizable cationic lipids as described herein, e.g., CICL-251, CICL-252, CICL-253, CICL-254, CICL-255, CICL-256, CICL-256, CICL-257, CICL-258, CICL-259, CICL-260, CICI-261, CICL-262, CICL-263, CICL-264, CICL-265, CICL-266, CICL-267, CICL-268, CICL-269, CICL-270, CICL-271, CICL-272, CICL-273, CICL-274, CICL-275, CICL-276, CICL-277, CICL-278, CICL-279, CICL-280, CICL-281, CICL-282, CICL-283, CICL-284, CICL-285, CICL-286, CICL-287, CICL-288, CICL-289, CICL-290, CICL-304, CICL-305, CICL-306, CICL-307, and CICL-308.

[0013] In certain aspects, this disclosure provides methods of synthesizing ionizable cationic lipids as described herein, e.g., CICL-253-154, CICL-253-155, CILC-253-157, CICL-253-159, CICL-253-160, CICL-253-167, CICL-255-154, CICL-255-155, CICL-255-157, CICL-255-159, CICL-255-160, CICL-255-167, CICL-258-154, CICL-258-155, CICL-258-157, CICL-258-159, CICL-258-160, CICL-258-167, CICL-260-154, CICL-260-155, CICL-260-157, CICL-260-159, CICL-260-160, CICL-260-167, CICL-261-154, CICL-261-155, CICL-261-157, CICL-261-159, CICL-261-160, CICL-261-167, CICL-264-154, CICL-264-166, CICL-264-157, CICL-264-159, CICL-264-160, CICL-264-167, CICL-266-154, CICL-266-155, CICL-266-157, CICL-266-159, CICL-266-160, CICL-266-167, CICL-268-154, CICL-268-155, CICL-268-157, CICL-268-159, CICL-268-160, CICL-268-167, CICL-270-154, CICL-270-155, CICL-270-157, CICL- 270-159, CICL-270-160, CICL-270-167, CICL-272-154, CICL-272-155, CICL-272-157, CICL-272-159, CICL-272-160, CICL-272-167, CICL-273-154, CICL-273-155, CICL-273-157, CICL-273-159, CICL-273-160, CICL-273-167, CICL-275-154, CICL-275-155, CICL-275-157, CICL-275-159, CICL-275-160, CICL-275-167, CICL-278-154, CICL-278-155, CICL-278-157, CICL-278-159, CICL-278-160, CICL-278-167, CICL-280-154, CICL-280-155, CICL-280-157, CICL-280-159, CICL-280-160, CICL-280-167, CICL-281-154, CICL-281-155, CICL-281-157, CICL-281-159, CICL-281-160, CICL-281-167, CICL-284-154, CICL-284-155, CICL-284-157, CICL-284-159, CICL-284-160, CICL-284-167, CICL-286-154, CICL-286-155, CICL-286-157, CICL-286-159, CICL-286-160, CICL-286-167, CICL-288-154, CICL-288-155, CICL-288-157, CICL-288-159, CICL-288-160, CICL-288-167, CICL-290-154, CICL-290-155, CICL-290-157, CICL-290-159, CICL-290-160, CICL-290-167, CICL-304-154, CICL-304-155, CICL-304-157, CICL-304-159, CICL-304-160, CICL-304-167, CICL-305-154, CICL-305-155, CICL-305-157, CICL-305-159, CICL-305-160, CICL-305-167, CICL-306-154, CICL-306-155, CICL-306-157, CICL-306-159, CICL-306-160, CICL-306-167, CICL-307-154, CICL-307-155, CICL-307-157, CICL-307-159, CICL-307-160, CICL-308-167, CICL-308-154, CICL-308-155, CICL-308-157, CICL-308-159, CICL-308-160, and CICL-308-167.

[0014] In certain aspects, this disclosure provides methods of synthesizing ionizable cationic lipids as described herein, e.g., CICL-252-164, CICL-254-164, CICL-256-164, CICL-257-164, CICL-259-164, CICL-262-164, CICL-263-164, CICL-265-164, CICL-267-164, CICL-269-164, CICL-271-164, CICL-274-164, CICL-276-164, CICL-277-164, CICL-279-164, CICL-282-164, CICL-283-164, CICL-285-164, CICL-287-164, and CICL-289-164.

[0015] In certain aspects, this disclosure provides intermediates and methods of synthesizing such intermediates useful in the synthesis of the ionizable cationic lipids as described herein.

[0016] In certain aspects, this disclosure provides lipid nanoparticles (LNPs) and targeted lipid nanoparticles (tLNPs) incorporating the ionizable cationic lipids disclosed herein.

[0017] In certain aspects, this disclosure provides methods of preparing LNPs and tLNPs as described herein.

[0018] In certain embodiments, this disclosure provides methods of delivering biologically active payload (e.g., nucleic acid molecules encoding a therapeutic agent) into a cell (such as a T cell or hematopoietic stem cell) comprising contacting the cell with an LNP or tLNP of this disclosure.

[0019] These and other features, objects, and advantages of this disclosure will become better understood from the description that follows. In the description, reference is made to the accompanying drawings, which form a part hereof and in which there is shown by way of illustration, not limitation, embodiments of this disclosure. The description of preferred embodiments is not intended to limit this disclosure to cover all modifications, equivalents, and alternatives. Reference should therefore be made to the claims recited herein for interpreting the scope of the disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The disclosure is better understood and features, aspects, and advantages other than those set forth above will become apparent when consideration is given to the following description of the drawings.

[0021] Figures 1A-1C depict the transfection rate (percentage of cells expressing the mRNA) versus expression level (as molecules of equivalent soluble fluorochrome (MESF)) for two tLNP compositions with CICL-251 or CICL-1, as indicated. The full lipid compositions are described in Table 6. Results in splenic T cells (Figure 1A), CD45' liver cells (Figure 1B) and liver Kupffer cells (CD45+ / CD11+ liver cells; Figure 1C) from C57BL / 6 mice administered the tLNPs are shown. The binding moiety of the tLNPs was an anti-CD5 antibody and the payload was an mRNA encoding mCherry (SEQ ID NO: 2). DETAILED DESCRIPTION

[0022] This disclosure provides constrained ionizable cationic lipids, referred to as ionizable cationic lipids throughout, methods for synthesizing them, as well as intermediates useful in synthesis of these lipids and methods of synthesizing the intermediates. This disclosure further provides ionizable cationic lipids as components of lipid nanoparticles (LNPs) that can be used for delivering a biologically active payload (e.g., nucleic acid molecules encoding a therapeutic agent) into cells in vivo or ex vivo. LNP compositions are also disclosed herein, including LNPs comprising a functionalized PEG-lipid to enable conjugation of a binding moiety to generate targeted LNPs (tLNPs); that is, LNPs containing a binding moiety that directs the tLNP to a desired tissue or cell type (e.g., immune cells such as T cells or stem cells such as hematopoietic stem cells (HSCs)). Also disclosed herein are methods of delivering therapeutic agents, such as a nucleic acid molecule, into a cell comprising contacting the cell with an LNP or tLNP of this disclosure. The LNP and tLNP of this disclosure can be used for in vivo, ex vivo, or extracorporeal transfection. Also disclosed herein are methods for preparing LNPs and tLNPs comprising the ionizable cationic lipids as described herein.

[0023] Prior to setting forth this disclosure in more detail, it may be helpful to provide abbreviations of certain terms used herein. Additional abbreviations are set forth throughout this disclosure. ABBREVIATIONS

[0024] Abbreviations used herein include: BOC2O - Di-tert-butyl dicarbonate DSPC - Distearoylphosphatidylcholine MesN - Trimethylamine CDCI3- Deuterochloroform DSPE - Distearoylphosphatidylethanolamine MeOH - Methanol GDI - Carbonyl diimidazole EDC-HCI - 1-Ethyl-3-(3'-dimethylaminopropyl)carbodiimide ■ HCI MeOTf - methyl trifluoromethanesulfonate CH3CN - Acetonitrile EtOAc - Ethyl acetate Pd / C - Palladium on carbon CHOL- Cholesterol EtOH - Ethanol PEG - Polyethylene glycol DMAP - 4- Dimethylaminopyridine Et20 - Diethyl ether PhCHs - Toluene DMG - Dimyristoyl glycerol Et3N - Triethylamine TFA - Trifluoroacetic acid DSG - Distearoyl glycerol MAL - Maleimide THF - Tetrahydrofuran

[0025] While this disclosure is capable of being embodied in various forms, the description below of several embodiments is made with the understanding that the disclosure is to be considered as an exemplification of the innovations disclosed herein and is not intended to limit the disclosure to the specific embodiments illustrated.

[0026] Headings are provided for convenience only and are not to be construed to limit the embodiments in any manner. Embodiments illustrated under any heading can be combined with embodiments illustrated under any other heading.

[0027] To the extent any materials incorporated herein by reference conflict with this disclosure, this disclosure controls. DEFINITIONS

[0028] Prior to setting forth this disclosure in more detail, definitions of certain terms to be used herein are provided. Additional definitions are set forth throughout this disclosure.

[0029] As used in the specification and claims, the singular form “a,” “an,” and “the” includes plural references unless the context clearly dictates otherwise. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components.

[0030] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[0031] The term “about” as used herein in the context of a number refers to a range centered on that number and spanning 10% less than that number and 10% more than that number. The term “about” used in the context of a range refers to an extended range spanning 10% less than that of the lowest number listed in the range and 10% more than the greatest number listed in the range. For any value modified by the word “about,” it should be understood to also disclose an embodiment wherein the value is not so modified.

[0032] Throughout this disclosure, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range of this disclosure relating to any physical feature, such as polymer subunits, size, or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. Throughout this disclosure, numerical ranges are inclusive of their recited endpoints, unless specifically stated otherwise.

[0033] Unless the context requires otherwise, throughout this specification and claims, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” As used herein, the terms “include” and “comprise” are used synonymously.

[0034] The phrase “at least one of’ when followed by a list of items or elements refers to an open-ended set of one or more of the elements in the list, which can, but does not necessarily, include more than one of the elements.

[0035] “Derivative,” as used herein, refers to a chemically or biologically modified version of a compound that is structurally similar to a parent compound and (actually or theoretically) derivable from that parent compound. Generally, a “derivative” differs from an “analogue” in that a parent compound can be the starting material to generate a “derivative,” whereas the parent compound is not necessarily used as the starting material to generate an “analogue.” A derivative can have different chemical or physical properties than the parent compound. For example, a derivative can be more hydrophilic or hydrophobic, or it can have altered reactivity as compared to the parent compound. Although a derivative can be obtained by physical (for example, biological or chemical) modification of the parent compound, a derivative can also be conceptually derived, for example, as when a protein sequence is designed based on one or more known sequences, an encoding nucleic acid is constructed, and the derived protein obtained by expression of the encoding nucleic acid.

[0036] As used herein “extracorporeal” is used with reference to cells, such as peripheral blood or bone marrow cells, harvested or extracted from the body and the manipulation or modification of those cells prior to their intended return (reinfusion). Manipulation and modification of cells generally relates to cell separation and washing procedures and exposure to activation agents (e.g., biological response modifiers (BRMs)) and transfection agents (e.g., LNPs, tLNPs), over a time interval of several hours, for example, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour; and in space to a single institution. Extracorporeal is used in contradistinction to ex vivo which, as used herein, includes more extensive manipulation including extended periods of cell culture and expansion, and / or refrigerated or cryogenic storage or shipment, over several days or longer.

[0037] As used herein “expansion” refers to proliferation of cells increasing their number. Activating agents can be used to stimulate proliferation (among other metabolic changes) but can also result in activation-induced death upon initial exposure so that there is no immediate expansion. For T cells treated in vitro with activating agents such as IL-2 or CD3 / CD28 activators, doubling time can be about 24 hours (which is fairly typical of mammalian cells in vitro generally); in vivo doubling time can be substantially shorter, depending on the presence and type of stimulation. Accordingly, during a limited time of extracorporeal manipulation, even when activating agents are used, such protocols will be effectively expansion-less.

[0038] As used herein an “exogenous protein” refers to a synthetic, recombinant, or other peptide or protein that is not produced by a wild-type cell of that type or is expressed at a lower level in a wild-type cell than in a cell containing the exogenous polypeptide, or that is administered to a subject rather than being produced inside the subject’s body. In some embodiments, an exogenous peptide is a peptide or protein encoded by a nucleic acid that was introduced into the cell, which nucleic acid is optionally not retained by the cell. As used herein “peptide” refers to a chain of amino acids less than 50 amino acids in length, while “protein” and “polypeptide refer to a chain of amino acids at least 50 amino acids in length.

[0039] As used herein “transfection” or “transfecting” refers to the introduction of nucleic acids into cells by non-viral methods. Transfection can be mediated by calcium phosphate, cationic polymers, magnetic beads, electroporation, and lipid-based reagents. In preferred embodiments disclosed herein transfection is mediated by solid lipid nanoparticles (LNP) including targeted LNP (tLNP) (which can also be used to deliver non-nucleic acid payloads into cells). The term transfection is used in distinction to transduction - transfer of genetic material from cell to cell or virus to cell - and transformation - the uptake of extracellular genetic material by the natural processes of a cell. As used herein, phrases such as “delivering a nucleic acid into a cell” are synonymous with transfection.

[0040] “Reprogramming,” as used herein with respect to cells, refers to changing the functionality of a cell. For example, reprogramming an immune cell can result in a change in antigenic specificity by causing expression of an exogenous T cell receptor (TCR), a chimeric antigen receptor (CAR), or an immune cell engager (“reprogramming agents”). Generally, T lymphocytes and natural killer (NK) cells can be reprogrammed with a TCR, a CAR, or an immune cell engager, while a CAR or an immune cell engager are typically used in reprogramming monocytes. As used herein with respect to stem cells, for example hematopoietic stem cells (HSC) or mesenchymal stem cells (MSC), “reprogramming” refers to correction or amelioration of a genetic defect (for example, a hemoglobinopathy) so that the modified or corrected gene and gene product are the reprogramming agents. Reprogramming can be transient or durable depending on the nature of the engineering agent.

[0041] “Engineering agent,” as used herein, refers to agents that confer the expression of a reprogramming agent by a cell, such as an immune cell, particularly a non-B lymphocyte or monocyte. Engineering agents can include nucleic acid molecules, including mRNA, that encode a reprogramming agent. Engineering agents can also include nucleic acid molecules that are or encode components of gene editing systems, such as RNA-guided nucleases, guide RNA, and nucleic acid templates for knocking-in a reprogramming agent or knockingout an endogenous antigen receptor. Gene editing systems comprise base-editors, primeeditors or gene-writers. RNA-guided nucleases include CRISPR nucleases such as Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-11, and CasX. For transient expression of a reprogramming agent, such as a CAR, an mRNA encoding the reprogramming agent can be used as the engineering agent. For durable expression of the reprogramming agent, such as an exogenous, modified, or corrected gene (and its gene product), the engineering agent can comprise mRNA-encoded RNA-directed nucleases, guide RNAs, nucleic acid templates, and other components of gene / genome editing systems.

[0042] Examples of gene editing components that are encoded by a nucleic acid molecule include an mRNA encoding an RNA-guided nuclease, a gene or base editing protein, a prime editing protein, a Gene Writer protein (e.g., a modified or modularized non-long terminal repeat (LTR) retrotransposon), a retrotransposase, an RNA writer, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a transposase, a retrotransposon, a reverse transcriptase (e.g., M-MLV reverse transcriptase), a nickase or inactive nuclease (e.g., Cas9, nCas9, dCas9), a DNA recombinase, a CRISPR nuclease (e.g., Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-11, CasX), a DNA nickase, a Cas9 nickase (e.g., D10A or H840A), or any fusion or combination thereof. Other components include a guide RNA (gRNA), a single guide RNA (sgRNA), a prime editing guide RNA (pegRNA), a clustered regularly interspaced short palindromic repeat (CRISPR) RNA (crRNA), a trans-activating clustered regularly interspaced short palindromic repeat (CRISPR) RNA (tracrRNA), ora DNA molecule to be inserted or serve as a template for double-strand break (DSB) repair at a specific genomic locus. Genome-, gene-, and base-editing technology are reviewed in Anzalone etal., Nature Biotechnology 38:824-844, 2020, Sakuma, Gene and Genome Editing 3-4:100017, 2022, and Zhou etal., MedComm 3(3):e155, 2022, each of which is incorporated by reference for all that they teach about the components and uses of this technology to the extent that it does not conflict with this disclosure.

[0043] “Conditioning agent,” as used herein, refers to a biological response modifier (BRM) that enhances the efficiency of engineering an immune cell, expands the number of immune cells available to be engineered or the number of engineered cells in a target tissue (for example, a tumor, fibrotic tissue, or tissue undergoing autoimmune attack), promotes activity of the engineered cell in a target tissue, or broadens the range of operative mechanisms contributing to a therapeutic immune reaction. A conditioning agent can be provided by delivering an encoding nucleic acid in a LNP or tLNP. Exemplary BRMs include cytokines, such as IL-7, IL-15, or IL-18. Conditioning agents can be provided as the agent itself or, when the conditioning agent is a peptide or protein, as a nucleic acid molecule encoding the conditioning agent.

[0044] “Immune cell,” as used herein, can refer to any cell of the immune system. However, particular aspects can exclude polymorphonuclear leukocytes and / or B cells, or be limited to non-B lymphocytes such as T cell and / or NK cells, or to monocytes such as dendritic cells and / or macrophages in their various forms.

[0045] As used herein, “lipid nanoparticle” (LNP) means a solid particle, as distinct from a liposome having an aqueous lumen. The core of an LNP, like the lumen of a liposome, is surrounded by a layer of lipid that can be, but is not necessarily, a continuous lipid monolayer, a bilayer, or multi-layer having three or more lipid layers.

[0046] As used herein, a “binding moiety” or “targeting moiety” refers to a protein, polypeptide, oligopeptide, peptide, carbohydrate, nucleic acid, or combination thereof that is capable of specifically binding to a target or multiple targets. A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule or another target of interest. Exemplary binding moieties of this disclosure include an antibody, a Fab', F(ab')2, Fab, Fv, rlgG, scFv, hcAbs (heavy chain antibodies), a single domain antibody, VHH, VNAR, sdAbs, nanobody, receptor ectodomains or ligandbinding portions thereof, or ligands (e.g., cytokines, chemokines). A “Fab” (fragment antigen binding) is the part of an antibody that binds to antigens and includes the variable region and CH1 of the heavy chain linked to the light chain via an inter-chain disulfide bond. In other embodiments, a binding moiety comprises a ligand-binding domain of a receptor or a receptor ligand. In some embodiments, a binding moiety can have more than one specificity including, for example, bispecific or multispecific binders. A variety of assays are known for identifying binding moieties of this disclosure that specifically bind a particular target, including Western blot, ELISA, and Biacore® analysis. A binding moiety, such as a binding moiety comprising immunoglobulin light and heavy chain variable domains (e.g., scFv), can be incorporated into a variety of protein scaffolds or structures as described herein, such as an antibody or an antigen binding fragment thereof, a scFv-Fc fusion protein, or a fusion protein comprising two or more of such immunoglobulin binding domains.

[0047] As used herein, “antibody” refers to a protein comprising an immunoglobulin domain having hypervariable regions determining the specificity with which the antibody binds antigen; so-called complementarity determining regions (CDRs). The term antibody can thus refer to intact or whole antibodies as well as antibody fragments and constructs comprising an antigen binding portion of a whole antibody. While the canonical natural antibody has a pair of heavy and light chains, camelids (camels, alpacas, llamas, etc.) produce antibodies with both the canonical structure and antibodies comprising only heavy chains. The variable region of the camelid heavy chain only antibody has a distinct structure with a lengthened CDR3 referred to as VHH or, when produced as a fragment, a nanobody. Antigen binding fragments and constructs of antibodies include F(ab)2, F(ab), minibodies, Fv, single-chain Fv (scFv), diabodies, and VH. Such elements can be combined to produce bi- and multi-specific reagents, such as T-cell engagers (for example, Bispecific T-Cell Engagers (BiTEs)) or other immune cell engagers. The term “monoclonal antibody” arose out of hybridoma technology but is now used to refer to any singular molecular species of antibody regardless of how it was originated or produced. Antibodies can be obtained through immunization, selection from a naive or immunized library (for example, by phage display), alteration of an isolated antibodyencoding sequence, or any combination thereof. Numerous antibodies that could be used as binding moieties are known in the art. An accepted source of information about antibodies for an International Non-proprietary Name (INN) has been proposed or recommended, including sequence information, is Wilkinson & Hale, 2022, MAbs 14(1):2123299, including its Supplementary Tables, which is incorporated by reference herein for all that it teaches about individual antibodies and the various antibody formats that can be constructed. U.S. Patent No. 11,326,182 and especially its Table 9 entitled “Cancer, Inflammation and Immune System Antibodies,” is a source of sequence and other information for a wide range of antibodies including many that do not have an INN and is incorporated herein by reference for all that it teaches about individual antibodies.

[0048] An antibody or other binding moiety (or a fusion protein thereof) “specifically binds” a target if it binds the target with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1 / M) equal to or greater than 105 M-1, while not significantly binding other components present in a test sample. Binding domains (or fusion proteins thereof) can be classified as “high affinity” binding domains (or fusion proteins thereof) and “low affinity” binding domains (or fusion proteins thereof). “High affinity” binding domains refer to those binding domains with a Ka of at least 108 M'1, at least 109 M'1, at least 1010 M'1, at least 1011 M'1, at least 1012 M'1, or at least 1013 M'1, preferably at least 108 M'1 or at least 109 M’1. “Low affinity” binding domains refer to those binding domains with a Ka of up to 108 M'1, up to 107 M’1, upto 106 M’1, upto 105 M'1. Alternatively, affinity can be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10'5 M to 10’ 13 M). Affinities of binding domain polypeptides and fusion proteins according to this disclosure can be readily determined using conventional techniques (see, e.g., Scatchard et al., 1949, Ann. N.Y. Acad. Sci. 51:660; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).

[0049] As used herein, “payload” refers to a negatively charged agent that can interact with cationic lipids, such as the ionizable cationic lipids of this disclosure, to become encapsulated within lipid nanoparticles comprising the cationic lipid. The negatively charged agent can be a biologically active small organic molecule, or a macromolecule such as a nucleic acid molecule, a carbohydrate, a peptide, or a polypeptide. In some embodiments, a payload can be one or more nucleic acid molecules, such as RNA or DNA, including mRNA and guide RNA (gRNA) molecules. In certain embodiments, a payload comprises an mRNA that encodes a biologically active molecule, such as a chimeric antigen receptor, a T cell receptor, an immune cell engager, or the like.

[0050] As used herein, “biologically active agent” refers to any substance that affects a metabolic or physiologic response in a living organism, cells, or cultured cells thereof, including genetically reprograming, permanently or transiently, such organism or cells.

[0051] As used herein, “therapeutic agent” is a substance, or a component of a combination of substances, the biological activity of which can potentially cure, ameliorate, stabilize, prevent, or otherwise beneficially impact a disease, pathological condition, or other disorder.

[0052] For simplicity, chemical moieties are defined and referred to throughout primarily as univalent chemical moieties (e.g., alkyl, aryl, and the like). Nevertheless, such terms can also be used to convey corresponding multivalent moieties under the appropriate structural circumstances clear to those skilled in the art. For example, while an “alkyl” moiety generally refers to a monovalent radical (e.g. CH3-CH2-), in certain circumstances a bivalent linking moiety can be “alkyl,” in which case those skilled in the art will understand the alkyl to be a divalent radical (e.g., -CH2-CH2-), which is equivalent to the term “alkylene.” (Similarly, in circumstances in which a divalent moiety is required and is stated as being “aryl,” those skilled in the art will understand that the term “aryl” refers to the corresponding divalent moiety, arylene.) All atoms are understood to have their normal number of valences for bond formation (i.e., 4 for carbon, 3 for nitrogen, 2 for oxygen, and 2, 4, or 6 for sulfur, depending on the oxidation state of the sulfur atom).

[0053] The term "alkyl" as employed herein refers to saturated straight and branched chain aliphatic groups having from 1 to 12 carbon atoms. As such, “alkyl” encompasses Ci, C2, C3, C4, C5, Ce, C7, Cs, C9, C10, C11 and Ci2 groups.

[0054] The term "alkenyl" as used herein means an unsaturated straight or branched chain aliphatic group with one or more carbon-carbon double bonds, having from 2 to 12 carbon atoms. As such, “alkenyl” encompasses C2, C3, C4, C5, Ce, C7, Cs, C9, C10, Cn and C12 groups.

[0055] In some embodiments, the hydrocarbon chain is unsubstituted. In other embodiments, one or more hydrogens of the alkyl or alkenyl group can be substituted with the same or different substituents.

[0056] Alkynoic refers to a carboxylic acid moiety comprising one or more carbon-carbon triple bonds. In some embodiments, hydrogens are unsubstituted. In other embodiments, one or more hydrogens of the alkynoic group can be substituted with the same or different substituents.

[0057] Amide refers to a carboxylic acid derivative comprising a carbonyl group of a carboxylic acid bonded to an amine moiety.

[0058] Ester refers to a carboxylic acid derivative comprising a carbonyl group bond to an alkyloxy group to form an ester bond -C(=O)-O-.

[0059] Head group refers to the hydrophilic or polar portion of a lipid.

[0060] For any of the chemical structures herein, a wavy bond (i.e., -^) indicates any relative or absolute stereo-configuration of the corresponding ring atom or a mixture thereof when not referring to a single molecule.

[0061] Sterol refers to a subgroup of steroids that contain at least one hydroxyl (OH) group. Examples of sterols include, without limitation, cholesterol, ergosterol, p-sitosterol, stigmasterol, stigmastanol, 20-hydroxycholesterol, 22-hydroxycholesterol, and the like.

[0062] As standard, the bond represented as a solid wedge extends above the plane and the bond represented as a dashed wedge extends below the plane when depicting absolute stereochemistry, throughout.

[0063] As compared to similar molecules in which a head group and two branching tails extend from a single central branchpoint nitrogen atom (e.g., a tertiary amine) and are unconnected to each other, in the constrained ionizable lipids described herein, a ring comprising a central branchpoint nitrogen atom (e.g., a heterocyclic ring) connects the two branching tail groups to each other, reducing their freedom of movement. As used herein, “constrained” refers to this limited freedom of movement of the two tail groups extending from a ring comprising a nitrogen atom. Ionizable Cationic Lipids

[0064] In certain aspects, the constrained ionizable cationic lipids of this disclosure have a structure of the formula M6, a stereoisomer thereof, or a mixture of such stereoisomers: M6 Y is O, S, NH, or NCH3; Zis 0, NH, or NCH3; each W is independently CH or N; each R2 is independently 0 or C=0, wherein at least one R2 is O; each R3 is independently 0 or C=0, wherein when a R2 is 0 then its adjoining R3 is C=0, or when a R2 is C=0 then its adjoining R3 is O; wherein when at least one R3 at a beta position of a W is 0, that W is CH; and wherein when both R3 groups at a beta position of a W are C=0, that W is CH or N; each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl; each A1, A2, A3, and A4 is independently (CH2)oor (CH2)i, provided that A1 is (CH2)i when A3 is (CH2)o; A1 is (CH2)o when A3 is (CH2)i; A2 is (CH2)i when A4 is (CH2)0; and A2 is (CH2)o when A4 is (CH2)i; and A5 is (CH2)o-4, CH=CH, or CH2-CH=CH-CH2.

[0065] It will be apparent to one skilled in the art that certain compounds of this disclosure can exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the disclosure. Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of these compounds are within the scope of the disclosure. Both the R and the S stereochemical isomers, as well as all mixtures thereof, are included within the scope of the disclosure.

[0066] The position of X can be referred to as the central branch point and the position of each W can be referred to as a distal branch point. Each of the disclosed ionizable cationic lipids has two tail groups extending from the central branch point and each tail group branches again at its distal branch point. The mobility of the two tail groups is constrained by the ring comprising the central branch point atom and the first one or two main chain atoms of each tail group.

[0067] As used herein, when a subscript has a value of “0”, the group is absent and the adjoining atoms are bonded to each other. For example, when A1 is (CH2)o, A1 is absent.

[0068] In various embodiments of M6, A1 through A4 are such that there are only two main chain atoms between the ring nitrogen and each nearest ester oxygen in the nearest tail group. For example, in various embodiments of M6, each A1, A2, A3, and A4 is independently (CH2)oor (CH2)i, provided that A1 is (CH2)i when A3 is (CH2)o; A1 is (CH2)o when A3 is (CH2)i; A2 is (CH2)i when A4 is (CH2)o; and A2 is (CH2)o when A4 is (CH2)i.

[0069] In certain embodiments of M6, A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)i-4 or CH2-CH=CH-CH2.

[0070] In certain embodiments of M6, A1 is (CH2)o, A2 is (CH2)i, A3 is (CH2)i, A4 is (CH2)o, and A5 is (CH2)i.

[0071] In certain embodiments of M6, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is (CH2)o.

[0072] In certain embodiments of M6, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5is(CH2)i.

[0073] In certain embodiments of M6, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is (CH2)2 or CH=CH.

[0074] In some embodiments of formula M6 as described herein, X is B -CH- \         i por examp|e jn some embodiments of formula M6, X is

[0075] In some embodiments of formula M6 as described herein, X is (CH2E3OCH3

[0076] In some embodiments of formula M6 as described herein, X is ...... (CH^OCHS

[0077] In some embodiments of formula M6 as described herein, X is —7            .In some embodiments X is            . YHCHv'h..—]

[0078] In some embodiments of formula M6 as described herein, X is ’

[0079] In some embodiments of formula M6 as described herein, X is Y-(CH^.

[0080] In some embodiments of formula M6 as described herein, X is

[0081] In some embodiments of formula M6 as described herein, X is Y^W^N b ' ■ \______ /

[0082] In some embodiments of formula M6 as described herein, X is Y~(CH2)M-^ NCHg

[0083] In some embodiments of formula M6 as described herein, X is

[0084] In some embodiments of formula M6 as described herein, X is .. ,...... ‘               x....... /

[0085] In some embodiments of formula M6 as described herein, X is Y-(CH 2WI—(z

[0086] In some embodiments of formula M6 as described herein, X is "4'         .

[0087] In some embodiments of formula M6, X is             v' . In some Z—Z — embodiments of formula M6, X is             . In some embodiments of formula M6, X is 2           ..A

[0088] In some embodiments of formula M6, X is            " . In some Z     N embodiments of formula M6, X is       v .

[0089] In some embodiments of formula M6, X is . In some "N embodiments of formula M6, X is . In some embodiments of formula M6, X is N- zz y >

[0090] In some embodiments of formula M6, X is            7 . In some ~ I > embodiments of formula M6, X is             . In some embodiments of formula M6, X is I >                                              “ I ) . In some embodiments of formula M6, X is z.....y y

[0091] In some embodiments of formula M6, X is                  . In some embodiments of formula M6 In some embodiments of formula M6, X is ’.....('']

[0092] In some embodiments of formula M6, X is                  . In some z y । embodiments of formula M6, X is             . In some embodiments of formula M6, X is

[0093] In some embodiments of formula M6, X is                    |n some embodiments of formula M6, X is In some embodiments of formula M6, X is z Y \

[0094] In some embodiments of formula M6, X is          V—-1   |n some r r \ embodiments of formula M6, X is                |n some embodiments of formula M6, X is Z--(CH^4—(   N—

[0095] In some embodiments of formula M6, X is                s---z . In some embodiments of formula M6, X is               . A I

[0096] In some embodiments of formula M6, X is            x—. in some embodiments of formula M6, X is

[0097] As described above, in some embodiments of formula M6, Y can be selected from O, S, NH, or NCH3. In some embodiments of formula M6, Y is O. In some other embodiments of formula M6, Y is S. CHS C”

[0098] In some embodiments of formula M6, X is               and Y is O. In some CH> embodiments of formula M6, X is             's and Y is S.

[0099] As described above, Z can be selected from O, NH, or NCH3. In some embodiments, Z is O. Z~(CH2)MX^ n—

[00100] In some embodiments of formula M6, X is              v and Z is O. In some embodiments of formula M6, X is             and Z is O. In some embodiments of Z . formula M6, X is                and Z is O. and Z is O. (CHV<-C >

[00101] In some embodiments of formula M6, X is            z and Z is O. In some Z, embodiments of formula M6, X is

[00102] In some embodiments of formula M6, X is X J some embodiments of formula M6, X is z y formula M6, X is                 and Z is O. and Z is O. In and Z is O. In some embodiments of IN

[00103] In some embodiments of formula M6, X is embodiments of formula M6, X is             and Z is O. In some embodiments of and Z is O. In some formula M6, X is             and Z is O. In some embodiments of formula M6, X is and Z is 0.

[00104] In some embodiments of formula M6, X is and Z is O. In some embodiments of formula M6, X is and Z is O. In some embodiments of formula M6, X is and Z is O. z. I I

[00105] In some embodiments of formula M6, X is                   and Z is O. In z.                      | some embodiments of formula M6, X is             and Z is O. In some embodiments of formula M6, X is                  and Z is O.

[00106] In some embodiments of formula M6, X is and Z is O. In some embodiments of formula M6 and Z is O. In some embodiments of formula M6, X is                 and Z is O. -(CH3)g-4 z' V \ I /

[00107] In some embodiments of formula M6, X is                   and Z is O. In z" -y ... I } some embodiments of formula M6, X is V-—' anc| y is O. In some embodiments / of formula M6, X is                   anc| z jS o Z—(CHfe.4—{   N—

[00108] In some embodiments of formula M6, X is                 x---r" and Z is O. In some embodiments of formula M6, X is               and Z is O. z' i y

[00109] In some embodiments of formula M6, X is                  j anc| z jS L / 1- O. In some embodiments of formula M6, X is                and Z is O.

[00110] As described above, for formula M6, each R1 is independently C7-C11 alkyl or C7-Cn alkenyl. In some embodiments of formula M6, each R1 is independently C7-C11 alkyl, e.g., C7-C10 alkyl, orC7-Cg alkyl. In certain embodiments of formula M6, each R1 is independently a linear C7-C11 alkyl, e.g., a linear C7-C10 alkyl, or a linear C7-C9 alkyl. In some embodiments of formula M6 as described herein, each R1 is independently (CH2)6-8CH3. In some of these and other embodiments, R1 is (CH2)7CH3. In some embodiments of formula M6, each R1 is independently a linear C7-C11 alkenyl, e.g., a linear C7-C10 alkenyl, or a linear C7-C9 alkenyl. For example, in some embodiments of formula M6, each R1 is a linear Cs alkenyl. In certain other embodiments of formula M6, each R1 is independently a branched C7-C11 alkyl, e.g., C7-C10 alkyl, orC7-Cg alkyl. For example, in some embodiments of formula M6, each R1 is a branched Cs alkyl. In certain embodiments of formula M6, each R1 is independently a branched C7-C11 alkenyl, e.g., C7-C10 alkenyl, or C7-Cg alkenyl. For example, in some embodiments of formula M6, each R1 is a branched Cs alkenyl. In some embodiments of formula M6, wherein R1 is a branched alkyl or alkenyl, the branch point is positioned so that ester carbonyls are not in an a position relative to the branch point, for example they are in a p position relative to the branch point.

[00111] In certain embodiments of formula M6 as described herein, each R1 is the same. In certain embodiments of formula M6, each R1 nearest a common branch point is the same, but those nearest a first common branch point differ from those nearest a second common branch point. In certain embodiments of formula M6, each R1 nearest a common branch point is different but the pair of R1 groups nearest a first common branch point is the same the pair nearest a second common branch point.

[00112] As described above, in various embodiments of formula M6 as described herein, each R2 is independently O or C=O, wherein at least one R2 is O and each R3 is independently O or C=O, wherein when a R2 is O then its adjoining R3 is C=O, or when a R2 is C=O then its adjoining R3 is O. Accordingly, together, each R2 and R3 group form an ester group. In some embodiments of formula M6 as described herein, in each tail group one R2 is O and its adjoining R3 is C=O, and the other R2 is C=O and its adjoining R3 is O. In some embodiments of formula M6 as described herein, in one tail group, one R2 is O and its adjoining R3 is C=O, and the other R2 is C=O and its adjoining R3 is O, and in the other tail group, both R2 groups are O and both R3 groups are C=O. In some embodiments of formula M6 as described herein, in one tail group one R2 is O and its adjoining R3 is C=O, and the other R2 is C=O and its adjoining R3 is O and in the other tail group both R2 groups are C=O and both R3 groups are O. In some embodiments of formula M6 as described herein, in one tail group, both R2 groups are 0 and both R3 groups are C=0, and in the other tail group both R2 groups are C=0 and both R3 groups are 0. In some embodiments of formula M6 as described herein, in each tail group both R2 groups are 0 and both R3 groups are C=0.

[00113] As described above, each W is independently CH or N. For example, in some embodiments, each W is CH. In some embodiments, each W is N. In some other embodiments, one W is CH and the other W is N. In certain embodiments, when both R3 groups at a beta position of a W are C=O, that W can be CH or N. For example, in certain other embodiments, when both R3 groups at a beta position of a W are C=O, that W is N. In certain embodiments, when both adjoining R3 groups at a beta position of a W are C=O, that Wis CH.

[00114] When used in biological systems, it is advantageous to use ionizable cationic lipids that avoid formation of formaldehyde upon degradation. Accordingly, in certain embodiments as described herein, the combination of a R3 at a beta position of W being O and W being N is avoided. For example, in other certain embodiments, when at least one R3 at a beta position of a W is O, that W is CH. In other certain embodiments, when both R3 groups at a beta position of a W are 0, that W is CH.

[00115] As described above, in some embodiments of formula M6 as described herein, in each tail group one R2 is O and the other R2 is C=O, and one R3 is C=O and the other R3 is O, respectively. Accordingly, in certain aspects, the constrained ionizable cationic lipids of this disclosure have a structure of formula M6-1, a stereoisomer thereof, or a mixture of such stereoisomers: M6-1 wherein X, A1, A2, A3, A4, A5, and R1, are as otherwise described herein.

[00116] As described above, in some embodiments of formula M6 as described herein, in one tail group one R2 is O and the other R2 is C=O, and one R3 is C=O and the other R3 is O, respectively, and in the other tail group both R2 groups are C=O and both R3 groups are O. Accordingly, in certain aspects, the constrained ionizable cationic lipids of this disclosure have a structure of formula M6-2A or formula M6-2B, a stereoisomer thereof, or a mixture of such stereoisomers: M6-2A wherein X, A1, A2, A3, A4, A5, and R1, are as otherwise described herein.

[00117] As described above, in some embodiments of formula M6 as described herein, in one tail group, one R2 is O and the other R2 is C=O, and one R3 is C=O and the other R3 is O, respectively, and in the other tail group, both R2 groups are O and both R3 groups are C=O. Accordingly, in certain aspects, the constrained ionizable cationic lipids of this disclosure have a structure of formula M6-3A or M6-3B, a stereoisomer thereof, or a mixture of such stereoisomers: M6-3A wherein X, A1, A2, A3, A4, A5, W, and R1, are as otherwise described herein.

[00118] As described above, in some embodiments of formula M6 as described herein, in one tail group, both R2 groups are O and both R3 groups are C=O, and in the other tail group, both R2 groups are C=O and both R3 groups are O. Accordingly, in certain aspects, the constrained ionizable cationic lipids of this disclosure have a structure of formula M6-4A or formula M6-4B, a stereoisomer thereof, or a mixture of such stereoisomers: M6-4A wherein X, A1, A2, A3, A4, A5, W, and R1, are as otherwise described herein.

[00119] As described above, in some embodiments as described herein, the ionizable cationic lipid has a structure of formula M6, wherein A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)i-4 or CH2-CH=CH-CH2. For example, in some embodiments as described herein, A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)i. In some embodiments as described herein, the ionizable cationic lipid has the structure: wherein X, W, R1, R2, and R3 are as described herein.

[00120] In certain embodiments of formula M6 as described herein, A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)2. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X, W, R1, R2, and R3 are as described herein.

[00121] In certain embodiments of formula M6 as described herein, A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)3. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X, W, R1, R2, and R3 are as described herein.

[00122] In certain embodiments of formula M6 as described herein, A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)4. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X, W, R1, R2, and R3 are as described herein.

[00123] In certain embodiments of formula M6 as described herein, A1 is (CH2)0, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is CH2-CH=CH-CH2. For example, in some embodiments, the ionizable cationic lipid has the structure: R1—R2 V3 R1-R2           V wherein X, W, R1, R2, and R3 are as described herein.

[00124] As described above, in some embodiments as described herein, the ionizable cationic lipid has a structure of formula M6, wherein A1 is (CH2)o, A2 is (CH2)i, A3 is (CH2)i, A4 is (CH2)o, and A5 is (CH2)i. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X, W, R1, R2, and R3 are as described herein.

[00125] As described above, in some embodiments as described herein, the ionizable cationic lipid has a structure of formula M6, wherein A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is (CH2)o- For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X, W, R1, R2, and R3 are as described herein.

[00126] As described above, in some embodiments as described herein, the ionizable cationic lipid has a structure of formula M6, wherein A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)0, A4 is (CH2)o, and A5 is (CH2)i. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X, W, R1, R2, and R3 are as described herein.

[00127] As described above, in some embodiments as described herein, the ionizable cationic lipid has a structure of formula M6 wherein A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)0, A4 is (CH2)o, and A5 is (CH2)2 or CH=CH. For example, in some embodiments as described herein, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is (CH2)2. In some embodiments as described herein, the ionizable cationic lipid has the structure: wherein X, W, R1, R2, and R3 are as described herein.

[00128] In certain embodiments of formula M6 as described herein, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is CH=CH. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X, W, R1, R2, and R3 are as described herein.

[00129] As described above, in some embodiments as described herein, the ionizable cationic lipid has a structure of formula M6, wherein A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)i-4 or CH2-CH=CH-CH2. For example, in some embodiments as described herein, A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)i. In some embodiments as described herein, the ionizable cationic lipid has the structure: wherein X, W, R2, and R3 are as described herein.

[00130] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . CH3 Y (CH2)2-^

[00131] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00132] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . ch3 Y (CH2)2-M'

[00133] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00134] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein.

[00135] In certain embodiments of formula M6, X is . CH3 Y (CH2)2-M' ^H3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00136] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2-^

[00137] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00138] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2-

[00139] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00140] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2-M'

[00141] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00142] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . ch3 Y (CH2)2-M'

[00143] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00144] In certain embodiments of formula M6 as described herein, A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)2. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X, W, R2, and R3 are as described herein.

[00145] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . CH3 Y (CH2)2-N'

[00146] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00147] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein.

[00148] In certain embodiments of formula M6, X is . CH3 Y (CH2)2-M' ^H3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: o

[00149] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . ch3 Y (CH2)2-M'

[00150] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00151] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . ch3 Y (CH2)2-

[00152] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00153] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . ch3 Y (CH2)2-N'

[00154] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00155] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2—

[00156] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00157] For example, in certain embodiments of formula M6, the ionizable cationic lipid has wherein X and W are as described herein. . ch3 Y (CH2)2—N'

[00158] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: o wherein W is as described herein.

[00159] In certain embodiments of formula M6 as described herein, A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)3. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X, W, R2, and R3 are as described herein.

[00160] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . CH3 Y (CH2)2—

[00161] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00162] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . CH3 Y (CH2)2-^

[00163] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00164] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . CH3 Y (CH2)2—

[00165] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00166] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2-^ ten In certain embodiments of formula M6, X is              3, wherein Y is O. certain embodiments, the ionizable cationic lipid has the structure: For example, in wherein W is as described herein.

[00167] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2—

[00168] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00169] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2—

[00170] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00171] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . ch3 Y (CH2)2-

[00172] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00173] In certain embodiments of formula M6 as described herein, A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)4. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X, W, R2, and R3 are as described herein.

[00174] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . ch3 Y (CH2)2-N'

[00175] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00176] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . CH3 Y (CH2)2—

[00177] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00178] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . CH3 Y (CH2)2—

[00179] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00180] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2-N'

[00181] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00182] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . ch3 Y (CH2)2-

[00183] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00184] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2-N'

[00185] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00186] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2-N'

[00187] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00188] In certain embodiments of formula M6 as described herein, A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is CH2-CH=CH-CH2. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X, W, R2, and R3 are as described herein.

[00189] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . CH3 Y (CH2)2-N'

[00190] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00191] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . CH3 Y (CH2)2-

[00192] In certain embodiments of formula M6, X is              3, wherein Y is O. example, in certain embodiments, the ionizable cationic lipid has the structure: For

[00193] For example, in certain embodiments of formula M6, the ionizable cationic lipid having the structure: wherein X is as described herein. . CH3 Y (CH2)2-M'

[00194] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00195] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . ch3 Y (CH2)2-M'

[00196] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00197] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2-M'

[00198] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00199] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2-M'

[00200] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00201] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . ch3 Y (CH2)2-

[00202] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00203] As described above, in some embodiments as described herein, the ionizable cationic lipid has a structure of formula M6, wherein A1 is (CH2)o, A2 is (CH2)i, A3 is (CH2)i, A4 is (CH2)o, and A5 is (CH2)i. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X, W, R2, and R3 are as described herein.

[00204] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . CH3 Y (CH2)2-^

[00205] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00206] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . CH3 Y (CH2)2-^

[00207] In certain embodiments of formula M6, X is              3, wherein Y is O. example, in certain embodiments, the ionizable cationic lipid has the structure: For

[00208] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein.

[00209] In certain embodiments of formula M6, X is . CH3 Y (CH2)2-M' ^H3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00210] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2-N'

[00211] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein .

[00212] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2—

[00213] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00214] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2-N'

[00215] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00216] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2—

[00217] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00218] As described above, in some embodiments as described herein, the ionizable cationic lipid has a structure of formula M6, wherein A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is (CH2)o. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X, W, R2, and R3 are as described herein.

[00219] For example, in certain embodiments of formula M6, the ionizable cationic lipid has wherein X is as described herein. CH3 Y (CH2)2-^ ten

[00220] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00221] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. CH3 Y (CH2)2-N' ten

[00222] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00223] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. CH3 Y (CH2)2-N' ten

[00224] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00225] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein.

[00226] In certain embodiments of formula M6, X is CH3 Y (CH2)2-N / bH3 wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00227] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein.

[00228] In certain embodiments of formula M6, X is CH3 Y (CH2)2-N' tH3 wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00229] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2-N'

[00230] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein .

[00231] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2-N'

[00232] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00233] As described above, in some embodiments as described herein, the ionizable cationic lipid has a structure of formula M6, wherein A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is (CH2)i. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X, W, R2, and R3 are as described herein.

[00234] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. CH3 Y (CH2)2-n'

[00235] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00236] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . CH3 Y (CH2)2-N'

[00237] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00238] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . ch3 Y (CH2)2-

[00239] In certain embodiments of formula M6, X is              3, wherein Y is O. example, in certain embodiments, the ionizable cationic lipid has the structure: For o

[00240] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2—

[00241] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00242] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2-

[00243] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00244] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2-N'

[00245] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00246] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2-N'

[00247] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00248] As described above, in some embodiments as described herein, the ionizable cationic lipid has a structure of formula M6 wherein A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is (CH2)2 or CH=CH. For example, in some embodiments as described herein, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is (CH2)2. In some embodiments as described herein, the ionizable cationic lipid has the structure: wherein X, W, R2, and R3 are as described herein.

[00249] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . CH3 Y (CH2)2-N'

[00250] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00251] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein.

[00252] In certain embodiments of formula M6, X is . CH3 Y (CH2)2-M' ^H3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00253] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . CH3 Y (CH2)2-

[00254] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00255] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2-

[00256] In certain embodiments of formula M6, X is              3, wherein Y is O. example, in certain embodiments, the ionizable cationic lipid has the structure: For wherein W is as described herein.

[00257] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2—

[00258] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00259] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2—

[00260] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00261] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2—

[00262] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00263] In certain embodiments of formula M6 as described herein, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is CH=CH. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X, W, R2, and R3 are as described herein.

[00264] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . CH3 Y (CH2)2-N' ten

[0001] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00265] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . CH3 Y (CH2)2-^

[0002] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00266] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X is as described herein. . CH3 Y (CH2)2-N' ten

[0003] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00267] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2-M'

[0004] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00268] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . ch3 Y (CH2)2—

[0005] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00269] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2— ten

[0006] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00270] For example, in certain embodiments of formula M6, the ionizable cationic lipid has the structure: wherein X and W are as described herein. . CH3 Y (CH2)2-

[0007] In certain embodiments of formula M6, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure: wherein W is as described herein.

[00271] In certain aspects of the ionizable cationic lipids of formula M6 as described herein, in both tail groups, both R2 groups are O and both R3 groups are C=O. For example, in certain aspects, the constrained ionizable cationic lipids of this disclosure have a structure of the formula M3, a stereoisomer thereof, or a mixture of such stereoisomers: ___                               (CH2)0-3^H3 l / XN-(CH2)0.3CH3   Y-(CH2)2-4-N wherein X is \— /              ,              (CH2)o-3 3 (CH2)2-3O^H3 Y-(CH2)2-4-Nv (CH2)2-3OCH3 Y is O, S, NH, or NCH3; Zis 0, NH, or NCH3; W is CH or N; and each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl; each A1, A2, A3, and A4 is independently (CH2)oor (CH2)i, provided that A1 is (CH2)i when A3 is (CH2)0; A1 is (CH2)0 when A3 is (CH2)i; A2 is (CH2)i when A4 is (CH2)0; and A2 is (CH2)0 when A4 is (CH2)i; A5 is (CH2)0-4, CH=CH, or CH2-CH=CH-CH2.

[00272] The position of X can be referred to as the central branch point and the position of each W can be referred to as a distal branch point. Each of the disclosed ionizable cationic lipids has two tail groups extending from the central branch point and each tail group branches again at its distal branch point. The mobility of the two tail groups is constrained by the ring comprising the central branch point atom and the first one or two main chain atoms of each tail group.

[00273] As used herein, when a subscript has a value of “0”, the group is absent. For example, when A1 is (CH2)o, A1 is absent. For example, in various embodiments of M3, each A1, A2, A3, and A4 is independently (CH2)oor (CH2)i, provided that A1 is (CH2)i when A3 is (CH2)o; A1 is (CH2)o when A3 is (CH2)i; A2 is (CH2)i when A4 is (CH2)o; and A2 is (CH2)o when A4 is (CH2)i.

[00274] In various embodiments of formula M3, A1 through A4 are such that there are only two main chain atoms between the ring nitrogen and each nearest ester oxygen in the nearest tail group.

[00275] In certain embodiments of formula M3, A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)i-4 or CH2-CH=CH-CH2.

[00276] In certain embodiments of formula M3, A1 is (CH2)o, A2 is (CH2)i, A3 is (CH2)i, A4 is (CH2)o, and A5 is (CH2)i.

[00277] In certain embodiments of formula M3, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is (CH2)o.

[00278] In certain embodiments of formula M3, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5is(CH2)i.

[00279] In certain embodiments of formula M3, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is (CH2)2 or CH=CH.

[0008] Different constituents for X, Y, Z, and R1 allow for tuning of cLogD and c-pKa to achieve a desired value of measured pKa within a LNP or a tLNP. For example, to make a . ch3 Y (CH2)2-N / lipid with a head group of              3 less basic, the following head groups could be used - (CH2)0.3CH3        y-(CH2)2.4-N / oh y-(CH2)2.4-N / S instead:     '—'               ,                    \ / Y-(CH2)2-4-Nr^NCH3 Y-(CH2)2_4NH^rA   Y-(CH2)2N \— /    ,             \= / , or

[0009] Conversely, to make a lipid with a head group of y-(ch2)3.4—i following head groups could be used instead: Y               ]     Y-(CH2)2_4— / (CH2)2OCH3 X(CH2)2OCH3 .         ch3 Y (CH2)2_ 'qH 3 more basic, the CH3              (CH2)l-3^H3 iV y-(ch2)2.4-n (CH2)1-3CH3

[00280] The addition of CH2 groups in X will tend to increase basicity of the lipid which in turn will tend to increase measured pKa. The addition of CH2 groups in R1 will tend to increase the lipophilicity (cLogD) of the lipid which in turn will tend to decrease measured pKa of the LNPor tLNP.

[00281] The use of further head groups to modify calculated pKa and target a desired pKa range is set out in Example 48.

[0010] In some embodiments of formula M3 as described herein, X is (CH2)0-3^H3                                                  CH3 Y-(CH2)2-4-N                                                  Y (CH2)2-M' (CH2)o-3ch3 por example, in some embodiments X is

[0011] In some embodiments of formula M3 as described herein, X is (CH2)0.3CH3

[0012] In some embodiments of formula M3 as described herein, X is (CH2)2-3OCH3 Y-(CH2)2-4-N (CH2)2-3OCH3

[0013] In some embodiments of formula M3 as described herein, X is (CH2)2-3OCH3 Y-(CH2)2-4-Nv (CH2)o-3CH3 YHCH-sh.—N   |

[00282] In some embodiments of formula M3 as described herein, X is ' Y-

[0014] ln some embodiments of formula M3 as described herein, X is

[0015] In some embodiments of formula M3 as described herein, X is

[0016] Y-(CH2)2.4- OH In some embodiments of formula M3 as described herein X is

[0017] In some embodiments of formula M3 as described herein, X is

[0018] Y-(CH2)2-4- In some embodiments of formula M3 as described herein X is

[0019] In some embodiments of formula M3 as described herein, X is

[0020] In some embodiments of formula M3 as described herein,

[00283] In some embodiments of formula M3 as described herein

[00284] In some embodiments of formula M3, X is                    . In some Z—< embodiments of formula M3, X is v . In some embodiments of formula M3, X is 2 N

[00285] In some embodiments of formula M3, X is               . In some £ A embodiments of formula M3, X is           .

[00286] In some embodiments of formula M3, X is . In some "N embodiments of formula M3, X is . In some embodiments of formula M3, X is N- zz y >

[00287] In some embodiments of formula M3, X is            7 . In some ~ I > embodiments of formula M3, X is             . In some embodiments of formula M3, X is In some embodiments of formula M3, X is z.....y y

[00288] In some embodiments of formula M3, X is                  . In some 2.. ,.-.. T T embodiments of formula M3, X is             . In some embodiments of formula M3, X is ’.....('']

[00289] In some embodiments of formula M3, X is                  . In some z y । embodiments of formula M3, X is             . In some embodiments of formula M3, X is

[00290] In some embodiments of formula M3, X is . In some embodiments of formula M3, X is In some embodiments of formula M3, X is

[00291] In some embodiments of formula M3, X is . In some embodiments of formula M3, X is . In some embodiments of formula M3, X is Y' z

[00292] In some embodiments of formula M3, X is . In some embodiments of formula M3, X is

[00293] In some embodiments of formula M3, X is . In some embodiments of formula M3, X is

[00294] As described above, in various embodiments of formula M3, Y can be selected from O, S, NH, or NCH3. In some embodiments, Y is O. In some other embodiments, Y is S.

[0021] In some embodiments, X is . ch3 Y (CH2)2-N / 'ch3 and Y is O. In some embodiments X is .         CH Y (CH2)2-N' bH 3 3 and Y is S.

[00295] As described above, Z can be selected from O, NH, or NCH3. In some embodiments, Z is O. ZXCH2)M-<^ —

[00296] In some embodiments of formula M3, X is              v" and Z is O. In z™ / x-- some embodiments of formula M3, X is             and Z is O. In some embodiments of Z - formula M3, X is                and Z is O. Z n (CH)c„X >

[00297] In some embodiments of formula M3, X is            z and Z is O. In some Z S x x embodiments of formula M3, X is z and Z is O. Z' I N— I /

[00298] In some embodiments of formula M3, X is                      and Z is O. In Z-~X X J some embodiments of formula M3, X is             and Z is O. In some embodiments of z y N_,„. formula M3, X is                 and Z is O. zz T '>

[00299] In some embodiments of formula M3, X is          * Y and Z is O. In some Z^X^y embodiments of formula M3, X is             and Z is O. In some embodiments of formula M3, X is             and Z is O. In some embodiments of formula M3, X is and Z is 0.

[00300] In some embodiments of formula M3, X is and Z is O. In some embodiments of formula M3, X is and Z is O. In some embodiments of formula M3, X is and Z is O. z. 1 I

[00301] In some embodiments of formula M3, X is                   and Z is O. In z.                      | some embodiments of formula M3, X is             and Z is O. In some embodiments of formula M3, X is                  and Z is O.

[00302] In some embodiments of formula M3, X is and Z is O. In some embodiments of formula M3, X is and Z is O. In some embodiments of formula M3, X is                 and Z is O. 1 > 1 -ALh2.10-4 z' V \ I /

[00303] In some embodiments of formula M3, X is                   and Z is O. In z" -y ... I } some embodiments of formula M3, X is V-—' anc| y is O. In some embodiments of formula M3, X is                   anc| z jS o Z—(CHfe.4—{   N—

[00304] In some embodiments of formula M3, X is                 x---r" and Z is O. In some embodiments of formula M3, X is               and Z is O. z' t y

[00305] In some embodiments of formula M3, X is                  j anc| z jS | N O. In some embodiments of formula M3, X is                and Z is O.

[00306] As described above, in various embodiments of formula M3, each R1 is independently C7-C11 alkyl or C7-C11 alkene. In some embodiments of formula M3, each R1 is independently C7-C11 alkyl, e.g., C7-C10 alkyl, or C7-C9 alkyl. In certain embodiments of formula M3, each R1 is independently a linear C7-C11 alkyl, e.g., a linear C7-C10 alkyl, or a linear C7-C9 alkyl. In some embodiments of formula M3 as described herein, each R1 is independently (CH2)6-8CH3. In some of these and other embodiments of formula M3, R1 is (CH2)7CH3. In some embodiments of formula M3, each R1 is independently a linear C7-C11 alkenyl, e.g., a linear C7-C10 alkenyl, or a linear C7-C9 alkenyl. For example, in some embodiments of formula M3, each R1 is a linear Cs alkenyl. In certain other embodiments of formula M3, each R1 is independently a branched C7-C11 alkyl, e.g., C7-C10 alkyl, or C7-C9 alkyl. For example, in some embodiments of formula M3, each R1 is a branched Cs alkyl. In certain embodiments of formula M3, each R1 is independently a branched C7-C11 alkenyl, e.g., C7-C10 alkenyl, orC7-C9 alkenyl. For example, in some embodiments of formula M3, each R1 is a branched Cs alkenyl. In some embodiments of formula M3, wherein R1 is a branched alkyl or alkenyl, the branch point is positioned so that ester carbonyls are not in an a position relative to the branch point, for example they are in a p position relative to the branch point.

[00307] In certain embodiments as described herein of formula M3, each R1 is the same. In certain embodiments of formula M3, each R1 nearest a common branch point is the same, but those nearest a first common branch point differ from those nearest a second common branch point. In certain embodiments of formula M3, each R1 nearest a common branch point is different but the pair of R1 groups nearest a first common branch point is the same the pair nearest a second common branch point.

[00308] As described above, W is CH or N. In certain embodiments, W is CH. In certain other embodiments, W is N.

[00309] As described above, in various embodiments of formula M6 as described herein, in both tail groups, both R2 groups are O and both R3 groups are C=O. For example, in certain aspects, the constrained ionizable cationic lipids of this disclosure have a structure of the formula M3. Accordingly, together, each R2 and R3 group form an ester group and in formula M3, each R3 is a C=O and at the beta position of the W. Various embodiments of formula M3 where each R3 is a C=O and at the beta position of the W are described below. Analogous embodiments where at least one R3 is O are described above.

[00310] As described above, in some embodiments as described herein, the ionizable cationic lipid has a structure of formula M3, wherein A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)i-4 or CH2-CH=CH-CH2. For example, in some embodiments as described herein, A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)i. In some embodiments as described herein, the ionizable cationic lipid has the structure: wherein X and W are as described herein.

[00311] For example, in certain embodiments of formula M3, W is CH and the ionizable . ch3 Y (CH2)2-wherein X is as described herein. In certain embodiments of formula M3, X is              3 , wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00312] For example, in certain embodiments of formula M3, W is N and the ionizable cationic lipid has the structure: . CH3 Y (CH2)2-N' ten wherein X is as described herein. In certain embodiments of formula M3, X is              3 , wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00313] In certain embodiments of formula M3 as described herein, A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)2. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X and W are as described herein.

[00314] For example, in certain embodiments of formula M3, W is CH and the ionizable cationic lipid has the structure: . CH3 Y (CH2)2-wherein X is as described herein. In certain embodiments of formula M3, X is              3 , wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00315] For example, in certain embodiments of formula M3, W is N and the ionizable cationic lipid has the structure: wherein X is as described herein. In certain embodiments of formula M3, X is . CH3 Y (CH2)2~ bH3 , wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00316] In certain embodiments of formula M3 as described herein, A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)3. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X and W are as described herein.

[00317] For example, in certain embodiments of formula M3, Wis CH the ionizable cationic lipid has the structure: . CH3 Y (CH2)2-N' ten wherein X is as described herein. In certain embodiments of formula M3, X is              3 , wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00318] For example, in certain embodiments of formula M3, W is N the ionizable cationic lipid has the structure: . CH3 Y (CH2)2-N' wherein X is as described herein. In certain embodiments of formula M3, X is              3 , wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00319] In certain embodiments of formula M3 as described herein, A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)4. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X and W are as described herein.

[00320] For example, in certain embodiments of formula M3, W is CH and the ionizable cationic lipid has the structure: . CH3 Y (CH2)2— ten wherein X is as described herein. In certain embodiments of formula M3, X is              3 , wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00321] For example, in certain embodiments of formula M3, W is N and the ionizable cationic lipid has the structure: . CH3 Y (CH2)2-N' ten wherein X is as described herein. In certain embodiments of formula M3, X is              3 , wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00322] In certain embodiments of formula M3 as described herein, A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is CH2-CH=CH-CH2. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X and W are as described herein.

[00323] For example, in certain embodiments of formula M3, W is CH and the ionizable cationic lipid has the structure: wherein X is as described herein. In certain embodiments of formula M3 X is . ch3 Y (CH2)2~ bH3 , wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00324] For example, in certain embodiments of formula M3, W is N and the ionizable cationic lipid has the structure: wherein X is as described herein. In certain embodiments of formula M3 . ch3 Y (CH2)2- X is            feH3 , wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00325] As described above, in some embodiments as described herein, the ionizable cationic lipid has a structure of formula M3, wherein A1 is (CH2)o, A2 is (CH2)i, A3 is (CH2)i, A4 is (CH2)o, and A5 is (CH2)i. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X and W are as described herein.

[00326] For example, in certain embodiments of formula M3, W is CH and the ionizable cationic lipid has the structure: . CH3 Y (CH2)2-N' ten wherein X is as described herein . In certain embodiments of formula M3, X is              3 , wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00327] For example, in certain embodiments of formula M3, W is N and the ionizable cationic lipid has the structure: . CH3 Y (CH2)2-N' ten wherein X is as described herein. In certain embodiments of formula M3, X is              3 , wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00328] As described above, in some embodiments as described herein, the ionizable cationic lipid has a structure of formula M3, wherein A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is (CH2)o. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X and W are as described herein.

[00329] For example, in certain embodiments of formula M3, W is CH and the ionizable cationic lipid has the structure: . ch3 Y (CH2)2-N' wherein X is as described herein. In certain embodiments of formula M3, X is              3 , wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00330] For example, in certain embodiments of formula M3, W is N and the ionizable cationic lipid has the structure: . ch3 Y (CH2)2-N' wherein X is as described herein. In certain embodiments of formula M3, X is              3 , wherein Y is O. For example, in certain embodiments, W is N and the ionizable cationic lipid has the structure:

[00331] As described above, in some embodiments as described herein, the ionizable cationic lipid has a structure of formula M3, wherein A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is (CH2)i. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X and W are as described herein.

[00332] For example, in certain embodiments of formula M3, W is CH and the ionizable cationic lipid has the structure: . CH3 Y (CH2)2-ten wherein X is as described herein. In certain embodiments of formula M3, X is              3 , wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00333] For example, in certain embodiments of formula M3, W is N and the ionizable cationic lipid has the structure: . CH3 Y (CH2)2-N' ten wherein X is as described herein. In certain embodiments of formula M3, X is              3 , wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00334] As described above, in some embodiments as described herein, the ionizable cationic lipid has a structure of formula M3 wherein A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)0, A4 is (CH2)o, and A5 is (CH2)2 or CH=CH. For example, in some embodiments as described herein, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is (CH2)2. In some embodiments as described herein, the ionizable cationic lipid has the structure: wherein X and W are as described herein.

[00335] For example, in certain embodiments of formula M3, W is CH and the ionizable cationic lipid has the structure: . CH3 Y (CH2)2— ten wherein X is as described herein. In certain embodiments of formula M3, X is              3 , wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00336] For example, in certain embodiments of formula M3, W is N and the ionizable cationic lipid has the structure: . CH3 Y (CH2)2— ten wherein X is as described herein. In certain embodiments of formula M3, X is              3 , wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00337] In certain embodiments of formula M3 as described herein, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is CH=CH. For example, in some embodiments, the ionizable cationic lipid has the structure: wherein X and W are as described herein.

[00338] For example, in certain embodiments of formula M3, W is CH and the ionizable cationic lipid has the structure: . ch3 Y (CH2)2-wherein X is as described herein. In certain embodiments of formula M3, X is              3 , wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00339] For example, in certain embodiments of formula M3, W is N and the ionizable cationic lipid has the structure: wherein X is as described herein. . CH3 Y (CH2)2-

[00340] In certain embodiments of formula M3, X is              3, wherein Y is O. For example, in certain embodiments, the ionizable cationic lipid has the structure:

[00341] In certain embodiments, an ionizable cationic lipid of this disclosure is substantially enantiomerically pure (e.g., 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 100% of one enantiomer). In certain embodiments, an ionizable cationic lipid of this disclosure is a racemic mixture. In certain embodiments, an ionizable cationic lipid of this disclosure is a mixture of two or more stereoisomers. In certain embodiments, at least two of the two or more stereoisomers are diastereomers. In certain embodiments, at least two of the two or more stereoisomers are enantiomers.

[00342] Ionizable cationic lipids as described herein, can be useful as a component of lipid nanoparticles for delivering nucleic acids, including DNA, mRNA, or siRNA into cells. The ionizable cationic lipids can have a c-pKa (calculated pKa) in the range of from about 6, 7, or 8 to about 9, 10, or 11. For example, in various embodiments as described herein, the ionizable cationic lipids have a c-pKa ranging from about 6 to about 10, about 7 to about 10, about 8 to about 10, about 8 to about 9, 6 to 10, 7 to 10, 8 to 10, or 8 to 9. In certain embodiments, the ionizable cationic lipids have a c-pKa ranging from about 8.2 to about 9.0 or from 8.2 to 9.0. In certain embodiments, the ionizable cationic lipids have a c-pKa ranging from about 8.4 to about 8.7 or from 8.4 to 8.7. The ionizable cationic lipids as described herein can have cLogD ranging from about 9 to about 18, for example, ranging from about 10 to about 18, or about 10 to about 16, to about 10 to about 14, or about 11 to about 18, or about 11 to about 15, or about 11 to about 14, or about 12 to about 14. The ionizable cationic lipids as described herein can have cLogD ranging from 9 to 18, for example, ranging from 10 to 18, or 10 to 16, to 10 to 14, or 11 to 18, or 11 to 15, or 11 to 14, or 12 to 14. In certain embodiments, the ionizable cationic lipids have a cLogD ranging from about 13.6 to about 14.4 or from 13.6 to 14.4. In certain embodiments, the ionizable cationic lipids as described herein can have a c-pKa ranging from about 8 to about 11 or from 8 to 11 and a cLogD ranging from about 9 to about 18 or from 9 to 18. For example, in certain embodiments, the ionizable cationic lipids have a c-pKa ranging from about 8.4 to about 8.7 or from 8.4 to 8.7 and cLogD ranging from about 13.6 to about 14.4 or from 13.6 to 14.4. These ranges can lead to a measured pKa in the LNP ranging from about 6 to about 7 or from 6 to 7, which facilitates ionization in an endosome after delivery into a cell.

[00343] Ionizable cationic lipids of this disclosure have a branched structure to give the lipid a conical rather than cylindrical three-dimensional shape and such structure helps promote endosomolytic activity when incorporated into an LNP. The greater the endosomolytic activity, the more efficient is release of the biologically active payload (e.g., one or more species of nucleic acid molecule).

[00344] Another consideration in designing or selecting a particular ionizable cationic lipid for use in an LNP for delivering nucleic acids (or other negatively charged payloads) into cells is the type of cell and, for in vivo delivery, locus within the body targeted for delivery. This is also influenced by pKa. Zhang et al. (iScience 27:109804, 2024) relates that measured pKa >9 can favor accumulation in the lungs, measured pKa of 2-6 can favor accumulation in spleen, and measured pKa of 6-7 can favor accumulation in liver. Thus, ionizable lipids with pKa across a wide range can be useful for delivery to various tissues. Of course, other factors can also impact what tissue or cells an LNP will deliver its payload to, including the use of a targeting moiety attached to the surface of the LNP.

[00345] In some embodiments, somewhat greater basicity can be desirable and can be obtained from ionizable cationic lipids with c-pKa and cLogD in the ranges disclosed herein. In some embodiments, cLogD of ionizable cationic lipids of this disclosure is about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or in a range bound by any pair of these values. Lipid design also accounts for potential biodegradability pathways of target lipids, such as by way of esterases in plasma, liver and other tissues. Another consideration in lipid design is the fate of fragments of ionizable lipids resulting from degradation, such as after esterase cleavage(s). Preferably, the resulting fragments are rapidly cleared from the body without the need for hepatic oxidative metabolism.

[00346] As used herein, cLogD is a calculated measure of lipophilicity that accounts for the state of ionization of the lipid molecule at a particular pH, which is a predictor of partitioning of the lipid between water and octanol as a function of pH. More specifically, cLogD is calculated at a specified pH based on cLogP and c-pKa. (LogP is the partition coefficient of a lipid molecule between aqueous (e.g., water) and lipophilic (e.g., octanol) phases). Numerous software packages are available to calculate cLogD values. When higher basicity of an ionizable lipid is desired, it should be balanced by greater lipophilicity as represented by a higher cLogD value. Balance of basicity and lipophilicity is needed for optimal functioning of the LNP for both stability of the particle and release of the biologically active payload (e.g., one or more species of nucleic acid molecule that can encode a therapeutic agent) upon uptake by a cell. Accordingly, for ionizable cationic lipids of this disclosure having a structure of formula M3 or M6, as R1 increases from Ce-C™, the increase overall lipophilicity of the ionizable cationic lipid will increase, as represented by cLogD. This can be balanced by alterations in X, which result in higher c-pKa based on the basicity of the head group. Each of the ionizable cationic lipid species described herein have a cLogD and c-pKa values within the desired range(s), as described herein. Specific cLogD and c-pKa values have been calculated using ACD Labs Structure Designer v 12.0 for ionic cationic lipids of the disclosure. cLogP was calculated using ACD Labs Version B; cLogD was calculated at pH 7.4. Table 1 shows cLogD and c-pKa for CICL-251 - CICL-290. Table 1. cLogD and c-pKa of Exemplary CICL Lipid c-LogD (pH 7.4) c-pKa CICL-252 13.76 8.42 CICL-253 13.76 8.42 CICL-251 13.84 8.42 CICL-254 13.84 8.42 CICL-255 13.84 8.42 CICL-256 13.84 8.42 CICL-257 13.76 8.42 CICL-258 13.76 8.42 CICL-259 14.05 8.42 CICL-260 14.05 8.42 CICL-261 13.89 8.50 CICL-262 13.89 8.50 CICL-263 14.50 8.50 CICL-264 14.50 8.50 CICL-265 13.80 8.49 CICL-266 13.80 8.49 CICL-267 15.04 8.49 CICL-268 15.04 8.49 CICL-269 15.03 8.50 CICL-270 15.03 8.50 CICL-271 13.96 8.42 CICL-272 13.96 8.42 CICL-273 14.04 8.42 CICL-274 14.04 8.42 CICL-275 14.04 8.42 CICL-276 14.04 8.42 CICL-277 13.96 8.42 CICL-278 13.96 8.42 CICL-279 14.24 8.42 Lipid c-LogD (pH 7.4) c-pKa CICL-280 14.24 8.42 CICL-281 14.09 8.50 CICL-282 14.09 8.50 CICL-283 14.70 8.50 CICL-284 14.70 8.50 CICL-285 14.06 8.49 CICL-286 14.06 8.49 CICL-287 15.24 8.49 CICL-288 15.24 8.49 CICL-289 15.22 8.50 CICL-290 15.22 8.50

[00347] In some embodiments, the ionizable cationic lipid has the structure CICL-252: CICL-252 is an example of a lipid as disclosed herein in which A1 and A2 are absent, A3, A4, and A5 are CH2, W is CH, and the tail groups are symmetrically placed relative to the ring nitrogen in a cis configuration. CICL-252 is symmetric with the two nominal cis configurations being superimposable, due to the tail groups being identical. Other embodiments include diastereomers of this compound (e.g., CICL-253), as well as mixtures including two or more of the various stereoisomers.

[00348] In some embodiments, the ionizable cationic lipid has the structure CICL-253: CICL-253 is an example of a lipid as disclosed herein in which A1 and A2 are absent, and A3, A4, and A5 are CH2, W is CH, and the tail groups are symmetrically placed relative to the ring nitrogen in a trans configuration. CICL-253 was synthesized as a racemate so that the structure drawn above indicates the relative stereochemistry for this lipid. Other embodiments include the absolute stereochemistry drawn above, its enantiomer, and mixtures of the enantiomers with various ratios. Other embodiments include diastereomers of these compounds (e.g., CICL-252), as well as mixtures including two or more of the various stereoisomers.

[00349] In some embodiments, the ionizable cationic lipid has the structure CICL-251: CICL-251 CICL-251 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-251 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios). Other embodiments include diastereomers of these compounds (e.g., CICL-254 and CICL-256), as well as mixtures including two or more of the various stereoisomers.

[00350] In some embodiments, the ionizable cationic lipid has the structure CICL-251-153: CICL-251-153 CICL-251-153 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-251-153 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00351] In some embodiments, the ionizable cationic lipid has the structure CICL-251-154: CICL-251-154 CICL-251-154 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-251-154 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00352] In some embodiments, the ionizable cationic lipid has the structure CICL-251-155: CICL-251-155 CICL-251-155 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-251-155 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00353] In some embodiments, the ionizable cationic lipid has the structure CICL-251-156: CICL-251-156 CICL-251-156 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-251-156 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00354] In some embodiments, the ionizable cationic lipid has the structure CICL-251-157: CICL-251-157 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-251-157 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00355] In some embodiments, the ionizable cationic lipid has the structure CICL-251-158: CICL-251-158 CICL-251-158 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-251-158 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00356] In some embodiments, the ionizable cationic lipid has the structure CICL-251-159: CICL-251-159 CICL-251-159 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-251-159 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00357] In some embodiments, the ionizable cationic lipid has the structure CICL-251-160: CICL-251-160 CICL-251-160 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-251-160 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00358] In some embodiments, the ionizable cationic lipid has the structure CICL-251-161: CICL-251-161 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-251-161 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00359] In some embodiments, the ionizable cationic lipid has the structure CICL-251-162: CICL-251-162 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-251-162 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00360] In some embodiments, the ionizable cationic lipid has the structure CICL-251-163: CICL-251-163 CICL-251-163 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-251-163 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00361] In some embodiments, the ionizable cationic lipid has the structure CICL-251-164: CICL-251-164 CICL-251-164 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-251-164 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00362] In some embodiments, the ionizable cationic lipid has the structure CICL-251-165: CICL-251-165 CICL-251-165 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-251-165 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00363] In some embodiments, the ionizable cationic lipid has the structure CICL-251-166: CICL-251-166 CICL-251-166 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-251-166 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00364] In some embodiments, the ionizable cationic lipid has the structure CICL-251-167: CICL-251-167 CICL-251-167 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-251-167 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00365] In some embodiments, the ionizable cationic lipid has the structure CICL-251-168: CICL-251-168 CICL-251-168 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-251-168 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00366] In some embodiments, the ionizable cationic lipid has the structure CICL-251-169: CICL-251-169 CICL-251-169 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-251-169 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00367] In some embodiments, the ionizable cationic lipid has the structure CICL-254: CICL-254 CICL-254 is an example of a lipid as disclosed herein in which A1 and A4 are absent, A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a cis configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-254 (CICL-256) and mixtures of the enantiomers (e.g., racemate, or other mixtures with various ratios). Other embodiments include diastereomers of this compound (e.g., CICL-255), as well as mixtures including two or more of the various stereoisomers.

[00368] In some embodiments, the ionizable cationic lipid has the structure CICL-255: CICL-255 CICL-255 is an example of a lipid as disclosed herein in which A1 and A4 are absent, A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-255 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios). Other embodiments include diastereomers of these compounds (e.g., CICL-254 and CICL-256), as well as mixtures including two or more of the various stereoisomers.

[00369] In some embodiments, the ionizable cationic lipid has the structure CICL-256: CICL-256 CICL-256 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a cis configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-256 (CICL-254) and mixtures of the enantiomers (e.g., racemate, or other mixtures with various ratios). Other embodiments include diastereomers of this compound (e.g., CICL-255), as well as mixtures including two or more of the various stereoisomers.

[00370] In some embodiments, the ionizable cationic lipid has the structure CICL-257: CICL-257 CICL-257 is an example of a lipid as disclosed herein in which A3, A4, and A5 are absent, A1 and A2 are CH2, W is CH, and the tail groups are symmetrically placed relative to the ring nitrogen in a cis configuration. CICL-257 is symmetric with the two nominal cis configurations being superimposable, due to the tail groups being identical. Other embodiments include diastereomers of this compound (e.g., CICL-258), as well as mixtures including two or more of the various stereoisomers.

[00371] In some embodiments, the ionizable cationic lipid has the structure CICL-258: CICL-258 CICL-258 is an example of a lipid as disclosed herein in which A3, A4, and A5 are absent, A1 and A2 are CH2, W is CH, and the tail groups are symmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-258 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios). Other embodiments include diastereomers of these compounds (e.g., CICL-257), as well as mixtures including two or more of the various stereoisomers.

[00372] In some embodiments, the ionizable cationic lipid has the structure CICL-259: CICL-259 CICL-259 is an example of a lipid as disclosed herein in which A1 and A2 are absent, A3 and A4 are CH2, A5 is CH2CH2, W is CH, and the tail groups are symmetrically placed relative to the ring nitrogen in a cis configuration. CICL-259 is symmetric with the two nominal cis configurations being superimposable, due to the tail groups being identical. Other embodiments include diastereomers of this compound (e.g., CICL-260), as well as mixtures including two or more of the various stereoisomers.

[00373] In some embodiments, the ionizable cationic lipid has the structure CICL-260: CICL-260 CICL-260 is an example of a lipid as disclosed herein in A1 and A2 are absent, A3 and A4 are CH2, A5 is CH2CH2, W is CH, and the tail groups are symmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-260 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios). Other embodiments include diastereomers of these compounds (e.g., CICL-259), as well as mixtures including two or more of the various stereoisomers.

[00374] In some embodiments, the ionizable cationic lipid has the structure CICL-261: CICL-261 CICL-261 is an example of a lipid as disclosed herein in which A1, A2, and A5 are CH2, and A3 and A4 are absent, W is CH, and the tail groups are symmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-261 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios). Other embodiments include diastereomers of these compounds (e.g., CICL-262), as well as mixtures including two or more of the various stereoisomers.

[00375] In some embodiments, the ionizable cationic lipid has the structure CICL-262: CICL-262 is an example of a lipid as disclosed herein in which A1, A2, and A5 are CH2, A3 and A4 are absent, W is CH, and the tail groups are symmetrically placed relative to the ring nitrogen in a cis configuration. CICL-262 is symmetric with the two nominal cis configurations being superimposable, due to the tail groups being identical. Other embodiments include diastereomers of this compound (e.g., CICL-261), as well as mixtures including two or more of the various stereoisomers.

[00376] In some embodiments, the ionizable cationic lipid has the structure CICL-263: CICL-263 is an example of a lipid as disclosed herein in which A1 and A2 are absent, A3 and A4, are CH2, A5 is CH2CH2CH2, W is CH, and the tail groups are symmetrically placed relative to the ring nitrogen in a cis configuration. CICL-263 is symmetric with the two nominal cis configurations being superimposable, due to the tail groups being identical. Other embodiments include diastereomers of this compound (e.g., CICL-264), as well as mixtures including two or more of the various stereoisomers.

[00377] In some embodiments, the ionizable cationic lipid has the structure CICL-264: CICL-264 CICL-264 is an example of a lipid as disclosed herein in which A1 and A2 are absent, A3 and A4, are CH2, A5 is CH2CH2CH2, W is CH, and the tail groups are symmetrically placed relative to the ring nitrogen in a trans configuration. CICL-264 was synthesized as a racemate, so that the structure drawn above indicates the relative stereochemistry for this lipid. Other embodiments include the absolute stereochemistry drawn above, its enantiomer, and mixtures of the enantiomers with various ratios. Other embodiments include diastereomers of these compounds (e.g., CICL-263), as well as mixtures including two or more of the various stereoisomers.

[00378] In some embodiments, the ionizable cationic lipid has the structure CICL-265: CICL-265 CICL-265 is an example of a lipid as disclosed herein in which A3 and A4 are absent, A1 and A2 are CH2, A5 is CH=CH, W is CH, and the tail groups are symmetrically placed relative to the ring nitrogen in a cis configuration. CICL-265 is symmetric with the two nominal cis configurations being superimposable, due to the tail groups being identical. Other embodiments include diastereomers of this compound (e.g., CICL-266), as well as mixtures including two or more of the various stereoisomers.

[00379] In some embodiments, the ionizable cationic lipid has the structure CICL-266: CICL-266 CICL-266 is an example of a lipid as disclosed herein in which A3 and A4 are absent, A1 and A2 are CH2, A5 is CH=CH, W is CH, and the tail groups are symmetrically placed relative to the ring nitrogen in a trans configuration. CICL-266 was synthesized as a racemate so that the structure drawn above indicates the relative stereochemistry for this lipid. Other embodiments include the absolute stereochemistry drawn above, its enantiomer and mixtures of the enantiomers with various ratios. Other embodiments include diastereomers of these compounds (e.g., CICL-265), as well as mixtures including two or more of the various stereoisomers.

[00380] In some embodiments, the ionizable cationic structure has the structure CICL-267: CICL-267 CICL-267 is an example of a lipid as disclosed herein in which A1 and A2 are absent, A3 and A4 are CH2, A5 is CH2CHCHCH2, W is CH, and the tail groups are symmetrically placed relative to the ring nitrogen in a cis configuration. CICL-267 is symmetric with the two nominal cis configurations being superimposable, due to the tail groups being identical. Other embodiments include diastereomers of this compound (e.g., CICL-268), as well as mixtures including two or more of the various stereoisomers.

[00381] In some embodiments, the ionizable cationic lipid has the structure CICL-268: CICL-268 is an example of a lipid as disclosed herein in which A1 and A2 are absent, A3 and A4 are CH2, A5 is CH2CHCHCH2, W is CH, and the tail groups are symmetrically placed relative to the ring nitrogen in a trans configuration. CICL-268 was synthesized as a racemate so the structure drawn above indicates the relative stereochemistry for this lipid. Other embodiments include the absolute stereochemistry drawn above, its enantiomer, and mixtures of the enantiomers with various ratios. Other embodiments include diastereomers of these compounds (e.g., CICL-267), as well as mixtures including two or more of the various stereoisomers.

[00382] In some embodiments, the ionizable cationic lipid has the structure CICL-269: CICL-269 is an example of a lipid as disclosed herein in which A1 and A2 are absent, A3 and A4 are CH2, A5 is CH2(CH2)2CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a cis configuration. CICL-269 is symmetric with the two nominal cis configurations being superimposable, due to the tail groups being identical. Other embodiments include diastereomers of this compound (e.g., CICL-270), as well as mixtures including two or more of the various stereoisomers.

[00383] In some embodiments, the ionizable cationic lipid has the structure CICL-270: O CICL-270 CICL-270 is an example of a lipid as disclosed herein in which A1 and A2 are absent, A3 and A4 are CH2, A5 is CH2(CH2)2CH2, W is CH, and the tail groups are symmetrically placed relative to the ring nitrogen in a trans configuration. CICL-270 was synthesized as a racemate so the structure drawn above indicates the relative stereochemistry for this lipid. Other embodiments include the absolute stereochemistry drawn above, its enantiomer, and mixtures of the enantiomers with various ratios. Other embodiments include diastereomers of these compounds (e.g., CICL-269), as well as mixtures including two or more of the various stereoisomers.

[00384] In some embodiments, the ionizable cationic lipid has the structure CICL-271: CICL-271 CICL-271 is an example of a lipid as disclosed herein in which A1 and A2 are absent, A3, A4, and A5 are CH2, W is N, and the tail groups are symmetrically placed relative to the ring nitrogen in a cis configuration. CICL-271 is symmetric with the two nominal cis configurations being superimposable, due to the tail groups being identical. Other embodiments include diastereomers of this compound (e.g., CICL-272), as well as mixtures including two or more of the various stereoisomers.

[00385] In some embodiments, the ionizable cationic lipid has the structure CICL-272: o CICL-272 CICL-272 is an example of a lipid as disclosed herein in which A1 and A2 are absent, and A3, A4, and A5 are CH2, W is N, and the tail groups are symmetrically placed relative to the ring nitrogen in a trans configuration. CICL-272 was synthesized as a racemate so the structure drawn above indicates the relative stereochemistry for this lipid. Other embodiments include the absolute stereochemistry drawn above, its enantiomer, and mixtures of the enantiomers with various ratios. Other embodiments include diastereomers of these compounds (e.g., CICL-271), as well as mixtures including two or more of the various stereoisomers

[00386] In some embodiments, the ionizable cationic lipid has the structure CICL-273: CICL-273 CICL-273 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is N, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-273 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios). Other embodiments include diastereomers of these compounds (e.g., CICL-274 and CICL-275), as well as mixtures including two or more of the various stereoisomers.

[00387] In some embodiments, the ionizable cationic lipid has the structure CICL-274: CICL-274 CICL-274 is an example of a lipid as disclosed herein in which A1 and A4 are absent, A2, A3, and A5 are CH2, W is N, and the tail groups are asymmetrically placed relative to the ring nitrogen in a cis configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-274 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios). Other embodiments include diastereomers of this compound (e.g., CICL-273), as well as mixtures including two or more of the various stereoisomers

[00388] In some embodiments, the ionizable cationic lipid has the structure CICL-275: CICL-275 CICL-275 is an example of a lipid as disclosed herein in which A1 and A4 are absent, A2, A3, and A5 are CH2, W is N, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-275 (CICL-273) and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios). Other embodiments include diastereomers of these compounds (e.g., CICL-274 and CICL-276), as well as mixtures including two or more of the various stereoisomers.

[00389] In some embodiments, the ionizable cationic lipid has the structure CICL-276: CICL-276 CICL-276 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is N, and the tail groups are asymmetrically placed relative to the ring nitrogen in a cis configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-276 (CICL-274) and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios). Other embodiments include diastereomers of this compound (e.g., CICL-273 and CICL-275) as well as mixtures including two or more of the various stereoisomers.

[00390] In some embodiments, the ionizable cationic lipid has the structure CICL-277: CICL-277 CICL-277 is an example of a lipid as disclosed herein in which A3, A4, and A5 are absent, A1 and A2 are CH2, W is N, and the tail groups are symmetrically placed relative to the ring nitrogen in a cis configuration. CICL-277 is symmetric with the two nominal cis configurations being superimposable, due to the tail groups being identical. Other embodiments include diastereomers of this compound (e.g., CICL-278), as well as mixtures including two or more of the various stereoisomers.

[00391] In some embodiments, the ionizable cationic lipid has the structure CICL-278: CICL-278 CICL-278 is an example of a lipid as disclosed herein in which A3, A4, and A5 are absent, A1 and A2 are CH2, W is N, and the tail groups are symmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-278 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios). Other embodiments include diastereomers of these compounds (e.g., CICL-277), as well as mixtures including two or more of the various stereoisomers.

[00392] In some embodiments, the ionizable cationic lipid has the structure CICL-279: CT 0 CICL-279 CICL-279 is an example of a lipid as disclosed herein in which A1 and A2 are absent, A3 and A4 are CH2, A5 is CH2CH2, Wis N, and the tail groups are symmetrically placed relative to the ring nitrogen in a cis configuration. CICL-279 is symmetric with the two nominal cis configurations being superimposable, due to the tail groups being identical. Other embodiments include diastereomers of this compound (e.g., CICL-280), as well as mixtures including two or more of the various stereoisomers.

[00393] In some embodiments, the ionizable cationic lipid has the structure CICL-280: 0^° CICL-280 CICL-280 is an example of a lipid as disclosed herein in A1 and A2 are absent, A3 and A4 are CH2, A5 is CH2CH2, W is N, and the tail groups are symmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-280 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios). Other embodiments include diastereomers of these compounds (e.g., CICL-279), as well as mixtures including two or more of the various stereoisomers.

[00394] In some embodiments, the ionizable cationic lipid has the structure of CICL-281: CICL-281 CICL-281 is an example of a lipid as disclosed herein in which A1, A2, and A5 are CH2, and A3 and A4 are absent, W is N, and the tail groups are symmetrically placed relative to the ring nitrogen in a trans configuration. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-281 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios). Other embodiments include diastereomers of these compounds (e.g., CICL-282), as well as mixtures including two or more of the various stereoisomers.

[00395] In some embodiments, the ionizable cationic lipid has the structure CICL-282: CICL-282 is an example of a lipid as disclosed herein in which A1, A2, and A5 are CH2, A3 and A4 are absent, Wis N, and the tail groups are symmetrically placed relative to the ring nitrogen in a cis configuration. CICL-282 is symmetric with the two nominal cis configurations being superimposable, due to the tail groups being identical. Other embodiments include diastereomers of this compound (e.g., CICL-281), as well as mixtures including two or more of the various stereoisomers.

[00396] In some embodiments, the ionizable cationic lipid has the structure CICL-283: CICL-283 is an example of a lipid as disclosed herein in which A1 and A2 are absent, A3 and A4, are CH2, A5 is CH2CH2CH2, W is N, and the tail groups are symmetrically placed relative to the ring nitrogen in a cis configuration. CICL-283 is symmetric with the two nominal cis configurations being superimposable, due to the tail groups being identical. Other embodiments include diastereomers of this compound (e.g., CICL-284), as well as mixtures including two or more of the various stereoisomers.

[00397] In some embodiments, the ionizable cationic lipid has the structure CICL-284: CICL-284 CICL-284 is an example of a lipid as disclosed herein in which A1 and A2 are absent, A3 and A4, are CH2, A5 is CH2CH2CH2, W is N, and the tail groups are symmetrically placed relative to the ring nitrogen in a trans configuration. CICL-284 was synthesized as a racemate so the structure drawn above indicates the relative stereochemistry for this lipid. Other embodiments include the absolute stereochemistry drawn above, its enantiomer, and mixtures of the enantiomers with various ratios. Other embodiments include diastereomers of these compounds (e.g., CICL-283), as well as mixtures including two or more of the various stereoisomers.

[00398] In some embodiments, the ionizable cationic lipid has the structure CICL-285: CICL-285 CICL-285 is an example of a lipid as disclosed herein in which A3 and A4 are absent, A1 and A2 are CH2, A5 is CH=CH, W is N, and the tail groups are symmetrically placed relative to the ring nitrogen in a cis configuration. CICL-285 is symmetric with the two nominal cis configurations being superimposable, due to the tail groups being identical. Other embodiments include diastereomers of this compound (e.g., CICL-286), as well as mixtures including two or more of the various stereoisomers.

[00399] In some embodiments, the ionizable cationic lipid has the structure CICL-286: CICL-286 CICL-286 is an example of a lipid as disclosed herein in which A3 and A4 are absent, A1 and A2 are CH2, A5 is CH=CH, W is N, and the tail groups are symmetrically placed relative to the ring nitrogen in a trans configuration. CICL-286 was synthesized as a racemate so the structure drawn above indicates the relative stereochemistry for this lipid. Other embodiments include the absolute stereochemistry drawn above, its enantiomer and mixtures of the enantiomers with various ratios. Other embodiments include diastereomers of these compounds (e.g., CICL-285), as well as mixtures including two or more of the various stereoisomers.

[00400] In some embodiments, the ionizable cationic lipid has the structure CICL-287: CICL-287 CICL-287 is an example of a lipid as disclosed herein in which A1 and A2 are absent, A3 and A4 are CH2, A5 is CH2CHCHCH2, Wis N, and the tail groups are symmetrically placed relative to the ring nitrogen in a cis configuration. CICL-287 is symmetric with the two nominal cis configurations being superimposable, due to the tail groups being identical. Other embodiments include diastereomers of this compound (e.g., CICL-288), as well as mixtures including two or more of the various stereoisomers.

[00401] In some embodiments, the ionizable cationic lipid has the structure CICL-288: O CICL-288 CICL-288 is an example of a lipid as disclosed herein in which A1 and A2 are absent, A3 and A4 are CH2, A5 is CH2CHCHCH2, Wis N, and the tail groups are symmetrically placed relative to the ring nitrogen in a trans configuration. CICL-288 was synthesized as a racemate so the structure drawn above indicates the relative stereochemistry for this lipid. Other embodiments include the absolute stereochemistry drawn above, its enantiomer, and mixtures of the enantiomers with various ratios. Other embodiments include diastereomers of these compounds (e.g., CICL-287), as well as mixtures including two or more of the various stereoisomers.

[00402] In some embodiments, the ionizable cationic lipid has the structure CICL-289: CICL-289 CICL-289 is an example of a lipid as disclosed herein in which A1 and A2 are absent, A3 and A4 are CH2, A5 is CH2(CH2)2CH2, W is N, and the tail groups are symmetrically placed relative to the ring nitrogen in a cis configuration. CICL-289 is symmetric with the two nominal cis configurations being superimposable, due to the tail groups being identical. Other embodiments include diastereomers of this compound (e.g., CICL-290), as well as mixtures including two or more of the various stereoisomers.

[00403] In some embodiments, the ionizable cationic lipid has the structure CICL-290: O CICL-290 CICL-290 is an example of a lipid as disclosed herein in which A1 and A2 are absent, A3 and A4 are CH2, A5 is CH2(CH2)2CH2, W is N, and the tail groups are symmetrically placed relative to the ring nitrogen in a trans configuration. CICL-290 was synthesized as a racemate so the structure drawn above indicates the relative stereochemistry for this lipid. Other embodiments include the absolute stereochemistry drawn above, its enantiomer, and mixtures of the enantiomers with various ratios. Other embodiments include diastereomers of these compounds (e.g., CICL-289), as well as mixtures including two or more of the various stereoisomers.

[00404] In some embodiments, the ionizable cationic lipid has the structure CICL-304: CICL-304 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. In each tail group, one R2 is O and one R2 is C=O, and one R3 is O and one R3 is C=O. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-304 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00405] In some embodiments, the ionizable cationic lipid has the structure CICL-305: CICL-305 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. In the first tail group (i.e., the 2S tail group), one R2 is O and one R2 is C=O, and one R3 is O and one R3 is C=O. In the second tail group (i.e., the 4R tail group), both R2 groups are O and both R3 groups are C=O. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-305 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00406] In some embodiments, the ionizable cationic lipid has the structure CICL-306: CICL-306 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. In the first tail group (i.e., the 2S tail group), both R2 groups are O and both R3 groups are C=O. In the second tail group (i.e., the 4R tail group), one R2 is O and one R2 is C=O, and one R3 is O and one R3 is C=O. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-306 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00407] In some embodiments, the ionizable cationic lipid has the structure CICL-307: CICL-307 CICL-307 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. In the first tail group (i.e., the 2S tail group), one R2 is O and one R2 is C=O, and one R3 is O and one R3 is C=O. In the second tail group (i.e., the 4R tail group), both R2 groups are C=O and both R3 groups are O. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-307 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00408] In some embodiments, the ionizable cationic lipid has the structure CICL-308: CICL-308 is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. In the first tail group (i.e., the 2S tail group), both R2 groups are C=O and both R3 groups are O. In the second tail group (i.e., the 4R tail group), one R2 is 0 and one R2 is C=0, and one R3 is 0 and one R3 is C=0. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-308 and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00409] In some embodiments, the ionizable cationic lipid has the structure CICL-308a: CICL-308a CICL-308a is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. In the first tail group (i.e., the 2S tail group), both R2 groups are C=O and both R3 groups are O. In the second tail group (i.e., the 4R tail group), both R2 groups are O and both R3 groups are C=O. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-308a and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00410] In some embodiments, the ionizable cationic lipid has the structure CICL-308b: CICL-308b is an example of a lipid as disclosed herein in which A1 and A4 are absent, and A2, A3, and A5 are CH2, W is CH, and the tail groups are asymmetrically placed relative to the ring nitrogen in a trans configuration. In the first tail group (i.e., the 2S tail group), both R2 groups are O and both R3 groups are C=O. In the second tail group (i.e., the 4R tail group), both R2 groups are C=O and both R3 groups are O. The structure drawn above indicates the absolute stereochemistry for this lipid. Other embodiments include the enantiomer of CICL-308b and mixtures of the enantiomers (e.g., a racemate, or other mixtures with various ratios).

[00411] In some embodiments, the ionizable cationic lipid has the structure of the following: CICL-253-154 CICL-253-159 CICL-253-160 CICL-253-157 CICL-253-167 t Jp ^° Y° ^Y^Y° CICL-255-154 pPY0 pP^opY° A AY Y^ V / ^o CICL-255-155 pY^Y° pY^OpY0 '-■^f CICL-255-157 ^p^Y0 ^p^^Yy p^y^Y0 ^p^o^Yy tototo to pY^Y° pY^°pY° r Yo YY'0'^ V / ^o CICL-255-159 y y^cr0 CICL-255-160 CICL-255-167 r^^°T° pAv to r p Y / p   Y 0   ° Y ^YA 'to pj^Y0 p^opY0 Y ( Yz^0 A pY^Y0 AyY ^Y^cA CICL-258-154 CICL-258-155 '^xoA' CICL-258-157 pP^Y0 to € Y Ppo Y >?y p< ° Q ° c^o Cp^ pY'Y0 poY>° Y r pp YY'u Vyo CICL-258-159 CICL-258-160 ^^^A^p-'Cj'0 CICL-258-167 pj"^Y° P’X ^Y\p ?b CICL-260-154 pY^Y0 yY \ Ay X^cYPc /    x ^Yz-O^0 CICL-260-155 AApA^ ^'Y / Y0 CICL-260-157 CICL-260-159 CICL-260-160 CICL-260-167 CICL-261-155 CICL-261-157 CICL-261-154 CICL-261-159 CICL-264-159 CICL-264-155 CICL-261-167 CICL-264-157 CICL-264-160 CICL-264-167 CICL-266-154 CICL-266-157 CICL-266-160 CICL-266-167 CICL-268-154 CICL-268-155 CICL-268-157 CICL-268-167 CICL-268-159 CICL-268-160 CICL-273-154 CICL-273-155 CICL-273-157 CICL-273-159 CICL-273-160 CICL-273-167 CICL-275-154 CICL-275-155 CICL-275-157 CICL-275-159 CICL-275-160 CICL-275-167 CICL-278-154 CICL-278-155 CICL-278-157 / -xj^Y0 ^r%x oajAO l^ / ^o CICL-278-159 CICL-278-160 CICL-278-167 a-o r^V0 3b bf XO o CICL-280-154 CICL-280-155 CICL-280-157 oa° J^°Y° yA A ^Wv-A^ V CICL-280-159 CICL-280-160 CICL-280-167 rxJ^V0 r-xJ^'V0 z. XN^ ^r^o^ / “V° A^VO0 OX AAX 0 o \       ° d     HA \''' / A^ X Xb CICL-281-154 CICL-281-155 CICL-281-157 rxJ^V0 y"V° r^^0?0 a^vA0 rv^X^'Y0 rAb> o OhAi '-AAaA ' ox AOO Ij A V^oY_^° CICL-281-159 CICL-281-160 CICL-281-167 CICL-284-154 CICL-284-155 CICL-284-157 CICL-284-159 CICL-284-160 CICL-284-167 CICL-286-154 CICL-286-155 CICL-286-157 CICL-286-160 CICL-286-167 CICL-304-157 CICL-304-154 CICL-304-155 CICL-305-159 CICL-305-155 CICL-305-160 CICL-305-167

[00412] In some embodiments, the ionizable cationic lipid has the structure of the following: CICL-252-164 CICL-254-164 CICL-256-164 CICL-259-164 CICL-262-164 / \ / "Vp Y° / v r'\—o> )—K — Y° o \ / \ CICL-263-164 / 0 I                          0 I / ¼ 0 r^°r° y ^O- CICL-265-164 / / \— / / ^-0 / --V                 )=^0 '— / ^0  \_ o \_7 CICL-267-164 o t— ' ' YO V o I                             0 o     A 0 o y^_0_ I iYY O ' \_ / \ CICL-269-164 Yx^cr^o CICL-271-164 CICL-274-164 Aa / Y CICL-276-164 r^^Y0 ^A^-oi0 CICL-277-164 Y^V0 Yy x> o CICL-279-164 - «v Po CICL-282-164 / \ J \_ 0 C 0 Y> Vy-, .AA YC> CICL-283-164 i YX o N JI A. o Y<> CICL-285-164 \— / ^-0 A€ / Z \— / / ^0 cr   r^Q o Y^nY YPP cA YCY CICL-287-164 CICL-289-164

[00413] In other aspects of this disclosure are provided intermediate lipids of the ionizable cationic lipids disclosed herein. In certain aspects, the lipid (e.g., intermediate lipid) of this disclosure have a structure of formula M6-5, a stereoisomer thereof, or a mixture of such stereoisomers: wherein each W is independently CH or N; each R2 is independently O or C=O, wherein at least one R2 is O; each R3 is independently O or C=O, wherein when a R2 is O then its adjoining R3 is C=O, or when a R2 is C=O then its adjoining R3 is O; wherein when at least one R3 at a beta position of W is O, W is CH; and wherein when both R3 groups at a beta position of W are C=O, W is CH or N; each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl; R4 is H or a protecting group; each A1, A2, A3, and A4 is independently (CH2)oor (CH2)i, provided that A1 is (CH2)i when A3 is (CH2)o; A1 is (CH2)o when A3 is (CH2)i; A2 is (CH2)i when A4 is (CH2)o; and A2 is (CH2)o when A4 is (CH2)i; A5 is selected from (CH2)o-4, CH=CH, or CH2-CH=CH-CH2.

[00414] In various embodiments as described herein, R4is H.

[00415] In various embodiments as described herein, R4is a protecting group (i.e., PGi). PGi can be selected from base labile or acid labile protecting groups as known in the art. For example, in some embodiments, R4is an acid labile protecting group such as t-butoxycarbonyl (BOG) or benzyloxycarbonyl (Cbz). In some other embodiments, R4 is a base labile protecting group such as a trimethylsilylethoxycarbonyl moiety. In various embodiments of formula M6-5, R1, R2, R3, A1, A2, A3, A4, and A5 are as otherwise described here.

[00416] For example, in various embodiments of formula M6-5, A1 through A4 are such that there are only two main chain atoms between the ring nitrogen and each nearest ester oxygen in the nearest tail group. For example, in various embodiments of M6, each A1, A2, A3, and A4 is independently (CH2)0or (CH2)i, provided that A1 is (CH2)i when A3 is (CH2)0; A1 is (CH2)0 when A3 is (CH2)i; A2 is (CH2)i when A4 is (CH2)o; and A2 is (CH2)o when A4 is (CH2)i.

[00417] In certain embodiments of formula M6-5, A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)i-4 or CH2-CH=CH-CH2.

[00418] In certain embodiments of formula M6-5, A1 is (CH2)o, A2 is (CH2)i, A3 is (CH2)i, A4 is (CH2)0, and A5 is (CH2)i.

[00419] In certain embodiments of formula M6-5, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)0, and A5 is (CH2)0.

[00420] In certain embodiments of formula M6-5, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)0, and A5is(CH2)i.

[00421] In certain embodiments of formula M6-5, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)0, and A5 is (CH2)2 or CH=CH.

[00422] In certain aspects, the lipid (e.g., intermediate lipid) of this disclosure have a structure of formula M6-6, a stereoisomer thereof, or a mixture of such stereoisomers: each W is independently CH or N; each R2 is independently 0 or C=O, wherein at least one R2 is O; each R3 is independently O or C=O, wherein when a R2 is O then its adjoining R3 is C=O, or when a R2 is C=O then its adjoining R3 is O; wherein when at least one R3 at a beta position of W is O, W is CH; and wherein when both R3 groups at a beta position of W are C=0, W is CH or N; each R1 is independently C7-C11 alkyl or C7-C11 alkenyl; each A1, A2, A3, and A4 is independently (CH2)oor (CH2)i, provided that A1 is (CH2)i when A3 is (CH2)o; A1 is (CH2)o when A3 is (CH2)i; A2 is (CH2)i when A4 is (CH2)o; and A2 is (CH2)o when A4 is (CH2)i; A5 is (CH2)o-4, CH=CH, or CH2-CH=CH-CH2.

[00423] In various embodiments as described herein, R1, R2, R3, A1, A2, A3, A4, and A5 are as otherwise described here.

[00424] For example, in various embodiments of formula M6-6, A1 through A4 are such that there are only two main chain atoms between the ring nitrogen and each nearest ester oxygen in the nearest tail group. For example, in various embodiments of M6, each A1, A2, A3, and A4 is independently (CH2)oor (CH2)i, provided that A1 is (CH2)i when A3 is (CH2)o; A1 is (CH2)o when A3 is (CH2)i; A2 is (CH2)i when A4 is (CH2)o; and A2 is (CH2)o when A4 is (CH2)i.

[00425] In certain embodiments of formula M6-6, A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)i-4 or CH2-CH=CH-CH2.

[00426] In certain embodiments of formula M6-6, A1 is (CH2)o, A2 is (CH2)i, A3 is (CH2)i, A4 is (CH2)o, and A5 is (CH2)i.

[00427] In certain embodiments of formula M6-6, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is (CH2)o.

[00428] In certain embodiments of formula M6-6, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5is(CH2)i.

[00429] In certain embodiments of formula M6-6, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is (CH2)2 or CH=CH.

[00430] In other aspects of this disclosure are provided intermediate lipids of the ionizable cationic lipids disclosed herein. In certain aspects, the lipid (e.g., intermediate lipid) of this disclosure have a structure of formula M3-1, a stereoisomer thereof, or a mixture of such stereoisomers: A3 A5 N-R4 ^A2 wherein WisCH orN; each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl; R4 is H or a protecting group; each A1, A2, A3, and A4 is independently (CH2)oor (CH2)i, A5 is selected from (CH2)o-4, CH=CH, or CH2-CH=CH-CH2.

[00431] In various embodiments as described herein, R4is H.

[00432] In various embodiments as described herein, R4is a protecting group (i.e., PGi). PGi can be selected from base labile or acid labile protecting groups as known in the art. For example, in some embodiments, R4is an acid labile protecting group such as t-butoxycarbonyl (BOG) or benzyloxycarbonyl (Cbz). In some other embodiments, R4 is a base labile protecting group such as a trimethylsilylethoxycarbonyl moiety. In various embodiments of formula M3-1, R1, A1, A2, A3, A4, and A5 are as otherwise described here.

[00433] For example, in various embodiments of formula M3-1, A1 through A4 are such that there are only two main chain atoms between the ring nitrogen and each nearest ester oxygen in the nearest tail group.

[00434] In certain embodiments of formula M3-1, A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)i.4 or CH2-CH=CH-CH2.

[00435] In certain embodiments of formula M3-1, A1 is (CH2)0, A2 is (CH2)i, A3 is (CH2)i, A4 is (CH2)0, and A5 is (CH2)i.

[00436] In certain embodiments of formula M3-1, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)0, A4 is (CH2)0, and A5 is (CH2)0.

[00437] In certain embodiments of formula M3-1, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)0, A4 is (CH2)0, and A5is(CH2)i.

[00438] In certain embodiments of formula M3-1, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)0, A4 is (CH2)0, and A5 is (CH2)2 or CH=CH.

[00439] In certain aspects, the lipid (e.g., intermediate lipid) of this disclosure have a structure of formula M3-2, a stereoisomer thereof, or a mixture of such stereoisomers: WisCH orN; each R1 is independently C7-C11 alkyl or C7-C11 alkenyl; each A1, A2, A3, and A4 is independently (CH2)oor (CH2)i, A5 is (CH2)o-4, CH=CH, or CH2-CH=CH-CH2.

[00440] In various embodiments as described herein, R1, A1, A2, A3, A4, and A5 are as otherwise described here.

[00441] For example, in various embodiments of formula M3-2, A1 through A4 are such that there are only two main chain atoms between the ring nitrogen and each nearest ester oxygen in the nearest tail group.

[00442] In certain embodiments of formula M3-2, A1 is (CH2)0, A2 is (CH2)0, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)i.4 or CH2-CH=CH-CH2.

[00443] In certain embodiments of formula M3-2, A1 is (CH2)o, A2 is (CH2)i, A3 is (CH2)i, A4 is (CH2)0, and A5 is (CH2)i.

[00444] In certain embodiments of formula M3-2, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)0, and A5 is (CH2)0.

[00445] In certain embodiments of formula M3-2, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)0, and A5is(CH2)i.

[00446] In certain embodiments of formula M3-2, A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)0, and A5 is (CH2)2 or CH=CH.

[00447] To promote biodegradability and minimize accumulation of ionizable cationic lipids of this disclosure, the fatty acid tails are designed to comprise esters in a position that minimizes steric hindrance of ester cleavage. For example, while a single fatty acid tail will tend to extend away from the ester carbonyl to provide the most energetically favorable position, the presence of two tails leads to the tails extending in opposite directions to provide the most energetically favorable conformation. Conformation is dynamic and the fatty acid tails can also move through less energetically favorable positions. For example, in certain embodiments, one of the tails can extend toward the carbonyl and sterically hinders cleavage of the ester. Accordingly, large branches immediately adjacent to the ester carbonyl were avoided in the design of the ionizable cationic lipids disclosed herein. For example, in some embodiments, there is at least one carbon atom between the ester carbonyl of a tail group and the branch point (i.e., W, as described herein). In certain embodiments, there are at least two atoms between the ester carbonyl of a tail group and the branch point (i.e., W, as described herein).

[00448] In positioning the ester(s) within the lipid, consideration was also given to potential degradation products to avoid generation of toxic compounds, such as formaldehyde. The configuration of the esters around W also can affect the rate and pathway of biodegradation of the lipid. When an R3 is O, esterase-mediated cleavage of the ester releases a carboxylic acid and a hydroxyl remains which can cyclize in an intramolecular transesterification forming a butyrolactone and releasing the core structure, a reaction known to proceed rapidly. For this mechanism to proceed it is important that the anteriorly located esters (i.e., the esters nearest to the central branchpoint nitrogen atom) not be oriented with its O closer to W than its C=O, as in such a structure the transesterification would be a simple intramolecular ester exchange and no piece of the molecule would be released. Accordingly, to promote biodegradation, it is advantageous for a methylene to be at an alpha position of W.

[00449] An advantage of ionizable cationic lipids of this disclosure is that, at least in part, the toxicity associated with quaternary ammonium cationic lipids can be avoided. Some LNPs containing quaternary ammonium lipids, which are effectively permanently cationic, have displayed a fatal hyperacute toxicity in laboratory animals. In contrast, the use of ionizable cationic lipids of this disclosure in an LNP obviates the need for quaternary ammonium cationic lipids and, thereby, can mitigate or avoid potential LNP toxicity. In certain embodiments, use of an LNP or tLNP of this disclosure causes no detectable toxicity to cells or in a subject. In certain embodiments, use of an LNP or tLNP of this disclosure causes no more than mild toxicity to cells or in a subject that is asymptomatic or induces only mild symptoms that do not require intervention. In certain embodiments, use of an LNP or tLNP of this disclosure causes no more than moderate toxicity to cells or in a subject which may impair activities of daily living that requires only minimal, local, or non-invasive interventions.

[00450] The relationship between the efficacy and toxicity of a drug is generally expressed in terms of therapeutic window and therapeutic index. Therapeutic window is the dose range from the lowest dose that exhibits a detectable therapeutic effect up to the maximum tolerated dose (MTD); the highest dose that will the desired therapeutic effect without producing unacceptable toxicity. Most typically, therapeutic index is calculated as the ratio of LD5o:ED5o when based on animal studies and TD5o:EDso when based on studies in humans (though this calculation could also be derived from animal studies and is sometimes called the protective index), where LD50, TD50, and ED50 are the doses that are lethal, toxic, and effective in 50% of the tested population, respectively. These concepts are applicable whether the toxicity is based on the active agent itself or some other component of the drug product, such as, for example, the LNP or its components. For any inherent level of toxicity of the disclosed lipids or LNPs themselves, an increase in the efficiency of delivering the nucleic acid into the cytoplasm will improve the therapeutic window or index, as an effective amount of a biologically active payload (e.g., one or more species of nucleic acid molecule) would be deliverable with a smaller dosage of LNP (and its component lipids).

[00451] Toxicities and adverse events can be graded according to a 5-point scale. A grade 1 or mild toxicity is asymptomatic or induces only mild symptoms; may be characterized by clinical or diagnostic observations only; and intervention is not indicated. A grade 2 or moderate toxicity may impair activities of daily living (such as preparing meals, shopping, managing money, using the telephone, etc.) but only minimal, local, or non-invasive interventions are indicated. Grade 3 toxicities are medically significant but not immediately lifethreatening; hospitalization or prolongation of hospitalization is indicated; activities of daily living related to self-care (such as bathing, dressing and undressing, feeding oneself, using the toilet, taking medications, and not being bedridden) may be impaired. Grade 4 toxicities are life-threatening and urgent intervention is indicated. Grade 5 toxicity produces an adverse event-related death. Thus, in various embodiments, by use of the disclosed LNP and tLNP a toxicity is confined to grade 2 or less, grade 1 or less, or produces no observed toxicity. Tolerability

[00452] Conventional LNPs deliver primarily to the liver. Liver toxicity has been the major dose-limiting parameter observed with LNP-containing pharmaceuticals. For example, ONPATTRO®, comprising the ionizable lipid MC3, has a NOAEL (no observed adverse effect level) of only 0.3 mg / kg for multiple dosing in rats. A benchmark LNP comprising the ionizable cationic lipid ALC-0315, used in the SARS-CoV-2 vaccine COMIRNATY®, caused elevated levels of liver enzymes and acute phase proteins at single doses of >1 mg / kg in the rat. Merely attaching an antibody to the benchmark LNP partially reverses that elevation and the reversal is greater if the antibody directs the LNP to some other tissue (that is, a tLNP). However, use of a highly biodegradable ionizable cationic lipid, CICL-1, similar to those disclosed herein, reduced delivery to the liver and associated liver enzyme and acute phase protein levels to a greater extent for LNP, antibody-conjugated LNP, and tLNP. Methods of Making Ionizable Cationic Lipids

[00453] Structural symmetries and convergent rather than linear synthesis pathways can be used to simplify the synthesis of the ionizable lipids of this disclosure.

[00454] In certain aspects, this disclosure provides methods for synthesizing an ionizable cationic lipid of formula M6 (e.g., CICL-304, CICL-305, CICL-306, CICL-307, CICL-308, CICL-XX1, and CICL-XX2).

[00455] In certain aspects, this disclosure provides methods for synthesizing an ionizable cationic lipid of formula M3 (e.g., CICL-251, CICL-252, CICL-253, CICL-254, CICL-255, CICL-256, CICL-256, CICL-257, CICL-258, CICL-259, CICL-260, CICI-261, CICL-262, CICL-263, CICL-264, CICL-265, CICL-266, CICL-267, CICL-268, CICL-269, CICL-270, CICL-271, CICL-272, CICL-273, CICL-274, CICL-275, CICL-276, CICL-277, CICL-278, CICL-279, CICL-280, CICL-281, CICL-282, CICL-283, CICL-284, CICL-285, CICL-286, CICL-287, CICL-288, CICL-289, and CICL-290).

[00456] Table 2 provides a summary of substituents in formula M3 for the ring structures of CICL-251 - CICL-290. Table 2. Exemplary CICL Structures Lipid head and ring structure A1 A2 A5 Ring Size A3 A4 X w (not drawn) I 0 9 CICL-252 \ Absent Absent ch2 4 ch2 ch2 o-ch2 ch2-N(CH3)2 CH o CICL-253 \ 3¾¼ Absent Absent ch2 4 ch2 ch2 o-ch2 ch2-N(CH3)2 CH r Y 0 >ko CICL-251 \ Absent ch2 ch2 5 ch2 Absent o-ch2 ch2-N(CH3)2 CH Y CICL-254 \ Absent ch2 ch2 5 ch2 Absent o-ch2 ch2-N(CH3)2 CH Lipid head and ring structure A1 A2 A5 Ring Size A3 A4 X w (not drawn) r X^O CICL-255 \ Absent ch2 ch2 5 ch2 Absent o-ch2 ch2-N(CH3)2 CH ° Of  O T cr^o Ki Ql > 0 1 Absent ch2 ch2 5 ch2 Absent o-ch2 ch2-N(CH3)2 CH X . CICL-257 \ ch2 ch2 Absent 5 Absent Absent o-ch2 ch2-N(CH3)2 CH X^O CICL-258 \ ch2 ch2 Absent 5 Absent Absent o-ch2 ch2-N(CH3)2 CH ° ) ^Kq7 CICL-259 \ Absent Absent CH2CH2 5 ch2 ch2 o-ch2 ch2-N(CH3)2 CH ^¢ / CICL-260 \ Absent Absent ch2ch2 5 ch2 ch2 o-ch2 ch2-N(CH3)2 CH c" CICL-261 \ ch2 ch2 ch2 6 Absent Absent o-ch2 ch2-N(CH3)2 CH Lipid head and ring structure A1 A2 A5 Ring Size A3 A4 X w (not drawn) a     ^^1 / CICL-262 \ ch2 ch2 ch2 6 Absent Absent o-ch2 ch2-N(CH3)2 CH y°) / —( 0 0  \    ^1 / ^c / CICL-263 \ Absent Absent ch2ch2-ch2 6 ch2 ch2 o-ch2 ch2-N(CH3)2 CH / —(   0 Q\, 0   '      N—N CICL-264 \ X Absent Absent ch2ch2-ch2 6 ch2 ch2 o-ch2 ch2-N(CH3)2 CH 0 CICL-265 \ ch2 ch2 CH=CH 7 Absent Absent o-ch2 ch2-N(CH3)2 CH O 0 Qv. CICL-266 \ ch2 ch2 CH=CH 7 Absent Absent o-ch2 ch2-N(CH3)2 CH d^0 CICL-267 \ Absent Absent CH2CH-chch2 7 ch2 ch2 0-CH2 ch2-N(CH3)2 CH Lipid head and ring structure A1 A2 A5 Ring Size A3 A4 X w (not drawn) 4k CICL-268 \ Absent Absent ch2ch-chch2 7 ch2 ch2 o-ch2 ch2-N(CH3)2 CH 4k CICL-269 \ Absent Absent ch2-(CH2)2-ch2 7 ch2 ch2 o-ch2 ch2-N(CH3)2 CH 4k yk CICL-270 \ Absent Absent ch2-(CH2)2-ch2 7 ch2 ch2 o-ch2 ch2-N(CH3)2 CH r 1 0 k / J 9 CICL-271 \ Absent Absent ch2 4 ch2 ch2 o-ch2 ch2-N(CH3)2 N O CICL-272 \ X^o Absent Absent ch2 4 ch2 ch2 o-ch2 ch2-N(CH3)2 N r V 0 k >ko CICL-273 \ Absent ch2 ch2 5 ch2 Absent o-ch2 ch2-N(CH3)2 N Lipid head and ring structure A1 A2 A5 Ring Size A3 A4 X w (not drawn) Y >^o CICL-274 \ Absent ch2 ch2 5 ch2 Absent o-ch2 ch2-N(CH3)2 N r CICL-275 \ Absent ch2 ch2 5 ch2 Absent o-ch2 ch2-N(CH3)2 N v,o t CICL-276 \ Absent ch2 ch2 5 ch2 Absent o-ch2 ch2-N(CH3)2 N X X CICL-277 \ ch2 ch2 Absent 5 Absent Absent o-ch2 ch2-N(CH3)2 N X^O CICL-278 \ ch2 ch2 Absent 5 Absent Absent o-ch2 ch2-N(CH3)2 N rA ° ^07 CICL-279 \ Absent Absent CH2CH2 5 ch2 ch2 o-ch2 ch2-N(CH3)2 N T^c / CICL-280 \ Absent Absent ch2ch2 5 ch2 ch2 o-ch2 ch2-N(CH3)2 N Lipid head and ring structure A1 A2 A5 Ring Size A3 A4 X w (not drawn) CICL-281 \ ch2 ch2 ch2 6 Absent Absent o-ch2 ch2-N(CH3)2 N 5—0 CICL-282 \ ch2 ch2 ch2 6 Absent Absent o-ch2 ch2-N(CH3)2 N p^ / CICL-283 \ Absent Absent ch2ch2-ch2 6 ch2 ch2 o-ch2 ch2-N(CH3)2 N p p^ / CICL-284 \ Absent Absent ch2ch2-ch2 6 ch2 ch2 o-ch2 ch2-N(CH3)2 N 0 >A0 Pa o pv^ A / j / 0 CICL-285 \ ch2 ch2 CH=CH 7 Absent Absent o-ch2 ch2-N(CH3)2 N O PP o Qp y^x° CICL-286 \ ch2 ch2 CH=CH 7 Absent Absent o-ch2 ch2-N(CH3)2 N Lipid head and ring structure A1 A2 A5 Ring Size A3 A4 X w (not drawn) CICL-287 \ Absent Absent ch2ch-chch2 7 ch2 ch2 o-ch2 ch2-N(CH3)2 N CICL-288 \ Absent Absent ch2ch-chch2 7 ch2 ch2 o-ch2 ch2-N(CH3)2 N CICL-289 \ Absent Absent ch2-(CH2)2-CH2 7 ch2 ch2 0-CH2 ch2-N(CH3)2 N CICL-290 \ Absent Absent ch2-(CH2)2-CH2 7 ch2 ch2 0-CH2 ch2-N(CH3)2 N

[00457] In certain embodiments, this disclosure provides methods for synthesizing an ionizable cationic lipid of formula M6 comprising the synthesis step as shown in Scheme M6, A' is an anion of an acid AH, and the rest of the substituents are defined the same as in formula M6. In certain embodiments, the method further comprises the synthesis step shown in Scheme 4-A. A particular stereoisomer of the ionizable cationic lipid of formula M6 can be prepared according to the synthesis method disclosed herein with starting materials and / or intermediates having the same stereochemistry. Formula M6 allows for the esters constituted by R2 and R3 to be oriented in either direction in various combinations among the 4 positions within the lipid where the R2 / R3 esters occur. Additionally, the tails containing the R2 / R3 esters attach to the ring at equivalent or non-equivalent positions depending on the particular species to be synthesized. More detailed synthetic schemes leading to each of the tail configurations encompassed by Formula M6 are set out in Schemes M3, M6-1, M6-2, M6-3, and M-4.

[00458] The lipid preparations, described in Schemes M6, M3, M6-1, M6-2, M6-3, and M-4 utilize the coupling of a mono-acid 2-A, 2-B, 2-C, or 2-D with a protected (PGi) diol 4-A. The nature of the A1, A2, A3, A4, and A5 sections of 4-A, when combined with the stereochemistry of the -A3-OH and -A4-OH moieties will result in the individual hydroxyl moieties either being identical / symmetric or regioisomeric / stereoisomeric. The coupling of 4-A with an excess (> 2 eq.) of 2-A, 2-B, 2-C, or 2-D will afford bis-esters 5. Lipid synthesis is completed upon removal of an acid-labile protecting group (PG1) with an acid HA, to give an amine salt, followed by a reaction with carbonyldiimidazole (CDI), giving imidazolecarboxyamides 7. Activation of the imidazolecarboxyamides 7 with methyl trifluoromethanesulfonate (MeOTf) and reaction of the resulting imidazolium salt with an alcohol, thiol or amine would give carbamate, thiocarbamate, or urea containing lipids M3, M6, and M6-1. The coupling of mono-acids 2-A, 2-B, 2-C, or 2D with diol 4-A where ca. 1 eq. of the acid is employed, gives mono-esters 8 and 9. In cases where nature of the A1, A2, A3, A4, and A5 sections of 4-A, when combined with the stereochemistry of the -A3-OH and -A4-OH moieties result in identical / symmetrical hydroxyl moieties, the result is symmetric, that is: mono-esters 8 and 9 are identical. The coupling of a different mono-acid with mono-ester 8 gives a single lipid with differentiated ester moieties. In cases where nature of the A1, A2, A3, A4, and A5 sections of 4-A, when combined with the stereochemistry of the -A3-OH and -A4-OH moieties results in the hydroxyl groups as regioisoimeric / stereoisomeric, the coupling of these diols with ca. 1 eq. of mono-acids 2-A, 2B, 2-C, or 2-D provides monoesters 8 and 9 which are not identical. Additional coupling with a different mono-acid then provides 2 final lipids, utilizing the chemistry described above, which are regioisomers / stereoisomers. 2-B Scheme M6

[00459] In certain embodiments, this disclosure provides methods for synthesizing an ionizable cationic lipid of formula M3 comprising the synthesis step as shown in Scheme M3, A’ is an anion of an acid AH, and the rest of the substituents are defined the same as in formula M3. In certain embodiments, the method further comprises the synthesis step shown in Scheme 4-A. A particular stereoisomer of the ionizable cationic lipid of formula M3 can be prepared according to the synthesis method disclosed herein with starting materials and / or intermediates having the same stereochemistry.

[00460] In certain embodiments, this disclosure provides methods for synthesizing an ionizable cationic lipid of formula M6-1 comprising the synthesis steps as shown in Scheme M6-1, A' is an anion of an acid AH, and the rest of the substituents are defined the same as in formula M3. In certain embodiments, the method further comprises the synthesis step shown in Scheme 4-A. A particular stereoisomer of the ionizable cationic lipid of formula M6- 1 can be prepared according to the synthesis method disclosed herein with starting materials and / or intermediates having the same stereochemistry. Scheme M6-1 M6-1

[00461] In certain embodiments, the method can further comprise synthesis of the diesteramine 6-B, 6-A, and / or 6-C comprising deprotecting a protected diester-amine 5-B, 5-A, and / or 5-C to provide the unprotected diester-amine 6-B, 6-A, and / or 6-C. The deprotection can be carried out under reductive (hydrogen and a catalyst), acidic or basic conditions, depending on the protecting group (PGi). For example, the deprotection step can be carried out under an acidic condition (e.g., in the presence of an acid HA, such as trifluoroacetic acid, TFA, when the protecting groups is an acid labile protecting group, e.g., t-butoxycarbonyl (BOC)) in an organic solution (e.g., CH2CI2) as illustrated in Schemes M6, M3, and M6-1. Reductive and basic removal of other PG1 groups, if desired, can be done in a manner so as to avoid undesired reaction paths and side products, as would be understood by the person of skill in the art.

[00462] In certain embodiments, the method further comprises synthesis of the protected diester-amine 5-B, 5-A, and / or 5-C comprising coupling an unprotected diester-acid 2-B, 2-A, and / or 2-C with a desired diol-amine protected by a protecting group (PG1) (diol-protected amine 4-A) to form protected diester-amine 5-B, 5-A, and / or 5-C. In certain embodiments, the coupling reaction is carried out in an organic solvent (e.g., acetonitrile) in the presence of a nucleophilic catalyst (e.g., DMAP) and an acidic catalyst (e.g., 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC-HCI)). In certain embodiments, PG1 can be t-butoxycarbonyl (-C(=O)-O-C(CH3)3), benzyl, trityl, tri methoxytrityl, benzyloxycarbonyl, or trimethylsilylethoxycarbonyl. Other PG1 and deprotection steps can be used as known to the person of ordinary skill in the art. For example, PG1 can be a be a benzyloxycarbonyl (Cbz) that can be removed in the presence of hydrogen and a Pd / C catalyst. As would be understood by the person of ordinary skilled the art, such removal can be done in a manner to avoid undesired reaction paths and side products. A base labile PG1, such as trimethylsilylethoxycarbonyl moiety, can also be used and be removed with nBuNH4 or HF-pyridine. As would be understood by the person of ordinary skilled the art, such reductive and basic removal can be done in a manner to avoid undesired reaction paths and side products.

[00463] In certain embodiments, this disclosure provides methods for synthesizing an ionizable cationic lipid of formula M6-2 comprising the synthesis steps as shown in Scheme M6-2, The use of ca. 1 equivalent of 2-D in an initial reaction with 4-A results in the formation of mono-ester isomers 8-D and 9-D if the nature of 4A is such that OH groups appended to A3 and A4 are non-equivalent / non-symmetrical. After the chromatographic separation of 8-D and 9-D, the use of a different coupling partner in the subsequent step (2-C) leads to the regioisomeric / stereoisomeric products 5-DC and 5-CD, and the rest of the substituents are defined the same as in formula M3, with protecting group removal utilizing acid HA and formation of the imidazolecarboxyamides 7-DC and 7-CD with GDI. In certain embodiments, the method further comprises the synthesis step shown in Scheme 4-A. Particular regioisomeric / stereoisomeric forms of the ionizable cationic lipids of formula M6-2A and M6- 2B can be prepared according to the synthesis method disclosed herein with starting materials and / or intermediates having the same stereochemistry. In the case of the utilization of a form of 4-A wherein the OH groups appended to A3 and A4 are equivalent / symmetrical: 8-D and 9-D would be identical, resulting in the production of a single ionizable cationic lipid M6-2 as M6-2A and M6-2B would be identical. SCHEME M6-2 2-D 1. HA 2. CDI 1. MeOTf 2. HX M6-2A

[00464] In certain embodiments, this disclosure provides methods for synthesizing an ionizable cationic lipid of formula M6-3 comprising the synthesis steps as shown in Scheme M6-3, The use of ca. 1 equivalent of 2-A in an initial reaction with 4-A results in the formation of mono-ester isomers 8-A and 9-A if the nature of 4A is such that OH groups appended to A3 and A4 are non-equivalent / non-symmetrical. After the chromatographic separation of 8-A and 9-A, the use of a different coupling partner in the subsequent step (2-C) leads to the regioisomeric / stereoisomeric products 5-AC and 5-CA, and the rest of the substituents are defined the same as in formula M3, with protecting group removal utilizing acid HA and formation of the imidazolecarboxyamides 7-AC and 7-CA with GDI. In certain embodiments, the method further comprises the synthesis step shown in Scheme 4-A. Particular regioisomeric / stereoisomeric forms of the ionizable cationic lipids of formula M6-3A and M6-3B can be prepared according to the synthesis method disclosed herein with starting materials and / or intermediates having the same stereochemistry. In the case of the utilization of a form of 4-A wherein the OH groups appended to A3 and A4 are equivalent / symmetrical: 8-A and 9-A would be identical, resulting in the production of a single ionizable cationic lipid M6-3 as M6-3A and M6-3B would be identical. Scheme M6-3 1. HA 2. CDI 1. HA 2. CDI

[00465] In certain embodiments, this disclosure provides methods for synthesizing an ionizable cationic lipid of formula M6-3 comprising the synthesis steps as shown in Scheme M6-4, The use of ca. 1 equivalent of 2-A in an initial reaction with 4-A results in the formation of mono-ester isomers 8-A and 9-A if the nature of 4A is such that OH groups appended to A3 and A4 are non-equivalent / non-symmetrical. After the chromatographic separation of 8-A and 9-A, the use of a different coupling partner in the subsequent step (2-D) leads to the regioisomeric / stereoisomeric products 5-AD and 5-DA, and the rest of the substituents are defined the same as in formula M3, with protecting group removal utilizing acid HA and formation of the imidazolecarboxyamides 7-AD and 7-DA with GDI. In certain embodiments, the method further comprises the synthesis step shown in Scheme 4-A. Particular regioisomeric / stereoisomeric forms of the ionizable cationic lipids of formula M6-4A and M6-4B can be prepared according to the synthesis method disclosed herein with starting materials and / or intermediates having the same stereochemistry. In the case of the utilization of a form of 4-A wherein the OH groups appended to A3 and A4 are equivalent / symmetrical: 8-A and 9-A would be identical, resulting in the production of a single ionizable cationic lipid M6-4 as M6-4A and M6-4B would be identical. Scheme M6-4 1. MeOTf 2. HX M6-4A

[00466] The synthesis steps shown in Schemes M6, M3, and M6-1 comprises reacting a diester-ammonium salt 6-B.6-A, and 6-C, respectively, with 1,1'-carbonyldiimidazole (CDI) to provide an imidazolecarboxyamide 7-B, 7-A, and 7-C, respectively; 7-B, 7-A, and 7-C are then reacted with MeOTf and then the activated imidazolecarboxyamide is coupled with a desired alcohol / thiol / amine (HX) to provide a corresponding carbamate / thiocarbamate / urea having the structure of formula M6M3, or M6-1, all substituents are defined the same as those in formula M6 and M3. The synthesis steps shown in Schemes M6-2, M6-3, and M6-4 comprises reacting protected amines 5-DC / 5-CD, 5-AC / 5-CA, and 5-AD / 5-DA respectively, with an acid HA followed by a reaction with GDI to provide imidazolecarboxyamides 7-DC / 7-CD, 7-AC / 7-CA, and 7-AD / 7-DA respectively, which are then reacted with MeOTf to give activated imidazolecarboxyamides which are coupled with a desired alcohol / thiol / amine to provide corresponding carbamates / thiocarbamates / ureas having the structures M6-2A / M6-2B, M6-3A / M6-3B, and M6-4A / M6-4B respectively.

[00467] In certain embodiments, the imidazolecarboxyamide 7-B, 7-A, 7-C, 7-DC / 7-CD, 7-AC / 7-CA, and / or 7-AD / 7-DA synthesis is carried out in an organic solvent (e.g., without limitation, CH2CI2) in the presence of a basic catalyst (e.g., without limitation, trimethylamine). In certain embodiments, the carbamate / thiocarbamate / urea synthesis can comprise first reacting the imidazolecarboxyamide 7-B, 7-A, 7-C, 7-DC / 7-CD, 7-AC / 7-CA, and / or 7-AD / 7-DA with methyl trifluoromethanesulfonate, then reacting with the desired alcohol / thiol / amine (HX) in the presence of a base (e.g., trimethylamine). The reaction can be carried out in an organic solvent (e.g., acetonitrile). In certain embodiments as described herein, HX is an alcohol, wherein X is as described herein. In certain embodiments as described herein, HX is a thiol, wherein X is as described herein. In certain embodiments as described herein, HX is an amine, wherein X is as described herein. Accordingly, in some embodiments, HX is an alcohol / thiol / amine that provides that desired X group to the lipids of formula M6 and M3. For example, in various embodiments as described herein, HX is selected from the group (CH2)0-3^H3 hy-(ch2)2.4-n         hn^ consisting of              (CH2)o-3 3,   \— / (CH2)2-3OCH3 HY-(CH2)2.4-N (CH2)2-3OCH3           hy-(ch2 HY-(CH2)2.4-f /      OH           HY-(CH2)2.4- HY-(CH2)2.4—HY-(CH2)2.4NH^AJ (CH2)2-3OCH3 N (CH> CH HY-(CH2)2,-N N-(CH2)o.3CH3              (CH2)o-3CH3 )2^^0       HY-(CH2)2.-O -l /                HY-(CH2)2-4-M / \ich3 f )                                 \ f ) HY-(CH2)2.4N-^A    hn^n ) ) and Z is 0, NH, NCH3. The various HX compounds disclosed herein are commercially available, are known in the scientific literature, or can be made using procedures familiar to the person of ordinary skill in the art, provided from commercial sources, or the general procedures described in the Examples below.

[00468] For example, in various embodiments, HX is selected from any one of HO          ho 131             132          133 ,                       ,or

[00469] In certain embodiments, the method further comprises synthesis of the diol-protected amine 4-A as shown in Scheme 4-A, comprising reacting a benzyl diol-amine 3-A with a dicarbonate (e.g., (PGi)2O such as di-tert-butyl dicarbonate (BOC2O)) in the presence of a catalyst (e.g., Pd(OH)2) and hydrogen to provide the diol-protected amine 4-A.

[00470] Scheme 4-A H2, Catalyst (PGi)2O OH / A3 W A5   'N—PG1 A3 HO-A4 «

[00471] The syntheses are described using specific solvents, but in all cases alternative solvents are known to the person of skill in the art. THF can be substituted, for example, without limitation, by DMF, diethyl ether, methyl t-butyl ether, dioxane, or2-methyl THF. Ethyl acetate can be substituted by, for example, without limitation, isopropyl acetate, THF, 2-methyl THF, dioxane, or methyl t-butyl ether. Dichloromethane can be substituted by, for example, without limitation, ethyl acetate, isopropyl acetate, THF, methyl t-butyl ether, 2-methyl THF, dioxane, or heptane. Methanol can be substituted by, for example, without limitation, ethanol, iso-propanol, or aqueous THF. Acetonitrile can be substituted by, for example, THF, 2-methyl THF, dichloromethane, ethyl acetate, isopropyl acetate, methyl t-butyl ether, or toluene.

[00472] Many general references providing commonly known chemical synthetic schemes and conditions useful for synthesizing materials / intermediates used in the synthesis of the disclosed compounds are available (see, e.g., Smith, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Eighth Edition, Wiley-lnterscience, 2019; or Furniss, Hannaford, Smith, Tatchelll, Vogel’s A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fifth Edition, New York: Longman, 1989).

[00473] Compounds as described herein can be purified by any of the means known in the art, including chromatographic means, such as HPLC, preparative thin layer chromatography, flash column chromatography and ion exchange chromatography. Any suitable stationary phase can be used, including normal and reversed phases as well as ionic resins. Most typically, the disclosed compounds are purified via silica gel and / or alumina chromatography. See, e.g., Still, Kahn, Mitra, J. Org. Chem. 1978, 43, 2923-292, Introduction to Modern Liquid Chromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, John Wiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl, Springer-Verlag, New York, 1969.

[00474] During any of the processes for preparation of the subject compounds, it can be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules concerned. This can be achieved by means of conventional protecting groups as described in standard works, such as J. F. W. McOmie, "Protective Groups in Organic Chemistry,” Plenum Press, London and New York 1973, in T P. G. M. Wuts, " Greene’s Protective Groups in Organic Synthesis,” Firth edition, Wiley, New York 2014, in "The Peptides"; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981, in "Methoden der organischen Chemie,” Houben-Weyl, 4.sup.th edition, Vol. 15 / 1, Georg Thieme Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, "Aminosauren, Peptide, Proteine,” Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982, and / or in Jochen Lehmann, "Chemie der Kohlenhydrate: Monosaccharide and Derivate,” Georg Thieme Verlag, Stuttgart 1974. The protecting groups can be removed at a convenient subsequent stage using methods known from the art.

[00475] The compounds disclosed herein can be made using procedures familiar to the person of ordinary skill in the art and as described herein. For example, compounds of structural formula M6 can be prepared according to Scheme M6, Scheme 4A, general procedures (see the Examples below), and / or analogous synthetic procedures. For example, compounds of structural formula M3 can be prepared according to Scheme M3, Scheme 4A, general procedures (see the Examples below), and / or analogous synthetic procedures. One of skill in the art can adapt the reaction sequences of Schemes M6, M3, and 4A, general procedures, and Examples described to fit the desired target molecule. Of course, in certain situations one of skill in the art will use different reagents to affect one or more of the individual steps or to use protected versions of certain of the substituents. Additionally, one skilled in the art would recognize that compounds of the disclosure can be synthesized using different routes altogether. Lipid Nanoparticles (LNPs) and Targeted LNPs (tLNPs)

[00476] In certain aspects, this disclosure provides an LNP comprising an ionizable cationic lipid of formula M3 and / or M6. In some embodiments, an LNP comprises an ionizable cationic lipid of formula M3 and / or M6 and a phospholipid, a sterol, a co-lipid, a PEGylated lipid, or a combination thereof. In certain embodiments, the PEG-lipids are not functionalized PEG-lipids. In other embodiments, the PEG-lipids are functionalized PEG-Lipids. In certain embodiments, the LNP comprises at least one PEG-lipid that is functionalized and at least PEG-lipid that is not functionalized.

[00477] In further aspects, this disclosure provides a targeted lipid nanoparticle (tLNP) comprising an ionizable cationic lipid of formula M3 and / or M6. In some embodiments, the aforementioned tLNP can further comprise one or more of a phospholipid, a sterol, a co-lipid, and a PEG-lipid, or a combination thereof, and a functionalized PEG-lipid. As used herein, “functionalized PEG-lipid” refers to a PEG-lipid in which the PEG moiety has been derivatized with a chemically reactive group that can be used for conjugating a targeting moiety to the PEG-lipid. The functionalized PEG-lipid can be reacted with a binding moiety (e.g., an antibody or Fab) after the LNP is formed, so that the binding moiety is conjugated to the PEG portion of the lipid. The conjugated binding moiety can thus serve as a targeting moiety for the tLNP.

[00478] In various embodiments, a binding moiety of a LNP or tLNP comprises an antigen binding domain, an antigen, a ligand-binding domain of a receptor, or a receptor ligand. In some embodiments, a binding moiety comprises a complete antibody, an F(ab)2, an Fab, a minibody, a single-chain Fv (scFv), a diabody, a VH domain, or a nanobody, such as a VHH or single domain antibody. In some embodiments, the receptor ligand is a carbohydrate, for example, a carbohydrate comprising terminal galactose or N-acetylgalactosamine units, which are bound by the asialoglycoprotein receptor. These binding moieties constitute means for LNP targeting. Some embodiments specifically include one or more of these binding moieties. Other embodiments specifically exclude one or more of these binding moieties. LNP and tLNP Compositions

[00479] The LNP composition contributes to the formation of stable LNPs and tLNPs, efficient encapsulation of a payload, protection of a payload from degradation until it is delivered into a cell, and promotion of endosomal escape of a payload into the cytoplasm. These functions are primarily independent of the specificity of the binding moiety (or moieties) serving to direct or bias a tLNP to a particular cell type(s). Additional LNP and tLNP compositions are generally disclosed in PCT / US2024 / 032141, filed 31 May 2024, published as WO2024 / 249954, and entitled Lipid Nanoparticle Formulations and Compositions, which is incorporated by reference for all that it teaches about the design, formation, characterization, properties, and use of LNPs and tLNPs.

[00480] The LNPs and / or tLNPs can include the various components in amounts sufficient to provide a nanoparticle with a desired shape, fluidity, and bio-acceptability as described herein. With respect to LNPs or tLNPs of this disclosure, in some embodiments, the LNPs (or tLNP) comprises at least one ionizable cationic lipid as described herein in an amount in the range of from about 35 to about 65 mol%, e.g., in an amount of from about 40 to about 65 mol%, about 40 to about 60 mol%. In some embodiments, the LNP or tLNP comprises about 58 mol% or 62 mol% ionizable cationic lipid. In some embodiments, is the LNP (or tLNP) comprises a phospholipid in an amount in the range of from about 7 to about 30 mol%, e.g., in an amount of from about 13 to about 30 mol%. In some embodiments, the LNP or tLNP comprises about 10 mol% phospholipid. In some embodiments, the LNP (or tLNP) comprises a sterol in an amount in the range of from about 20 to about 50 mol%, e.g., in an amount in the rage of from about 20 to about 45 mol%, or about 30 to about 50 mol%, or about 30 to about 45 mol%. In some embodiments, the LNP or tLNP comprises about 30.5, 26.5, or 23.5 mol% sterol. In some embodiments, the LNP (or tLNP) comprises at least one co-lipid in an amount in the range of from about 1 to about 30 mol%. In some embodiments, the LNP (or tLNP) comprises at least one unfunctionalized PEG-lipid in an amount of from 0 to about 5 mol%, e.g., in the range of amount 0 to about 3 mol%, or about 0.1 to about 5 mol%, or about 0.5 to about 5 mol%, or about 0.5 to about 3 mol%. In some embodiments, the LNP or tLNP comprises about 1.4 mol% unfunctionalized PEG-lipid. In some embodiments, the LNP or tLNP comprises at least one functionalized PEG-lipid in an amount in the range of from about 0.1 to about 5 mol%, e.g., in the range of from about 0.1 to 0.3 mol%. In some embodiments, the LNP or tLNP comprises about 0.1 mol% functionalized PEG-lipid. In some embodiments, the functionalized PEG-lipid is conjugated to a binding moiety.

[00481] In certain aspects, this disclosure provides an LNP or tLNP, wherein the LNP or tLNP comprises about 35 mol% to about 65 mol% of an ionizable cationic lipid, about 0.5 mol% to about 3 mol% of a PEG-lipid (including non-functionalized PEG-lipid and optionally a functionalized PEG-lipid), about 7 mol% to about 13 mol% of a phospholipid, and about 30 mol% to about 50 mol% of a sterol. In some embodiments, an LNP or tLNP comprises a payload with a net negative charge for example, a peptide, a polypeptide, a protein, a small molecule, or a nucleic acid molecule, and combinations thereof. A payload is generally encompassed by or in the interior of an LNP or tLNP. As disclosed herein dosages always refer to the amount of payload being provided. In some embodiments, a payload comprises one or more species of nucleic acid molecule. For tLNP encapsulating mRNA dosages are typically in the range of 0.05 to 5 mg / kg without regard for recipient species. In some embodiments, the dosage is in the range of 0.1 to 1 mg / kg.

[00482] With respect to LNPs or tLNPs of this disclosure, in some embodiments, the ratio of total lipid to nucleic acid is about 10:1 to about 50:1 on a weight basis. In some embodiments, the ratio of total lipid to nucleic acid is about 10:1, about 20:1, about 30:1, or about 40:1 to about 50:1, or 10:1 to 20:1, 30:1,40:1 or 50:1, or any range bound by a pair of these ratios. The ratio of lipid to nucleic acid can also be reported as an N / P ratio, the ratio of positively chargeable lipid amine (N = nitrogen) groups to negatively-charged nucleic acid molecule phosphate (P) groups. In some instances, the N / P ratio is from about 3 to about 9, about 3 to about 7, about 3 to about 6, about 4 to about 6, about 5 to about 6, or about 6. In some instances, the N / P ratio is from 3 to 9, 3 to 7, 3 to 6, 4 to 6, 5 to 6, or 6. In some embodiments, an LNP or tLNP comprises about 40 mol% to about 62 mol% ionizable cationic lipid. In some embodiments, an LNP or tLNP comprises about 1 mol% to about 2 mol% total PEG-lipid. In certain embodiments, an LNP or tLNP comprises about 0.1 mol% to about 0.3 mol%, for example about 0.1 mol%, about 0.2 mol%, or about 0.3 mol% functionalized PEG-lipid. In some embodiments, a binding moiety is conjugated to functionalized PEG-lipid. In certain instances, a tLNP is an LNP that further comprises an antibody (for example, a whole IgG) as the binding moiety which is present at an antibody:mRNA ratio (w / w) of about 0.3 to about 1.0 w / w.

[00483] Due to physiologic and manufacturing constraints LNP or tLNP, particles with a hydrodynamic diameter of about 50 to about 150 nm are desirable for in vivo use. Accordingly, in some embodiments, the LNP or tLNP has a hydrodynamic diameter of 50 to 150 nm and in some embodiments the hydrodynamic diameter is <120, <110, <100, or <90 nm. Uniformity of particle size is also desirable with a polydispersity index (PDI) of <0.2 (on a scale of 0 to 1) being acceptable. Both hydrodynamic diameter and polydispersity index are determined by dynamic light scattering (DLS). Particle diameter as assessed from cryo-transmission electron microscopy (Cryo-TEM) can be smaller than the DLS-determined value. Phospholipids

[00484] As would be understood by the person or ordinary skill in the art, phospholipids are amphiphilic molecules. Due to the amphiphilic nature of phospholipids, these molecules are known to form bilayers and by including them in the LNPs and tLNPs, as described herein, they can provide membrane formation, stability, and rigidity. As used herein, phospholipids include a hydrophilic head group, including a functionalized phosphate group, and two hydrophobic tail groups derived from fatty acids. For example, in various embodiments as described herein, the phospholipids include a phosphate group functionalized with ethanolamine, choline, glycerol, serine, or inositol. As described above, the phospholipid includes two hydrophobic tail groups derived from fatty acids. These hydrophobic tail groups can be derived from unsaturated or saturated fatty acids. For example, the hydrophobic tail groups can be derived from a C12-C20 fatty acid.

[00485] With respect to LNPs or tLNPs of this disclosure, in various embodiments, a phospholipid comprises dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), or 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC), or a combination thereof. In various embodiments, the phospholipid is selected from the group comprising dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), or 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC). In some embodiments, the phospholipid is distearoylphosphatidylcholine (DSPC). Phospholipids can contribute to formation of a membrane, whether monolayer, bilayer, or multi-layer, surrounding the core of the LNP or tLNP. Additionally, phospholipids such as DSPC, DMPC, DPPC, DAPC impart stability and rigidity to membrane structure. Phospholipids, such as DOPE, impart fusogenicity. Further phospholipids, such as DMPG, which attains negative charge at physiologic pH, facilitates charge modulation. Thus, phospholipids constitute means for facilitating membrane formation, means for imparting membrane stability and rigidity, means for imparting fusogenicity, and means for charge modulation.

[00486] In some embodiments, an LNP or tLNP has about 7 mol% to about 13 mol% phospholipid, about 7 mol% to about 10 mol% phospholipid, or about 10 mol% to about 13 mol% phospholipid. In certain embodiments, an LNP has about 7 mol%, about 10 mol%, or about 13 mol% phospholipid. In certain instances, the phospholipid is DSPC. In certain instances, the phospholipid is DAPC. Sterols

[00487] The disclosed LNP and tLNP comprise a sterol. Sterol refers to a subgroup of steroids that contain at least one hydroxyl (OH) group. More specifically, a gonane derivative with an OH group substituted for an H at position 3, or said differently, but equivalently, a steroid with an OH group substituted for an H at position 3. Examples of sterols include, without limitation, cholesterol, ergosterol, p-sitosterol, stigmasterol, stigmastanol, 20-hydroxycholesterol, 22-hydroxycholesterol, and the like. With respect to LNPs ortLNPs of this disclosure, in various embodiments, a sterol is cholesterol, 20-hydroxycholesterol, 22-hydroxycholesterol, or a phytosterol. In further embodiments, the phytosterol comprises campesterol, sitosterol, or stigmasterol, or combinations thereof. In preferred embodiments, the cholesterol is not animal-sourced but is obtained by synthesis using a plant sterol as a starting point. LNPs incorporating C-24 alkyl (such as methyl or ethyl) phytosterols have been reported to provide enhanced gene transfection. The length of the alkyl tail, the flexibility of the sterol ring, and polarity related to a retain C-3 -OH group are important to obtaining high transfection efficiency. While p-sitosterol and stigmasterol performed well, vitamin D2, D3 and calcipotriol, (analogs lacking intact body of cholesterol) and betulin, lupeol ursolic acid and olenolic acid (comprising a 5th ring) should be avoided. Sterols serve to fill space between other lipids in the LNP or tLNP and influence LNP or tLNP shape. Sterols also control fluidity of lipid compositions, reducing temperature dependence. Thus, sterols such as cholesterol, 20-hydroxycholesterol, 22-hydroxycholesterol, campesterol, fucosterol, p-sitosterol, and stigmasterol constitute means for controlling LNP shape and fluidity or sterol means for increasing transfection efficiency. In designing a lipid composition fora LNP ortLNP, in some embodiments, sterol content can be chosen to compensate for different amounts of other types of lipids, for example, ionizable cationic lipid or phospholipid.

[00488] In some embodiments, an LNP or tLNP has about 27 mol% or about 30 mol% to about 50 mol% sterol, or about 30 mol% to about 38 mol% sterol. In certain embodiments, an LNP or tLNP has about 30.5 mol%, about 33.5 mol%, or about 37.5 mol% sterol. In certain instances, the sterol is cholesterol. In certain embodiments, the sterol is a mixture of sterols, for example, cholesterol and p-sitosterol or cholesterol and 20-hydroxycholesterol. In some instances, the sterol is about 25 mol% 20-hydroxycholesterol and about 75 mol% cholesterol. In some instances, the sterol is about 25 mol% p-sitosterol and about 75 mol% cholesterol. In some instances, the sterol is about 50 mol% p-sitosterol and about 50 mol% cholesterol. In certain embodiments, an LNP or tLNP has 27 mol% or 30 mol% to 50 mol% sterol or 30 mol% to 38 mol% sterol. In further embodiments, an LNP or tLNP has 30.5 mol%, 33.5 mol%, or 37.5 mol% sterol. In certain instances, a sterol is cholesterol. In certain embodiments, a sterol is a mixture of sterols, for example, cholesterol and p-sitosterol or cholesterol and 20-hydroxycholesterol. In some instances, a sterol is 25 mol% 20-hydroxycholesterol and 75 mol% cholesterol. In further instances, a sterol is 25 mol% p-sitosterol and 75 mol% cholesterol. In still further instances, a sterol is 50 mol% p-sitosterol and 50 mol% cholesterol.

[00489] With respect to LNPs ortLNPs of this disclosure, in some embodiments, a co-lipid is absent or comprises an ionizable lipid, anionic or cationic. A co-lipid can be used to adjust various properties of an LNP or tLNP, such as surface charge, fluidity, rigidity, size, stability, and the like properties. In some embodiments, a co-lipid is an ionizable lipid, such as cholesterol hemisuccinate (CHEMS) or an ionizable lipid of this disclosure. In some embodiments, a co-lipid is a charged lipid, such as a quaternary ammonium head group containing lipid. In some embodiments, a quaternary ammonium head group containing lipid comprises 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium            (DOTMA),            or            3p-(N-(N’,N’- Dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), or combinations thereof. In certain embodiments, these compounds a chloride, bromide, mesylate, or tosylate salt. As described above, when quaternary ammonium head group containing lipids are included in LNPs or tLNPs, fatal hyperacute toxicity in laboratory animals has been observed. Accordingly, when the co-lipid is a quaternary ammonium head group containing lipid, the quaternary ammonium head group containing lipid is present it makes up no more than 50 mol% of the total cationic lipid, for example, from 5 to 50% of the total cationic lipid. For illustration, if an LNP or tLNP were to have cationic lipid content of 70 mol% and 5 to 50 mol% of the total cationic lipid as quaternary ammonium lipid, the LNP or tINP would have from 3.5 mol% quaternary ammonium lipid and 66.5 mol% ionizable cationic lipid to 35 mol% each of quaternary ammonium lipid and ionizable cationic lipid.

[00490] When the disclosed ionizable lipids of formula M3 and / or M6 have a measured pKa ranging from about 6 to about 7 or from 6 to 7, they can contribute substantial endosomal release activity to an LNP or tLNP containing the ionizable lipid. More acidic or basic ionizable lipids of formula M3 and / or M6 can contribute surface charge and thus serve as a co-lipid as described immediately above. In such cases, it can be advantageous to incorporate another lipid with fusogenic activity into an LNP or tLNP of this disclosure. Surface charge is known to influence the tissue tropism of LNPs or tLNPs; for example, positively charged LNPs or tLNPs have shown a tropism for spleen and lung. PEG-Lipids

[00491] With respect to an LNP or tLNP of this disclosure, a PEG-lipid is a lipid conjugated to a polyethylene glycol (PEG). In some embodiments as described herein, the PEG-lipid is a C14-C20 lipid conjugated with a PEG. For example, in various embodiments as described herein, the PEG-lipid is a C14-C20 lipid conjugated with a PEG, or a C14-C18 lipid conjugated with a PEG, or a C14-C16 lipid conjugated with a PEG. In certain embodiments as described herein, the PEG-lipid is a fatty acid conjugated with a PEG. The fatty acid of the PEG-lipid can have a variety of chain lengths. For each, in some embodiments, the PEG-lipid is a fatty acid conjugated with PEG, wherein the fatty acid chain length is in the range of C14-C20 (e.g., in the range of C14-C18, or C14-C16). PEG-lipids with fatty acid chain lengths less than C14 are too rapidly lost from the LNP or tLNP while those with chain lengths greater than C20 are prone to difficulties with formulation.

[00492] In certain aspects, the LNP comprises one or more PEG-lipids and / or functionalized PEG-lipids; when both a functionalized and unfunctionalized PEG-lipid, the PEG-lipid present they can be the same or different; and one or more ionizable cationic lipids; the LNP can further comprise a phospholipid, a sterol, a co-lipid, or any combination thereof. The term “functionalized PEG-lipid” refers to a PEG-lipid in which the PEG moiety has been derivatized with a chemically reactive group that can be used for conjugating a targeting moiety to the PEG-lipid. The functionalized PEG-lipid can be reacted with a binding moiety so that the binding moiety is conjugated to the PEG portion of the lipid. The conjugated binding moiety can thus serve as a targeting moiety for the LNP to constitute a tLNP. In some embodiments, the binding moiety is conjugated to the functionalized PEG-lipid after an LNP comprising the functionalized PEG-lipid is formed. In other embodiments, the binding moiety is conjugated to the PEG-lipid and then the conjugate is inserted into a previously formed LNP.

[00493] In certain embodiments, the LNP is a tLNP comprising one or more functionalized PEG-lipids that has been conjugated to a binding moiety. In certain embodiments, the tLNP also comprises PEG-lipids not functionalized or conjugated with a binding moiety. In some embodiments, the functionalization is a maleimide. In some embodiments the functionalization is a bromomaleimide or bromomaleimide amide, alkynylamide, or alkynylimide moiety at the terminal hydroxyl end of the PEG moiety. In some embodiments, the binding moiety comprises an antibody or antigen binding portion thereof. In some embodiments, the binding moiety is a polypeptide comprising a binding domain and an N- or C-terminal extension comprising an accessible thiol group. In some embodiments, the conjugation linkage comprises a reaction product of a thiol in the binding moiety with a functionalized PEG-lipid. In some embodiments, the functionalization is a maleimide, azide, alkyne, dibenzocyclooctyne (DBCO), bromomaleimide or bromomaleimide amide, alkynylamide, or alkynylimide. In some embodiments, the binding moiety comprises an antibody or antigen binding portion thereof. In some embodiments, the binding moiety is a polypeptide comprising a binding domain and an N- or C-terminal extension comprising an accessible thiol group.

[00494] PEG can be made in a large range of sizes. In certain embodiments, the PEG of the disclosed LNP and tLNP is PEG-1000 to PEG-5000. It is to be understood that polyethylene preparations of these sizes are polydisperse and that the nominal size indicates an approximate average molecular weight of the distribution. Taking the molecular weight of an individual repeating unit of (OCH2CH2)n to be 44, a PEG molecule with n=22 would have a molecular weight of 986, with n=45 a molecular weight of 1998, and with n=113 a molecular weight of 4990. n=22 to 113 is used to represent PEG-lipids incorporating PEG moieties in the range of PEG-1000 to PEG-5000 such as PEG-1000, PEG-1500, PEG-2000, PEG-2500, PEG-3000, PEG-3500, PEG-4000, PEG-4500, and PEG-5000, although some molecules from preparations at the average molecular weight boundaries will have an n outside that range. For individual preparations n=22 is used to represent PEG-lipids incorporating PEG moieties from PEG-1000, n=45 is used to represent PEG-lipids incorporating PEG moieties from PEG-2000 n=67 is used to represent PEG-lipids incorporating PEG moieties from PEG-3000, n=90 is used to represent PEG-lipids incorporating PEG moieties from PEG-4000, n=113 is used to represent PEG-lipids incorporating PEG moieties from PEG-5000. Some embodiments incorporate PEG moieties in a range bounded by any pair of the foregoing values of n or average molecular weight. In some embodiments of the PEG-lipid, a PEG is of 500-5000 or 1000-5000 Da molecular weight (MW). For example, in some embodiments, the PEG of the PEG-lipid has a molecular weight in the range of 1500-5000 Da or 2000-5000 Da. In some embodiments as described herein, the PEG-lipid has a molecular weight in the range of 500-4000 Da, or 500-3000 Da, or 1000-4000 Da, or 1000-3000, or 1000-2500, or 1500-4000, or 1500-3000, or 1500-2500 Da. In some embodiments, the PEG unit has a MW of 2000 Da (sometime abbreviated as PEG(2k)). Some embodiments incorporate PEG moieties of PEG-1000, PEG-2000, or PEG-5000. Certain embodiments comprise a DSG-PEG, for example, DSG-PEG-2000. Certain embodiments comprise a DSPE-PEG, for example, DSPE-PEG-2000. Certain embodiments comprise both DSG-PEG-2000 and / or DSPE-PEG2000.

[00495] In some embodiments, the PEG moiety is PEG-500 to PEG-5000 such as PEG-500, PEG-1000, PEG-1500, PEG-2000, PEG-2500, PEG-3000, PEG-3500, PEG-4000, PEG-4500, and PEG-5000. In some instances, the PEG moiety is PEG-2000. In some embodiments, the PEG unit has a MW of 2000 Da.

[00496] Common PEG-lipids fall into two classes diacyl glycerols and diacyl phospholipids. Examples of diacyl glycerol PEG-lipids include DMG-PEG (1,2-dimyristoyl-glycero-3-methoxypolyethylene glycol), DPG-PEG (1,2-dipalmitoyl-glycero-3-methoxypolyethylene glycol), DSG-PEG (1,2-distearoyl-glycero-3-methoxypolyethylene glycol), and DOG-PEG (1,2-dioleoyl-glycero-3-methoxypolyethylene glycol). Examples of diacyl phospholipids include DMPE-PEG (1,2-dimyristoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol), DPPE-PEG (1,2-dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol), DSPE-PEG (1,2-distearoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol), and DOPE-PEG (1,2-dioleoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol).

[00497] In some embodiments, the MW2000 PEG-lipid (e.g., a PEG-lipid comprising a PEG of a molecular weight of 2000 Da) comprises DMG-PEG2000 (1,2-dimyristoyl-glycero-3- methoxypolyethylene     glycol-2000),     DPG-PEG2000 methoxypolyethylene     glycol-2000),     DSG-PEG2000 methoxypolyethylene     glycol-2000),      DOG-PEG2000 methoxypolyethylene    glycol-2000),     DMPE-PEG200 phosphoethanolamine-3-methoxypolyethylene glycol-2000), (1,2-dipalmitoyl-glycero-3- (1,2-distearoyl-glycero-3- (1,2-dioleoyl-glycero-3- (1,2-dimyristoyl-glycero-3- DPPE-PEG2000 (1,2- dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DSPE-PEG2000 (1,2-distearoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000),          DOPE-PEG2000         (1,2-dioleoyl-glycero-3-phosphoethanolamine-3- methoxypolyethylene glycol-2000), or combinations thereof. In some embodiments, the PEG unit has a MW of 2000 Da. In some embodiments, the MW2000 PEG-lipid comprises DMrG- PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DPrG-PEG2000 (1,2-dipalmitoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DSrG-PEG2000 (1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DorG-PEG2000 (1,2-dioleoyl-glycero-3-methoxypolyethylene-rac-glycol-2000), DMPEr-PEG200 (1,2-dimyristoyl-rac-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DPPEr-PEG2000 (1,2-dipalmitoyl-rac-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DSPEr-PEG2000                   (1,2-distearoyl-rac-glycero-3-phosphoethanolamine-3- methoxypolyethylene glycol-2000), DGPEr-PEG2000 (1,2-dioleoyl-rac-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), or combinations thereof. The glycerol in these lipids is chiral. Thus, in some embodiments, the PEG-lipid is racemic. Alternatively, optically pure antipodes of the glycerol portion can be employed, that is, the glycerol portion is homochiral. As used herein with respect to glycerol moieties, optically pure means >95% of a single enantiomer (D or L). In some embodiments, the enantiomeric excess is >98%. In some embodiments, the enantiomeric excess is >99%. Additional PEG-lipids, including achiral PEG-lipids built on a symmetric dihydroxyacetone scaffold, a symmetric 2-(hydroxymethyl)butane-1,4-diol, or a symmetric glycerol scaffold, are disclosed in U.S. Provisional Application No. 63 / 362,502, filed on April 5, 2022, and PCT / US2023 / 017648 filed on April 5, 2023 (WO 2023 / 196445), both entitled PEG-Lipids and Lipid Nanoparticles, which are incorporated by reference in their entirety.

[00498] The above examples are presented as methoxypolyethylene glycols, but the terminus need not necessarily be methoxyl. With respect to any of the PEG-lipids that have not been functionalized, in alternative embodiments, the PEG moiety of the PEG lipids can terminate with a methoxyl, a benzyloxyl, a 4-methoxybenzyloxyl, or a hydroxyl group (that is, an alcohol). The terminal hydroxyl facilitates functionalization. The methoxyl, benzyloxyl, and 4-methoxybenzyloxyl groups are advantageously provided for PEG-lipid that will be used without functionalization as a component of the LNP. However, all four of these alternatives are useful as the (non-functionalized) PEG-lipid component of LNPs. The 4-methoxybenzyloxyl group, often used as a protecting group during synthesis of the PEG-lipid, is readily removed to generate the corresponding hydroxyl group. Thus, the 4-methoxybenzyloxyl group offers a convenient path to the alcohol when it is not synthesized directly. The alcohol is useful for being functionalized, prior to incorporation of the PEG-lipid into an LNP, so that a binding moiety can be conjugated to it as a targeting moiety for the LNP (making it a tLNP). As used herein, the terminus of the PEG moiety, and similar constructions, refers to the end of the PEG moiety that is not attached to the lipid.

[00499] A PEG-moiety provides a hydrophilic surface on the LNP, inhibiting aggregation or merging of LNP, thus contributing to their stability and reducing polydispersity, i.e. reducing the heterogeneity of a dispersion of LNPs. Additionally, a PEG moiety can impede binding by the LNP, including binding to plasma proteins. These plasma proteins include apoE which is understood to mediate uptake of LNP by the liver so that inhibition of binding can lead to an increase in the proportion of LNP reaching other tissues. These plasma proteins also include opsonins so that inhibition of binding reduces recognition by the reticuloendothelial system. The PEG-moiety can also be functionalized to serve as an attachment point for a targeting moiety. Conjugating a cell- or tissue-specific binding moiety to the PEG-moiety enables a tLNP to avoid the liver and bind to its target tissue or cell type, greatly increasing the proportion of LNP that reaches the targeted tissue or cell type. PEG-lipid can thus serve as means for inhibiting LNP binding, and PEG-lipid conjugated to a binding moiety can serve as means for LNP-targeting.

[00500] As used herein, the term “functionalized PEG-lipid” and similar constructions refer generally to both the unreacted and reacted entities. The lipid composition of a LNP can be described referencing the reactive species even after conjugation has taken place (forming a tLNP). For example, a lipid composition can be described as comprising DSPE-PEG-maleimide and can be said to further comprise a binding moiety without explicitly noting that upon reaction to form the conjugate the maleimide will have been converted to a succinimide (or hydrolyzed succinimide). Similarly, if the reactive group is bromomaleimide, after conjugation it will be maleimide. These differences of chemical nomenclature for the unreacted and reacted species are to be implicitly understood even when not explicitly stated. Certain embodiments comprise a DSG-PEG, for example, DSG-PEG-2000. Certain embodiments comprise a functionalized DSPE-PEG, for example, functionalized DSPE-PEG-2000. Certain embodiments comprise both DSG-PEG-2000 and functionalized DSPE-PEG-2000. In some instances, the functionalized PEG-lipid is functionalized with a maleimide moiety, for example, DSPE-PEG-2000-MAL. Analogously, in certain embodiments, DSG-PEG serves as the functionalized or functionalized and non-functionalized PEG-lipid.

[00501] In certain embodiments, the PEG-lipid and / or functionalized PEG-lipid comprises a scaffold selected from Formula S1, Formula S2, Formula S3, or Formula S4: Formula S1                            Formula S2 Formula S3                           Formula S4 wherein represents the points of ester connection with a fatty acid, and represents the point of ester (S1) or ether (S2, S3, and S4) formation with the PEG moiety. In some embodiments, the fatty acid esters are C14-C20 straight-chain alkyl fatty acids. In some embodiments, the PEG moiety is functionalized and the fatty acid esters are C16-C20 straightchain alkyl fatty acids. For example, the straight-chain alkyl fatty acid is C14, C15, C16, C17, C18, C19, or C20. In some embodiments, the fatty acid esters are C14-C20 symmetric branched-chain alkyl fatty acids. For example, the branched-chain alkyl fatty acid is C14, C15, C16, C17, C18, C19, or 620- By symmetric it is meant that each alkyl branch has the same number of carbons. In some embodiments, the branch is at the 3, 4, 5, 6, or 7 position of the fatty acid ester. The synthesis and use of PEG-lipids built on scaffolds S1-S4 is disclosed in WO2023 / 196445A1 which is incorporated by reference for all that it teaches about PEG-lipids and their use.

[00502] Some embodiments of the disclosed ionizable cationic lipids have head groups with small (<250 Da) PEG moieties. These lipids are not what is meant by the term PEG-lipid as used herein. These small PEG moieties are generally too small to impede binding to a similar extent as the larger PEG moieties of the PEG-lipids disclosed above, though they will impact the lipophilicity of ionizable cationic lipid. Moreover, the PEG-lipids are understood to be primarily located in an exterior facing lamella whereas much of the ionizable cationic lipid is in the interior of the LNP.

[00503] In certain embodiments, a functionalized PEG-lipid of a LNP or tLNP of this disclosure comprises one or more fatty acid tails, each of which is no shorter than C16 nor longer than C20 for straight-chain fatty acids. For branched chain fatty acids, tails no shorter than C14 fatty acids nor longer than C20 are acceptable. In some embodiments, fatty acid tails are C16. In some embodiments, the fatty acid tails are C18. In some embodiments, the functionalized PEG-lipid comprises a dipalmitoyl lipid. In some embodiments, the functionalized PEG-lipid comprises a distearoyl lipid. The fatty acid tails serve as means to anchor the PEG-lipid in the tLNP to reduce or eliminate shedding of the PEG-lipid from the tLNP. This is a useful property for the PEG-lipid whether or not it is functionalized but has greater significance for the functionalized PEG-lipid as it will have a targeting moiety attached to it and the targeting function could be impaired if the PEG-lipid (with the conjugated binding moiety, such as an antibody) were shed from the tLNP.

[00504] In some embodiments, an LNP or tLNP comprises about 0.5 mol% to about 3 mol% or 0.5 mol% to 3 mol% PEG-lipid comprising functionalized and non-functionalized PEG-lipid. In certain embodiments, an LNP or tLNP comprises DSG-PEG. In other embodiments, an LNP or tLNP comprises DMG-PEG or DPG-PEG. In certain embodiments, an LNP or tLNP comprises DSPE-PEG. In some embodiments, the functionalized and non-functionalized PEG-lipids are not the same PEG-lipid, for example, the non-functionalized PEG-lipid can be a diacylglycerol and the functionalized PEG-lipid a diacyl phospholipid. tLNP with such mixtures have reduced expression in the liver, possibly due to reduced uptake. In certain embodiments the functionalized PEG-lipid is DSPE-PEG and the non-functionalized PEG-lipid is DSG-PEG. In some embodiments, an LNP or tLNP comprises about 0.4 mol% to about 2.9 mol% or about 0.9 mol% to about 1.4 mol% non-functionalized PEG lipid. In certain embodiments, an LNP or tLNP comprises about 1.4 mol% or 1.4 mol% non-functionalized PEG lipid. In some embodiments, an LNP or tLNP comprises about 0.1 mol% to about 0.3 mol% or 0.1 mol% to 0.3 mol% functionalized lipid. In some instances, the functionalized lipid is DSPE-PEG. In certain instances, an LNP or tLNP comprises about 0.1 mol%, about 0.2 mol%, or about 0.3 mol% DSPE-PEG. In certain instances, an LNP or tLNP comprises 0.1 mol%, 0.2 mol%, or 0.3 mol% DSPE-PEG. In certain instances, the functionalized PEG-lipid is conjugated to a binding moiety. As used herein, the phrase “is conjugated to” and similar constructions are meant to convey a state of being, that is, a structure, and not a process, unless context dictates otherwise. Conjugation

[00505] Any suitable chemistry can be used to conjugate the binding moiety to the PEG of the PEG-lipid, including maleimide (see Parhiz et al., J. Controlled Release 291:106, 2018) and click (see Kolb et al., Angewandte Chemie International Edition 40(11):2004, 2001; and Evans, Australian J. Chern. 60(6):384, 2007) chemistries. Reagents for such reactions include lipid-PEG-maleimide, lipid-PEG-cysteine, lipid-PEG-alkyne, lipid, PEG- dibenzocyclooctyne (DBCO), and lipid-PEG-azide. Further conjugations reactions make use of lipid-PEG-bromo maleimide, lipid-PEG-alkylnoic amide, PEG-alkynoic imide, and lipid-PEG-alkyne reactions, as disclosed in U.S. Provisional Application No. 63 / 362,502, filed on April 5, 2022, and PCT / US2023 / 017648 filed on April 5, 2023 (WO 2023 / 196445), both entitled PEG-Lipids and Lipid Nanoparticles, which are incorporated by reference in their entirety. On the binding moiety side of the reaction can be used an existing cysteine sulfhydryl, or the protein derivatized by adding a sulfur containing carboxylic acid, for example, to the epsilon amino of a lysine to react with a maleimide, bromomaleimide, alkylnoic amide, or alkynoic imide. In certain embodiments, to modify an epsilon amino of a binding moiety lysine to react with a maleimide functionalized PEG-lipid, the binding moiety (e.g., an antibody) can be reacted with N-succinimidyl S-acetylthioacetate (SATA). SATA is then deprotected, for example, using 0.5 M hydroxylamine followed by removal of the unreacted components by G-25 Sephadex Quick Spin Protein columns (Roche Applied Science, Indianapolis, IN). The reactive sulfhydryl group on the binding moiety is then conjugated to maleimide moieties on LNPs of the disclosure using thioether conjugation chemistry. Alternatively, an alkyne can be added to a sulfhydryl or an epsilon amino of a lysine to participate in a click chemistry reaction.

[00506] Purification can be performed using Sepharose CL-4B gel filtration columns (Sigma-Aldrich). tLNPs (LNPs conjugated with a targeting antibody) can be stored frozen at -80°C until needed. Others have conjugated antibody to free functionalized PEG-lipid and then incorporated the conjugated lipid into pre-formed LNP. However, it was found that this procedure is more controllable and produces more consistent results.

[00507] There are also several approaches to site-specific conjugation. Particularly but not exclusively suitable for truncated forms of antibody, C-terminal extensions of native or artificial sequences containing a particularly accessible cysteine residue are commonly used. Partial reduction of cysteine bonds in an antibody, for example, with tris(2-carboxy)phosphine (TCEP), can also generate thiol groups for conjugation which can be site-specific under defined conditions with an amenable antibody fragment. Alternatively, the C-terminal extension can contain a sortase A substrate sequence, LPXTG (SEQ ID NO: 1) which can then be functionalized in a reaction catalyzed by sortase A and conjugated to the PEG-lipid, including through click chemistry reactions (see, for example, Moliner-Morro et al., Biomolecules 10(12):1661, 2020 which is incorporated by reference herein for all that it teaches about antibody conjugations mediated by the sortase A reaction and / or click chemistry). The use of click chemistry for the conjugation of a targeting moiety, such as various forms of antibody, is disclosed, for example, in WO2024 / 102,770 which is incorporated by reference in its entirety for all that it teaches about the conjugation of targeting moieties to LNPs that is not inconsistent with this disclosure.

[00508] For whole antibody and other forms comprising an Fc region, site-specific conjugation to either (or both) of two specific lysine residues (Lys248 and Lys288) can be accomplished without any change to or extension of the native antibody sequence by use of one of the AJICAP® reagents (see, for example, Matsuda et al., Mol. Pharmaceutics 18:4058, 2021 and Fujii et al., Bioconjugate Chern. 34:728, 2023, which are incorporated by reference herein for all that they teach regarding conjugation of antibodies with AJICAP reagents). AJICAP reagents are modified affinity peptides that bind to specific loci on the Fc and react with an adjacent lysine residue to form an affinity peptide conjugate of the antibody. The peptide is then cleaved with base to leave behind a thiol-functionalized lysine residue which can then undergo conjugation through maleimide or haloamide reactions, for example). Functionalization with azide or dibenzocyclooctyne (DBCO) for conjugation by click chemistry is also possible. This and similar technology are further described in US 2020 / 0190165 (corresponding to WO 2018 / 199337), US 2021 / 0139549 (corresponding to WO 2019 / 240287) and US 2023 / 0248842 (corresponding to WO 2020 / 184944) which are incorporated by reference in their entirety for all that they teach about such modified affinity peptides and their use.

[00509] Accordingly, in some embodiments the binding moiety is conjugated to the PEG moiety of the PEG-lipid through a thiol modified lysine residue. In some embodiments, the conjugation is through a cysteine residue in a native or added antibody sequence. In other embodiments, the conjugation is through a sortase A substrate sequence. In still other embodiments, the conjugation is through a specific lysine residue (Lys248 or Lys288) in the Fc region. Binding Moieties

[00510] The tLNP of the various disclosed aspects comprise a binding moiety, such as an antibody or antigen binding domain thereof or a cell surface receptor ligand. As used herein, a “binding moiety” or “targeting moiety” refers to a protein, polypeptide, oligopeptide or peptide, carbohydrate, nucleic acid, or combinations thereof capable of specifically binding to a target or multiple targets. A binding domain includes any naturally occurring, synthetic, semisynthetic, or recombinantly produced binding partner for a biological molecule or another target of interest. Exemplary binding moieties of this disclosure include an antibody, a Fab', F(ab')2, Fab, Fv, rlgG, scFv, hcAb (heavy chain antibody), a single domain antibody, VHH, VNAR, sdAb, nanobody, receptor ectodomain or ligand-binding portions thereof, or ligand (e.g., cytokines, chemokines). An “Fab” (antigen binding fragment) is the part of an antibody that binds to antigens and includes the variable region and CH1 of the heavy chain linked to the light chain via an inter-chain disulfide bond. In other embodiments, a binding moiety comprises a ligand-binding domain of a receptor or a receptor ligand. In some embodiments, a binding moiety can have more than one specificity including, for example, bispecific or multispecific binders. A variety of assays are known for identifying binding moieties of this disclosure that specifically bind a particular target, including Western blot, ELISA, biolayer interferometry, and surface plasmon resonance. A binding moiety, such as a binding moiety comprising immunoglobulin light and heavy chain variable domains (e.g., scFv), can be incorporated into a variety of protein scaffolds or structures as described herein, such as an antibody or an antigen binding fragment thereof, a scFv-Fc fusion protein, or a fusion protein comprising two or more of such immunoglobulin binding domains.

[00511] The fundamental ability of the tLNP to deliver a payload into the cytoplasm of a cell is agnostic with respect to, and does not depend upon, a particular binding specificity. Of course, a binding moiety is a determinant of which cells a payload is delivered into. There are many known antibodies with specificity for one or another cell surface marker associated with particular cell type(s) that could be used as the target of the binding moiety on a disclosed tLNP and there are several sources that have compiled such information. An excellent source of information about antibodies for which an International Non-proprietary Name (INN) has been proposed or recommended is Wilkinson & Hale, MAbs 14(1):2123299, 2022, including its Supplementary Tables, which is incorporated by reference herein for all that it teaches about individual antibodies and the various antibody formats that can be constructed. US11,326,182 and especially its Table 9 Cancer, Inflammation and Immune System Antibodies, is a source of sequence and other information for a wide range of antibodies including many that do not have an INN and is incorporated herein by reference for all that it teaches about individual antibodies. Sequence information is not always readily available for antibodies mentioned in the art, even when commercially available. This is not necessarily an impediment to their use. Where the antibody or a cell line is commercially available or obtainable from its originator it can be used as the binding moiety of tLNP without any need for sequence information. Even where sequence information is needed, it is well within the capabilities of the skilled artisan to sequence the antibody protein (or have it done by a contract laboratory) so that the antibody’s variable region can be incorporated into a scFv, a diabody, a minibody, or some other antibody format, or be humanized. In choosing among available antibodies in the art for the development of an agent to be used in humans, a human antibody is preferred to a humanized antibody is preferred to a non-human antibody, other factors being equal. Other factors can include stability and ease of production of the antibody, affinity of the antibody, and cross-reactivity for the cognate antigen in model species to be used in product development.

[00512] In some embodiments, a binding moiety can be an antibody or an antigen-binding portion thereof; an antigen; a ligand-binding domain of a receptor; or a receptor ligand. In some embodiments, a binding moiety can have more than one specificity including, for example, bispecific or multispecific binders.

[00513] In some embodiments, a binding moiety comprises an antibody or an antigenbinding portion thereof. As used herein, “antibody” refers to a protein comprising an immunoglobulin domain having hypervariable regions determining the specificity with which the antibody binds antigen, termed complementarity determining regions (CDRs). The term antibody can thus refer to intact or whole antibodies as well as antibody fragments and constructs comprising an antigen binding portion of a whole antibody. While the canonical natural antibody has a pair of heavy and light chains, camelids (from camels, alpacas, llamas, etc.) produce antibodies with both the canonical structure and antibodies comprising only heavy chains. The variable region of the camelid heavy chain-only antibody has a distinct structure with a lengthened CDR3 referred to as VHH or, when produced as a fragment, a nanobody. Antigen binding fragments and constructs of antibodies include F(ab)2, F(ab), minibodies, Fv, single-chain Fv (scFv), diabodies, and VH. Such elements can be combined to produce bi- and multi-specific reagents, including various immune cell engagers, such as BiTEs (bi-specific T-cell engagers). The term “monoclonal antibody” arose out of hybridoma technology but is now used to refer to any singular molecular species of antibody regardless of how it was originated or produced. Antibodies can be obtained through immunization, selection from a naive or immunized library (for example, by phage display), alteration of an isolated antibody-encoding sequence, or any combination thereof. Numerous antibodies that can be used as binding moieties are known in the art. An excellent source of information about antibodies for an International Non-proprietary Name (INN) has been proposed or recommended, including sequence information, is Wilkinson & Hale, 2022, MAbs 14(1):2123299, including its Supplementary Tables, which is incorporated by reference herein for all that it teaches about individual antibodies and the various antibody formats that can be constructed. U.S. Patent No. 11,326,182 and especially its Table 9 entitled “Cancer, Inflammation and Immune System Antibodies,” is a source of sequence and other information for a wide range of antibodies including many that do not have an INN and is incorporated herein by reference for all that it teaches about individual antibodies. WO2024040195A1 is also a source of sequence and other information for a wide range of antibodies with specificity for various cell surface antigens of immune system cells and cancer cells and is incorporated herein by reference for all that it teaches about individual antibodies and the antigens they bind.

[00514] An antibody or other binding moiety (or a fusion protein thereof) “specifically binds” a target if it binds the target with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1 / Molar or 1 / M) equal to or greater than 105 M-1, while not significantly binding other components present in a test sample. Binding domains (or fusion proteins thereof) can be classified as “high affinity” binding domains (or fusion proteins thereof) and “low affinity” binding domains (or fusion proteins thereof). “High affinity” binding domains refer to those binding domains with a Ka of at least 108 M'1, at least 109 M'1, at least 1010 M’1, at least 1011 M'1, at least 1012 M'1, or at least 1013 M'1, preferably at least 108 M'1 or at least 109 M'1. “Low affinity” binding domains refer to those binding domains with a Ka of up to 108 M’1, up to 107 M’1, up to 106 M’1, up to 105 M-1. Alternatively, affinity can be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10’5 M to 10’13 M). Affinities of binding domain polypeptides and fusion proteins according to this disclosure can be readily determined using conventional techniques (see, e.g., Scatchard et al., 1949, Ann. N.Y. Acad. Sci. 51:660; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).

[00515] A diabody is a type of scFv dimer in which each chain consists of the VH and VL regions connected by a small peptide linker that is too short to allow pairing between the two domains of the same chain. This arrangement forces the VH of one chain to pair with the VL of a second chain thereby forming a bivalent, and often bispecific, dimer. A BiTE is a fusion protein having two scFvs of different antibodies, usually an antibody for a tumor-associated antigen and antibody for CD3, on a single peptide chain, thus forming a cytolytic synapse between T cells and target antigen-bearing cells. The term "antigen-binding portion" can refer to a portion of an antibody as described that possesses the ability to specifically recognize, associate, unite, or combine with a target molecule. An antigen-binding portion includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a specific antigen. Thus, antibodies and antigen-binding portions thereof constitute means for binding to the surface molecule on a cell. In various embodiments, the cell can be an immune cell, a leukocyte, a lymphocyte, a monocyte, a stem cell, an HSC or an MSC, according to the specificity of the antibody.

[00516] In some embodiments, the antibody or antigen-binding portion thereof can be derived from a mammalian species, for example, mice, rats, or human. Antibody variable regions can be those arising from one species, or they can be chimeric, containing segments of multiple species possibly further altered to optimize characteristics such as binding affinity or low immunogenicity. For human applications, it is desirable that the antibody has a human sequence. In the cases where the antibody or antigen-binding portion thereof is derived from a non-human species, the antibody or antigen-binding portion thereof can be humanized to reduce immunogenicity in a human subject. For example, if a human antibody of the desired specificity is not available, but such an antibody from a non-human species is, the non-human antibody can be humanized, e.g., through CDR grafting, in which the CDRs from the nonhuman antibody are placed into the respective positions in a framework of a compatible human antibody. Less preferred is an antibody in which only the constant region of the non-human antibody is replaced with human sequence. Such antibodies are commonly referred to as chimeric antibodies in distinction to humanized antibodies.

[00517] In some embodiments, the antibody or antigen-binding portion thereof is non-immunogenic. In some embodiments, the antibody can be modified in its Fc region to reduce or eliminate secondary functions, such as FcR engagement, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complementdependent cytotoxicity (CDC); this is often referred to as an Fc silenced antibody.

[00518] A binding moiety density on the LNP (or tLNP) can be defined according to the ratio of antibody (binder) to mRNA (w / w) either based on the amount of antibody input in the conjugation reaction or as measured in the LNP or (tLNP) after conjugation. For an intact antibody (e.g., whole IgG), in some embodiments, preferred ratios are about 0.3 to about 1.0, about 0.3 to about 0.7, about 0.3 to about 0.5, about 0.5 to about 1.0, and about 0.5 to about 0.7 for either the input or final measured binder ratio. In certain embodiments, a LNP (or tLNP) has an antibody ratio of 0.3 to 1.0, 0.3 to 0.7, 0.3 to 0.5, 0.5 to 1.0, and 0.5 to 0.7 for either the input or final measured binder ratio. In some embodiments, if the binder is different in size from an intact antibody (for example a scFv, diabody, or minibody, etc.) the w / w ratio is adjusted for the different size of the binder.

[00519] In certain embodiments, a LNP or tLNP comprise a binding moiety that is a F(ab’) or F(ab’) analog. F(ab’) and F(ab’)-like formats offer certain advantages as targeting moieties for a tLNP. Although any antibody fragment with a structure similar to or derived from that of a classical, proteolytically produced F(ab’) is often referred to as an F(ab’), the term “F(ab’) analog” has been adopted herein to refer to engineered sequences comprising amino acid substitutions and / or that have been truncated and to distinguish them from the paradigmatic natural sequence. F(ab’) are smaller than whole antibodies which can be advantageous in manufacturing. When used as a targeting moiety on a tLNP their antigen binding domain is further from the LNP surface than, for example, a scFv, which can facilitate interaction with the target cell surface. F(ab’) molecules have cysteine residues in the partial hinge region that can be readily conjugated to a functionalized PEG-lipid (for example, a maleimide-functionalized PEG-lipid). Moreover, the F(ab’) can be engineered so that there is unique accessible cysteine enabling for site-specific conjugation which is desirable for product consistency. This can be accomplished with recombinant DNA technology by truncating the hinge region of the F(ab’) or by changing cysteine residues to another amino acid, such as serine, or both.

[00520] The hinge region cysteines can form a cystine with another F(ab’) molecule forming an F(ab’)2 which would make the cysteine unavailable for conjugation to an LNP (more specifically, a functionalized lipid thereof). This can be prevented by processing the F(ab’) under mildly reducing conditions, however, this poses a risk of disrupting the interchain disulfide bond between CL and CH1. That risk can be obviated by relocating the interchain bond to a less accessible region in the molecule.

[00521] Some aspects combine constant regions of an F(ab’) or F(ab’) analog with a humanized immunoglobulin antigen binding domain derived from the anti-CD8a antibody CT8 as disclosed herein. Humanized anti-CD8 antigen binding domains and antibodies and F(ab’) analog design useful as LNP targeting moieties are disclosed, for example, in International Patent Application No. PCT / US2024 / 060426, filed December 16, 2024, which is incorporated for reference for all that it teaches about anti-CD8 antigen binding domains and antibodies and F(ab’) analog designs.

[00522] Some aspects combine constant regions of an F(ab’) analog with the antigen binding domain of an anti-CD8 antibody. In some embodiments, the anti-CD8 antigen binding domain recognizes the CT8 epitope. In some embodiments, the anti-CD8 antigen binding domain is derived from YTC182.20, TRX2, or CT8. In some embodiments, the anti-CD8 antigen binding domain comprises a humanized immunoglobulin antigen binding domain derived from the anti-CD8a antibody CT8 as disclosed herein.

[00523] In some aspects, an F(ab’) analog engineered as disclosed herein is conjugated to an LNP but is generic with respect to the variable domains of the F(ab’) analog and its specificity.

[00524] In some aspects, an F(ab’) or F(ab’) analog constant regions are combined with the antigen binding domain of an anti-CD8 antibody which is conjugated to an LNP. In some embodiments, the anti-CD8 antigen binding domain recognizes the CT8 epitope. In some embodiments, the anti-CD8 antigen binding domain is derived from YTC182.20, TRX2, or CT8. In some embodiments, the anti-CD8 antigen binding domain comprises a humanized immunoglobulin antigen binding domain derived from the anti-CD8a antibody CT8 as disclosed herein.

[00525] With respect to these forgoing aspects, in some embodiments, the F(ab’) analog, as appropriate, comprises a relocated interchain disulfide bond, for example, a Ck S162C substitution paired with an lgG1 or lgG4 CH1 F174C substitution. In further embodiments, one, the other, or both cysteines involved in forming the native interchain disulfide bond are mutated, for example, Ck C214S, lgG1 C233S, or lgG4 CH1 C127S.

[00526] In certain embodiments, a LNP or tLNP comprises a binding moiety derived from an anti-CD40*t antibody, an anti-LRRC15t:|: antibody, an anti-CTSK antibody, an anti-ADAM12* antibody, an anti-ITGA11 antibody, an anti-FAP*1"* antibody, an anti-NOX4 antibody, an anti-SGCD antibody, an anti-SYNDIG1 antibody, an anti-CDHU* antibody, an anti-PLPP4 antibody, an anti-SLC24A2 antibody, an anti-PDGFRB** antibody, an anti-THYl* antibody, an anti-ANTXRI* antibody, an anti-GAS1 antibody, an anti-CALHM5 antibody, an anti-SDCI** antibody, an anti-HER2*t:|: antibody, an anti-TROP2*t:|: antibody, an anti-MSLN** antibody, an anti-Nectin4t:|: antibody, or an anti-MUC16*t:|: antibody. In further embodiments, a LNP (or tLNP) comprises a binding moiety specific for an immune cell antigen selected from CD1, CD2***, CD3***, CD4***, CD5**, CD7**, CD8* CD11b*, CD14**, CD16, CD25**, CD26**, CD27***, CD28***, CD30***, CD32*, CD38***, CD39*. CD40***, CD40L (CD154)***, CD44**, CD45**, CD64**, CD62**, CD68, CD69*. CD73« CD80**, CD83*. CD86**, CD95*. CD103* CD119* CD126* CD137 (41BB)« CD150* CD153* CD161* CD166* CD183 (CXCR3)*, CD183 (CXCR5)*, CD223 (LAG-3)***, CD254*. CD275*, CD45RA, CTLA-4****, DEC205, 0X40*, PD-1***, GITR* TIM-3***, , FasL**, IL18R1, ICOS (CD278)*, leu-12, TCR*. TLR1, TLR2**, TLR3**, TLR4«, TLR6, TREM2* NKG2D* CCR, CCR1 (CD191)*, CCR2 (CD192)*«, CCR4(CD194)***, CCR6(CD196)*, CCR7* low affinity IL-2 receptor**, IL-7 receptor* IL-12 receptor*, IL-15 receptor*, IL-18 receptor*, and IL-21 receptor*. In further embodiments, a tLNP comprises a binding moiety specific for an HSC surface molecule selected from CD117*, CD34**, CD44**, CD90 (Thy1)*, CD105* CD133* BMPR2* and Sca-1; or specific for an MSG surface molecules selected from CD70**, CD105* CD73* Stro-1* SSEA-3* SSEA-4* CD271* CD146* GD2***, SUSD2, Stro-4, MSCA-1, CD56* CD200***, PODXL* CD13* CD29**, CD44**, and CD10*. In various embodiments, a binding moiety is an antibody or antigen-binding portion thereof. (* indicates that exemplary antibodies with the indicated specificity from which a binding moiety could be derived can be found in US Patent 11,326,182B2 Table 9 or 10. *indicates that exemplary antibodies with the indicated specificity from which a binding moiety could be derived can be found in Wilkinson & Hale, 2022. Both references cited and incorporated by reference above. * indicates that exemplary antibodies with the indicated specificity from which a binding moiety could be derived can be found in the Therapeutic Antibody Database (TABS) at tabs.craic.com). Other suitable antibodies can be found in Appendix A, or in International Patent Publication No. WO2024040195A1, filed August 17, 2023, each of which is incorporated herein by reference for all that it teaches about individual antibodies and the antigens they bind.

[00527] The following paragraphs provide non-exhaustive examples of known antibodies that bind to cell surface markers / antigens on immune cells (lymphocytes and monocytes) and stem cells (HSC and MSC). These antibodies or the antigen binding domains thereof can be used as binding moieties to target the disclosed LNP. Similarly, these antibodies can contribute their antigen binding domains to immune cell reprogramming agents such as CARs and ICEs. While typically an immune cell reprogramming agent is expressed in an immune cell, one call also express a biological response modifier (conditioning agent) or an immune cell reprogramming agent, such as an ICE, in a tumor cell. The immune and stem cell surface markers that can serve as a targeted antigen of a tLNP can also usefully be a target of an immune cell reprogramming agent when the cell expressing that antigen has a role in the pathology of some disease or condition. Collectively these antibodies and polypeptides comprising the antigen binding domains thereof constitute means for binding cell surface markers or means for binding immune and stem cells.

[00528] In some embodiments, CD2 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD2 antibody. CD2 contains three well-characterized epitopes (T11.1, T11.2, and T11.3 / CD2R). T11.3 / CD2R are membrane proximal and exposure is increased upon T cell activation and CD2 clustering. Accordingly, in some such embodiments, the anti-CD2 antigen binding domain is derived from, RPA-2.10; OKT11, UMCD2, 0.1, and 3T4-8B5 (T11.1 epitope); 9.6 and 1OLD2-4C1 (T11.2 epitope); 1Mono2A6 (T11.3 epitope), siplizumab (T11.2 / T11.3 epitope), HuMCD2, TS2 / 18, TS1 / 8, AB75, LT-2, T6.3, MEM-65, OTI4E4, or an antigen-binding portion thereof. Additionally, the ligand of CD2, CD58 (LFA-3) can be used as a CD2 binding moiety as can alefacept, a CD58-Fc fusion. Each of these constitutes a means for binding CD2 (Li et al., 1996, J Mol Biol. 263:209-26; Binder etal., 2020, Front Immunol. 9:11:1090).

[00529] In some embodiments, CD3 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD3 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from muromonab-CD3 (OKT3), teplizumab, otelixizumab, visilizumab, cevostamab, teclistamab, elranatamab pavurutamab, vibecotamab, odronextamab, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD3.

[00530] In some embodiments, CD4 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD4 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from ibalizumab, inezetamab, semzuvolimab, zanolimumab, tregalizumab, UB-421, priliximab, MTRX1011A, cedelizumab, clenoliximab, keliximab, M-T413, TRX1, hB-F5, MAX.16H5, IT208, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD4.

[00531] In some embodiments, CD5 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD5 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from 5D7, UCHT2, L17F12, H65, HE3, OKT1, MAT304, as well as those disclosed in WO1989006968, WO2008121160, US8,679,500, WO2010022737, WO2019108863, WO2022040608, or WO2022127844, each of which is incorporated by reference for all that they teach about anti-CD5 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD5.

[00532] In some embodiments, CD7 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD7 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from TH-69, 3A1E, 3A1F, Huly-m2, WT1, YTH3.2.6, T3-3A1, grisnilimab, as well as those disclosed in LIS10,106,609, WO2017213979, WO2018098306, US11447548, WO2022136888, WO2020212710, WO2021160267, WO2022095802, WO2022095803, WO2022151851, or WO2022257835 each of which is incorporated by reference for all that they teach about anti-CD7 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD7.

[00533] In some embodiments, CD8 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD8 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from crefmirlimab (IAB22M), 3B5, SP-16, LT8, 17D8, MEM-31, MEM-87, RIV11, UCHT4, YTC182.20, RPA-T8, OKT8, SK1, 51.1, TRX2, MT807-R1, HIT8a, C8 / 144B, RAVB3, SIDI8BEE, BU88, EPR26538-16, 2ST8.5H7, as well as those disclosed in US10,414,820, WO2015184203, WO2017134306, WO2019032661, WO2020060924, US10,730,944, WO2019033043, WO2021046159, WO2021127088, WO2022081516, US11,535,869, or WO2023004304 each of which is incorporated by reference for all that they teach about anti-CD8 antibodies and their properties, or an antigen-binding portion thereof. Additionally, humanized anti-CD8 antibodies are described in U.S. Provisional Patent Application Number 63 / 610,917, filed on December 15, 2023, and U.S. Provisional Patent Application Number 63 / 654,930, filed on May 31,2024, .S. Provisional Patent Application Number 63 / 708,461, filed on October 17, 2024, and International Patent Application Number PCT / US2024 / 060426, filed on December 16, 2024, each of which is incorporated by reference for all that it teaches about these humanized anti-CD8 antibodies and their properties, or an antigen-binding portion thereof. Each of the foregoing anti-CD8 antibodies constitutes a means for binding CD8.

[00534] In some embodiments, a tLNP is targeted to CD10 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD10 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from the one produced by the hybridoma represented by Accession No. NITE BP-02489 (disclosed in WO2018235247 which is incorporated by reference for all that they teach about anti-CD10 antibodies and their properties), FR4D11, or REA877, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD10.

[00535] In some embodiments, CD11b is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD11 b antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from ASD141 or MAB107 as well as those disclosed in US20150337039, US10,738,121, WO2016197974, US10,919,967, or WO2022147338 each of which is incorporated by reference for all that they teach about anti CD11b antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD11b.

[00536] In some embodiments, CD13 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD13 antibody. CD13 is also known as aminopeptidase N (APN). Accordingly, in some such embodiments, the antigen binding domain is derived from MT95-4 or Nbl57 (disclosed in WO2021072312 which is incorporated by reference for all that they teach about anti-CD13 antibodies and their properties), as well as those disclosed in WO2023037015 which is incorporated by reference for all that it teaches about anti-CD13 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD13.

[00537] In some embodiments, CD14 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD14 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from atibuclimab or r18D11 as well as those disclosed in WO2018191786 or WO2015140591 each of which is incorporated by reference for all that they teach about anti-CD14 antibodies and their properties, or an antigenbinding portion thereof. Each of these constitutes a means for binding CD14.

[00538] In some embodiments, CD16a is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD16a antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from AFM13, sdA1, sdA2, or hu3G8-5.1-N297Q as well as those disclosed in US11535672, WO2018158349, WO2007009065, US10385137,  WO2017064221, US10,758,625, WO2018039626, WO2018152516, WO2021076564, WO2022161314, or WO2023274183 each of which is incorporated by reference for all that they teach about anti-CD16A antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD16a.

[00539] In some embodiments, CD25 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD25 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from daclizumab, basiliximab, camidanlumab, tesirine, inolimomab, RO7296682, HuMax-TAC, CYT-91000, STI-003, RTX-003, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD25.

[00540] In some embodiments, CD28 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD28 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from GN 1412, acazicolcept, lulizumab, prezalumab, theralizumab, FR104CD, and davoceticept, as well as those disclosed in US8,454,959, US8,785,604, US11,548,947, US11,530,268, US11,453,721, US11,591,401, WO2002030459, WO2002047721, US20170335016, US20200181260, US11608376, WO2020127618, WO2021155071, or WO2022056199 each of which is incorporated by reference for all that they teach about anti-CD28 antibodies and their properties, or an antigenbinding portion thereof. Each of these constitutes a means for binding CD28.

[00541] In some embodiments, CD29 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD29 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from OS2966, 6D276, 12G10, REA1060, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD29.

[00542] In some embodiments, CD32A is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD32A antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from VIB9600, humanized IV.3, humanized AT-10, or MDE-8 as well as those disclosed in US9,688,755, US9,284,375, US9,382,321, US11306145, or WO2022067394 each of which is incorporated by reference for all that they teach about anti-CD32A antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD32A.

[00543] In some embodiments, CD34 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD34 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from h4C8, 9C5, 2E10, 5B12, REA1164, C5B12, C2e10, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD34.

[00544] In some embodiments, CD40 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD40 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from cifurtilimab, sotigalimab, iscalimab, dacetuzumab, selicrelumab, bleselumab, lucatumumab, or mitazalimab as well as those disclosed in US10633444, each of which is incorporated by reference for all that they teach about anti-CD40 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD40.

[00545] In some embodiments, CD44 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD44 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from RO5429083, VB6-008, PF-03475952, or RG7356, as well as those disclosed in WO2008144890, US8,383,117, WO2008079246, US20100040540, WO2015076425, US9,220,772, US20140308301, WO2020159754, WO2021160269, WO2021178896, WO2022022749, WO2022022720, or WO2022243838, each of which is incorporated by reference for all that they teach about anti-CD44 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD44.

[00546] In some embodiments, CD45 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD45 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from apamistamab, BC8-B10, as well as those disclosed in WO2023183927, WO2023235772, US7,825,222, WO2017009473, WO2021186056,   US9,701,756,   US9,701,756,  WO2020092654,  WO2022040088, WO2022040577, WO2022064191, WO2022063853, or WO2024064771, each of which is incorporated by reference for all that they teach about anti-CD45 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD45.

[00547] In some embodiments, CD56 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD56 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from lorvotuzumab, adcitmer, or promiximab, as well as those disclosed in WO2012138537, US10,548,987, US10,730,941, or US20230144142, each of which is incorporated by reference for all that they teach about anti-CD56 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD56.

[00548] In some embodiments, CD64 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD64 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from HuMAb 611 or H22 as well as those disclosed in US7,378,504, WO2014083379, US20170166638, or WO2022155608 each of which is incorporated by reference for all that they teach about anti-CD64 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD64.

[00549] In some embodiments, CD68 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD68 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from KI-M7, PG-M1, 514H12, ABM53F5, 3F7C6, 3F7D3, Y1 / 82A, EPR20545, CDLA68-1, LAMP4-824, or an antigenbinding portion thereof. Each of these constitutes a means for binding CD68.

[00550] In some embodiments, CD70 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD70 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from cusatuzumab, vorsetuzumab, MDX-1203, MDX-1411, AMG-172, SGN-CD70A, ARX305, PRO1160, as well as those disclosed in US9,765,148, US8,124,738, IS10,266,604, WO2021138264, US9,701,752, US10,108,123, WO2014158821, US10,689,456, WO2017062271, US11,046,775, US11,377,500, WO2021055437, WO2021245603, WO2022002019, WO2022078344, WO2022105914, WO2022143951, WO2023278520, WO2022226317, WO2022262101, US11,613,584, or WO2023072307, each of which is incorporated by reference for all that they teach about anti-CD70 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD70.

[00551] In some embodiments, CD73 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD73 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from oleclumab, uliledlimab, mupadolimab, AK119, IBI325, BMS-986179, NZV930, JAB-BX102, Sym024, TB19, TB38, HBM1007, 3F7, mAb19, Hu001-MMAE, IPH5301, or INCA00186, as well as those disclosed in US9,938,356, US10,584,169, WO2022083723, WO2022037531, WO2021213466, WO2022083049,   US10,822,426,  WO2021259199,   US10,100,129, US11,312,783, US11,174,319, US11,634,500, WO2021138467, WO2017118613, US9,388,249, WO2020216697,  US11180554, US11,530,273, WO2019173692, WO2019170131, US11,312,785, WO2020098599, WO2020143836, WO2020143710, US11,034,771, US11,299,550, WO2020253568, WO2021017892, WO2021032173, WO2021032173, WO2021097223, WO2021205383, WO2021227307, WO2021241729, WO2022096020, WO2022105881, WO2022179039, WO2022214677, or WO2022242758, each of which is incorporated by reference for all that they teach about anti-CD73 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD73.

[00552] In some embodiments, CD90 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD90 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from REA897, OX7, 5E10, K117, L127, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD90.

[00553] In some embodiments, CD105 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD105 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from carotuximab, TRC205, or huRH105, as well as those disclosed in US8,221,753, US9,926,375, WO2010039873, WO2010032059, WO2012149412, WO2015118031, WO2021118955, US20220233591, or US20230075244, each of which is incorporated by reference for all that they teach about anti-CD105 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD105.

[00554] In some embodiments, CD117 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD117 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from briquilimab, barzolvolimab, CDX-0158, LOP628, MGTA-117, NN2101, CK6, JSP191, Ab85, 104D2, or SR1, as well as those disclosed in US7.915.391, WO2022159737, US9540443, WO2015050959, US9,789,203,    US8,552,157,    US10,406,179,    US9,932,410,    WO2019084067, WO2020219770, US10,611,838, WO2020076105, WO2021107566, US11,208,482, WO2021044008, WO2021099418, WO2022187050, or WO2023026791, WO2021188590, each of which is incorporated by reference for all that they teach about anti-CD117 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD117.

[00555] In some embodiments, CD133 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD133 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from AC133, 293C3, CMab-43, or RW03, as well as those disclosed in WO2018045880, US8,722,858, US9,249,225, WO2014128185, US10,711,068, US10,106,623, WO2018072025, or WO2022022718, each of which is incorporated by reference for all that they teach about anti-CD133 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD133.

[00556] In some embodiments, CD137 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD137 antibody. CD137 is also known as 4-1BB. Accordingly, in some such embodiments, the antigen binding domain is derived from YH004, urelumab (BMS-663513), utomilumab (PF-05082566), ADG106, LVGN6051, PRS-343, as well as those disclosed in WO2005035584, WO2012032433, WO2017123650, US11,203,643, US11,242,395, US11,555,077, US20230067770, US11,535,678, US11.440.966, WO2019092451, US10.174.122, US11,242,385, US10.716.851, WO2020011966, WO2020011964, or US11,447,558, each of which is incorporated by reference for all that they teach about CD137 antibodies and their properties, or an antigenbinding portion thereof. Each of these constitutes a means for binding CD137.

[00557] In some embodiments, CD146 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD146 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from imaprelimab, ABX-MA1, huAA98, M2H, or IM1-24-3, as well as those disclosed in US10,407,506, US10,414,825, US6,924,360,   US9,447,190,   WO2014000338,   US9,782,500,   WO2018220467, US11,427,648, WO2019133639, WO2019137309, WO2020132190, or WO2022082073, each of which is incorporated by reference for all that they teach about CD146 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD146.

[00558] In some embodiments, CD166 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD166 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from praluzatamab, AZN-L50, REA442, or AT002, as well as those disclosed in US10,745,481, US11,220,544, or WO2008117049, each of which is incorporated by reference for all that they teach about CD166 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD166.

[00559] In some embodiments, CD200 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD200 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from samalizumab, OX-104, REA1067, B7V3V2, HPAB-0260-YJ, or TTI-CD200, as well as those disclosed in WO2007084321 or WO2019126536, each of which is incorporated by reference for all that they teach about CD200 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD200.

[00560] In some embodiments, CD205 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD205 antibody. CD205 is also known as DEC205. Accordingly, in some such embodiments, the antibody comprises 3G9-2D2 (a component of CDX-1401) or LY75_A1 (a component of MEN1309) as well as those disclosed in US8,236,318, US10,081,682, or US11,365,258, each of which is incorporated by reference for all that they teach about anti-CD205 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD205.

[00561] In some embodiments, CD271 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD271 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from REA844 or REAL709 as well as those disclosed in WO2022166802 which is incorporated by reference for all that it teaches about anti-CD271 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD271.

[00562] In some embodiments, BMPR2 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-BMPR2 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from TAB-071 CL (Creative Biolabs catalog no.) as well as those disclosed in US11,292,846 or WO2021174198, each of which is incorporated by reference for all that they teach about anti-BMPR2 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding BMPR2.

[00563] In some embodiments, claudin 18.2 (CLDN 18.2) is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-claudin 18.2 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from zolbetuximab, osemitamab, RC118, IBI-343, AZD0901, M108, SYSA1801, TORL-2-307-ADC, LM-302, ASKB589, gresonitamab, SPX-101, SKB315, Q-1802, GIVASTOMIG, LCAR-C18S, SOT102, CT041 as well as those disclosed in WO2013167259, WO2021032157, WO2021254481, WO2022007808, WO2021008463, WO2022111616, WO2018006882, WO2020147321, WO2019219089, US20200040101, WO2020025792, WO2020139956, WO2020135201, US20240228610, WO2021218874, WO2021027850, WO2021129765, WO2022068854, WO2021111003, each of which is incorporated by reference for all that it teaches about anti- claudin 18.2 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding claudin 18.2.

[00564] In some embodiments, CTLA-4 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CTLA-4 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from botensilimab, ipilimumab, nurulimab, quavonlimab, tremelimumab, zalifrelimab, ADG116, ADG126, ADU-1604, AGEN 1181, BCD-145, BMS-986218, BMS-986249, BT-007, CS1002, GIGA-564, HBM4003, IBI310 JK08, JMW-3B3, JS007, KD6001, KN044, ONC-392, REGN4659, TG6050, XTX101, YH001, or an antigen-binding portion thereof. Each of these constitutes a means for binding CTLA-4.

[00565] In some embodiments, GD2 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-GD2 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from dinutuximab, ganglidiximab, naxitamab, nivatrotamab, EMD 273063, hu14.18k322A, MORAb-028, 3F8BiAb, BCD-245, KM666, ATL301, Ektomab, as well as those disclosed in US9,777,068, US9,315,585, WO2004055056, US9,617,349,    US9,493,740,    US20210002384,    US8507657, WO2001023573, WO2012071216, WO2018010846, US8,951,524, WO2023280880, US9,856,324, WO2015132604, WO2017055385, WO2019059771, WO2020020194, or an antigen-binding portion thereof. Each of these constitutes a means for binding GD2.

[00566] In some embodiments, GITR is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-GITR antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from ragifilimab, TRX518, MK-4166, AMG 228, MEDI1873, BMS-986156, REGN6569, ASP1951, MK-1248, FRA154, GWN323, JNJ-64164711, ATOR-1144, or an antigen-binding portion thereof. Each of these constitutes a means for binding GITR.

[00567] In some embodiments, a low affinity IL-2 receptor is a targeted cell surface antigen (CD122 and / or CD132) and a binding moiety comprises the antigen binding domain of an antiIL-2 receptor antibody. Accordingly, in some such embodiments, the antiCD122 antibody comprises ANV419, FB102, MiK-Beta-1 and the anti CD122 antibodies disclosed in WO2011127324, WO2017021540, WO2022212848, WO2022221409, WO2023078113, US20230272090, WO2024073723, or an antigen-binding portion thereof. Accordingly, in some such embodiments, the anti-CD132 antibody comprises REGN7257 and the anti-CD132 antibodies disclosed in WO2020160242, WO2017021540, WO2022212848, WO2023078113, US20230272089, or an antigen-binding portion thereof. Each of these constitutes a means for binding the low affinity IL-2 receptor (CD122 or CD132, as appropriate),

[00568] In some embodiments, a high affinity IL-2 receptor is a targeted cell surface antigen (CD25) and a binding moiety comprises the antigen binding domain of an anti-IL-2 receptor antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from daclizumab, basiliximab, camidanlumab, vopitug, inolimomab, HuMAx-TAC, Xenopax, STI-003, RA8, RTX-003, and the anti-CD25 antibodies disclosed in WO2023031403, WO2006108670, WO2019175223, WO2019175215, WO2019175226, WO2004045512, WO2022104009, WO2020102591, or an antigen-binding portion thereof. Each of these constitutes a means for binding the high affinity IL-2 receptor (CD25).

[00569] In some embodiments, IL-7 receptor (CD127) is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-IL-7 receptor antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from PF-06342674, GSK2618960, OSE-127, lusvertikimab, bempikibart, and the anti-CD127 antibodies disclosed in WO2011104687, WO2011094259, WO2013056984, WO2015189302, WO2017062748, WO2020154293, WO2020254827, WO2021222227, WO2023201316, or an antigen-binding portion thereof. Each of these constitutes a means for binding the CD127.

[00570] In some embodiments, IL-12 receptor is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-IL-12 receptor antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from CBYY-10413, REA333, or an antigen-binding portion thereof. Each of these constitutes a means for binding the IL-12 receptor.

[00571] In some embodiments, IL-15 receptor a is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-IL-15 receptor a antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from MAB1472-100, MAB5511, JM7A4, 5E3E1, JM7A4, 2639B, or an antigen-binding portion thereof. Each of these constitutes a means for binding the IL-15 receptor a.

[00572] In some embodiments, IL-18 receptor a is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-IL-18 receptor a antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from H44, or an antigen-binding portion thereof. Each of these constitutes a means for binding the IL-18 receptor a.

[00573] In some embodiments, IL-21 receptor is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-IL-21 receptor antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from 1D1C2, 19F5, 18A5, REA233, or an antigen-binding portion thereof. Each of these constitutes a means for binding the IL-21 receptor a.

[00574] In some embodiments, LAG-3 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-LAG-3 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from relatlimab, tebotelimab, favezelimab, fianlimab, miptenalimab, HLX26, ieramilimab, GSK2831781, INCAGN2385, RO7247669, encelimab, FS118, SHR-1802, Sym022, IBI110, IBI323, bavunalimab, EMB-02, ABL501, INCA32459, AK129, or an antigen-binding portion thereof. Each of these constitutes a means for binding LAG-3.

[00575] In some embodiments, MSCA-1 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti- MSCA-1 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from REAL219, W8B2, X9C3, or an antigen-binding portion thereof. Each of these constitutes a means for binding MSCA-1.

[00576] In some embodiments, OX40 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-OX40 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from MEDI6469, ivuxolimab, rocatinlimab, GSK3174998, BMS-986178, vonlerizumab, INCAGN1949, tavolimab, BGB-A445, INBRX-106, BAT6026, telazorlimab, ATOR-1015, MEDI6383, cudarolimab, FS120, HFB301001, EMB-09, HLX51, Hu222, ABM193, or an antigen-binding portion thereof. Each of these constitutes a means for binding OX40.

[00577] In some embodiments, PD-1 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-PD-1 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from nivolumab, pembrolizumab, camrelizumab, torpalimab, sintilimab, tislelizumab, cemiplimab, spartalizumab, serplulimab, cadonilimab, penpulimab, dostarlimab, zimberelimab, retifanlimab, pucotenlimab, pidilizumab, pidilizumab, balstilimab, ezabenlimab, AK112, geptanolimab, cetrelimab, prolgolimab, tebotelimab, sasanlimab, SG001, vudalimab, MEDI5752, rulonilimab, peresolimab, IBI318, budigalimab, MEDI0680, pimivalimab, QL1706, AMG 404, RO7121661, lorigerlimab, nofazinlimab, sindelizumab, or an antigen-binding portion thereof. Each of these constitutes a means for binding PD-1.

[00578] In some embodiments, PODXL is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-PODXL antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from MAI1738, HPAB-3334LY, HPAB-MO612-YC, REA246, REA157, or an antigen-binding portion thereof. Each of these constitutes a means for binding PODXL.

[00579] In some embodiments, Sca-1 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-Sca-1 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from CPP32 4-1-18, 2D4-C9-F1, AMM22070N, or an antigen-binding portion thereof. Each of these constitutes a means for binding SCA-1.

[00580] In some embodiments, SSEA-3 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-SSEA-3 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from MC631,2A9, 8A7, ND-742, 3H420, as well as those disclosed in US11,643,456 or WO2021138378, each of which is incorporated by reference for all that they teach about anti-SSEA-3 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding SSEA-3.

[00581] In some embodiments, SSEA-4 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-SSEA-4 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from ch28 / 11, REA101, MC-813-70, ND-942-80, as well as those disclosed in US11,446,379, US10,273,295, US11,643,456, WO2019190952, or WO2021044039, each of which is incorporated by reference for all that they teach about anti-SSEA-4 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding SSEA-4.

[00582] In some embodiments, Stro-1 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-Stro-1 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from STRO-1, TLISP-2, as well as those disclosed in LIS20130122022, which is incorporated by reference for all that it teaches about anti-Stro-1 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding Stro-1.

[00583] In some embodiments, Stro-4 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-Stro-4 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from STRO-4, efungumab, 4C5, as well as those disclosed in US7,722,869, US20110280881, US9,115,192, US10,273,294, US10,457,726, WO2023091148, each of which is incorporated by reference for all that they teach about anti-Stro-4 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding Stro-4 (also known as heat shock protein-90).

[00584] In some embodiments, SLISD2 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-SUSD2 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from REA795, CBXS-3571, CBXS-1650, CBXS-1989, CBXS-1671, CBXS1990, CBXS-3676, 1279B, EPR8913(2), W5C5, or an antigen-binding portion thereof. Each of these constitutes a means for binding SLISD2.

[00585] In some embodiments, TIM-3 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-TIM-3 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from TQB2618, sabatolimab, cobolimab, RO7121661, INCAGN02390, AZD7789, surzebiclimab, LY3321367, Sym023, BMS-986258, SHR-1702, LY3415244, LB1410, or an antigen-binding portion thereof. Each of these constitutes a means for binding TIM-3.

[00586] In some embodiments, TREM2 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-TREM2 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from PI37012 as well as those disclosed in US10,508,148, US10,676,525, WO2017058866, US11,186,636, US11,124,567, WO2020055975, US11,492,402, WO2020121195, WO2023012802, WO2021101823, WO2023047100, WO2022032293, WO2022241082, WO2023039450, or WO2023039612, each of which is incorporated by reference for all that they teach about anti-TREM2 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding TREM2.

[00587] In some embodiments, G protein-coupled receptor, class C, group 5, member D (GPRC5D) is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-GPRC5D antibody. Accordingly, in some such embodiments, the antigen binding domain of an anti-GPRC5D antibody is derived from talquetamab, forimtamig, BMS-986393, IBI-3003, QLS32015, SIM0500, or EPR28376-41, or is disclosed in WO2018017786, WO2016090329, WO2022174813, WO2023236889, WO2018147245, WO2024079015, WO2019154890, WO2021018859, WO2021018925, WO2020092854, WO2024031091, WO2020148677, WO2022175255, WO2022222910, WO2022247804, WO2022247756, WO2023078382, WO2023125728, WO2023143537, WO2024046239, or WO2024131962 each of which is incorporated by reference for all that they teach about anti-GPRC5D antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding GPRC5D.

[00588] In some embodiments, FCRL5 (CD307E) is a targeted cell surface antigen and binding moiety comprises the antigen binding domain of an anti-FCRL5 antibody. Accordingly, in some such embodiments, the antigen binding domain of an anti-FCRL5 antibody is derived from cevostamab, 2A10H7, 307307, 2A10D6, EPR27365-87, EPR26948-19, or EPR26948-67, oris disclosed in WO2016090337, WO2017096120, WO2022263855, or WO2024047558 each of which is incorporated by reference for all that they teach about anti-FCRL5 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding FCRL5.

[00589] In some embodiments, LRRC15 is a targeted cell surface antigen and binding moiety comprises the antigen binding domain of an anti-LRRC15 antibody. Accordingly, in some such embodiments, the antigen binding domain of an anti-LRRC15 antibody is derived from samrotamab or DLINP19 or is disclosed in WO2005037999, WO2021022304, WO2024081729, WO2021102332, WO2021202642, WO2022157094, or WO2024158047, each of which is incorporated by reference for all that they teach about anti-LRRC15 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding LRRC15.

[00590] In still further embodiments, a tLNP is targeted to a tumor cell. In some embodiments, the tumor cell expresses one of the antigens described above and the tLNP is targeted to antigen expressing tumors using the same means as described above. In other embodiments the tLNP is targeted to some other tumor antigen, such as those enumerated in U.S. Provisional Application No. 63 / 371,742, filed on August 17, 2022, entitled CONDITIONING FOR IN VIVO IMMUNE CELL ENGINEERING which is incorporated by reference for all that it teaches about the delivery of nucleic acids into tumor cells using tLNP that is not inconsistent with this disclosure. Nucleic Acid

[00591] In certain embodiments, the disclosed LNP and tLNP comprise a payload comprising or consisting of one or more nucleic acid species. In some embodiments, the LNP or tLNP payload comprises only one nucleic acid species while in other embodiments the LNP or tLNP payload comprises multiple nucleic acid species, for example, 2, 3, or 4 nucleic acid species. For example, in embodiments in which the payload comprises a nucleic acid encoding a CAR or immune cell engager (ICE), the payload can comprise or consist of 1) a single nucleic acid species encoding a single species of CAR or ICE, 2) a single nucleic acid species encoding 2 or more species of CAR or ICE (or a mixture of CAR and ICE) such as a bicistronic or multicistronic mRNA in which each CAR and / or ICE has specificity for a same target antigen, 3) a single nucleic acid species encoding 2 or more species of CAR or ICE (or a mixture of CAR and ICE) such as a bicistronic or multicistronic mRNA in which at least one CAR and / or ICE has specificity for a different target antigen than the other(s), 4) two or more nucleic acid species encoding 2 or more species of CAR or ICE (or a mixture of CAR and ICE) in which each CAR and / or ICE has specificity for a same target antigen, 5) two or more nucleic acid species encoding 2 or more species of CAR or ICE (or a mixture of CAR and ICE) in which at least one CAR and / or ICE has specificity for a different target antigen than the other(s). When two or more CAR and / or ICE have specificity for a same target antigen, they can have specificity for different epitopes of the same target antigen. Further variations will be apparent to one of skill in the art (e.g., multiple bi- or multicistronic nucleic acids, nucleic acids encoding a TCR and the like). The nucleic acid can be RNA or DNA. The nucleic acid can be multicistronic, for example, bicistronic.

[00592] In some embodiments, LNPs or tLNPs of this disclosure further comprise a nucleic acid payload. In various embodiments, a nucleic acid is an mRNA, a self-replicating RNA (also known as self-amplifying RNA), a circular RNA, a siRNA, a miRNA, DNA, a gene editing component (for example, a guide RNA, a tracr RNA, an sgRNA), a gene writing component, an mRNA encoding a gene or base editing protein, a zinc-finger nuclease, a TALEN, a CRISPR nuclease, such as Cas9, a DNA molecule to be inserted or serve as a template for repair), and the like, or a combination thereof. In some embodiments, the nucleic acid comprises small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotide (ASO). In some embodiments, the nucleic acid comprises a self-replicating RNA or a circular RNA. In some embodiments, the mRNA encodes a reprogramming agent or comprises or encodes a conditioning agent. In some embodiments, the RNA (linear mRNA, circular, or selfreplicating) comprises an miRNA binding site. In some embodiments, an mRNA encodes a chimeric antigen receptor (CAR). In other embodiments, an mRNA encodes a gene-editing or base-editing or gene writing protein. In some embodiments, a nucleic acid is a guide RNA. In some embodiments, an LNP or tLNP comprises both a gene- or base-editing or gene writing protein-encoding mRNA and one or more guide RNAs. CRISPR nucleases can have altered activity, for example, modifying the nuclease so that it is a nickase instead of making doublestrand cuts or so that it binds the sequence specified by the guide RNA but has no enzymatic activity. Base-editing proteins are often fusion proteins comprising a deaminase domain and a sequence-specific DNA binding domain (such as an inactive CRISPR nuclease).

[00593] In some embodiments, the reprogramming agent comprises an immune receptor (for example, a chimeric antigen receptor or a T cell receptor) or an immune cell engager (for example, a bispecific T cell engager (BiTE), a bispecific killer cell engager (BiKE), a trispecific kill cell engager (TriKE), a dual affinity retargeting antibody (DART), a TRIDENT (linking two DART units or a DART unit and a Fab domain), a macrophage engager (e.g., BiME), an innate cell engager, and the like).

[00594] In some embodiments, the nucleic acid is an RNA, for example, mRNA, and the RNA comprises at least one modified nucleoside. In some embodiments, the modified nucleoside is pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methyluridine, N6-methyladenosine, 2’-O-methyluridine, or 2-thiouridine. In certain embodiments, all of the uridines are substituted with a modified nucleoside. Further disclosure of modified nucleosides and their use can be found in U.S. Patent No. 8,278,036 which is incorporated herein by reference for those teachings.

[00595] In some embodiments, the reprogramming agent encodes or is a gene / genome editing component. In some embodiments, the gene / genome editing component is a guide RNA for an RNA-directed nuclease or other nucleic acid editing enzyme, clustered regularly interspaced short palindromic repeat RNA (crisprRNA), a trans-activating clustered regularly interspaced short palindromic repeat RNA (tracrRNA). In some embodiments, the gene / genome editing component is a nucleic acid-encoded enzyme, such as RNA-guided nuclease, a gene or base editing protein, a zinc finger nuclease (ZFN), a transcription activator-1 ike effector nuclease (TALEN), a meganuclease, a transposase, or a CRISPR nuclease (e.g., Cas9 or Cas 12, etc.). In some embodiments, the gene / genome editing component is DNA to be inserted or that serves as a template in gene or genome editing for example a template for repair of a double-strand break.

[00596] In some embodiments comprising multiple agents, the nucleic acid can be multicistronic. In other embodiments comprising multiple agents or components, each agent or component is encoded or contained is a separate nucleic acid species. In some embodiments involving multiple payload nucleic acid species, two or more nucleic acid species are packaged together in a single LNP species. In other embodiments, a subset of the payload nucleic acid species to be delivered, (e.g., a single nucleic acid species) is packaged in one LNP or tLNP species while another subset of the nucleic acid species is packaged in another LNP or tLNP species. The different (t)LNP species can differ by only the payload they contain. The different (t)LNP species can be combined in a single formulation or pharmaceutical composition for administration. Methods of Making an LNP or tLNP

[00597] In some aspects, this disclosure provides a method of making a LNP or tLNP comprising mixing of an aqueous solution of a nucle...

Claims

1. An ionizable cationic lipid having a structure of formula M6, a stereoisomer thereof, or a mixture of such stereoisomers,wherein X is ---7M6Y-OzJm-H.....¥<H2)2.4~f /  )Y is O, S, NH, or NCH3;Zis 0, NH, or NCH3;each W is independently CH or N;each R2 is independently 0 or C=0, wherein at least one R2 is O;each R3 is independently 0 or C=0,wherein when a R2 is 0 then its adjoining R3 is C=0, or when a R2 is C=0 then its adjoining R3 is O;wherein when at least one R3 at a beta position of a W is 0, that W is CH; andwherein when both R3 groups at a beta position of a W are C=0, that W is CH or N;each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl;whereinA1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)i-4 or CH2-CH=CH-CH2; orA1 is (CH2)o, A2 is (CH2)i , A3 is (CH2)i , A4 is (CH2)0, and A5 is (CH2)i; orA1 is (CH2)i , A2 is (CH2)i , A3 is (CH2)0, A4 is (CH2)0, and A5 is (CH2)0; orA1 is (CH2)i , A2 is (CH2)i , A3 is (CH2)0, A4 is (CH2)0, and A5 is (CH2)i; orA1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)0, A4 is (CH2)0, and A5 is (CH2)2 or CH=CH.

2. The ionizable cationic lipid of claim 1, wherein in each tail group one R2 is 0 and its adjoining R3 is C=0, and the other R2 is C=0, and its adjoining R3 is 0.

3. The ionizable cationic lipid of claim 1, wherein in one tail group, one R2 is 0 and its adjoining R3 is C=0, and the other R2 is C=0 and its adjoining R3 is 0, and in the other tail group, both R2 groups are 0 and both R3 groups are C=0.

4. The ionizable cationic lipid of claim 1, wherein in one tail group, one R2 is O and its adjoining R3 is C=O and the other R2 is C=O and its adjoining R3 is O, and in the other tail group both R2 groups are C=0 and both R3 groups are 0.

5. The ionizable cationic lipid of claim 1, wherein in one tail group, both R2 groups are 0 and both R3 groups are C=0, and in the other tail group, both R2 groups are C=0 and both R3s are 0.

6. The ionizable cationic lipid of claim 1, wherein in both tail groups, both R2 groups are 0 and both R3 groups are C=0.

7. The ionizable cationic lipid of any one of claims 1-5, wherein at least one R3 at a beta position of a W is 0 and that W is CH.

8. The ionizable cationic lipid of any one of claims 1, 4, or 5, wherein both R3 groups at a beta position of a W is 0 and that W is CH.

9. The ionizable cationic lipid of any one of claims 1,3,5, or 6, wherein both R3 groups at a beta position of a W are C=0 and that W is N.

10. The ionizable cationic lipid of any one of claims 1, 3, 5, or 6, wherein both R3 groups at a beta position of a W are C=0 and that W is CH.

11. The ionizable cationic lipid of any one of claims 7-10, wherein A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)i-4 or CH2-CH=CH-CH2.

12. The ionizable cationic lipid of claim 11, wherein A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5is(CH2)i.13 The ionizable cationic lipid of claim 11, wherein A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)2.

14. The ionizable cationic lipid of claim 11, wherein A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)3.

15. The ionizable cationic lipid of claim 7, wherein A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is (CH2)4.

16. The ionizable cationic lipid of claim 11, wherein A1 is (CH2)o, A2 is (CH2)o, A3 is (CH2)i, A4 is (CH2)i, and A5 is CH2-CH=CH-CH2.

17. The ionizable cationic lipid of any one of claims 7-10, wherein A1 is (CH2)o, A2 is (CH2)i, A3 is (CH2)i, A4 is (CH2)o, and A5 is (CH2)i.

18. The ionizable cationic lipid of any one of claims 7-10, wherein A1 is (CH2)i , A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is (CH2)o.

19. The ionizable cationic lipid of any one of claims 7-10, wherein A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is (CH2)i.

20. The ionizable cationic lipid of any one of claims 7-10, wherein A1 is (CH2)i, A2 is (CH2)i, A3 is (CH2)o, A4 is (CH2)o, and A5 is (CH2)2 or CH=CH.

21. The ionizable cationic lipid of any one of claims 7-20, wherein A1 through A4 are such that there are only two main chain atoms between the ring nitrogen and each nearest ester oxygen in the nearest tail group.(CH2)0-3^H3Y-(CH2)2-4-Nx22. The ionizable cationic lipid of any one of claims 7-20 wherein X is             (CH2)o-3CH3. CH3 Y (CH2)2-^23. The ionizable cationic lipid of claim 22 wherein X is               3.

24. The ionizable cationic lipid of any one of claims 7-23 wherein Y is O.

25. The ionizable cationic lipid of any one of claims 7-23 wherein Y is S.

26. The ionizable cationic lipid of any one of claims 7-23, wherein Y is NH.

27. The ionizable cationic lipid of any one of claims 7-23, wherein Y is NCH3.

28. The ionizable cationic lipid of any one of claims 7-21, wherein Z is O.

29. The ionizable cationic lipid of any one of claims 7-21, wherein Z is NH.

30. The ionizable cationic lipid of any one of claims 7-21, wherein Z is NCH3.

31. The ionizable cationic lipid of any one of claims 7-31 wherein R1 is (CH2)7CH3.

32. The ionizable cationic lipid of claim 6 having the structure:

33. The ionizable cationic lipid of claim 32 having the structure:

34. The ionizable cationic lipid of claim 33 having the structure CICL-252:

35. The ionizable cationic lipid of claim 33 having the structure CICL-253:, as a racemate, any other mixture of enantiomers, or either individual enantiomer.

36. The ionizable cationic lipid of claim 6 having the structure37. The ionizable cationic lipid of claim 36 having the structure38. The ionizable cationic lipid of claim 37 having the structure CICL-251:CICL-251, its enantiomer, or a racemate or othermixture comprising CICL-255.

39. The ionizable cationic lipid of claim 37 having the structure CICL-254:CICL-25440. The ionizable cationic lipid of claim 37 having the structure CICL-255:CICL-25541. The ionizable cationic lipid of claim 37 having the structure CICL-256:CICL-256or a racemate or other mixturecomprising CICL-254.

42. The ionizable cationic lipid of claim 6 having the structure43. The ionizable cationic lipid of claim 42 having the structure44. The ionizable cationic lipid of claim 43 having the structure CICL-257:

45. The ionizable cationic lipid of claim 43 having the structure CICL-258:CICL-258or a racemate, or other mixturecomprising CICL-257.

46. The ionizable cationic lipid of claim 6 having the structure47. The ionizable cationic lipid of claim 46 having the structure48. The ionizable cationic lipid of claim 47 having the structure CICL-259:

49. The ionizable cationic lipid of claim 47 having the structure CICL-260:mixture thereof.

50. The ionizable cationic lipid of claim 6 having the structure51. The ionizable cationic lipid of claim 50 having the structure52. The ionizable cationic lipid of claim 51 having the structure CICL-261:mixture of enantiomers.its enantiomer, a racemate, any other53. The ionizable cationic lipid of claim 51 having the structure CICL-262:

54. The ionizable cationic lipid of claim 6 having the structure:

55. The ionizable cationic lipid of claim 54 having the structure:

56. The ionizable cationic lipid of claim 55 having the structure CICL-263:

57. The ionizable cationic lipid of claim 55 having the structure CICL-264:a racemate, any other mixture ofenantiomers, or either individual enantiomer.

58. The ionizable cationic lipid of claim 6 having the structure:

59. The ionizable cationic lipid of claim 58 having the structure:

60. The ionizable cationic lipid of claim 59 having the structure CICL-265:

61. The ionizable cationic lipid of claim 59 having the structure CICL-266:enantiomers, or either individual enantiomer.

62. The ionizable cationic lipid of claim 6 having the structure:

63. The ionizable cationic lipid of claim 62 having the structure:

64. The ionizable cationic lipid of claim 63 having the structure CICL-267:CICL-26765. The ionizable cationic lipid of claim 63 having the structure CICL-268:CICL-268a racemate, any other mixture ofenantiomers, or either individual enantiomer.

66. The ionizable cationic lipid of claim 6 having the structure:

68. The ionizable cationic lipid of claim 67 having the structure CICL-269:

69. The ionizable cationic lipid of claim 67 having the structure CICL-270:or either individual enantiomer., a racemate, any other mixture of enantiomers,70. The ionizable cationic lipid of claim 6 having the structure:

71. The ionizable cationic lipid of claim 70 having the structure:

72. The ionizable cationic lipid of claim 71 having the structure CICL-271:

73. The ionizable cationic lipid of claim 71 having the structure CICL-272:, a racemate, any other mixture of enantiomers, or either individual enantiomer.

74. The ionizable cationic lipid of claim 6 having the structure:

75. The ionizable cationic lipid of claim 74 having the structure:

76. The ionizable cationic lipid of claim 75 having the structure CICL-273:enantiomers, or either individual enantiomer.a racemate, any other mixture of77. The ionizable cationic lipid of claim 75 having the structure CICL-274:CICL-27478. The ionizable cationic lipid of claim 75 having the structure CICL-275:CICL-275a racemate, any other mixture ofenantiomers, or either individual enantiomer.

79. The ionizable cationic lipid of claim 75 having the structure CICL-276:CICL-27680. The ionizable cationic lipid of claim 6 having the structure:

81. The ionizable cationic lipid of claim 80 having the structure:

83. The ionizable cationic lipid of claim 81 having the structure CICL-278:OCICL-278a racemate, any other mixture ofenantiomers, or either individual enantiomer.

84. The ionizable cationic lipid of claim 6 having the structure:

85. The ionizable cationic lipid of claim 84 having the structure:

86. The ionizable cationic lipid of claim 85 having the structure CICL-279:

87. The ionizable cationic lipid of claim 85 having the structure CICL-280:a racemate, any other mixture ofenantiomers, or either individual enantiomer.

88. The ionizable cationic lipid of claim 6 having the structure:

89. The ionizable cationic lipid of claim 88 having the structure:

90. The ionizable cationic lipid of claim 89 having the structure CICL-281:or either individual enantiomer.a racemate, any other mixture of enantiomers,91. The ionizable cationic lipid of claim 89 having the structure CICL-282:

92. The ionizable cationic lipid of claim 6 having the structure:

93. The ionizable cationic lipid of claim 92 having the structure:

94. The ionizable cationic lipid of claim 93 having the structure CICL-283:

95. The ionizable cationic lipid of claim 93 having the structure CICL-284:or either individual enantiomer., a racemate, any other mixture of enantiomers,96. The ionizable cationic lipid of claim 6 having the structure:

97. The ionizable cationic lipid of claim 96 having the structure:

98. The ionizable cationic lipid of claim 97 having the structure CICL-285:

99. The ionizable cationic lipid of claim 97 having the structure CICL-286:a racemate, any other mixture ofenantiomers, or either individual enantiomer.

100. The ionizable cationic lipid of claim 6 having the structure:

101. The ionizable cationic lipid of claim 100 having the structure:

102. The ionizable cationic lipid of claim 101 having the structure CICL-287:CICL-287103. The ionizable cationic lipid of claim 101 having the structure CICL-288:CICL-288or either individual enantiomer., a racemate, any other mixture of enantiomers,104. The ionizable cationic lipid of claim 6 having the structure:

105. The ionizable cationic lipid of claim 104 having the structure:

106. The ionizable cationic lipid of claim 105 having the structure CICL-289:

107. The ionizable cationic lipid of claim 105 having the structure CICL-290:or either individual enantiomer., a racemate, any other mixture of enantiomers,108. A lipid nanoparticle (LNP), comprising at least one ionizable cationic lipid of any one of claims 1-107.

109. A targeted lipid nanoparticle (tLNP), comprising at least one ionizable cationic lipid of any one of claims 1-107 and a functionalized PEG-lipid, wherein the functionalized PEG-lipid has been conjugated with a binding moiety.

110. The LNP of claim 108 or the tLNP of claim 109, further comprising one or more of a phospholipid, a sterol, a co-lipid, an unfunctionalized and / or a functionalized PEG-lipid, or combinations thereof.

111. The LNP or tLNP of claim 110, wherein the phospholipid comprises dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidyl glycerol (DM PG), dipalmitoyl phosphatidylcholine (DPPC), or 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC), or a combination thereof.

112. The LNP or tLNP of claim 110 or 111, wherein the sterol comprises cholesterol, campesterol, sitosterol, stigmasterol, or combinations thereof.

113. The LNP or tLNP of any one of claims 110-112, wherein the co-lipid comprises cholesterol hemisuccinate (CHEMS) or a quaternary ammonium head group containing lipid.

114. The LNP or tLNP of claim 113, wherein the quaternary ammonium head group containing lipid comprises 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium (DOTMA), or 3p-(N-(N',N'-Dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), or combinations thereof.

115. The LNP of any one of claims 110-114 or the tLNP of any one of claims 109-114, wherein the unfunctionalized PEG-lipid and / or the functionalized PEG-lipid comprises a PEG moiety of 1000-5000 Da molecular weight (MW).

116. The LNP of any one of claims 110-115 or the tLNP of any one of claims 109-115, wherein the unfictionalized PEG-lipid and / or the functionalized PEG-lipid comprises fatty acids with a fatty acid chain length of C14-C18.

117. The LNP of any one of claims 110-116 or the tLNP of any one of claims 109-116, wherein the unfunctionalized PEG-lipid and / or the functionalized PEG-lipid comprises DMG-PEG2000 (1,2-dimyristoyl-rglycero-3-methoxypolyethylene glycol-2000), DPG-PEG2000 (1,2-dipalmitoyl-glycero-3-methoxypolyethylene glycol-2000), DSG-PEG2000 (1,2-distearoyl-glycero-3-methoxypolyethylene glycol-2000), DOG-PEG2000 (1,2-dioleoyl-glycero-3-methoxypolyethylene glycol-2000), DMPE-PEG200 (1,2-dimyristoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DPPE-PEG2000 (1,2-dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DSPE-PEG2000                    (1,2-distearoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DOPE-PEG2000 (1,2-dioleoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), or combinations thereof.

118. The LNP or tLNP of any one of claims 110-17, comprising a phospholipid in an amount in the range from 7 to 30 mol%.

119. The LNP or tLNP of any one of claims 110-1118, comprising a sterol in an amount in the range from 20 to 45 mol%.

120. The LNP or tLNP of any one of claims 110-119, comprising at least one co-lipid in an amount in the range from 1 to 30 mol%.

121. The LNP or tLNP of any one of claims 110-120, comprising at least one unfunctionalized PEG-lipid in an amount in the range from 0.1 to 5 mol%.

122. The LNP or tLNP of any one of claims 110-121, comprising at least one functionalized PEG-lipid in an amount in the range from 0.1 to 5 mol%.

123. The tLNP of any one of claims 109-122, wherein the binding moiety comprises an antigen, a ligand-binding domain of a receptor, a receptor ligand, an antibody, or an antigen binding domain of an antibody.

124. The LNP of any one of claims 108 or 110-122 or tLNP of any one of claims 109-123, further comprising a nucleic acid molecule payload.

125. The LNP or tLNP of claim 124, wherein the weight ratio of total lipid to nucleic acid molecule is 10:1 to 50:1.

126. The LNP or tLNP of claim 124, wherein the N / P ratio is from 3 to 9.

127. The LNP or tLNP of any one of claims 124-126, wherein the nucleic acid molecule is an mRNA.

128. The tLNP of any one of claims 124-127, wherein the binding moiety is a whole antibody and the ratio of antibody to nucleic acid molecule is from about 0.3 to about 1.0 (w / w).

129. The tLNP of any one of claims 109-127, wherein the binding moiety is a F(ab’) or F(ab’) analog.

130. A method of delivering a nucleic acid into a cell comprising contacting the cell with the LNP or tLNP of any one of claims 124-129.

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