Ionizable lipids, lipid nanoparticles for mRNA delivery and methods of making the same

Abstract

Provided herein are compositions including ionizable lipids and lipid nanoparticles comprising the ionizable lipids. The ionizable lipids may have a general structure according to formula I: (I). Lipid nanoparticles generally include the ionizable lipid according to formula (I); a helper lipid; a sterol; and a PEGylated lipid conjugate. The ionizable lipids and lipid nanoparticles may be used to carrier cargo for a vaccine.

Description

2024-031 / 10738-1181-1-IONIZABLE LIPIDS, LIPID NANOPARTICLES FOR MRNA DELIVERY AND METHODS OF MAKING THE SAMECROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 601,330, filed November 21, 2023, the entire contents of which are incorporated herein by reference.TECHNICAL FIELD

[0002] The present disclosure generally relates to ionizable lipids, lipid nanoparticles, and methods of making and using the same.BACKGROUND

[0003] One of the major challenges in the field of targeted delivery of biologically active substances is their instability and low cell penetrating potential, as well as their susceptibility to enzymatic degradation. This has created challenges in the development of therapies utilizing nucleic acid molecules, in particular RNA molecules.

[0004] In that respect, lipid-based nanoparticle compositions such as lipoplexes and liposomes have been used as packaging vehicles for biologically active substances to allow transport into cells and / or intracellular compartments. These lipid-based nanoparticle compositions typically comprise a mixture of different lipids such as ionizable lipids, helper lipids, structural lipids (such as sterols or cholesterol), and lipid conjugates.

[0005] Emerging clinical therapies, particularly nucleic acid-based vaccines, require drug delivery systems, such lipid nanoparticles, that can encapsulate and deliver a variety of cargo molecules. Accordingly, a need exists to develop new lipids and / or nanoparticles to better deliver the therapy.SUMMARY

[0006] Disclosed herein are ionizable lipids, lipid nanoparticles including the ionizable lipids and use of the ionizable lipids as a carrier for cargo in a vaccine.2024-031 / 10738-1181-2-

[0007] In one embodiment, the present disclosure relates to a composition including at least one ionizable lipid according to Formula (I),(Formula (I)), wherein ni is from 0-8 carbons; n2 is from 2-8 carbons; ns is from 2-8 carbons; is from 2-8 carbons; and ns is from 0-8 carbons. Ri is selected from:Each X is independently selected from:2024-031 / 10738-1181-3-Y is selected from:Each R2 is independently selected from hydrogen, Cl -Cl 8 alkyl groups, or Cl -Cl 8 alkenyl groups. Each R3 is independently selected from hydrogen, Cl -Cl 8 alkyl groups, or Cl -Cl 8 alkenyl groups.

[0008] In some embodiments, the present disclosure relates to lipid nanoparticle compositions having an ionizable lipid a helper lipid; a sterol; and a PEGylated lipid conjugate.

[0009] In some embodiments, the present disclosure relates to use of the ionizable lipids and / or the lipid nanoparticles in a vaccine.

[0010] These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description, appended claims, and accompanying drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Though the specification concludes with claims particularly pointing out and distinctly claiming the invention, it is believed that the present invention will be better understood from the following description taken in conjunction with the accompanying drawings.

[0012] FIG. 1 depicts a synthesis schematic of an exemplary ionizable lipid, according to one or more aspects shown and described herein.

[0013] FIG. 2 depicts a schematic representation of the general structure of ionizable lipids, according to one or more aspects shown and described herein.

[0014] FIG. 3 A depicts the chemical structure of lipid 1.

[0015] FIG. 3B depicts theJH NMR spectrum for lipid 1.

[0016] FIG. 3C depicts the13C NMR spectrum for lipid 1.2024-031 / 10738-1181-4-

[0017] FIG. 4A depicts the chemical structure of lipid 2.

[0018] FIG. 4B depicts theJH NMR spectrum for lipid 2.

[0019] FIG. 4C depicts the13C NMR spectrum for lipid 2.

[0020] FIG. 5 A depicts the chemical structure of lipid 3.

[0021] FIG. 5B depicts theJH NMR spectrum for lipid 3.

[0022] FIG. 5C depicts the13C NMR spectrum for lipid 3.

[0023] FIG. 6A depicts the chemical structure of lipid 4.

[0024] FIG. 6B depicts theJH NMR spectrum for lipid 4.

[0025] FIG. 6C depicts the13C NMR spectrum for lipid 4.

[0026] FIG. 7 A depicts the chemical structure of lipid 5.

[0027] FIG. 7B depicts theJH NMR spectrum for lipid 5.

[0028] FIG. 7C depicts the13C NMR spectrum for lipid 5.

[0029] FIG. 8 A depicts the chemical structure of lipid 6.

[0030] FIG. 8B depicts theJH NMR spectrum for lipid 6.

[0031] FIG. 8C depicts the13C NMR spectrum for lipid 6.

[0032] FIG. 9A depicts the chemical structure of lipid 7.

[0033] FIG. 9B depicts theJH NMR spectrum for lipid 7.

[0034] FIG. 9C depicts the13C NMR spectrum for lipid 7.

[0035] FIG. 10A depicts the chemical structure of lipid 8.

[0036] FIG. 10B depicts theJH NMR spectrum for lipid 8.

[0037] FIG. 10C depicts the13C NMR spectrum for lipid 82024-031 / 10738-1181-5-

[0038] FIG. 11A depicts the chemical structure of lipid 9.

[0039] FIG. 1 IB depicts theJH NMR spectrum for lipid 9.

[0040] FIG. 11C depicts the13C NMR spectrum for lipid 9.

[0041] FIG. 12A depicts the chemical structure of lipid 10.

[0042] FIG. 12B depicts theJH NMR spectrum for lipid 10.

[0043] FIG. 12C depicts the13C NMR spectrum for lipid 10.

[0044] FIG. 13 A depicts the chemical structure of lipid 11.

[0045] FIG. 13B depicts theJH NMR spectrum for lipid 11.

[0046] FIG. 13C depicts the13C NMR spectrum for lipid 11.

[0047] FIG. 14A depicts the chemical structure of lipid 12.

[0048] FIG. 14B depicts theJH NMR spectrum for lipid 12.

[0049] FIG. 14C depicts the13C NMR spectrum for lipid 12.

[0050] FIG. 15A depicts the chemical structure of lipid 13.

[0051] FIG. 15B depicts theJH NMR spectrum for lipid 13.

[0052] FIG. 15C depicts the13C NMR spectrum for lipid 13.

[0053] FIG. 16A depicts the chemical structure of lipid 14.

[0054] FIG. 16B depicts theJH NMR spectrum for lipid 14.

[0055] FIG. 16C depicts the13C NMR spectrum for lipid 14.

[0056] FIG. 17A depicts the chemical structure of lipid 15.

[0057] FIG. 17B depicts theJH NMR spectrum for lipid 15.

[0058] FIG. 17C depicts the13C NMR spectrum for lipid 15.2024-031 / 10738-1181-6-

[0059] FIG. 18A depicts the chemical structure of lipid 16.

[0060] FIG. 18B depicts the 'H NMR spectrum for lipid 16.

[0061] FIG. 18C depicts the13C NMR spectrum for lipid 16.

[0062] FIG. 19A depicts the chemical structure of lipid 17.

[0063] FIG. 19B depicts the 'H NMR spectrum for lipid 17.

[0064] FIG. 19C depicts the13C NMR spectrum for lipid 17.

[0065] FIG. 20A depicts the chemical structure of lipid 18.

[0066] FIG. 20B depicts theJH NMR spectrum for lipid 18.

[0067] FIG. 20C depicts the13C NMR spectrum for lipid 18.

[0068] FIG. 21A depicts the chemical structure of lipid 19.

[0069] FIG. 2 IB depicts theJH NMR spectrum for lipid 19.

[0070] FIG. 21C depicts the13C NMR spectrum for lipid 19.

[0071] FIGS. 22A-22D depict lipid nanoparticle characterization data demonstrating hydrodynamic size (FIG. 22A), PDI (FIG. 22B), surface charge (FIG. 22C), and mRNA encapsulation efficiency as measured by Ribogreen® assay for LNPs with different ionizable lipids containing FLuc mRNA (FIG. 22D).

[0072] FIG. 23 graphically depicts the apparent pKa of LNPs measured using INS assay.

[0073] FIG. 24 graphically depicts in vitro FLuc mRNA delivery efficiency of LNPs to JurkatT cells after 20-hour incubation.

[0074] FIG. 25 graphically depicts showing whole-body bioluminescence flux of female BALB / c mice at different time points following intravenous injection of different LNPs.

[0075] FIGS. 26A-26D depict lipid nanoparticle characterization data demonstrating2024-031 / 10738-1181-7- hydrodynamic size (FIG. 26A), PDI (FIG. 26B), surface charge (FIG. 26C), and mRNA encapsulation efficiency as measured by Ribogreen® assay for LNPs with different ionizable lipids containing FLuc mRNA (FIG. 26D).

[0076] FIG. 27 graphically depicts the apparent pKa of LNPs measured using TNS assay.

[0077] FIG. 28 graphically depicts in vitro FLuc mRNA delivery efficiency of LNPs to JurkatT cells after 20-hour incubation.

[0078] FIG. 29 graphically depicts the whole-body bioluminescence flux of female BALB / c mice at different time points following intravenous injection of different LNPs.

[0079] FIGS. 30A-30D depict lipid nanoparticle characterization data demonstrating hydrodynamic size (FIG. 30 A), PDI (FIG. 30B), surface charge (FIG. 30C), and mRNA encapsulation efficiency as measured by Ribogreen ® assay for LNPs with different ionizable lipids containing FLuc mRNA (FIG. 30D).

[0080] FIG. 31 graphically depicts the apparent pKa of LNPs measured using TNS assay.

[0081] FIG. 32 graphically depicts in vitro FLuc mRNA delivery efficiency of LNPs to JurkatT cells after 20-hour incubation.

[0082] FIGS. 33A-33C depict bioluminescence flux data including whole-body bioluminescence flux of female BALB / c mice at different time points following intravenous injection of different LNPs (FIG. 33A) and total bioluminescence flux of isolated liver (FIG. 33B) and spleen (FIG. 33C) 24 hours post-injection.

[0083] FIGS. 34A-34B graphically depict in vitro eGFP mRNA delivery efficiency of LNPs to Jurkat T cells after 20-hour incubation using transfection efficiency determined by flow cytometry (FIG. 34A) and mean fluorescent intensity (FIG. 34B). Data represents mean ± s.d.(n=3); ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001, *** p < 0.0001. Significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test.

[0084] FIGS. 35A-35B graphically depict in vitro eGFP mRNA delivery efficiency of LNPs to Jurkat T cells after 20-hour incubation using transfection efficiency determined by flow2024-031 / 10738-1181-8- cytometry (FIG. 35 A) and mean fluorescent intensity (FIG. 35B) Data represents mean ± s.d. (n=3); ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001, *** p < 0.0001. Significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test.

[0085] The exemplifications set out herein illustrate at least one embodiment of the present disclosure, and such exemplifications are not to be construed as limiting the scope of the present disclosure in any manner.DETAILED DESCRIPTION

[0086] Features and advantages of the invention will now be described with occasional reference to specific embodiments. However, the invention may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art.

[0087] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting.

[0088] As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms, including “at least one,” unless the content clearly indicates otherwise. “Or” means “and / or.” As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. It will be further understood that the terms “comprises” and / or “comprising,” or “includes” and / or “including” when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof. It is to be further understood that where descriptions of various embodiments use the term “comprising,” and / or “including” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of’ or “consisting of.” The term “or a combination thereof’ means a combination including at least one of the foregoing elements.2024-031 / 10738-1181-9-

[0089] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth as used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated, the numerical properties set forth in the specification and claims are approximations that may vary depending on the desired properties sought to be obtained in embodiments of the present invention. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. One of ordinary skill in the art will understand that any numerical values inherently contain certain errors attributable to the measurement techniques used to ascertain the values.

[0090] It should be understood that every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, examples include from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0091] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 25 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 25 may comprise 1 to 5, 1 to 10, 1 to 15, and 1 to 20 in one direction, or 25 to 20, 25 to 15, 25 to 10, and 25 to 5 in the other direction.

[0092] As used herein, the terms "improve," "increase," "inhibit,” "reduce," or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement2024-031 / 10738-1181-10- in a particular system (e.g., in a single subject) under otherwise comparable conditions absent presence of (e.g., prior to and / or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.

[0093] As used herein, the term "expression" of a nucleic acid sequence refers to the generation of any gene product from the nucleic acid sequence. In some embodiments, a gene product can be a transcript. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end formation); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0094] The term “independently selected from,” as used herein, is intended to mean that the referenced groups can be the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, the phrase “X1, X2, and X3are independently selected from noble gases” would include the scenario where X1, X2, and X3are all the same, where X1, X2, and X3are all different, and where X1and X2are the same but X3is different.

[0095] The term “subject” as used herein refers to any living organism to which a pharmaceutical can be administered. The term subject includes, but is not limited to, humans, nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult, child, and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.

[0096] As used herein, the term “pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

[0097] As used herein, the term “pharmaceutically acceptable excipient, carrier, or diluent”2024-031 / 10738-1181-11- or the like refer to an excipient, carrier, or diluent that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.

[0098] The term “pharmaceutically acceptable salt” as used herein refers to pharmaceutically acceptable organic or inorganic salts of an ionizable lipid of the present disclosure. Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate “mesylate,” ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 1,1’- methylene-bis-(2- hydroxy-3 -naphthoate)) salts, alkali metal (e.g, sodium and potassium) salts, alkaline earth metal (e.g, magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counter ion. The counter ion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counter ion.

[0099] As used herein, the term “lipid encapsulated” is meant to refer to a lipid particle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g, an anti-sense oligonucleotide (ASO), mRNA, siRNA, close ended DNA (ceDNA), viral vector, etc.), with full encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acid is fully encapsulated in the lipid particle (e.g., to form a nucleic acid containing lipid particle).

[0100] Unless otherwise stated, the structures depicted and described herein include all isomeric (e.g., enantiomeric, diastereomeric, and geometric) forms of the structure; for example, tautomers, R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Additionally, unless otherwise stated, the structures depicted and described herein include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement2024-031 / 10738-1181-12- of hydrogen by deuterium or tritium, or the replacement of a carbon by a13C- or14C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools or as therapeutic agents.

[0101] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that any particular order be inferred. Any recited single or multiple feature or aspect in any one claim can be combined or permuted with any other recited feature or aspect in any other claim or claims.Ionizable Lipids

[0102] As used herein, the term “ionizable lipid” is refers to a lipid, e.g., cationic lipid, having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, at or above physiological pH. It will be understood by one of ordinary skill in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. In some aspects, an ionizable lipid is characterized by three portions: an amine head, a linker and a hydrophobic tail.

[0103] As shown in FIG. 2, ionizable lipids of the present disclosure generally have a structure according to Formula I:(Formula I).

[0104] In some aspects, m is from 0-8 carbons. In some aspects ni is zero carbons, 1 carbon, 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, or 8 carbons. In some aspects n22024-031 / 10738-1181-13- is from 2-8 carbons. Optionally, n2 is 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, or 8 carbons. In some aspects, ns is from 2-8 carbons, Optionally, ns is 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, or 8 carbons. In some aspects n4 is from 2-8 carbons. Optionally, n4 is 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, or 8 carbons. In some aspects, ns is from 0-8 carbons. In some aspects ns is zero carbons, 1 carbon, 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, or 8 carbons.

[0105] In some aspects, each X is independently selected from:

[0106] In some aspects, each X is independently selected from:

[0107] In some aspects, Y is selected from:

[0108] In some aspects, Y is selected from:

[0109] In some aspects, Y is2024-031 / 10738-1181-14-

[0110] In some aspects

[0111] In some aspects Ri is selected from:

[0112] In some aspects Ri is selected from:

[0113] In some aspects

[0114] In some aspects Ri is2024-031 / 10738-1181-15-

[0115] In some aspects, each R2 is independently selected from hydrogen, Cl -Cl 8 alkyl groups or Cl -Cl 8 alkenyl groups. For example, each R2 may be independently selected from hydrogen, methyl, ethyl, ethanyl, propyl, propenyl, butyl, butenyl, pentyl, pentenyl, hexyl, hexenyl, heptyl, heptenyl octyl, octenyl, nonyl, nonenyl, decyl, decenyl, undecyl, undecenyl, dodecyl, dodecenyl, tridecyl, tridecenyl, tetradecyl, tetradecenyl, pentadecyl, pentadecenyl, hexadecyl, hexadecenyl, heptadecyl, heptadecenyl, octydecyl, and octadecenyl.

[0116] In some aspects, each R3 is independently selected from hydrogen, Cl -Cl 8 alkyl groups or C1-C18 alkenyl groups. For example, each R3 may be independently selected from hydorgen, methyl, ethyl, ethane, propyl, propenyl, butyl, butenyl, pentyl, pentenyl, hexyl, hexenyl, heptyl, heptenyl octyl, octenyl, nonyl, nonenyl, decyl, decenyl, undecyl, undecenyl, dodecyl, dodecenyl, tridecyl, tridecenyl, tetradecyl, tetradecenyl, pentadecyl, pentadecenyl, hexadecyl, hexadecenyl, heptadecyl, heptadecenyl, octydecyl, and octadecenyl.

[0117] In some aspects, each R3 is independently selected from hydrogen, methyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, heptadecyl, hexadecyl, and octadecyl. In some aspects, each R3 is independently selected from hydrogen, hexyl, and octyl. In some aspects, each R3 is independently selected from hydrogen and hexyl. In some aspects, each R3 is independently selected from hydrogen and octyl. In some aspects, an ionizable lipid has 2 hydrogen R3 and two hexyl R3. In some aspects, an ionizable lipid has 2 hydrogen R3 and two octyl R3. In some aspects, an ionizable lipid has 3 hydrogen R3 and one octyl R3. In some aspects, an ionizable lipid has 3 hydrogen R3 and one hexyl R3.

[0118] According to some embodiments of any of the aspects or embodiments herein, the ionizable lipid is selected from any one of the lipids in Table 1 or a pharmaceutically acceptable salt thereof.Table 12024-031 / 10738-11812024-031 / 10738-11812024-031 / 10738-11812024-031 / 10738-1181-19-

[0119] In some aspects, the ionizable lipid is any of Lipids 1-19. In some aspects, the ionizable lipid is selected from Lipid 1, Lipid 4, Lipid 7, Lipid 10, Lipid 13, Lipid 14, Lipid 15,2024-031 / 10738-1181-20-Lipid 16, Lipid 17, Lipid 18, or Lipid 19. In some aspects, the ionizable lipid is selected from Lipid 7, Lipid 10, Lipid 13, Lipid 14, Lipid 15, Lipid 16, Lipid 17, Lipid 18, or Lipid 19. In some aspects, the ionizable lipid is Lipid 1. In some aspects, the ionizable lipid is Lipid 2. In some aspects, the ionizable lipid is Lipid 3. In some aspects, the ionizable lipid is Lipid 4. In some aspects, the ionizable lipid is Lipid 5. In some aspects, the ionizable lipid is Lipid 6. In some aspects, the ionizable lipid is Lipid 7. In some aspects, the ionizable lipid is Lipid 8. In some aspects, the ionizable lipid is Lipid 9. In some aspects, the ionizable lipid is Lipid 10. In some aspects, the ionizable lipid is Lipid 11. In some aspects, the ionizable lipid is Lipid 12. In some aspects, the ionizable lipid is Lipid 13. In some aspects, the ionizable lipid is Lipid 14. In some aspects, the ionizable lipid is Lipid 15. In some aspects, the ionizable lipid is Lipid 16. In some aspects, the ionizable lipid is Lipid 17. In some aspects, the ionizable lipid is Lipid 18. In some aspects, the ionizable lipid is Lipid 19. In some aspects, the ionizable lipid includes a mixture of any of Lipids 1-19.

[0120] In some aspects, ionizable lipids of the present disclosure are synthesized according to the scheme shown in FIG. 1.

[0121] In some aspects, ionizable lipids are synthesized by reacting an acyl chloride, an alkyl halide, a diaminoalkyl, or an aminoethanolic alkyl such as 2-aminoethanol chloride with a fatty alcohol in trimethylamine and dichloromethane to generate a first reaction product (1-R). The reaction can be performed with or without stirring. The reaction may proceed for any appropriate amount of time with or without monitoring. In aspects, the amount of time includes, for example, about 0-96 hours, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, and 96, or any range having endpoints defined by any two of the aforementioned values.

[0122] Illustrative acyl chlorides include, but are not limited to, acryloyl chloride, acetyl chloride, adipoyl chloride, anisoyl chloride, azelaoyl chloride, benzoyl chloride, butyryl chloride, chloroacetyl chloride, glutaryl chloride, hepatnoyl chloride, hexanoyl chloride, isobutyryrl chloride, lauyroyl chloride, malonyl chloride, methacrylol chloride, octanoyl chloride, oxalyl2024-031 / 10738-1181-21- chloride, pentanoyl chloride, pimeloyl chloride, pivaloyl chloride, propionyl chloride, thionyl chloride, thioaceyl chloride, and the like, though any suitable acyl chloride is contemplated and possible.

[0123] As shown in FIG. 1, the fatty alcohol is denoted HO-R. In some aspects the fatty alcohol is saturated. In other aspects, the fatty acid is unsaturated. Illustrative fatty alcohols include, but are not limited to, methanol, ethanol, propanol, butanol, heptanoyl, hexanol, octanoyl, nonanoyl, decanol, undecanol, dodecanol, oleyl alcohol, linoleyl alcohol, tert-butyl alcohol, tertamyl alcohol, enanthic alcohol, capryl alcohol, pelargoinc alcohol, capric alcohol, undecyl alcohol, lauryl alcohol, tridecyl alcohol, myristyl alcohol, pentadecyl alcohol, cetyl alcohol, palmitoleyl alcohol, heptadecyl alcohol, stearyl alcohol, nonadecyl alcohol, arachidyl alcohol, heneicosyl alcohol, behenyl alcohol, erucyl alcohol, lignoceryl alcohol, and ceryl alcohol, though any suitable fatty alcohol is contemplated and possible.

[0124] In aspects, the first reaction product can be extracted using an appropriate solvent. Illustrative examples of solvents include, but are not limited to, polar aprotic solvents (e.g. dichloromethane (DCM), dimethyl sulfoxide (DMSO), ethyl acetate, acetone, dimethylformamide (DMF), acetonitrile, nitromethane, propylene carbonate, etc.), nonpolar hydrocarbon solvents (e.g. pentane, hexane, benzene, heptane, toluene, etc.), nonpolar ether solvents (e.g. diethyl ether, tetrahydrofuran, etc.), nonpolar chlorocarbon solvents (e.g. chloroform, etc.), polar protic solvents (e.g. ammonia, formic acid, n-butanol, isopropyl alcohol, n-propanol, ethanol, methanol, acetic acid, water etc.), or combinations thereof.

[0125] Monitoring of the reaction may include, for example, thin-layer chromatography, Fourier-transform infrared spectroscopy (FTIR), Ultraviolet-visible spectroscopy (UV-Vis), nuclear magnetic resonance (NMR), temperature monitoring, pH monitoring, and the like, though any method of monitoring known in the art is contemplated and possible.

[0126] In some aspects, the first reaction product is allowed to dry after extraction. In aspects, drying of the product is effected by any acceptable method, including, but not limited to, evaporation at ambient temperature, use of a heat source (e.g., a steam bath, hot plate, sand bath, oven, etc.), rotary evaporation, or gas blow-down.

[0127] In aspects, the first reaction product is reacted with an amino alcohol to generate a second reaction product (2-R). In some aspects, this reaction is performed with stirring. In other2024-031 / 10738-1181-22- aspects, it is performed without stirring. In aspects, this reaction can occur at temperatures above ambient temperature, including, but not limited to, about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 °C. In some aspects, this reaction can occur at about ambient temperature. In some aspects, this reaction can occur below ambient temperature, including, but not limited to, about 20, 15, 10, 5, or 0 °C. The reaction may proceed for any appropriate amount of time with or without monitoring.

[0128] In some aspects, a suitable amino alcohol has 2-8 carbons, optionally alkyl or alkenyl. Illustrative amino alcohols include, but are not limited to ethanolamines, (i.e. 2- aminoethanol), methanol amine, dimethylethanolamine, N-methylethanolamine, aminopropanol, aminobutanol, aminopentanol, aminohexanol, aminoheptanol, aminooctanol, and / or aminomethyl propanol, thought any suitable amino alcohol is contemplated and possible.

[0129] One or more solvents are added to extract the second reaction product. In aspects, the second reaction product is allowed to dry. In some embodiments, the second reaction product has the structure of Formula II:(Formula II).

[0130] In some aspects, each X is independently selected from:

[0131] In some aspects, Y is independently selected from:2024-031 / 10738-1181-23-

[0132] In some aspects, each R2 is independently selected from C0-C18 alkyl groups or COCIS alkenyl groups. For example, each R2 may be independently selected from hydorgen, methyl, ethyl, ethane, propyl, propenyl, butyl, butenyl, pentyl, pentenyl, hexyl, hexenyl, heptyl, heptenyl octyl, octenyl, nonyl, nonenyl, decyl, decenyl, undecyl, undecenyl, dodecyl, dodecenyl, tridecyl, tridecenyl, tetradecyl, tetradecenyl, pentadecyl, pentadecenyl, hexadecyl, hexadecenyl, heptadecyl, heptadecenyl, octadecyl, and octadecenyl.

[0133] In some aspects, each R3 is independently selected from C0-C18 alkyl groups or COCIS alkenyl groups. For example, each R2 may be independently selected from hydorgen, methyl, ethyl, ethane, propyl, propenyl, butyl, butenyl, pentyl, pentenyl, hexyl, hexenyl, heptyl, heptenyl octyl, octenyl, nonyl, nonenyl, decyl, decenyl, undecyl, undecenyl, dodecyl, dodecenyl, tridecyl, tridecenyl, tetradecyl, tetradecenyl, pentadecyl, pentadecenyl, hexadecyl, hexadecenyl, heptadecyl, heptadecenyl, octadecyl, and octadecenyl.

[0134] In aspects, the second reaction product is used in the synthesis of an ionizable lipid. The second reaction product is reacted with a carboxylic acid derivative, optionally in the presence of one or more solvents. Optionally, for example in aspects where the second reaction product is dried, the second reaction product is dissolved in the solvent. Exemplary solvents include DCM and / or DMF, optionally DCM. In aspects, a carboxyl activating agent, such as l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) or N, N'-dicyclohexyl carbodiimide (DCC), is added to the reaction solution. In some embodiments, an esterification catalyst, such as 4- dimethylaminopyridine (DMAP) is added to the reaction solution. The reaction can be performed with or without stirring. The reaction may proceed for any appropriate amount of time with or without monitoring. The reaction may proceed for any appropriate amount of time with or without monitoring. The desired reaction product(s) can be separated by any method known in the art, such as column chromatography, to separate the reaction product.

[0135] In some aspects, the carboxylic acid derivative is added following the addition of the carboxyl activating agent and / or the esterification catalyst. In some embodiments, the carboxylic2024-031 / 10738-1181-24- acid derivative includes an R” group. Optionally, the R” group reacts with the second reaction product to yield the ionizable lipid. For example, in aspects where the second reaction product has a structure of Formula II, the R” group may react with the Y group to yield the ionizable lipid. Optionally, R” is selected from:Lipid Nanoparticles

[0136] In some aspects of this disclosure, the ionizable lipids disclosed herein, optionally those identified in Table 1 may be incorporated into lipid nanoparticles (LNPs). In some aspects, the lipid nanoparticles may be used to deliver cargo molecules (e.g. polypeptides, nucleic acids, small molecules, etc.) alone or as packaged in a deliverable pharmaceutical composition, such as a vaccine. Lipid nanoparticles may include one or more ionizable lipids, helper lipids, sterol and / or conjugated lipid components along with nucleic acid and / or polypeptide cargo of interest.

[0137] In some aspects, lipid nanoparticles comprise one or more ionizable lipids as described herein. In some aspects, lipid nanoparticles comprise one or more helper lipids as described herein. In some embodiments, lipid nanoparticles comprise one or more sterols as described herein. In some embodiments, lipid nanoparticles comprise one or more conjugate linker lipids as described herein.

[0138] LNPs may be used in some aspects to carry and / or deliver cargo to a subject or a portion thereof such as a cell or cellular compartment. DNA and RNA vaccines utilizing such LNPs share many similarities, but each targets different cellular environments. For example, DNA vaccines target and are used in the nucleus of a cell, whereas RNA vaccines target and are expressed in the cytosol. This makes mRNA vaccines easier to deliver, yet both may capitalize on the success of recent advances in LNP formulations and sometimes other modifications to the nucleic acid cargo itself that may improve overall function.2024-031 / 10738-1181-25-

[0139] As used herein, the term “nanoparticle” refers to a particle having dimensions on a scale of less than about 1000 nm. Routinely, nanoparticles have any one structural feature on a scale of less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm or less than about 100 nm. In exemplary embodiments, a nanoparticle is a particle having one or more dimensions of the order of about 10-500 nm. In other exemplary embodiments, a nanoparticle is a particle having one or more dimensions of the order of about 10-1000 nm. A spherical nanoparticle would have a diameter, for example, of between 10-100 nm or 10-1000 nm.

[0140] The term “particle size” or “particle diameter” refers to the mean diameter of the particles in a sample, as measured by dynamic light scattering (DLS), multiangle light scattering (MALS), nanoparticle tracking analysis, or comparable techniques. It will be understood that a dispersion of lipid nanoparticles as described herein will not be of uniform size but can be described by the average diameter and, optionally, the polydispersity index.

[0141] In some embodiments, lipid nanoparticles described herein have an average particle diameter that is about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185, nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, 225 nm, 230 nm, 235 nm, 240 nm, 245 nm, 250 nm, 255 nm, 260 nm, 265 nm, 270 nm, 275 nm, 280 nm, 285, nm, 290 nm, 295 nm, 300 nm, 305 nm, 310 nm, 315 nm, 320 nm, 325 nm, 330 nm, 335 nm, 340 nm, 345 nm, 350 nm, 355 nm, 360 nm, 365 nm, 370 nm, 375 nm, 380 nm, 385, nm, 390 nm, 395 nm, 400 nm, 405 nm, 410 nm, 415 nm, 420 nm, 425 nm, 430 nm, 435 nm, 440 nm, 445 nm, 450 nm, 455 nm, 460 nm, 465 nm, 470 nm, 475 nm, 480 nm, 485, nm, 490 nm, 495 nm, 500 nm, or any range having endpoints defined by any two of the aforementioned values. For example, in some embodiments, lipid nanoparticles described herein have an average particle diameter from about 100 nm to about 200 nm. Optionally, the lipid nanoparticles described herein have an average particle diameter of less than about 200 nm.

[0142] In some embodiments, the lipid nanoparticles described herein have a polydispersity index of less than 0.5, less than 0.4, less than 0.3, less than 0.2, or less than 0.1. Optionally, the lipid nanoparticles described herein have a polydispersity index of less than 0.3.2024-031 / 10738-1181-26-

[0143] The zeta potential of a nanoparticle composition may be used to indicate the electrokinetic potential of the composition. For example, the zeta potential may describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a nanoparticle composition may be from about -20 mV to about +20 mV, from about -20 mV to about +15 mV, from about -20 mV to about +10 mV, from about -20 mV to about +5 mV, from about -20 mV to about 0 mV, from about -20 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0144] In some aspects, the ionizable lipid is any of Lipids 1-19. In some aspects, the ionizable lipid is selected from Lipid 1, Lipid 4, Lipid 7, Lipid 9, Lipid 10, Lipid 13, Lipid 14, Lipid 15, Lipid 16, Lipid 17, Lipid 18, or Lipid 19. In some aspects, the ionizable lipid is selected from Lipid 7, Lipid 9, Lipid 10, Lipid 13, Lipid 14, Lipid 15, Lipid 16, Lipid 17, Lipid 18, or Lipid 19. In some aspects, the ionizable lipid is selected from Lipid 7, Lipid 10, Lipid 13, Lipid 14, Lipid 15, Lipid 16, Lipid 17, Lipid 18, or Lipid 19. In some aspects, the ionizable lipid is selected from Lipid 7, Lipid 9, Lipid 10, Lipid 16, or Lipid 18. In some aspects, the ionizable lipid is selected from Lipid 7, Lipid 10, Lipid 16, or Lipid 18. In some aspects, the ionizable lipid is Lipid 1. In some aspects, the ionizable lipid is Lipid 2. In some aspects, the ionizable lipid is Lipid 3. In some aspects, the ionizable lipid is Lipid 4. In some aspects, the ionizable lipid is Lipid 5. In some aspects, the ionizable lipid is Lipid 6. In some aspects, the ionizable lipid is Lipid 7. In some aspects, the ionizable lipid is Lipid 8. In some aspects, the ionizable lipid is Lipid 9. In some aspects, the ionizable lipid is Lipid 10. In some aspects, the ionizable lipid is Lipid 11. In some aspects, the ionizable lipid is Lipid 12. In some aspects, the ionizable lipid is Lipid 13. In some aspects, the ionizable lipid is Lipid 14. In some aspects, the ionizable lipid is Lipid 15. In some aspects, the ionizable lipid is Lipid 16. In some aspects, the ionizable lipid is Lipid 17. In some aspects, the ionizable lipid is Lipid 18. In some aspects, the ionizable lipid is Lipid 19. In some aspects, the ionizable lipid includes a mixture of any of Lipids 1-19.2024-031 / 10738-1181-27-

[0145] In some embodiments, ionizable lipids constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the total lipids in a suitable lipid solution by weight or by molar percent. In some embodiments, ionizable lipid(s) constitute(s) about 30- 70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30- 40%, about 35-50%, about 35-45%, about 35-40%, about 40-60%, about 45-60%, about 50-60%, about 55-60, about 40-65%, about 45-65%, about 50-65%, about 55-65%, about 60-65%) of the total lipid mixture by weight or by molar percent.

[0146] In some embodiments, lipid nanoparticles contain a mixture of ionizable and / or cationic lipids in combination with any of the above ionizable lipids for the formation of lipid nanoparticles. Suitable cationic lipids include, but are not limited to N-[l-(2,3-dioleyloxy)propyl]- N,N,N-trimethylammonium chloride (DOTMA), 5-carboxyspermylglycinedioctadecylamide (DOGS) 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-propanaminium (DOSPA), l,2-Dioleoyl-3-Dimethylammonium-Propane (DODAP), l,2-Dioleoyl-3- Trimethylammonium-Propane (DOTAP), l,2-distearyloxy-N,N-dimethyl-3 -aminopropane (DSDMA), l,2-dioleyloxy-N,N-dimethyl-3 -aminopropane (DODMA), l,2-dilinoleyloxy-N,N- dimethyl-3 -aminopropane (DLinDMA), 1,2-dilinolenyl oxy -N,N-dimethyl-3 -aminopropane (DLenDMA), N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N- dimethylarnrnonium bromide (DDAB), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N- hydroxyethyl ammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan- 4-oxy)-l-(cis,cis-9, 12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)- 3 '-oxapentoxy)-3 -dimethyl -1 -(cis, ci s-9', l-2'-octadecadienoxy)propane (CpLinDMA), N,N- dimethyl-3,4-di oleyl oxybenzylamine (DMOBA), l,2-N,N'-di oleyl carbamyl-3- dimethylaminopropane or (DOcarbDAP_, 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), l,2-N,N'-Dilinoleylcarbamyl-3 -dimethylaminopropane (DLincarbDAP), 1,2- Dilinoleoylcarbamyl-3 -dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-di oxolane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- di oxolane (DLin-K-XTC2-DMA), 2-(2,2-di((9Z,12Z)-octadeca-9,l 2-dien-l-yl)-l,3-dioxolan-4- yl)-N,N-dimethylethanamine (DLin-KC2-DMA), DLin-MC3-DMA, (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino) butanoate (MC3), or mixtures thereof.

[0147] In some embodiments, ionizable lipids in combination with the cationic lipids constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the total lipids in a suitable lipid solution by weight or by molar percent. In some2024-031 / 10738-1181-28- embodiments, ionizable lipid(s) constitute(s) about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, about 35-40%, about 40-60%, about 45-60%, about 50-60%, about 55-60, about 40-65%, about 45-65%, about 50-65%, about 55-65%, about 60-65%) of the total lipid mixture by weight or by molar percent.

[0148] In embodiments, the LNPs further comprise a non-cationic, helper lipid. The helper lipid may serve to increase fusogenicity and / or increase stability of the LNP during formation. As used herein, the phrase “helper lipid” refers to any neutral, zwitterionic or anionic lipid. Accordingly, the helper lipid may be a neutral uncharged, zwitterionic, or anionic lipid. As used herein, the term “neutral lipid” is meant to refer to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

[0149] As used herein, the term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N- glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyl oleyolphosphatidyl glycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0150] Helper lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidyl ethanolamine (DOPE), 2-diphytanoyl-sn-glycero-3- phosphatidylethanolamine (DPyPE), di stearoyl -sn-glycero- phosphoethanolamine, palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl -phosphatidylethanolamine(POPE), di oleoyl -phosphatidyl ethanolamine 4-(N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl -phosphatidyl ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, l-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), hydrogenated soy2024-031 / 10738-1181-29- phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), egg sphingomyelin (ESM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoylphosphatidylethanolamine (DEPE), l,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (DPHyPE); lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, or a combinations thereof. Optionally, the helper lipid is DSPC and / or DOPE. Optionally, the helper lipid is DSPC. Optionally, the helper lipid is DOPE.

[0151] In some embodiments, helper lipids may constitute at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the total lipids in a suitable lipid solution by weight or by molar percent. In some embodiments, helper lipid(s) constitute(s) about 5-25% (e.g., about 5-20%, about 5-15%, about 5-10%, about 10-25%, about 10-20%, about 10-15%, about 15-25%, about 15-20%, or about 20-25%) of the total lipids in a suitable lipid solution by weight or by molar percent.

[0152] In some embodiments, the LNPs can further comprise a component, such as a sterol, to provide membrane integrity and stability of the lipid particle. Illustrative examples of sterols include, but are not limited to, cholesterol, ergosterol, campesterol, oxysterol, antrosterol, desmosterol, nicasterol, sitosterol, stigmasterol, derivatives and variants thereof, and mixtures of the foregoing. In some embodiments, the sterol is cholesterol or a derivative or variant thereof. Non-limiting examples of cholesterol derivatives include 5a-cholestanol, 5P-coprostanol, cholesteryl-(2’-hydroxy)-ethyl ether, cholesteryl-(4’-hydroxy)-butyl ether, 6-ketocholestanol; 5a- cholestane, cholestenone, 5a-cholestanone, 5P-cholestanone, cholesteryl decanoate, 25- hydroxycholesterol (25-OH), 20a-hydroxycholesterol (20a-OH), 27-hydroxycholesterol, 6-keto- 5a- hydroxycholesterol, 7-ketocholesterol, 7-hydroxy cholesterol, 7a-hydroxycholesterol, 7 -25- dihydroxycholesterol, beta-sitosterol, stigmasterol, brassicasterol, campesterol, or combinations thereof.

[0153] In some embodiments, sterols constitute at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the total lipids in a suitable lipid solution by weight or by molar percent. In2024-031 / 10738-1181-30- some embodiments, sterols constitute about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipids in a suitable lipid solution by weight or by molar percent.

[0154] In some embodiments, LNPs may further include a lipid conjugate. As used herein, the term “lipid conjugate” is meant to refer to a conjugated lipid that inhibits aggregation of LNPs. Such lipid conjugates include, but are not limited to, polyethylene glycol (PEG)-lipid conjugates such as, e.g., PEG coupled to dialkyloxypropyls (e.g, PEG-DAA conjugates), PEG coupled to diacylglycerols (e.g, PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides, ionizable PEG lipids, polyoxazoline (POZ)-lipid conjugates, polyamide oligomers (e.g, ATTA-lipid conjugates), and mixtures thereof. PEG or POZ can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG or the POZ to a lipid may be used, such as non-ester containing linker moieties and ester-containing linker moieties. In certain embodiments, non-ester containing linker moieties, such as amides or carbamates, are used.

[0155] In some embodiments, lipid conjugates include a PEG-modified lipid. In embodiments, the PEGylated lipid may be used to enhance lipid nanoparticle colloidal stability in vitro and circulation time in vivo. Illustrative PEG-lipids for use in LNPs include, but are not limited to PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG, PEG- DSPE, dimystyrlglycerol (PEG-DMG), 1,2-dipalmitoyl-rac-glycerol, methoxypolyethylene glycol (DPG-PEG), 1,2-distearoyl-rac- glycero-3 -methylpolyoxyethylene (DSG-PEG). In some embodiments, the lipid conjugate is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol- 2000 (DMG-PEG). In some embodiments, a lipid conjugate has an average molecular mass from about 500 Da to about 5000 Da.

[0156] Lipid conjugates may constitute at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20% of the total lipids in a suitable lipid solution by weight or by molar. In some embodiments, lipid conjugates constitute about 1-5% (e.g., about 1-2%, about 1-3%, about 1-4%, about 2-5%, about 2-4%, about 2-3%, about 3-5%, about 3-4%, or about 4-5%) of the total lipids in a suitable lipid solution by weight or by molar percent.2024-031 / 10738-1181-31-

[0157] A suitable lipid solution may contain a mixture of desired lipids at various concentrations. In some embodiments, a lipid nanoparticle is formulated by mixing the ionizable lipid, helper lipid, sterol, and conjugated lipid at a molar ratio of 40: 10:48:2, respectively.

[0158] For example, a suitable lipid solution may contain a mixture of desired lipids at a total concentration of or greater than about 0.1 mg / ml, 0.5 mg / ml, 1.0 mg / ml, 2.0 mg / ml, 3.0 mg / ml, 4.0 mg / ml, 5.0 mg / ml, 6.0 mg / ml, 7.0 mg / ml, 8.0 mg / ml, 9.0 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, or 100 mg / ml. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration ranging from about 0.1-100 mg / ml, 0.25-50 mg / ml, 1.0-20 mg / ml, 1.0-70 mg / ml, 1.0-60 mg / ml, 1.0-50 mg / ml, 1.0-40 mg / ml, 1.0-30 mg / ml, 1.0-20 mg / ml, 1.0-15 mg / ml, 1.0-10 mg / ml, 1.0-9 mg / ml, 1.0-8 mg / ml, 1.0-7 mg / ml, 1.0-6 mg / ml, or 1.0-5 mg / ml. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration up to about 100 mg / ml, 90 mg / ml, 80 mg / ml, 70 mg / ml, 60 mg / ml, 50 mg / ml, 40 mg / ml, 30 mg / ml, 20 mg / ml, 10 mg / ml, 5 mg / mL, 4 mg / mL, 3 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, or 0.1 mg / mL.

[0159] In some embodiments, the ionizable lipid is included in the lipid solution in a molar percentage from about 25% to about 70%, including 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, and 70%, or any range having endpoints defined by any two of the aforementioned values.

[0160] In embodiments, the helper lipid is included in the lipid solution in a molar percentage from about 5% to about 40%, including 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, and 40% or any range having endpoints defined by any two of the aforementioned values.

[0161] In embodiments, the sterol is included in the lipid solution in a molar percentage from about 20% to about 60%, including about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60%, or any range having endpoints defined by any two of the aforementioned values.2024-031 / 10738-1181-32-

[0162] In embodiments, the lipid conjugate is included in the lipid solution in a molar percentage from about 1% to about 20%, including 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20%, or any range having endpoints defined by any two of the aforementioned values.

[0163] In some embodiments, the lipid nanoparticle solution includes a cryoprotectant. Optionally, the cryoprotectant is included in the lipid solution in a weight percentage from about 2% to about 20%, including about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20%, or any range having endpoints defined by any two of the aforementioned values. Optionally, the cryoprotectant is a sugar. Illustrative, non-limiting cryoprotectants include sucrose, glucose, trehalose, mannitol, raffinose, combinations thereof, and the like. In some embodiments, the cryoprotectant is sucrose.

[0164] In some embodiments, the cargo includes a nucleic acid (e.g., DNA, RNA, e.g., mRNA). The nucleic acid may be at a concentration between about 50 pg / ml and about 5 mg / mL, optionally about 50 pg / ml to about 200 pg per ml of the aqueous solution (e.g., about 50 pg / ml, about 60 pg / ml, about 70 pg / ml, about 80 pg / ml, about 90 pg / ml, about 100 pg / ml, about 110 pg / ml, about 120 pg / ml, about 130 pg / ml, about 140 pg / ml, about 150 pg / ml, about 175 pg / ml, or about 200 pg / ml). All of or a portion of the nucleic acid may be encapsulated in the lipid nanoparticles. In some embodiments, the method yields a nucleic acid encapsulation efficiency of at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater). In some embodiments, the method yields a nucleic acid encapsulation efficiency of at least 85%. In some embodiments, the method yields a nucleic acid encapsulation efficiency of at least 90%. In some embodiments, the method yields a nucleic acid encapsulation efficiency from about 85% to about 99%.

[0165] Generally, the LNPs are prepared at a molar ratio between the amine group of the ionizable lipid and the phosphate group of the mRNA, from about 5: 1 to 60: 1. In some embodiments, the lipid to nucleic acid ratio (mass / mass ratio; w / w ratio) can be in the range of from about 1 : 1 to about 60: 1, from about 1 : 1 to about 20: 1, from about 1 : 1 to about 19: 1, from about 1 : 1 to about 18: 1, from about 1 : 1 to about 17: 1, from about 1 : 1 to about 16: 1, from about 1 : 1 to about 15: 1, from about 1 : 1 to about 14: 1, from about 1 : 1 to about 13: 1, from about 1 : 1 to about 12: 1, from about 1 : 1 to about 10: 1, from about 1 : 1 to about 9: 1, from about 1 : 1 to about 8: 1, from about 1 : 1 to about 7: 1, from about 1 : 1 to about 6: 1, from about 1 : 1 to about 5: 1, from2024-031 / 10738-1181-33- about 3: 1 to about 15: 1, from about 4: 1 to about 15: 1, from about 5: 1 to about 15: 1, about 6: 1 to about 15: 1, from about 7: 1 to about 15: 1, from about 8: 1 to about 15: 1, from about 9: 1 to about 15: 1, from about 5: 1 to about 10: 1, from about 6: 1 to about 10: 1, from about 7: 1 to about 10: 1, from about 8: 1 to about 10: 1, or from about 9: 1 to about 10: 1. Optionally, the LNP is prepared with a ratio of 8: 1 between the amine group of the ionizable lipid and the phosphate group of the cargo RNA.

[0166] The LNPs can be prepared with the cargo at a volume ratio with the lipid solution, such that the lipid solution: cargo ratio is from about 1 : 1 to 10: 1, including 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 and 10: 1, or any range having endpoints defined by any two of the aforementioned values.EXAMPLES

[0167] The following examples are given by way of illustration and are in no way intended to limit the scope of the present disclosure.Example 1: Characterization of Ionizable Lipids

[0168] Lipid 1

[0169] As depicted in FIG. 3 A, Lipid 1 was synthesized using the general scheme shown in FIG. 1 and as described herein. Lipid 1 was characterized by proton nuclear magnetic resonance on a 400 MHz spectrometer in CDCh, the results of which are depicted in FIG. 3B. In particular, the data reported includes: chemical shift (5 ppm), multiplicity, integration, and coupling constant (Hz). 'HNMR (400 MHz, CDCh) 8 5.41 - 5.29 (m, 4H), 4.11 (q, J = 9.0, 7.6 Hz, 2H), 4.05 (t, J = 6.8 Hz, 4H), 2.83 (t, J = 7.2 Hz, 6H), 2.71 (t, J = 6.1 Hz, 2H), 2.43 (t, J = 7.2 Hz, 4H), 2.29 (s, 4H), 2.01 (q, J = 6.6 Hz, 10H), 1.92 (dd, J = 13.6, 3.6 Hz, 2H), 1.79 (qd, J = 10.8, 5.5 Hz, 2H), 1.61 (t, J = 7.0 Hz, 4H), 1.28 (d, J = 12.8 Hz, 44H), 0.88 (t, J = 6.8 Hz, 6H).

[0170] Lipid 1 was also characterized by carbon- 13 nuclear magnetic resonance one a 101 MHz spectrometer in CDCh, the results of which are depicted in FIG. 3C. In particular, the data reported includes the chemical shift (5 ppm).13C NMR (101 MHz, CDCh) 6 174.90, 174.75, 172.41, 129.97, 129.77, 64.64, 62.50, 60.36, 54.84, 52.14, 49.79, 46.21, 40.23, 32.95.

[0171] Lipid 22024-031 / 10738-1181-34-

[0172] As depicted in FIG. 4A, Lipid 2 was synthesized using the general scheme shown in FIG. 1 and as described herein. Lipid 2 was characterized by proton nuclear magnetic resonance on a 400 MHz spectrometer in CDCh, the results of which are depicted in FIG. 4B. In particular, the data reported includes: chemical shift (5 ppm), multiplicity, integration, and coupling constant (Hz). ‘H NMR (400 MHz, CDCh) 8 5.41 - 5.29 (m, 4H), 4.13 (t, J = 6.1 Hz, 2H), 4.05 (t, J = 6.8 Hz, 4H), 2.83 (t, J = 7.2 Hz, 4H), 2.72 (t, J = 6.2 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H), 2.46 (dt, J = 18.3, 7.2 Hz, 6H), 2.25 (s, 6H), 2.01 (q, J = 6.3 Hz, 8H), 1.61 (t, J = 7.0 Hz, 4H), 1.30 (t, J = 12.2 Hz, 44H), 0.88 (t, J = 6.7 Hz, 6H).

[0173] Lipid 2 was also characterized by carbon- 13 nuclear magnetic resonance one a 101 MHz spectrometer in CDCh, the results of which are depicted in FIG. 4C. In particular, the data reported includes the chemical shift (5 ppm).13C NMR (101 MHz, CDCh) 6 172.42, 172.33, 129.98, 129.78, 64.63, 62.51, 54.72, 52.09, 49.83, 45.24, 32.93, 32.81, 31.91, 29.77, 29.75, 29.52, 29.44, 29.32, 29.26, 29.25, 28.63, 27.22, 27.20, 25.93, 22.68, 14.10.

[0174] Lipid 3

[0175] As depicted in FIG. 5A, Lipid 3 was synthesized using the general scheme shown in FIG. 1 and as described herein. Lipid 3 was characterized by proton nuclear magnetic resonance on a 400 MHz spectrometer in CDCh, the results of which are depicted in FIG. 5B. In particular, the data reported includes: chemical shift (5 ppm), multiplicity, integration, and coupling constant (Hz). 'H NMR (400 MHz, CDCh) 6 5.50 - 5.19 (m, 4H), 4.11 (t, J = 6.1 Hz, 2H), 4.06 (td, J = 6.8, 4.0 Hz, 4H), 2.83 (t, J = 7.2 Hz, 4H), 2.71 (t, J = 6.2 Hz, 2H), 2.43 (t, J = 7.2 Hz, 4H), 2.35 (t, J = 7.4 Hz, 4H), 2.27 (d, J = 1.9 Hz, 6H), 2.00 (p, J = 8.1, 7.1 Hz, 8H), 1.86 - 1.77 (m, 2H), 1.61 (t, J = 7.0 Hz, 4H), 1.29 (q, J = 7.7, 4.7 Hz, 44H), 0.88 (t, J = 6.7 Hz, 6H).

[0176] Lipid 3 was also characterized by carbon- 13 nuclear magnetic resonance one a 101 MHz spectrometer in CDCh, the results of which are depicted in FIG. 5C. In particular, the data reported includes the chemical shift (5 ppm).13C NMR (101 MHz, CDCh) 6 173.56, 173.33, 172.43, 129.98, 129.78, 64.64, 64.57, 62.43, 58.74, 58.69, 52.10, 49.83, 45.17, 32.93, 32.60, 32.02, 31.91, 31.88, 29.77, 29.75, 29.70, 29.66, 29.53, 29.45, 29.42, 29.32, 29.26, 29.22, 28.63, 27.22, 27.20, 25.93, 22.68, 22.60, 14.11.

[0177] Lipid 42024-031 / 10738-1181-35-

[0178] As depicted in FIG. 6A, Lipid 4 was synthesized using the general scheme shown in FIG. 1 and as described herein. Lipid 4 was characterized by proton nuclear magnetic resonance on a 400 MHz spectrometer in CDCh, the results of which are depicted in FIG. 6B. In particular, the data reported includes: chemical shift (5 ppm), multiplicity, integration, and coupling constant (Hz). ‘H NMR (400 MHz, CDCh) 8 5.36 (qd, J = 11.2, 9.5, 4.0 Hz, 8H), 4.13 (t, J = 6.1 Hz, 2H), 4.05 (t, J = 6.8 Hz, 4H), 2.89 (dt, J = 12.1, 4.2 Hz, 2H), 2.83 (t, J = 7.2 Hz, 4H), 2.77 (t, J = 6.5 Hz, 4H), 2.71 (t, J = 6.1 Hz, 2H), 2.43 (t, J = 7.1 Hz, 4H), 2.37 (s, 4H), 2.23 (d, J = 15.2 Hz, 2H), 2.10 - 1.95 (m, 10H), 1.86 (ddt, J = 14.3, 10.4, 5.2 Hz, 2H), 1.62 (q, J = 6.9 Hz, 4H), 1.38 - 1.25 (m, 32H), 0.89 (t, J = 6.7 Hz, 6H).

[0179] Lipid 4 was also characterized by carbon- 13 nuclear magnetic resonance one a 101 MHz spectrometer in CDCh, the results of which are depicted in FIG. 6C. In particular, the data reported includes the chemical shift (5 ppm).13C NMR (101 MHz, CDCh) 6 174.40, 172.37, 130.19, 130.04, 128.02, 127.90, 64.62, 62.59, 54.45, 52.14, 49.78, 45.80, 32.93, 31.51, 29.64, 29.43, 29.33, 29.24, 28.62, 27.47, 27.21, 27.19, 25.91, 25.62, 22.56, 14.06.

[0180] Lipid 5

[0181] As depicted in FIG. 7A, Lipid 5 was synthesized using the general scheme shown in FIG. 1 and as described herein. Lipid 5 was characterized by proton nuclear magnetic resonance on a 400 MHz spectrometer in CDCh, the results of which are depicted in FIG. 7B. In particular, the data reported includes: chemical shift (5 ppm), multiplicity, integration, and coupling constant (Hz).XH NMR (400 MHz, CDCh) 6 5.47 - 5.26 (m, 8H), 4.12 (t, J= 6.1 Hz, 2H), 4.05 (t, J= 6.8 Hz, 4H), 2.83 (t, J= 7.2 Hz, 4H), 2.77 (t, J= 6.4 Hz, 4H), 2.71 (t, J= 6.1 Hz, 2H), 2.64 (t, J= 7.7 Hz, 2H), 2.49 (s, 6H), 2.42 (dt, J= 11.8, 7.1 Hz, 6H), 2.05 (q, J = 6.8 Hz, 8H), 1.95 (p, J = 7.3 Hz, 2H), 1.62 (q, J= 6.9 Hz, 4H), 1.38 - 1.26 (m, 32H), 0.89 (t, J= 6.8 Hz, 6H).

[0182] Lipid 5 was also characterized by carbon- 13 nuclear magnetic resonance one a 101 MHz spectrometer in CDCh, the results of which are depicted in FIG. 7C. In particular, the data reported includes the chemical shift (5 ppm).13C NMR (101 MHz, CDCh) 6 172.40, 172.07, 130.19, 130.05, 128.02, 127.91, 64.62, 62.60, 54.51, 52.07, 49.87, 49.82, 45.00, 32.97, 32.92, 32.47, 31.52, 29.64, 29.43, 29.34, 29.24, 28.62, 27.21, 27.20, 25.92, 25.63, 22.56, 14.06.

[0183] Lipid 62024-031 / 10738-1181-36-

[0184] As depicted in FIG. 8A, Lipid 6 was synthesized using the general scheme shown in FIG. 1 and as described herein. Lipid 6 was characterized by proton nuclear magnetic resonance on a 400 MHz spectrometer in CDCh, the results of which are depicted in FIG. 8B. In particular, the data reported includes: chemical shift (5 ppm), multiplicity, integration, and coupling constant (Hz). ‘H NMR (400 MHz, CDCh) 8 5.47 - 5.26 (m, 8H), 4.12 (t, J= 6.1 Hz, 2H), 4.05 (t, J= 6.8 Hz, 4H), 2.83 (t, J= 7.2 Hz, 4H), 2.77 (t, J= 6.4 Hz, 4H), 2.71 (t, J= 6.1 Hz, 2H), 2.64 (t, J= 7.7 Hz, 2H), 2.49 (s, 6H), 2.42 (dt, J= 11.8, 7.1 Hz, 6H), 2.05 (q, J = 6.8 Hz, 8H), 1.95 (p, J = 7.3 Hz, 2H), 1.62 (q, J= 6.9 Hz, 4H), 1.38 - 1.26 (m, 32H), 0.89 (t, J= 6.8 Hz, 6H).

[0185] Lipid 6 was also characterized by carbon- 13 nuclear magnetic resonance one a 101 MHz spectrometer in CDCh, the results of which are depicted in FIG. 8C. In particular, the data reported includes the chemical shift (5 ppm).13C NMR (101 MHz, CDCh) 6 172.75, 172.41, 130.22, 130.07, 128.03, 127.91, 64.66, 62.66, 58.02, 52.08, 49.81, 44.22, 32.91, 31.53, 31.36, 29.65, 29.44, 29.35, 29.25, 28.63, 27.22, 27.20, 25.93, 25.63, 22.57, 21.31, 14.07.

[0186] Lipid 7

[0187] As depicted in FIG. 9A, Lipid 7 was synthesized using the general scheme shown in FIG. 1 and as described herein. Lipid 7 was characterized by proton nuclear magnetic resonance on a 400 MHz spectrometer in CDCh, the results of which are depicted in FIG. 9B. In particular, the data reported includes: chemical shift (5 ppm), multiplicity, integration, and coupling constant (Hz). 'H NMR (400 MHz, CDCh) 6 4.13 (t, J = 6.1 Hz, 2H), 3.96 (d, J = 5.8 Hz, 4H), 2.83 (q, J = 7.5 Hz, 6H), 2.72 (t, J = 6.1 Hz, 2H), 2.44 (t, J = 7.3 Hz, 4H), 2.28 (s, 4H), 2.03 (t, J = 11.5 Hz, 2H), 1.92 (dt, J = 13.7, 3.7 Hz, 2H), 1.78 (dtd, J = 14.3, 11.0, 3.7 Hz, 2H), 1.66 - 1.54 (m, 2H), 1.27 (d, J = 2.8 Hz, 48H), 0.88 (t, J = 6.7 Hz, 12H).

[0188] Lipid 7 was also characterized by carbon- 13 nuclear magnetic resonance one a 101 MHz spectrometer in CDCh, the results of which are depicted in FIG. 9C. In particular, the data reported includes the chemical shift (5 ppm).13C NMR (101 MHz, CDCh) 6 174.81, 172.53, 67.35, 62.43, 54.92, 52.09, 49.77, 46.30, 40.34, 37.29, 32.89, 31.90, 31.83, 31.21, 29.98, 29.63, 29.59, 29.32, 28.14, 26.70, 26.65, 22.68, 22.65, 14.11.

[0189] Lipid 82024-031 / 10738-1181-37-

[0190] As depicted in FIG. 10 A, Lipid 8 was synthesized using the general scheme shown in FIG. 1 and as described herein. Lipid 8 was characterized by proton nuclear magnetic resonance on a 400 MHz spectrometer in CDCh, the results of which are depicted in FIG. 10B. In particular, the data reported includes: chemical shift (5 ppm), multiplicity, integration, and coupling constant (Hz). 'H NMR (400 MHz, CDCh) 8 4.13 (t, J = 6.2 Hz, 2H), 3.97 (d, J = 5.8 Hz, 4H), 2.84 (t, J = 7.3 Hz, 4H), 2.72 (t, J = 6.2 Hz, 2H), 2.64 (t, J = 7.2 Hz, 2H), 2.47 (dt, J = 19.7, 7.2 Hz, 6H), 2.26 (s, 6H), 1.61 (t, J = 5.8 Hz, 2H), 1.27 (d, J = 2.9 Hz, 48H), 0.88 (t, J = 6.7 Hz, 12H).

[0191] Lipid 8 was also characterized by carbon- 13 nuclear magnetic resonance one a 101 MHz spectrometer in CDCh, the results of which are depicted in FIG. 10C. In particular, the data reported includes the chemical shift (5 ppm).13C NMR (101 MHz, CDCh) 6 172.53, 172.17,67.35, 62.54, 54.61, 52.02, 49.80, 45.11, 37.29, 32.85, 32.62, 31.91, 31.83, 31.22, 29.98, 29.63, 29.59, 29.33, 26.70, 26.65, 22.68, 22.66, 14.11.

[0192] Lipid 9

[0193] As depicted in FIG. 11 A, Lipid 9 was synthesized using the general scheme shown in FIG. 1 and as described herein. Lipid 9 was characterized by proton nuclear magnetic resonance on a 400 MHz spectrometer in CDCh, the results of which are depicted in FIG. 1 IB. In particular, the data reported includes: chemical shift (5 ppm), multiplicity, integration, and coupling constant (Hz). 'HNMR (400 MHz, CDCh) 6 4.13 (t, J= 6.1 Hz, 2H), 3.97 (d, J= 5.8 Hz, 4H), 2.84 (t, J = 7.3 Hz, 4H), 2.78 - 2.62 (m, 4H), 2.52 (s, 6H), 2.43 (dt, J= 13.4, 7.1 Hz, 6H), 1.96 (q, J= 7.5 Hz, 2H), 1.68 - 1.54 (m, 2H), 1.35 - 1.21 (m, 48H), 0.88 (t, J= 6.6 Hz, 12H).

[0194] Lipid 9 was also characterized by carbon- 13 nuclear magnetic resonance one a 101 MHz spectrometer in CDCh, the results of which are depicted in FIG. 11C. In particular, the data reported includes the chemical shift (5 ppm).13C NMR (101 MHz, CDCh) 6 172.69, 172.52,67.36, 62.66, 57.98, 52.01, 49.77, 44.16, 37.28, 32.83, 31.90, 31.82, 31.31, 31.21, 29.97, 29.62, 29.58, 29.32, 26.70, 26.65, 22.67, 22.65, 21.21, 14.10.

[0195] Lipid 10

[0196] As depicted in FIG. 12 A, Lipid 10 was synthesized using the general scheme shown in FIG. 1 and as described herein. Lipid 10 was characterized by proton nuclear magnetic resonance on a 400 MHz spectrometer in CDCh, the results of which are depicted in FIG. 12B.2024-031 / 10738-1181-38-In particular, the data reported includes: chemical shift (5 ppm), multiplicity, integration, and coupling constant (Hz). 'H NMR (400 MHz, CDCI3) 8 4.86 (p, J = 6.3 Hz, 2H), 4.13 (t, J = 6.1 Hz, 2H), 2.83 (q, J = 9.1, 8.2 Hz, 6H), 2.72 (t, J = 6.2 Hz, 2H), 2.43 (t, J = 7.3 Hz, 4H), 2.27 (s, 4H), 2.01 (dt, J = 11.3, 5.7 Hz, 2H), 1.92 (dt, J = 13.6, 3.7 Hz, 2H), 1.85 - 1.71 (m, 2H), 1.50 (q, J = 6.3 Hz, 8H), 1.26 (s, 48H), 0.88 (t, J = 6.7 Hz, 12H).

[0197] Lipid 10 was also characterized by carbon- 13 nuclear magnetic resonance one a 101 MHz spectrometer in CDCI3, the results of which are depicted in FIG. 12C. In particular, the data reported includes the chemical shift (6 ppm).13C NMR (101 MHz, CDCI3) 6 174.83, 172.13, 74.49, 62.56, 54.93, 52.00, 49.94, 46.32, 40.36, 34.10, 33.10, 31.87, 29.56, 29.51, 29.27, 28.17, 25.33, 22.67, 14.10.

[0198] Lipid 11

[0199] As depicted in FIG. 13A, Lipid 11 was synthesized using the general scheme shown in FIG. 1 and as described herein. Lipid 11 was characterized by proton nuclear magnetic resonance on a 400 MHz spectrometer in CDCI3, the results of which are depicted in FIG. 13B. In particular, the data reported includes: chemical shift (6 ppm), multiplicity, integration, and coupling constant (Hz). 'H NMR (400 MHz, CDCI3) 6 4.86 (p, J = 6.3 Hz, 2H), 4.13 (t, J= 6.1 Hz, 2H), 2.84 (t, J= 7.4 Hz, 4H), 2.73 (t, J = 6.2 Hz, 2H), 2.62 (t, J= 7.2 Hz, 2H), 2.46 (dt, J = 20.6, 7.3 Hz, 6H), 2.23 (s, 6H), 1.50 (q, J= 6.1 Hz, 8H), 1.26 (s, 48H), 0.88 (t, J= 6.7 Hz, 12H).

[0200] Lipid 11 was also characterized by carbon-13 nuclear magnetic resonance one a 101 MHz spectrometer in CDCI3, the results of which are depicted in FIG. 13C. In particular, the data reported includes the chemical shift (6 ppm).13C NMR (101 MHz, CDCI3) 6 172.29, 172.11, 74.47, 62.56, 60.36, 54.70, 51.94, 49.95, 45.22, 34.10, 33.04, 32.78, 31.85, 29.54, 29.50, 29.26, 25.32, 22.65, 14.18, 14.08.

[0201] Lipid 12

[0202] As depicted in FIG. 14 A, Lipid 12 was synthesized using the general scheme shown in FIG. 1 and as described herein. Lipid 12 was characterized by proton nuclear magnetic resonance on a 400 MHz spectrometer in CDCI3, the results of which are depicted in FIG. 14B. In particular, the data reported includes: chemical shift (6 ppm), multiplicity, integration, and coupling constant (Hz). 'H NMR (400 MHz, CDCI3) 6 4.86 (p, J = 6.3 Hz, 2H), 4.12 (t, J= 6.22024-031 / 10738-1181-39-Hz, 2H), 2.84 (t, J= 7.4 Hz, 4H), 2.72 (t, J= 6.2 Hz, 2H), 2.43 (t, J= 7.4 Hz, 4H), 2.35 (t, J= 7.4 Hz, 4H), 2.27 (s, 6H), 1.82 (p, J= 7.4 Hz, 2H), 1.50 (t, J= 6.3 Hz, 8H), 1.26 (s, 48H), 0.88 (t, J= 6.7 Hz, 12H).

[0203] Lipid 12 was also characterized by carbon-13 nuclear magnetic resonance one a 101 MHz spectrometer in CDCh, the results of which are depicted in FIG. 14C. In particular, the data reported includes the chemical shift (5 ppm).13C NMR (101 MHz, CDCI3) 5 173.30, 172.14,74.49, 62.49, 58.68, 51.95, 49.95, 45.16, 34.10, 33.05, 31.86, 29.55, 29.51, 29.26, 25.33, 22.66, 22.59, 14.10 .

[0204] Lipid 13

[0205] As depicted in FIG. 15 A, Lipid 13 was synthesized using the general scheme shown in FIG. 1 and as described herein. Lipid 13 was characterized by proton nuclear magnetic resonance on a 400 MHz spectrometer in CDCI3, the results of which are depicted in FIG. 15B. In particular, the data reported includes: chemical shift (5 ppm), multiplicity, integration, and coupling constant (Hz). 'H NMR (400 MHz, CDCh) 8 4.86 (p, J = 6.2 Hz, 2H), 4.14 (t, J= 6.2 Hz, 2H), 3.12 - 2.98 (m, 1H), 2.85 (q, J = 8.5 Hz, 5H), 2.73 (t, J = 6.3 Hz, 2H), 2.70 - 2.62 (m, 2H), 2.61 - 2.48 (m, 1H), 2.43 (t, J= 7.4 Hz, 4H), 2.38 (s, 3H), 2.11 (q, J = 7.3 Hz, 2H), 1.50 (t, J= 6.3 Hz, 8H), 1.26 (s, 48H), 0.88 (t, J= 6.7 Hz, 12H).

[0206] Lipid 13 was also characterized by carbon- 13 nuclear magnetic resonance one a 101 MHz spectrometer in CDCh, the results of which are depicted in FIG. 15C. In particular, the data reported includes the chemical shift (5 ppm).13C NMR (101 MHz, CDCh) 6 174.75, 172.11,74.50, 62.87, 58.76, 55.93, 51.95, 49.97, 42.55, 41.81, 34.10, 33.07, 31.86, 29.55, 29.51, 29.27, 28.25, 25.33, 22.66, 14.10.

[0207] Lipid 14

[0208] As depicted in FIG. 16 A, Lipid 14 was synthesized using the general scheme shown in FIG. 1 and as described herein. Lipid 14 was characterized by proton nuclear magnetic resonance on a 400 MHz spectrometer in CDCh, the results of which are depicted in FIG. 16B. In particular, the data reported includes: chemical shift (5 ppm), multiplicity, integration, and coupling constant (Hz). 'HNMR (400 MHz, CDCh) 6 5.44 - 5.25 (m, 4H), 4.86 (p, J= 6.3 Hz, 1H), 4.13 (t, J= 6.1 Hz, 2H), 4.05 (t, J= 6.8 Hz, 2H), 2.96 - 2.57 (m, 10H), 2.43 (q, J= 6.8 Hz,2024-031 / 10738-1181-40-4H), 2.31 - 2.24 (m, 4H), 2.05 (q, J = 6.8 Hz, 6H), 1.92 (dt, J = 12.1, 3.8 Hz, 2H), 1.79 (qd, J = 10.7, 5.4 Hz, 2H), 1.61 (p, J= 6.9 Hz, 2H), 1.50 (q, J= 6.1 Hz, 4H), 1.28 (d, J= 17.4 Hz, 40H), 0.90 - 0.85 (m, 9H).

[0209] Lipid 14 was also characterized by carbon-13 nuclear magnetic resonance one a 101 MHz spectrometer in CDCh, the results of which are depicted in FIG. 16C. In particular, the data reported includes the chemical shift (5 ppm).13C NMR (101 MHz, CDCI3) 5 174.79, 172.42, 172.13, 130.21, 130.07, 128.03, 127.92, 74.51, 64.64, 62.54, 54.90, 52.08, 49.96, 49.93, 49.75, 46.28, 40.30, 34.10, 33.12, 32.91, 31.87, 31.53, 29.70, 29.66, 29.56, 29.51, 29.45, 29.35, 29.27, 28.64, 28.10, 27.22, 27.20, 25.92, 25.63, 25.33, 22.66, 22.57, 14.10, 14.07.

[0210] Lipid 15

[0211] As depicted in FIG. 17 A, Lipid 15 was synthesized using the general scheme shown in FIG. 1 and as described herein. Lipid 15 was characterized by proton nuclear magnetic resonance on a 400 MHz spectrometer in CDCI3, the results of which are depicted in FIG. 17B. In particular, the data reported includes: chemical shift (5 ppm), multiplicity, integration, and coupling constant (Hz). 'HNMR (400 MHz, CDCh) 8 5.43 - 5.30 (m, 4H), 4.86 (p, J= 6.1 Hz, 1H), 4.14 (t, J= 6.2 Hz, 2H), 4.05 (t, J= 6.8 Hz, 2H), 3.06 (p, J= 7.8 Hz, 1H), 2.91 - 2.80 (m, 4H), 2.72 (tt, J= 19.4, 7.3 Hz, 6H), 2.55 (q, J= 8.3, 7.9 Hz, 2H), 2.51 - 2.31 (m, 7H), 2.18 - 1.98 (m, 6H), 1.61 (p, J= 6.9 Hz, 2H), 1.49 (t, J= 6.2 Hz, 4H), 1.28 (d, J= 17.5 Hz, 40H), 0.88 (td, J = 6.7, 4.6 Hz, 9H).

[0212] Lipid 15 was also characterized by carbon- 13 nuclear magnetic resonance one a 101 MHz spectrometer in CDCh, the results of which are depicted in FIG. 17C. In particular, the data reported includes the chemical shift (5 ppm).13C NMR (101 MHz, CDCh) 6 174.72, 172.42, 172.12, 130.22, 130.08, 128.03, 127.92, 74.53, 64.64, 62.85, 58.73, 55.92, 53.41, 52.03, 49.99, 49.78, 42.54, 41.80, 34.10, 33.10, 32.89, 31.87, 31.53, 29.66, 29.56, 29.51, 29.45, 29.35, 29.27, 28.64, 28.24, 27.23, 27.21, 25.93, 25.64, 25.34, 22.67, 22.57, 14.11, 14.07.

[0213] Lipid 16

[0214] As depicted in FIG. 18 A, Lipid 16 was synthesized using the general scheme shown in FIG. 1 and as described herein. Lipid 3 was characterized by proton nuclear magnetic resonance on a 400 MHz spectrometer in CDCh, the results of which are depicted in FIG. 18B. In particular,2024-031 / 10738-1181-41- the data reported includes: chemical shift (5 ppm), multiplicity, integration, and coupling constant (Hz). 'H NMR (400 MHz, CDC13) 8 4.14 (t, J = 6.1 Hz, 2H), 3.97 (d, J = 5.8 Hz, 4H), 3.00 (s, 1H), 2.85 (h, J = 8.9 Hz, 5H), 2.76 - 2.64 (m, 4H), 2.56 (q, J = 8.4, 7.9 Hz, 1H), 2.45 (t, J = 7.2 Hz, 4H), 2.40 (s, 3H), 2.12 (q, J = 7.2 Hz, 2H), 1.61 (t, J = 5.6 Hz, 2H), 1.27 (s, 48H), 0.88 (t, J =6.6 Hz, 12H).

[0215] Lipid 16 was also characterized by carbon- 13 nuclear magnetic resonance one a 101 MHz spectrometer in CDCI3, the results of which are depicted in FIG. 4C. In particular, the data reported includes the chemical shift (6 ppm).13C NMR (101 MHz, CDCI3) 6 174.66, 172.51, 67.35, 62.77, 58.67, 55.88, 52.04, 49.80, 42.52, 41.77, 37.29, 32.85, 31.90, 31.83, 31.21, 29.97, 29.62, 29.58, 29.32, 28.24, 26.70, 26.65, 22.67, 22.65, 14.10.

[0216] Lipid 17

[0217] As depicted in FIG. 19 A, Lipid 17 was synthesized using the general scheme shown in FIG. 1 and as described herein. Lipid 17 was characterized by proton nuclear magnetic resonance on a 400 MHz spectrometer in CDCI3, the results of which are depicted in FIG. 19B. In particular, the data reported includes: chemical shift (6 ppm), multiplicity, integration, and coupling constant (Hz). 'H NMR (400 MHz, CDCI3) 6 4.10 (t, J = 6.2 Hz, 2H), 4.03 (t, J = 6.7 Hz, 6H), 3.57 (t, J = 5.3 Hz, 1H), 2.84 (dt, J = 9.2, 4.2 Hz, 2H), 2.65 (t, J = 6.2 Hz, 2H), 2.52 (t, J = 7.6 Hz, 2H), 2.42 (t, J = 7.4 Hz, 4H), 2.28 (dd, J = 14.7, 6.8 Hz, 10H), 2.19 - 2.03 (m, 2H), 1.93 (dt, J = 12.3, 3.9 Hz, 2H), 1.86 - 1.74 (m, 2H), 1.59 (dt, J = 10.9, 7.0 Hz, 11H), 1.44 (dq, J = 29.8,7.6 Hz, 6H), 1.36 - 1.18 (m, 50H), 0.86 (t, J = 6.6 Hz, 9H).

[0218] Lipid 17 was also characterized by carbon- 13 nuclear magnetic resonance one a 101 MHz spectrometer in CDCI3, the results of which are depicted in FIG. 19C. In particular, the data reported includes the chemical shift (6 ppm).13C NMR (101 MHz, CDCI3) 6 174.69, 173.78, 173.69, 64.50, 64.45, 62.66, 58.12, 55.82, 54.69, 54.50, 53.71, 52.25, 46.06, 34.33, 34.20, 31.90, 29.59, 29.58, 29.52, 29.32, 29.26, 28.65, 27.85, 27.02, 26.97, 26.84, 26.31, 25.93, 24.95, 24.78, 22.67, 14.10.

[0219] Lipid 18

[0220] As depicted in FIG. 20A, Lipid 18 was synthesized using the general scheme shown in FIG. 1 and as described herein. Lipid 18 was characterized by proton nuclear magnetic2024-031 / 10738-1181-42- resonance on a 400 MHz spectrometer in CDCI3, the results of which are depicted in FIG. 20B. In particular, the data reported includes: chemical shift (5 ppm), multiplicity, integration, and coupling constant (Hz). 'H NMR (400 MHz, CDCI3) 8 4.06 (t, J = 6.6 Hz, 2H), 3.97 (d, J = 5.8 Hz, 4H), 2.89 - 2.71 (m, 6H), 2.42 (dt, J = 10.8, 7.3 Hz, 6H), 2.29 (s, 4H), 2.05 (t, J = 11.4 Hz, 2H), 1.93 (dd, J = 13.6, 3.8 Hz, 2H), 1.80 (qd, J = 10.8, 5.5 Hz, 2H), 1.62 (q, J = 7.4, 7.0 Hz, 4H), 1.44 (q, J = 7.4 Hz, 2H), 1.27 (d, J = 3.7 Hz, 52H), 0.88 (t, J = 6.6 Hz, 12H).

[0221] Lipid 18 was also characterized by carbon- 13 nuclear magnetic resonance on a 101 MHz spectrometer in CDCI3, the results of which are depicted in FIG. 20C. In particular, the data reported includes the chemical shift (6 ppm).13C NMR (101 MHz, CDCI3) 6 174.95, 172.83, 67.26, 64.42, 54.89, 53.66, 49.20, 46.26, 40.38, 37.28, 32.57, 31.89, 31.82, 31.23, 29.96, 29.62, 29.57, 29.31, 28.66, 28.12, 27.13, 27.06, 26.70, 26.65, 25.91, 22.67, 22.65, 14.09.

[0222] Lipid 19

[0223] As depicted in FIG. 21 A, Lipid 19 was synthesized using the general scheme shown in FIG. 1 and as described herein. Lipid 19 was characterized by proton nuclear magnetic resonance on a 400 MHz spectrometer in CDCI3, the results of which are depicted in FIG. 21B. In particular, the data reported includes: chemical shift (6 ppm), multiplicity, integration, and coupling constant (Hz). 'H NMR (400 MHz, CDCI3) 6 5.72 (s, 1H), 3.96 (d, J = 5.8 Hz, 4H), 3.23 (q, J = 6.7 Hz, 2H), 3.01 (dt, J = 12.0, 4.2 Hz, 2H), 2.77 (t, J = 7.3 Hz, 4H), 2.48 - 2.32 (m, 10H), 2.26 - 2.09 (m, 2H), 2.00 - 1.89 (m, 2H), 1.89 - 1.74 (m, 2H), 1.68 - 1.56 (m, 2H), 1.54 - 1.37 (m, 4H), 1.27 (d, J = 3.6 Hz, 52H), 0.88 (t, J = 6.7 Hz, 12H).

[0224] Lipid 19 was also characterized by carbon- 13 nuclear magnetic resonance one a 101 MHz spectrometer in CDCI3, the results of which are depicted in FIG. 21C. In particular, the data reported includes the chemical shift (6 ppm).13C NMR (101 MHz, CDCI3) 6 174.51, 172.89, 67.29, 54.61, 53.46, 49.19, 45.84, 39.31, 37.28, 32.56, 31.90, 31.83, 31.23, 29.97, 29.62, 29.58, 29.54, 29.32, 28.35, 27.07, 26.89, 26.73, 26.70, 26.66, 22.67, 22.65, 14.10.Example 2: Preparation of lipid nanoparticles

[0225] Various lipid nanoparticles were formulated for mRNA delivery and their cellular uptake and transfection efficiency in vitro and in vivo were evaluated. LNPs were prepared by combining ionizable lipid, helper lipid (either l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine2024-031 / 10738-1181-43-[DOPE] or distearoylphosphatidylcholine [DSPC]), cholesterol, and 1,2-dimyristoyl-rac-glycero- 3 -methoxypolyethylene glycol-2000 (DMG-PEG) in ethanol at a concentration of 2 mg / mL and a molar ratio of 40: 10:48:2

[0226] A molar ratio of 8: 1 was used for the amine groups of the ionizable lipid to the phosphate groups of FLuc mRNA (Trilink Biotechnologies, L-7202). Using a microfluidic device, a 3 : 1 volume ratio of mRNA in citrate buffer (pH 5) to lipid mixture was mixed, and the resulting LNP solution was concentrated using an Amicon filter (MWCO: 100,000 Da) to remove ethanol and exchange the buffer with PBS.

[0227] Encapsulation efficiency of mRNA was assessed using the QUANT-IT™ Ribogreen® RNA assay (Invitrogen). The LNPs were characterized for particle size and surface charge using dynamic light scattering and zeta potential measurements. Particle size and surface charge were measured using a Nanobrook Omni analyzer.Example 3: Characterization of Lipid Nanoparticles

[0228] Lipid nanoparticles were formulated using Lipids 4 and 8 along with cholesterol, DSPC, and DMG-PEG. The LNPs were characterized alongside SM-102 as a positive control. The results of this characterization are depicted in FIG. 22. These LNPs showed a range of particle sizes from -150-175 nm. The surface charge of the LNPs was close to neutral, in contrast with SM102, which has a surface charge of - 5 mV. The polydispersity index of the LNPs was less than 0.3. The mRNA encapsulation efficiency measured by Ribogreen® assay was greater than 85% for all the LNPs.

[0229] Further, the LNPs formed using Lipids 4 and 8, were tested using a TNS assay to determine the apparent pKa of the LNPs (FIG. 23). SM-102 LNPs had a pKa of -6.5 while LNPs containing Lipids 4 and 8 had a pKa of less than 6.

[0230] Lipid nanoparticles were formulated using Lipids 10 and 11 along with cholesterol, DSPC, and DMG-PEG. The LNPs were characterized alongside SM-102 as a positive control. The results of this characterization are depicted in FIG. 26. Similar to the above, LNPs containing these lipids had particle sizes of less than 300 nm, PDI of less than 0.3, and near-neutral surface2024-031 / 10738-1181-44- charge. Encapsulation efficiency was greater than 90%, confirmed by Ribogreen® assay. The TNS assay indicated a pKa of ~6.5 for SM-102 LNPs, while Lipids 10 and 11 exhibited a pKa of less than 6 (FIG. 27).

[0231] Lipid nanoparticles were formulated using Lipids 7, 9, 12, and 13 along with cholesterol, DSPC, and DMG-PEG. The LNPs were characterized. These results are depicted in FIG. 30. These LNPs had particle sizes less than 200 nm, a PDI of less than 0.3, and a near-neutral surface charge. The encapsulation efficiency of mRNA was greater than 90% for all formulations. The TNS assay results showed a pKa of ~6.5 for SM-102 LNPs, while LNPs containing Lipids 9, 12, and 13 exhibited a pKa of less than 6, with Lipid 7 showing a pKa of ~6 (FIG. 31).Example 4: In Vitro Transfection Efficiency

[0232] In transfection studies, cells were seeded at a density of 40,000 cells / well in 96-well plates, and formulations containing 100 ng of FLuc or eGFP mRNA were added. After incubation at 37°C for 20 hours, the cells were washed and centrifuged at 300 * g. After the cells were diluted in PBS, flow cytometry analysis was performed to quantify the transfection efficiency. Luciferase expression was quantified using the Bright-GloTM luciferase assay (Promega) following the manufacturer’s protocol, using a luminometer. Quant-it™ RiboGreen RNA Assay (Thermo Fisher) was used to determine encapsulation efficiency. eGFP expression in human Jurkat T cells was measured by flow cytometry (BD LSRFortessa™ Cell Analyzer), gating for live cells. Jurkat T cells, representing T lymphocytes, are particularly challenging to transfect.

[0233] LNPs as described above were tested for their in vitro FLuc mRNA delivery to Jurkat T cells. LNPs containing SM-102 showed a luminescence intensity of ~105, while LNPs containing Lipids 4 and 8 had a luminescence intensity of less than 104(FIG. 24). Lipid nanoparticles formulated with Lipids 10 and 11 were analyzed for their in vitro FLuc mRNA delivery to Jurkat T cells, as shown in FIG. 28. LNPs containing SM-102 showed a luminescence intensity of ~106while LNPs containing Lipids 10 and 11 had a luminescence intensity of less than 105. LNPs containing Lipids 7, 9, 12, and 13 were tested for their in vitro FLuc mRNA delivery to Jurkat T cells as shown in FIG 32.. LNPs containing SM-102 demonstrated a luminescence intensity of ~105while LNPs containing Lipids 7, 9, 12, and 13 had a luminescence intensity of less than 104.2024-031 / 10738-1181-45-

[0234] Lipid nanoparticles using various ionizable lipids, with DOPE as a helper lipid were compared with various controls, including STEMFECT™ transfection reagent and lipid nanoparticles containing SM-102, ALC-0315, and MC3. The transfection efficiency is demonstrated in FIG. 34A-34B. As shown, LNP formulations containing Lipid 1, Lipid 4, Lipid 6, Lipid 7, Lipid 9, Lipid 10, Lipid 13, Lipid 16, Lipid 17, and Lipid 18 outperformed the StemFect transfection reagent. Lipids 7, 9, 10, 16, and 18 demonstrated transfection efficiencies comparable to SM-102, ALC-0315, and MC3 LNP formulations. Further, the MFI of the formulations with Lipids 6, 7, 9, 10, 16, and 18 demonstrated a higher MFI than ALC-0315 and MC3. Higher MFI in LNP formulations indicates better payload encapsulation and more eGFP expression as a results of efficient transfection and endosomal escape, which is important in delivering sufficient nucleic acids, especially in systemic or in vivo applications.

[0235] Lipid nanoparticles were formulated using Lipids 1, 4, 6, 7, 9, 10, 13, 16, 17, and 18 along with cholesterol, DSPC and DMG-PEG, with eGFP mRNA cargo. These lipid nanoparticles were compared with various controls, including STEMFECT™ transfection reagent and lipid nanoparticles containing SM-102, ALC-0315, and MC3. The transfection efficiency is demonstrated in FIG. 35A-35B. As shown, LNP formulations containing Lipid 1, Lipid 4, Lipid 6, Lipid 7, Lipid 9, Lipid 10, Lipid 16, and Lipid 18 outperformed the StemFect transfection reagent. Lipids 4, 7, and 16 demonstrated transfection efficiencies comparable to SM-102, ALC- 0315, and MC3 LNP formulations. Further, the MFI of the formulations with Lipid 4 demonstrated a higher MFI than SM-102. ALC-0315 and MC3. Lipids 1, 6, and 7, demonstrated a higher MFI than ALC-0315 and MC3.Example 5: In vivo delivery of Lipid Nanoparticles

[0236] Female BALB / c mice aged 7-9 weeks (Strain #: 000651, 18-22 grams in body weight) were purchased from the Jackson Laboratory. All animals were housed in pathogen-free conditions and were provided with water and food ad libitum. All procedures involving animals were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council of the National Academies, USA) and were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati.

[0237] Different LNPs were formulated by mixing the ionizable lipids, DSPC, cholesterol, and DMG-PEG dissolved in ethanol at a molar ratio of 40: 10:48:2, respectively. A molar ratio of2024-031 / 10738-1181-46-8: 1 was used between the amine group of the ionizable lipid and the phosphate group of the FLuc mRNA (Trilink Biotechnologies, L-7202). mRNA diluted in 10 mM citrate buffer (pH 5.0) was mixed with the lipid mixture at a 3 : 1 volume ratio using a microfluidic device. The solution was concentrated using an Amicon filter (MWCO: 100,000 Da) to remove the ethanol and exchange the buffer with PBS.

[0238] mRNA-LNPs diluted in Dulbecco’s PBS were injected i.v. (0.1 mg / ml) into mice via the tail vein using 26 g, 1 ml syringes (Vetrijec) after gentle warming of the animals using a heat lamp for 3 minutes at a dosage of 0.5 mg / kg.

[0239] BALB / c mice were injected with D-Luciferin sodium Salt (GoldBio, catalog #: LUCNA) intraperitoneally (i.p.) 10 min before the imaging time point. Luciferin was diluted using PBS to a concentration of 15 mg / ml and was injected at a dosage of 150 mg / kg. Three minutes before imaging, mice were placed in a chamber filled with 3% isoflurane and 97% oxygen. Once the mice were completely anesthetized, they were moved into isoflurane- delivering nosecones in the imaging chamber positioned supine and maintained on 3% isoflurane and 97% oxygen. Images were acquired using IVIS Spectrum (PerkinElmer). After wholebody imaging, animals were returned to their cage for recovery or were euthanized by CO2 administration followed by cervical dislocation. Different organs including liver, spleen, lungs, heart, kidneys, etc. were removed and isolated for ex vivo imaging. The different organs were placed on non-luminescent paper in the imaging chamber and images were acquired. Ex vivo imaging of organs was completed within 25 min of luciferin administration. All images were quantified by region of interest for total flux using Living Image Software Version 4.7.4 (Perkin Elmer).

[0240] LNPs formulated with firefly luciferase-encoding mRNA cargo, using the ionizable lipid, cholesterol, DSPC, and DMG-PEG were delivered to BALB / c mice at a dosage of 0.5 mg / kg via intravenous administration. The luminescence signal was measured at three different time points: 3, 6, and 24 hours post-injection.

[0241] As shown in FIG. 25, LNPs containing SM-102 showed a total flux of -1011p / sec / cm2 / sr. LNPs containing Lipid 4 showed a total flux of ~107p / sec / cm2 / sr. Comparatively, LNPs containing Lipid 8 showed a higher total flux of greater than 1010p / sec / cm2 / sr. As shown2024-031 / 10738-1181-47- in FIG. 29, LNPs containing SM-102 showed a total flux of -IO11p / sec / cm2 / sr. LNPs containing Lipid 10 showed a total flux of -1011p / sec / cm2 / sr. LNPs containing Lipid 11 showed a total flux of ~ 109p / sec / cm2 / sr .

[0242] As shown in FIG. 33A, LNPs containing SM-102 showed a total flux of -1011p / sec / cm2 / sr. LNPs containing Lipid 7 showed a total flux of -1011p / sec / cm2 / sr. LNPs containing Lipid 13 showed a total flux of ~1O10p / sec / cm2 / sr. LNPs containing Lipids 9 and 12 showed a total flux of ~109p / sec / cm2 / sr.

[0243] After euthanizing the mice 24 hours post-injection, the organs were harvested from the mice. The total flux was measured from the liver (FIG. 33B) and spleen (FIG. 33C). The total flux measured ex vivo demonstrated that LNPs containing Lipid 7 showed the highest flux in liver and spleen amongst the ionizable lipids, with similar results as SM-102, followed by LNPs containing Lipid 13, Lipid 12, and Lipid 9.Further Examples:

[0244] A first item of the present disclosure, either alone or in combination with any other item herein, concerns a composition comprising at least one ionizable lipid according to Formula (I) or a pharmaceutically-acceptable salt thereof,(Formula (I)), wherein ni is from 0-8 carbons; n2 is from 2-8 carbons; ns is from 2-8 carbons; is from 2-8 carbons; and ns is from 0- 8 carbons. Ri is selected from:2024-031 / 10738-1181-48-Each X is independently selected from:Each R2 is independently selected from hydrogen, Cl -Cl 8 alkyl groups, or Cl -Cl 8 alkenyl groups. Each R3 is independently selected from hydrogen, Cl -Cl 8 alkyl groups, or Cl -Cl 8 alkenyl groups.2024-031 / 10738-1181-49-

[0245] A second item of the present disclosure, either alone or in combination with any other item herein, concerns a composition comprising an ionizable lipid wherein wherein Ri is selectedfrom

[0246] A third item of the present disclosure, either alone or in combination with any other item herein, concerns a composition wherein each R3 is independently selected from hydrogen, methyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, heptadecyl, hexadecyl, and octadecyl.

[0247] A fourth item of the present disclosure, either alone or in combination with any other item herein, concerns a composition wherein each R3 is independently selected from hydrogen, hexyl, and octyl.

[0248] A fifth item of the present disclosure, either alone or in combination with any other item herein, concerns a composition wherein the ionizable lipid is selected from the group consisting of:2024-031 / 10738-1181-50-2024-031 / 10738-1181-51-2024-031 / 10738-1181-52-2024-031 / 10738-1181and combinations thereof.

[0249] A sixth item of the present disclosure, either alone or in combination with any other item herein, concerns a composition further comprising a helper lipid; a sterol; and a PEGylated lipid conjugate, wherein the composition forms lipid nanoparticles.

[0250] A seventh item of the present disclosure, either alone or in combination with any other item herein, concerns a composition wherein the ionizable lipid is selected from the group2024-031 / 10738-1181-54- consisting of: Lipid 1, Lipid 2, Lipid 3, Lipid 4, Lipid 5, Lipid 6, Lipid 7, Lipid 8, Lipid 9, Lipid 10, Lipid 11, Lipid 12, Lipid 13, Lipid 14, Lipid 15, Lipid 16, Lipid 17, Lipid 18, and Lipid 19.

[0251] An eighth item of the present disclosure, either alone or in combination with any other item herein, concerns a composition wherein the ionizable lipid is selected from Lipid 1, Lipid 4, Lipid 7, Lipid 10, Lipid 13, Lipid 14, Lipid 15, Lipid 16, Lipid 17, Lipid 18, or Lipid 19.

[0252] A ninth item of the present disclosure, either alone or in combination with any other item herein, concerns a composition, wherein the ionizable lipid is selected from Lipid 7, Lipid 10, Lipid 13, Lipid 14, Lipid 15, Lipid 16, Lipid 17, Lipid 18, or Lipid 19.

[0253] A tenth eighth item of the present disclosure, either alone or in combination with any other item herein, concerns a composition, wherein the helper lipid is selected from the group consisting of: l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero- 3 -phosphocholine (DPPC), l,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleyl- sn-glycero-3-phosphotidyl choline (DOPC) l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-dimyristoyl-sn-glycero-3 -phosphoethanolamine (DMPE), and 1,2-dioleoyl-sn- glycero-3-phospho-(l '-rac-glycerol) (DOPG).

[0254] An eleventh item of the present disclosure, either alone or in combination with any other item herein, concerns a composition wherein the helper lipid is DOPE and / or DSPC.

[0255] An twelfth item of the present disclosure, either alone or in combination with any other item herein, concerns a composition, wherein the sterol is cholesterol or a derivative thereof.

[0256] A thirteenth item of the present disclosure, either alone or in combination with any other item herein, concerns a composition wherein the PEGylated lipid conjugate is PEGylated myristoyl diglyceride (PEG-DMG).

[0257] A fourteenth item of the present disclosure, either alone or in combination with any other item herein, concerns a composition, wherein the lipid nanoparticle at least partially encapsulates a cargo molecule.2024-031 / 10738-1181-55-

[0258] A fifteenth item of the present disclosure, either alone or in combination with any other item herein, concerns a composition wherein the cargo molecule is a nucleic acid.

[0259] A sixteenth item of the present disclosure, either alone or in combination with any other item herein, concerns a composition, wherein the nucleic acid is mRNA.

[0260] A seventeenth item of the present disclosure, either alone or in combination with any other item herein, concerns a composition, further comprising a pharmaceutically acceptable excipient.

[0261] An eighteenth item of the present disclosure, either alone or in combination with any other item herein, concerns a composition, wherein the composition is formulated for administration by injection or infusion.

[0262] A nineteenth item of the present disclosure, either alone or in combination with any other item herein, concerns a composition wherein the ionizable lipid comprises from about 40- 60% mole percent, the helper lipid comprises from about 10-20% molar percent, the sterol comprises from about 30-50% mole percent; and the conjugate lipid comprises from about 1-5% mole percent.

[0263] A twentieth item of the present disclosure, either alone or in combination with any other item herein, concerns a composition for use as a vaccine.

[0264] The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “40 mm” is intended to mean “about 40 mm”.

[0265] Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent2024-031 / 10738-1181-56- that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

[0266] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

2024-031 / 10738-1181-57-CLAIMS1. A composition comprising at least one ionizable lipid according to Formula (I),(Formula (I)), wherein: is from 0-8 carbons; n2 is from 2-8 carbons; ns is from 2-8 carbons; n4 is from 2-8 carbons; ns is from 0-8 carbons; Ri is selected from:2024-031 / 10738-1181-58- each X is independently selected from:each R2 is independently selected from hydrogen, Cl -Cl 8 alkyl groups, or Cl -Cl 8 alkenyl groups; and each R3 is independently selected from hydrogen, Cl -Cl 8 alkyl groups, or Cl -Cl 8 alkenyl groups.

2. The composition of claim 1, wherein Ri is selected from3. The composition of claim 1, wherein each R3 is independently selected from hydrogen, methyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, heptadecyl, hexadecyl, and octadecyl.

4. The composition of claim 1, wherein each R3 is independently selected from hydrogen, hexyl, and octyl.

5. The composition of claim 1, wherein the ionizable lipid is selected from the group consisting of:2024-031 / 10738-1181-59-2024-031 / 10738-1181-60-2024-031 / 10738-1181-61-2024-031 / 10738-1181-62-and combinations thereof.

6. The composition of claim 1, further comprising: a helper lipid;2024-031 / 10738-1181-63- a sterol; and a PEGylated lipid conjugate, wherein the composition forms lipid nanoparticles.

7. The composition of claim 6, wherein the ionizable lipid is selected from the group consisting of: Lipid 1, Lipid 2, Lipid 3, Lipid 4, Lipid 5, Lipid 6, Lipid 7, Lipid 8, Lipid 9, Lipid 10, Lipid 11, Lipid 12, Lipid 13, Lipid 14, Lipid 15, Lipid 16, Lipid 17, Lipid 18, and Lipid 19.

8. The composition of claim 7, wherein the ionizable lipid is selected from Lipid 1, Lipid 4, Lipid 7, Lipid 10, Lipid 13, Lipid 14, Lipid 15, Lipid 16, Lipid 17, Lipid 18, or Lipid 19.

9. The composition of claim 7, wherein the ionizable lipid is selected from Lipid 7, Lipid10. Lipid 13, Lipid 14, Lipid 15, Lipid 16, Lipid 17, Lipid 18, or Lipid 19.

10. The composition of claim 6, wherein the helper lipid is selected from the group consisting of: l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero- 3 -phosphocholine (DPPC), l,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleyl- sn-glycero-3-phosphotidyl choline (DOPC) l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-dimyristoyl-sn-glycero-3 -phosphoethanolamine (DMPE), and 1,2-dioleoyl-sn- glycero-3-phospho-(l '-rac-glycerol) (DOPG).

11. The composition of claim 6, wherein the helper lipid is DOPE and / or DSPC.

12. The composition of claim 6, wherein the sterol is cholesterol or a derivative thereof.

13. The composition of claim 6, wherein the PEGylated lipid conjugate is PEGylated myristoyl diglyceride (PEG-DMG).

14. The composition of claim 6, wherein the lipid nanoparticle at least partially encapsulates a cargo molecule.

15. The composition of claim 14, wherein the cargo molecule is a nucleic acid.

16. The composition of claim 15, wherein the nucleic acid is mRNA.

17. The composition of claim 6, further comprising a pharmaceutically acceptable excipient.

18. The composition of claim 17, wherein the composition is formulated for administration by injection or infusion.2024-031 / 10738-1181-64-19. The composition of claim 6, wherein the ionizable lipid comprises from about 40-60% mole percent, the helper lipid comprises from about 10-20% molar percent, the sterol comprises from about 30-50% mole percent; and the conjugate lipid comprises from about 1-5% mole percent.

20. The composition of claim 1 or claim 6, for use in a vaccine.

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