EP4626858A1 - Novel ionizable lipids and lipid nanoparticles comprising the same - Google Patents

Abstract

Provided herein are an ionizable lipid compound, a lipid nanoparticle comprising the ionizable lipid compound, a composition comprising an mRNA formulated in the lipid nanoparticle, and a method of delivering an mRNA to a subject or a cell by administering the composition including an mRNA formulated in the lipid nanoparticle to the subject or cell.

Description

TITLE OF INVENTION

NOVEL IONIZABLE LIPIDS AND LIPID NANOPARTICLES

COMPRISING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Serial No. 63/429,596, filed December 2, 2022, and U.S. Provisional Application Serial No. 63/447,508, filed February 22, 2023, the content of which are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

Provided herein are an ionizable lipid, a lipid nanoparticle (LNP) formed therefrom, a composition including an mRNA formulated in the lipid nanoparticle, and a method of delivering an mRNA to a subject or cell comprising administering said composition.

BACKGROUND

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.

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 or phospholipids, structural lipids (such as sterols or cholesterol), and lipid conjugates.

Emerging clinical therapies, particularly nucleic acid-based vaccines, require drug delivery systems, such as 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

In one embodiment, a compound having the structure of Formula (I) or a pharmaceutically acceptable salt thereof,

wherein. Ri is a C2-C18 alkyl group or C2-C18 alkenyl group, optionally substituted with R2; R2 is a C2-C18 alkyl group or C2-C18 alkenyl group; R3 is a C2-C18 alkyl group; R4 is a C2-C6 alkyl group; R5 is a C2-C18 alkyl group; Re is a C2-C18 alky l group or C2-C18 alkenyl group, optionally substituted with R7; R7 is a C2-C18 alkyl group or C2-C18 alkenyl group; and Rs and R9 are independently hydrogen or a C1-C2 alkyl group.

Preferably, Ri is a C5-C15 alkyl group or C5-C15 alkenyl group, optionally substituted with R2; R2 is a C2-C12 alkyl group or C2-C12 alkenyl group; R3 is a C2-C13 alkyd group; R4 is a C2-C6 alky l group; R5 is a C2-C13 alkyd group; Re is a C5-C15 alkyl group or C5-C15 alkenyl group, optionally substituted with R7; R7 is a C2-C 12 alkyl group or C2-C12 alkenyl group; and Rs and R9 are independently hydrogen or a C1-C2 alkyd group.

More preferably, Ri is a C9-C11 alkyd group, optionally substituted with R2; R2 is a Ce- Cs alkyd group; R3 is a C4-C7 alkyd group; R4 is a C2-C6 alkyl group; R5 is a C4-C7 alkyd group; Re is a C9-C11 alkyd group, optionally substituted with R7; R7 is a Ce-Cs alkyd group; and Rs and R9 are independently hydrogen or a C1-C2 alkyl group.

In one embodiment, the compound having the structure of Formula (I) may be selected from the group consisting of the following compounds:

[Table 1] having any one of the structure of Formula (I). Formula (1-1). Formula (1-2), Formula (1-3), Formula (1-4), Formula (1-5), Formula (1-6), Formula (1-7), Formula (1-8), Formula (1-9), Formula (I- 10), Formula (1-11), Formula (1-12), Formula (1-13), Formula (1-14) or Formula (I- 15) or a pharmaceutically acceptable salt thereof is provided.

In one embodiment, said lipid nanoparticle may further comprise a phospholipid; a sterol; and a PEGylated lipid conjugate.

In one embodiment, the phospholipid may be selected from the group consisting of: l,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-Dipalmitoyl-sn-glycero-3-Phosphatidylcholine (DPPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), and 1, 2-dioleoyl-sn-glycero-3-phospho- rac-(l -glycerol) sodium salt (DOPG).

In one embodiment, the sterol may be cholesterol or a derivative thereof.

In one embodiment, the PEGylated lipid conjugate may be PEGylated myristoyl di glyceride (PEG-DMG).

In one embodiment, the ionizable lipid compound having any one of the structure of Formula (I), Formula (1-1), Formula (1-2), Formula (1-3), Formula (1-4), Formula (1-5), Formula (1-6), Formula (1-7), Formula (1-8), Formula (1-9), Formula (1-10), Formula (1-11), Formula (1-12), Formula (1-13), Formula (1-14) or Formula (1-15) may constitute about 30-60 mole%, the phospholipid may constitute about 5-15 mole%, the sterol may constitute about 30- 55 mole%, and the PEGylated lipid conjugate may constitute about 0.5-5 mole% of the total lipids in the lipid nanoparticle.

In one embodiment, the ionizable lipid compound may constitute about 35-55 mole%, the phospholipid may constitute about 7-12 mole%, the sterol may constitute about 35-50 mole%, and the PEGylated lipid conjugate may constitute about 0.5-3 mole% of the total lipids in the lipid nanoparticle.

In one embodiment, a composition comprising mRNA formulated in the lipid nanoparticle is provided.

In one embodiment, a method of delivering an mRNA to a subject or cell comprising administering the composition to the subj ect or cell is provided.

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

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, in which:

FIG. 1 is a graph showing the transfection efficiency of LNPs comprising different ionizable lipids in Jurkat T cells after 20-hour incubation using 100 ng eGFP mRNA.

FIG. 2 is a graph showing the transfection efficiency of LNPs comprising different ionizable lipids in Jurkat T cells after 20-hour incubation using 100 ng FLuc mRNA. FIG. 3 is a graph showing (a) particle size, and (b) surface charge of LNPs comprising different ionizable lipids.

FIG. 4 is a graph showing (a) total body flux expressed by luciferase 6 hours after the administration of LNPs comprising different ionizable lipids intravenously, and total flux expressed by (b) liver, (c) spleen, and (d) lymph nodes following the isolation of organs after euthanizing the mice 6 hours after the administration of LNPs.

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

DETAILED DESCRIPTION

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.

DEFINITIONS

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.

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. 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.

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.

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.

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 measurement 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.

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 may be a transcript. In some embodiments, a gene product may be a polypeptide. In some embodiments, expression of a nucleic acid sequence may involve 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.

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.

The term "independently" 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 X³ are independently hydrogen or a C1-C2 alkyl group" would include the scenario where X¹, X², and X³ are all the same, where X¹, X², and X³ are all different, and w here X¹ and X² are the same but X³ is different.

The tenn "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. 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.

As used herein, the tenn "pharmaceutically acceptable excipient, carrier, or diluent" 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.

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 phannaceutically 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.

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 may be fully encapsulated in the lipid particle (e.g., to form a nucleic acid containing lipid particle).

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 replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools or as therapeutic agents.

1. Ionizable lipid compound

The present disclosure provides a novel ionizable lipid compound. In one embodiment, the present disclosure provides a compound having the following structure of Formula (I) or a pharmaceutically acceptable salt thereof: wherein. Ri is a C2-C18 alkyl group or C2-C18 alkenyl group, optionally substituted with R2; R2 is a C2-C18 alkyl group or C2-C18 alkenyl group; R3 is a C2-C18 alkyl group; R4 is a C2-C6 alkyl group; R5 is a C2-C18 alkyl group; Re is a C2-C18 alky l group or C2-C18 alkenyl group, optionally substituted with R7; R7 is a C2-C18 alkyl group or C2-C18 alkenyl group; and Rs and R9 are independently hydrogen or a C1-C2 alkyl group.

Preferably, Ri is a C5-C15 alkyl group or C5-C15 alkenyl group, optionally substituted with R2; R2 is a C2-C12 alkyl group or C2-C12 alkenyl group; R3 is a C2-C13 alkyd group; R4 is a C2-C6 alky l group; R5 is a C2-C13 alkyd group; Re is a C5-C15 alkyl group or C5-C15 alkenyl group, optionally substituted with R7; R7 is a C2-C12 alkyl group or C2-C12 alkenyl group; and Rs and R9 are independently hydrogen or a C1-C2 alkyd group.

More preferably, Ri is a C9-C11 alkyd group, optionally substituted with R2; R2 is a Ce- Cs alkyd group; R3 is a C4-C7 alkyd group; R4 is a C2-C6 alkyl group; R5 is a C4-C7 alkyd group; Re is a C9-C11 alkyd group, optionally substituted with R7; R7 is a Ce-Cs alkyl group; and Rs and R9 are independently hydrogen or a C1-C2 alkyl group.

In some embodiment, the compound having structure of Formula (I) may be selected from the group consisting of the following compounds: [Table 2] 2. Lipid nanoparticle

The present disclosure also provides a lipid nanoparticle comprising the compound having any one of the structures selected from the group consisting of Formula (T), Formula (I- 1), Formula (1-2), Formula (1-3), Formula (1-4), Formula (1-5), Formula (1-6), Formula (1-7), Formula (1-8), Formula (1-9), Formula (1-10), Formula (1-11), Formula (1-12), Fomiula (1-13), Formula (1-14) and Formula (1-15). For example, the lipid nanoparticle may further comprise a phospholipid, a sterol, and a PEGylated lipid conjugate.

2.1. Neutral lipids (Phospholipids)

In some embodiments, the lipid component of a lipid nanoparticle may include one or more neutral lipids such as phospholipids including one or more (poly) unsaturated lipids, as a helper lipid. Without being bound by the theory, it is contemplated that phospholipids may assemble into one or more lipid bilayers structures.

Exemplary helper lipids or phospholipids that can form part of the present lipid nanoparticle may include but are not limited to 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1.2-dilinoleoyl-sn- glycero-3 -phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18: 0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3- phosphocholine, l,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl- sn-glycero-3-phosphocholine, l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (4ME 16.0 PE also referred herein as 1,2-DPyPE). l,2-distearoyl-sn-glycero-3 -phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3 -phosphorac^ 1 -glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-lcarboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, I -stearioyl-2-oleoylphosphatidy ethanol amine (SOPE). l,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE), and sphingomyelin (SM).

In one embodiment, the phospholipid may be phosphatidylcholine (PC), phosphatidylethanolamine (PE) phosphatidylserine (PS), phosphatidic acid (PA), or phosphatidylglycerol (PG).

In certain embodiments, a lipid nanoparticle may include at least one phospholipid selected from l,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-Dipalmitoyl-sn- glycero-3-Phosphatidylcholine (DPPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), and 1, 2-dioleoyl-sn- glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG). In certain embodiments, a lipid nanoparticle may include DOPE or DSPC.

In one embodiment, the lipid nanoparticle may include from about 5% to about 15% on a molar basis of the phospholipids e.g., from about 5 to about 12%, from about 7 to about 12%, from about 7 to about 15%, or about 5%, about 10%, or about 15% on a molar basis.

2.2. Polymer Conjugated Lipids

In some embodiments, the lipid component of a lipid nanoparticle may include one or more polymer conjugated lipids, such as PEGylated lipid (PEG-lipids) conjugates. Without being bound by the theory, it is contemplated that a polymer conjugated lipid component in a lipid nanoparticle can improve colloidal stability and/or reduce protein absorption of the nanoparticles.

Exemplary polymer conjugated lipids that can be used in connection with the present disclosure may include but are not limited to PEGylated phosphatidylethanolamines, PEGylated phosphatidic acids, PEGylated ceramides, PEGylated dialkylamines, PEGylated diacylglycerols, PEGylated dialkylglycerols, and mixtures thereof. For example, a PEG-lipid may be l,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (PEG-DMG also referred herein as DMG-PEG). PEG-1, 2-Dilauroyl-sn-glycero-3-phosphoethanolamine (PEG-DLPE), PEG-DMPE, PEG-DPPC, PEG-DSPE, Ceramide-PEG2000, or Chol-PEG2000.

In one embodiment, the lipid nanoparticle may include the PEG-lipids at a molar ratio of from about 0.5% to about 5% e.g., from about 0.5 to about 3%, from about 1 to about 5%, from about 1 to about 3%, or about 0.5%, about 1%. about 1.5%, about 2%, about 2.5%, or about 3% of the total lipids.

2.3. Structural Lipids In some embodiments, the lipid component of a lipid nanoparticle may include one or more structural lipids. Without being bound by the theory, it is contemplated that structural lipids can stabilize the amphiphilic structure of a nanoparticle, such as but not limited to the lipid bilayer structure of a nanoparticle.

Exemplary structural lipids that can be used in connection with the present disclosure may include but are not limited to sterol, for example, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alphatocopherol, and mixtures thereof. In certain embodiments, the structural lipid may be cholesterol. In some embodiments, the structural lipid may include cholesterol or a corticosteroid (such as prednisolone, dexamethasone, prednisone, or hydrocortisone), or a combination thereof. In one embodiment, the lipid nanoparticles provided herein may comprise a steroid or steroid analogue. In one embodiment, the steroid or steroid analogue may be cholesterol.

In one embodiment, the lipid nanoparticle may include the structural lipids at a molar ratio of from about 30% to about 55% e.g., from about 30 to about 50%, from about 35 to about 55%, from about 35 to about 50%, or about 38%, about 38.5%, about 39%, about 40%, about 45%, about 47%, or about 48% of the total lipids.

2.4. Ionizable lipids

In some embodiments, the lipid component of a lipid nanoparticle may include one or more ionizable lipids. In one embodiment, the lipid nanoparticle may comprise at least one ionizable lipid compound having any one of the structures selected from the group consisting of Formula (I), Formula (1-1), Formula (1-2), Formula (1-3), Formula (1-4), Formula (1-5), Formula (1-6), Formula (1-7), Fonnula (1-8), Formula (1-9), Formula (1-10), Formula (1-11), Formula (1-12), Formula (1-13), Formula (1-14) and Formula (1-15). In one embodiment, the lipid nanoparticle may include one or more other ionizable lipids which are known in the art, in addition to the ionizable lipids described above. Exemplary ionizable lipids that can be used in connection with the present disclosure may include but are not limited to 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-di oxolane (DLin-KC2-DMA), dilinoleyl-methyl-4- dimethyl aminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

In one embodiment, the lipid nanoparticle may include the ionizable lipids at a molar ratio of from about 30% to about 60% e.g.. from about 35 to about 55%, from about 38 to about 52%, from about 35 to about 50%, from about 40 to about 55%, from about 40 to about 50%, or about 35%, about 40%, about 45%, about 50%, or about 55% of the total lipids.

In one embodiment, the lipid nanoparticle may comprise (i) at least one ionizable lipid compound having any one of the structures selected from the group consisting of Formula (I), Formula (1-1), Formula (1-2). Formula (1-3), Formula (1-4), Formula (1-5), Formula (1-6), Formula (1-7), Formula (1-8), Formula (1-9), Formula (1-10), Formula (1-11), Formula (1-12), Formula (1-13), Formula (1-14) and Formula (1-15), (ii) at least one phospholipid, e.g., 1,2- distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), l,2-Dipalmitoyl-sn-glycero-3- Phosphatidylcholine (DPPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dioleoyl-sn-glycero-3-phosphochohne (DOPC), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE) or 1, 2-dioleoyl-sn-glycero-3-phospho- rac-(l -glycerol) sodium salt (DOPG), (iii) at least one PEG-lipid, e.g., PEG-DMG, and (iv) at least one structural lipid, e.g., cholesterol, in a molar ratio of about 30-60% ionizable lipid : 5- 15% phospholipid : 0.5-5% PEG-lipid : 30-55% structural lipid.

In one embodiment, the lipid nanoparticle may comprise (i) at least one ionizable lipid compound having any one of the structures selected from the group consisting of Formula (I), Formula (1-1), Formula (1-2), Formula (1-3), Formula (1-4), Formula (1-5), Formula (1-6), Formula (1-7), Formula (1-8), Formula (1-9), Formula (1-10), Fonnula (1-11), Formula (1-12), Formula (1-13), Formula (1-14) and Formula (1-15), (ii) at least one phospholipid, e.g., 1,2- distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), l,2-Dipalmitoyl-sn-glycero-3- Phosphatidylcholine (DPPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dioleoyl-sn-glycero-3 -phosphocholine (DOPC), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), or 1, 2-dioleoyl-sn-glycero-3-phospho- rac-(l -glycerol) sodium salt (DOPG), (iii) at least one PEG-lipid, e g., PEG-DMG, and (iv) at least one structural lipid, e.g., cholesterol, in a molar ratio of about 35-55% ionizable lipid : 7- 12% phospholipid : 0.5-3% PEG-lipid : 35-50% structural lipid.

In one embodiment, the lipid nanoparticle may be a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm). In some embodiments, the lipid nanoparticle has a mean diameter of about 50-300 nm, or about 50-200 nm.

3. Composition comprising mRNA formulated in the lipid nanoparticle

The present disclosure also provides a composition comprising an mRNA formulated in the lipid nanoparticle which comprises the compound having any one of the structures selected from the group consisting of Formula (I), Formula (1-1), Formula (1-2), Formula (1-3), Formula (1-4), Formula (1-5), Formula (1-6), Formula (1-7), Formula (1-8), Formula (1-9), Formula (1-10), Formula (1-11), Formula (1-12), Formula (1-13), Formula (1-14) and Formula (1-15). In one embodiment, in the above composition, mRNA may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting mRNA from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response.

In some embodiments, the LNPs may be prepared at a molar ratio between the amine group of the ionizable lipid (N) and the phosphate group of the mRNA (P) from about 4: 1 to about 20: 1, e g., from about 5: 1 to about 10: 1, or from about 6: 1 to about 8: 1. In some embodiments, the LNPs may be prepared at an N/P ratio about 4-20, e.g., about 5-10, or 6-8.

In some embodiment, the composition may additionally include a pharmaceutically acceptable carrier which is suitable for delivering an mRNA to a suitable in vivo or ex vivo site. Such a carrier can include, but is not limited to, an adjuvant, an excipient, etc.

In another embodiment, the composition may additionally include other therapeutic ingredients or adjuvants. In one embodiment, the composition may include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The composition can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

4. Method of delivering mRNA to a subject or cell

The present disclosure also provides a method of delivering mRNA to a subject or cell comprising administering the composition comprising an mRNA formulated in the lipid nanoparticle which comprises the compound having any one of the structures selected from the group consisting of Formula (I), Formula (1-1), Formula (1-2), Formula (1-3), Formula (1-4), Formula (1-5), Formula (1-6), Formula (1-7), Formula (1-8), Formula (1-9). Formula (1-10), Formula (1-11), Formula (1-12), Formula (1-13), Formula (1-14) and Formula (1-15) to the subject or cell.

In some embodiment, the cell may be a mammalian cell, such as, but not limited to, a human cell. In another embodiment, the cell may be, but is not limited to, a nerve cell, a muscle cell, a bone cell, a gland cell, a blood cell, or a reproductive cell. For example, the cell can be a T cell, a B cell, a macrophage, an epithelial cell, a chondrocyte or a stem cell.

In one embodiment, the composition of the present disclosure may be administered to a subject by any suitable route. In some embodiments, the composition of the present disclosure may be administered by one or more of a variety of routes, including parenteral (e.g., subcutaneous, intracutaneous, intravenous, intraperitoneal, intramuscular, intraarticular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion technique), oral, trans- or intra-dermal, interdermal, rectal, intravaginal, topical (e.g. by powders, ointments, creams, gels, lotions, and/or drops), mucosal, nasal, buccal, enteral, vitreal, intratumoral, sublingual, intranasal; by intratracheal instillation, bronchial instillation, and/or inhalation; as an oral spray and/or powder, nasal spray, and/or aerosol, and/or through a portal vein catheter. In some embodiments, a composition may be administered intravenously, intramuscularly, intradermally, intra-arterially, intratumorally, subcutaneously, or by inhalation. In some embodiments, the composition of the present disclosure may be administered intramuscularly. The present disclosure encompasses the delivery of composition of the present disclosure by any appropriate route taking into consideration likely advances in the sciences of drug delivery. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the pharmaceutical composition including one or more mRNAs (e.g., its stability in various bodily environments such as the bloodstream and gastrointestinal tract), and the condition of the patient (e.g., whether the patient is able to tolerate particular routes of administration).

In one embodiment, the composition of the present disclosure may be delivered, localized and/or concentrated in a specific location using the delivery methods described as follows. As anon-limiting example, a subject may be administered an empty polymeric particle prior to, simultaneously with or after delivering the composition of the present disclosure to the subject. The empty polymeric particle undergoes a change in volume once in contact with the subject and becomes lodged, embedded, immobilized or entrapped at a specific location in the subject.

In another embodiment, the composition of the present disclosure may be formulated in an active substance release system. For instance, the active substance release system may comprise at least one nanoparticle bonded to an oligonucleotide inhibitor strand which is hybridized with a catalytically active nucleic acid and a compound bonded to at least one substrate molecule bonded to a therapeutically active substance (e.g., polynucleotides described herein), where the therapeutically active substance is released by the cleavage of the substrate molecule by the catalytically active nucleic acid.

In another embodiment, the lipid nanoparticle of the present disclosure may comprise an inner core comprising a non-cellular material and an outer surface comprising a cellular membrane. The cellular membrane may be derived from a cell or a membrane derived from a virus. In another embodiment, the composition of the present disclosure may be formulated in porous nanoparticle-supported lipid bilayers (protocells). In another embodiment, the composition of the present disclosure may be formulated in polymeric nanoparticles which have a high glass transition temperature.

In one embodiment, the lipid nanoparticles of the present disclosure may be geometrically engineered to modulate macrophage and/or the immune response. In some embodiments, the geometrically engineered particles may have varied shapes, sizes and/or surface charges in order to incorporated the polynucleotides of the present disclosure for targeted delivery such as, but not limited to, pulmonary delivery. Other physical features the geometrically engineering particles may include, but are not limited to, fenestrations, angled arms, asymmetry' and surface roughness, charge which can alter the interactions with cells and tissues.

In one embodiment, the lipid nanoparticle of the present disclosure may be a nanoparticle-nucleic acid hybrid structure having a high density nucleic acid layer. The lipid nanoparticle of the present disclosure may comprise a nucleic acid such as, but not limited to, polynucleotides described herein and/or known in the art.

In one embodiment, at least one of the lipid nanoparticles of the present disclosure may be embedded in the core of a nanostructure or coated with a low density porous 3-D structure or coating which is capable of carrying or associating with at least one payload within or on the surface of the nanostructure.

In another embodiment, the composition of the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 10 mg/kg, from about 0.001 mg/kg to about 10 mg/kg, from about 0.005 mg/kg to about 10 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 10 mg/kg, from about 2 mg/kg to about 10 mg/kg, from about 5 mg/kg to about 10 mg/kg, from about 0.0001 mg/kg to about 5 mg/kg, from about 0.001 mg/kg to about 5 mg/kg, from about 0.005 mg/kg to about 5 mg/kg, from about 0.01 mg/kg to about 5 mg/kg, from about 0. 1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 5 mg/kg, from about 2 mg/kg to about 5 mg/kg, from about 0.0001 mg/kg to about 1 mg/kg, from about 0.001 mg/kg to about 1 mg/kg, from about 0.005 mg/kg to about 1 mg/kg, from about 0.01 mg/kg to about 1 mg/kg, or from about 0. 1 mg/kg to about 1 mg/kg in a given dose, where a dose of 1 mg/kg provides 1 mg of mRNA or nanoparticle per 1 kg of subj ect body weight. In particular embodiments, a dose of about 0.005 mg/kg to about 5 mg/kg of mRNA or nanoparticle of the disclosure may be administrated.

A dose may be administered one or more rimes per day, in the same or a different amount, to obtain a desired level of mRNA expression and/or effect (e.g., a therapeutic effect). The desired dosage may be delivered, for example, three times a day, two times a day. once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). In some embodiments, a single dose may be administered, for example, prior to or after a surgical procedure or in the instance of an acute disease, disorder, or condition. The specific therapeutically effective, prophylactically effective, or otherwise appropriate dose level for any particular patient will depend upon a variety of factors including the severity and identify of a disorder being treated, if any; the one or more mRNAs employed; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific pharmaceutical composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific pharmaceutical composition employed; and like factors well known in the medical arts.

In one embodiment, the effective amount of the present composition comprising mRNA as formulated in the lipid nanoparticle, may be as low as 10 pg, administered for example as a single dose or as two 5 pg doses. In some embodiments, the effective amount may be a total dose of 10 pg-300 pg. For example, the effective amount may be a total dose of 10 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg. 50 pg, 55 pg, 60 pg, 65 pg, 70 pg, 75 pg, 80 pg, 85 pg. 90 pg, 95 pg, 100 pg, 110 pg, 120 pg, 130 pg, 140 pg, 150 pg, 160 pg, 170 pg, 180 pg, 190 pg or 200 pg, 210 pg, 220 pg, 230 pg, 240 pg, 250 pg, 260 pg, 270 pg, 280 pg, 290 pg or 300 pg. In some embodiments, the effective amount may be a total dose of 10 pg-300 pg. In some embodiments, the effective amount may be a total dose of 30 pg-100 pg or 50 pg-200 pg.

In some embodiments, the composition of the present disclosure may be administered twice (e g., Day 0 and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0 and 9 months later. Day 0 and 12 months later, Day 0 and 18 months later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0 and 10 years later) at a total dose of or at dosage levels sufficient to deliver a total dose of 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg. 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg. 0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg. Higher and lower dosages and frequency of administration may be encompassed by the present disclosure. For example, the composition comprising mRNA formulated in the lipid nanoparticle may be administered three or four times.

EXAMPLES

The following examples are given by way of illustration and are in no way intended to limit the scope of the present disclosure.

General synthesis process of Lipids 1 to 15

Lipids 1 to 15 were synthesized according to the following general scheme:

Preparation of Intermediates

1. Preparation of Intermediate A-l: l-[benzyl({3-[benzyl(2-hydroxybutyl)amino]propyl})amino]butan-2-ol

To a solution of Nl,N3-Dibenzylpropane-l,3-diamine in methanol, ethyl oxirane (4 eq.) was added and stirred at 50 °C for 16 hours. The solution was concentrated in a rotary evaporator to obtain l-[benzyl({3-[benzyl(2-hydroxybutyl)amino]propyl})arnino]butan-2-ol.

2. Preparation of Intermediate B-l:

1 -( {3- [(2-hydroxy butyl )amino | propyl }amino)butan-2-ol

To a solution of l-[benzyl({3-[benzyl(2-hydroxybutyl)amino]propyl})amino]butan-2- ol (Intermediate A-l) in methanol, a catalytic amount of Pd/C was added in a reactor. The reactor was charged with hydrogen gas and stirred for 16 hours at room temperature. After the reaction was completed, the solution was filtered and concentrated in a rotary evaporator to obtain l-({3-[(2-hydroxybutyl)amino]propyl}amino)butan-2-ol.

3. Preparation of Intermediate A-2: l-[benzyl({2-[benzyl(2-hydroxybutyl)amino]ethyl})amino]butan-2-ol

To a solution of benzyl [2-(benzylamino)ethyl] amine in methanol, ethyl oxirane (4 eq.) was added and stirred at 50 °C for 16 hours. The solution was concentrated in a rotary evaporator to obtain l-[benzyl({2-[benzyl(2-hydroxybutyl)amino]ethyl})amino]butan-2-ol.

4. Preparation of Intermediate B-2: l-({2-[(2-hydroxybutyl)amino]ethyl}amino)butan-2-ol

To a solution of l-[benzyl({2-[benzyl(2-hydroxybutyl)amino]ethyl})amino]butan-2- ol (Intermediate A-2) in methanol, a catalytic amount of Pd/C was added in a reactor. The reactor was charged with hydrogen gas and stirred for 16 hours at room temperature. After the reaction was completed, the solution was filtered and concentrated in a rotary evaporator to obtain l-({2-[(2-hydroxybutyl)amino]ethyl}amino)butan-2-ol.

5. Preparation of Intermediate A-3: l-[benzyl({3- [benzyl(2-hydroxypropyl)amino] propyl})amino] propan-2-ol

To a solution of Nl,N3-Dibenzylpropane-l,3-diamine in methanol, methyl oxirane (4 eq.) was added and stirred at 50 °C for 16 hours. The solution was concentrated in a rotary evaporator to obtain l-[benzyl({3-[benzyl(2-hydroxypropyl)amino]propyl})amino]propan-2- ol.

6. Preparation of Intermediate B-3: l-({3-[(2-hydroxypropyl)amino]propyl}amino)propan-2-ol

To a solution of l-[benzyl({3-[benzyl(2- hydroxypropyl)amino]propyl})amino]propan-2-ol (Intermediate A-3) in methanol, a catalytic amount of Pd/C was added in a reactor. The reactor was charged with hydrogen gas and stirred for 16 hours at room temperature. After the reaction w as completed, the solution w as filtered and concentrated in a rotary' evaporator to obtain l-({3-[(2- hydroxypropyl)amino]propyl}amino)propan-2-ol.

7. Preparation of Intermediate A-4: l-[benzyl({2-[benzyl(2-hydroxypropyl)amino]ethyl})amino]propan-2-oI

To a solution of benzyl[2-(benzylamino)ethyl]amine in methanol, methyl oxirane (4 eq.) was added and stirred at 50 °C for 16 hours. The solution was concentrated in a rotary evaporator to obtain l-[benzyl({2-[benzyl(2-hydroxypropyl)amino]ethyl})amino]propan-2-ol.

8. Preparation of Intermediate B-4: l-({2-[(2-hydroxypropyl)amino]ethyl}amino)propan-2-ol

To a solution of l-[benzyl({2-[benzyl(2-hydroxypropyl)amino]ethyl})amino]propan- 2-ol (Intermediate A-4) in methanol, a catalytic amount of Pd/C was added in a reactor. The reactor was charged wi th hydrogen gas and stirred for 16 hours at room temperature. After the reaction was completed, the solution was filtered and concentrated in a rotary evaporator to obtain 1 -( { 2- [(2-hy droxy propyl)amino] ethyl } amino)propan-2-ol .

9. Preparation of Intermediate A-5:

2,2'-(propane-l,3-diylbis(benzylazanediyl))bis(ethan-l-ol)

To a solution of Nl,N3-Dibenzylpropane-l,3-diamine in methanol, oxirane (4 eq.) was added and stirred at 50 °C for 16 hours. The solution was concentrated in a rotary⁷ evaporator to obtain 2,2'-(propane-l,3-diylbis(benzylazanediyl))bis(ethan-l-ol).

10. Preparation of Intermediate B-5:

2,2'-(propane-l,3-diylbis(azanediyl))bis(ethan-l-ol)

To a solution of 2,2'-(propane-l,3-diylbis(benzylazanediyl))bis(ethan-l-ol) (Intermediate A-5) in methanol, a catalytic amount of Pd/C was added in a reactor. The reactor was charged with hydrogen gas and stirred for 16 hours at room temperature. After the reaction was completed, the solution was filtered and concentrated in a rotary evaporator to obtain 2,2'-(propane-l,3-diylbis(azanediyl))bis(ethan-l-ol).

11. Preparation of Intermediate A-6:

2,2'-(ethane-l,2-diylbis(benzylazanediyl))bis(ethan-l-ol)

To a solution of benzyl[2-(benzylamino)ethyl]amine in methanol, oxirane (4 eq.) was added and stirred at 50 °C for 16 hours. The solution was concentrated in a rotary evaporator to obtain 2.2'-(ethane-l ,2-diylbis(benzylazanediyl))bis(ethan-l -ol).

12. Preparation of Intermediate B-6:

2,2'-(ethane-l,2-diylbis(azanediyl))bis(ethan-l-ol)

To a solution of 2,2'-(ethane-l,2-diylbis(benzylazanediyl))bis(ethan-l-ol) (Intermediate A-6) in methanol, a catalytic amount of Pd/C was added in a reactor. The reactor was charged with hydrogen gas and stirred for 16 hours at room temperature. After the reaction was completed, the solution was filtered and concentrated in a rotary evaporator to obtain 2,2'-(ethane-l,2-diylbis(azanediyl))bis(ethan-l-ol).

13. Preparation of Intermediate C-l: 2-hexyldecyl 6-bromohexanoate

To a solution of 2-hexyldecanol (1.2 eq.), 6-bromohexanoic acid (1 eq.), l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (1.5 eq.) and 4-dimethylaminopyridine (0.2 eq.) was added in di chloromethane. The reaction was stirred at room temperature for 16 hours. The solution was diluted with dichloromethane and was extracted using brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain 2-hexyldecyl 6-bromohexanoate.

14. Preparation of Intermediate C-2: 2-hexyldecyl 5-bromopentanoate

To a solution of 2-hexyldecanol (1.2 eq.), 5-bromopentanoic acid (1 eq.), 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide (1.5 eq.) and 4-dimethylaminopyridine (0.2 eq.) was added in di chloromethane. The reaction was stirred at room temperature for 16 hours. The solution was diluted with dichloromethane and was extracted using brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was punfied using silica gel chromatography to obtain 2-hexyldecyl 5-bromopentanoate.

15. Preparation of Intermediate C-3: heptadecan-9-yl 8-bromooctanoate

To a solution of heptadecan-9-ol (1.2 eq.), 8-bromooctanoic acid (1 eq.), l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (1.5 eq.) and 4-dimethylaminopyridine (0.2 eq.) was added in di chloromethane. The reaction was stirred at room temperature for 16 hours. The solution was diluted with dichloromethane and was extracted using brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain heptadecan-9-yl 8-bromooctanoate.

16. Preparation of Intermediate C-4: undecyl 6-bromohexanoate To a solution of undecan-l-ol (1.2 eq.), 6-bromohexanoic acid (1 eq.), l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (1.5 eq.) and 4-dimethylaminopyridine (0.2 eq.) were added in di chloromethane. The reaction was stirred at room temperature for 16 hours. The solution was diluted with dichloromethane and was extracted using brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain undecyl 6-bromohexanoate.

17. Preparation of Intermediate D-l:

2-hexyldecyl 6- [(2-hyd roxy butyl)( {3- [(2- hydroxybutyl)amino]propyl})amino]hexanoate

A mixture of l-({3-[(2-hydroxybutyl)amino]propyl}amino)butan-2-ol (1 eq.) (Intermediate B-l) and 2-hexyldecyl 6-bromohexanoate (1 eq.) (Intennediate C-l) was stirred in ethanol at 60 °C for 16 hours. The solution was diluted in ethyl acetate and extracted using sodium bicarbonate and brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain 2-hexyldecyl 6-[(2-hydroxybutyl)({3-[(2-hydroxybutyl)amino]propyl})amino]hexanoate.

18. Preparation of Intermediate D-2: heptadecan-9-yl 8-[(2-hydroxybutyl)({3-[(2- hydroxybutyl)amino]propyl})amino]octanoate

A mixture of l-({3-[(2-hydroxybutyl)amino]propyl}amino)butan-2-ol (1 eq.) (Intermediate B-l) and heptadecan-9-yl 8-bromooctanoate (1 eq.) (Intermediate C-3) was stirred in ethanol at 60°C for 16 hours. The solution was diluted in ethyl acetate and extracted using sodium bicarbonate and brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain heptadecan-9-yl 8-[(2-hydroxybutyl)({3-[(2- hydroxybutyl)amino] propyl })amino] octanoate.

19. Preparation of Intermediate D-3: heptadecan-9-yl 8- [(2-hydroxypropyl)( {3- [(2- hydroxy propyl )amino | propyl })amino] octanoate

A mixture of l-({3-[(2-hydroxypropyl)amino]propyl}amino)propan-2-ol (1 eq.) (Intermediate B-3) and heptadecan-9-yl 8-bromooctanoate (1 eq.) (Intermediate C-3) was stirred in ethanol at 60 °C for 16 hours. The solution was diluted in ethyl acetate and extracted using sodium bicarbonate and brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain heptadecan-9-yl 8-|(2-hydroxypropyl)( j3-|(2- hydroxypropyl)amino] propyl })amino] octanoate.

Example 1: Lipid 1

To a solution of 2-hexyldecyl 6-t(2-hydroxybutyl)({3-[(2- hydroxybutyl)amino]propyl})amino]hexanoate (1 eq.) (Intermediate D-l) in ethanol, 2- hexyldecyl 5-bromopentanoate (1.5 eq.) (Intermediate C-2) andN,N-diisopropylethylamine (3 eq.) were added and stirred for 16 hours at 60 °C. The solution was diluted in ethyl acetate and extracted using sodium bicarbonate and brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain 2-hexyldecyl 6-{[3-({5-[(2-hexyldecyl)oxy]-5-oxopentyl}(2- hydroxybutyl)amino)propyl](2-hydroxybutyl)amino}hexanoate (Lipid 1).

Chemical Formula: C54H109N2C . MS (ESI): m/z (MH⁺) 881.8284.

'HNMR (400 MHz, CDCh) 6 3.97 (d, J = 5.8 Hz, 4H), 3.63 (s, 2H), 2.52 (d, J = 26.7 Hz. 10H), 2.38 - 2.28 (m, 4H), 1.69 - 1.39 (m, 14H), 1.27 (d, J = 2.9 Hz, 52H). 0.98 (Id, J = 7.4, 2.2 Hz, 6H). 0.88 (1. J = 6.6 Hz. 12H).

¹³C NMR (101 MHz, CDCls) 5 173.80, 173.62, 77.23, 70.08, 68.60, 68.45, 68.20,

67.24, 67.17, 66.11, 60.54, 60.13, 54.25, 54.07, 52.55, 52.47, 37.29, 34.24, 34.04, 31.90, 31.82,

31.25, 29.97, 29.62, 29.57, 29.31, 29.24, 27.88, 27.79, 26.93, 26.70, 26.66, 26.00, 24.83, 22.79, 22.68, 22.65. 21.83, 15.20. 14.11, 9.99.

Example 2: Lipid 2

To a solution of 2-hexyldecyl 6-[(2-hydroxybutyd)({3-[(2- hydroxybutyl)amino]propyl})amino]hexanoate(l eq.) (Intermediate D-l) in ethanol, heptadecan-9-yl 8-bromooctanoate (1.5 eq.) (Intermediate C-3) and N,N- diisopropylethylamine (3 eq.) were added and stirred for 16 hours at 60 °C. The solution was diluted in ethyl acetate and extracted using sodium bicarbonate and brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain heptadecan-9-yl 8-{[3-({6-[(2-hexyldecyl)oxy]-6- oxohexyl}(2-hydroxybutyl)amino)propyl](2-hydroxybutyl)amino} octanoate (Lipid 2). 'H NMR (400 MHz. CDCI3) 5 4.86 (p, J = 6.2 Hz, 1H), 3.97 (d, J = 5.8 Hz, 2H), 3.71 - 3.57 (m, 2H), 2.80 - 2.39 (m, 1 OH), 2.29 (dt, J = 14.6, 7.5 Hz, 4H), 1 .74 (q, J = 7.2 Hz, 2H), 1.63 (qt, J = 7.9, 6.0, 4.5 Hz, 6H), 1.54 - 1.40 (m, 11H), 1.27 (dd, J = 7.3, 4.0 Hz, 58H), 0.98

(t, J = 7.4 Hz, 6H), 0.88 (td, J = 6.9, 2.2 Hz, 12H).

¹³C NMR (101 MHz. CDCh) 5 173.81, 173.58, 74.13, 68.55, 68.43, 68.19. 68.08, 67. 16, 60.55, 60.18, 54.43, 54.27, 53.91, 52.56, 52.50, 52.38, 52.28, 37.29, 34.64, 34.25, 34.23,

34.13, 31.90, 31.86, 31.81, 31.25, 29.96, 29.62, 29.56, 29.53, 29.50, 29.31, 29.23, 29.20, 29.15,

29.12, 27.87, 27.79, 27.77, 27.23, 26.94, 26.69, 26.65, 26.21, 25.31, 25.06, 25.04, 24.85, 24.83,

22.67, 22.66. 22.64, 14.10. 9.99.

Example 3: Lipid 3

To a solution of heptadecan-9-yl 8-[(2-hydroxybutyl)({3-[(2- hydroxybutyl)amino] propyl })amino] octanoate (1 eq.) (Intermediate D-2) in ethanol, undecyl 6-bromohexanoate (1.5 eq.) (Intermediate C-4) and N,N-diisopropylethylamine (3 eq.) were added and stirred for 16 hours at 60 °C. The solution was diluted in ethyl acetate and extracted using sodium bicarbonate and brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain heptadecan-9-yl 8-[(2-hydroxybutyl)({3-[(2-hydroxybutyl)[6-oxo-6- (undecyloxy)hexyl] amino] propyl })amino] octanoate (Lipid 3).

Chemical Formula: C53H107N2C . MS (ESI): m/z (MH⁺) 867.8148.

'H NMR (400 MHz, CDCE) 5 4.86 (p, J = 6.3 Hz, 1H), 4.05 (t, J = 6.8 Hz, 2H), 3.68 (s, 2H), 2.88 - 2.44 (m, 10H), 2.29 (dt, J = 12. 1, 7.4 Hz, 4H), 1.78 (s, 2H), 1.70 - 1.56 (m, 6H), 1.56 - 1.39 (m, 12H), 1.38 - 1.05 (m, 50H), 0.98 (tt, J = 7.5, 2.0 Hz, 6H), 0.88 (t, J = 6.6 Hz, 9H).

¹³C NMR (101 MHz, CDCE) 5 173.85, 173.58, 74.16, 70.41, 68.53, 66.09, 64.53, 64.46, 60.55, 54.28, 34.64, 34.34, 34.14, 31.91, 31.87, 29.71, 29.60, 29.54, 29.51, 29.47, 29.33, 29.27, 29.24, 29. 13, 28.65, 27.81, 26.87, 25.93, 25.82, 25.32, 25.05, 24.87, 24.78, 22.69, 22.67, 15.23, 14.11. 9.98. Example 4: Lipid 4

To a solution of l-({3-[(2-hydroxybutyl)amino]propyl}amino)butan-2-ol (1 eq.) (Intermediate B-l) in ethanol, 2-hexyldecyl 6-bromohexanoate (2.2 eq.) (Intermediate C-l) and N,N-diisopropylethylamine (3 eq.) were added and stirred at 60 °C for 16 hours. The solution was diluted in ethyl acetate and extracted using sodium bicarbonate and brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain 2-hexyldecyl 6-{[3-({6-[(2- hexyldecyl)oxy]-6-oxohexyl}(2-hydroxybutyl)amino)propyl](2- hydroxybutyl)amino}hexanoate (Lipid 4).

Chemical Formula: C55H111N2C .MS (ESI): m/z (MH⁺) 895.8476.

’HNMR (400 MHz, CDCI3) 5 3.97 (d, J = 5.8 Hz, 4H), 3.61 (dq, J = 15.3, 7.9, 7.3 Hz, 2H), 2.74 - 2.39 (m, 10H), 2.31 (t, J = 7.4 Hz, 4H), 1.65 (dq, J = 15.2, 7.2 Hz, 8H), 1.56 - 1.38 (m. 8H), 1.27 (d. J = 3.3 Hz. 54H), 0.98 (Id, J = 7.4. 1.9 Hz. 6H), 0.88 (t, J = 6.6 Hz, 12H).

¹³C NMR (101 MHz, CDCL) 5 173.79, 173.76, 68.49, 68.14, 67.16, 60.54, 60.20, 54.27, 53.90, 52.52, 52.32, 37.28, 34.24, 34.21, 31.89, 31.81, 31.25, 29.95, 29.61, 29.55, 29.30, 27.86, 27.78, 26.93, 26.91, 26.69, 26.64, 26.12, 25.81, 24.84, 24.81, 22.66, 22.64, 14.10, 9.99, 9.97.

Example 5: Lipid 5

To a solution of l-({3-[(2-hydroxybutyl)amino]propyl}amino)butan-2-ol (1 eq.) (Intermediate B-l) in ethanol, 2-hexyldecyl 5-bromopentanoate (2.2 eq.) (Intermediate C-2) and N,N-diisopropylethylamine (3 eq.) were added and stirred at 60 °C for 16 hours. The solution was diluted in ethyl acetate and extracted using sodium bicarbonate and brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain 2-hexyldecyl 5-{[3-({5-[(2- hexyldecyl)oxy]-5-oxopentyl}(2-hydroxybutyl)amino)propyl](2- hydroxybutyl)amino}pentanoate (Lipid 5).

Chemical Formula: C53H107N2O6L MS (ESI): m/z (MH⁺) 867.8122.

'H NMR (400 MHz, CDCI3) 8 3.97 (d, J = 5.8 Hz, 4H), 3.57 (ddt, J = 15.3, 9.9, 4.7 Hz. 2H), 2.63 (tq, J = 15.0, 7.8, 7.4 Hz, 4H), 2.53 - 2.37 (m. 6H), 2.33 (t, J = 7.3 Hz, 4H), 1.63 (ddd. J = 16.2, 9.2. 6.0 Hz, 8H). 1.56 - 1.36 (m, 8H). 1.36 - 1.22 (m, 50H), 0.97 (td, J = 7.5, 1.8 Hz, 6H), 0.88 (t, J = 6.6 Hz, 12H).

¹³C NMR (101 MHz, CDCls) 8 173.60, 173.58, 68.56, 68.25, 67.21, 60.58, 60.27, 54.08, 53.79, 52.44, 52. 17, 37.28, 34.05, 34.02, 31.89, 31.81, 31.24, 29.95, 29.61, 29.55, 29.30,

27.80, 27.74. 26.69, 26.64. 26.07, 25.83, 24.29, 22.79, 22.77, 22.66, 22.64, 14.09, 9.98.

Example 6: Lipid 6

To a solution of l-({3-[(2-hydroxybutyl)amino]propyl}amino)butan-2-ol (1 eq.) (Intermediate B-l) in ethanol, heptadecan-9-yl 8-bromooctanoate (2.2 eq.) (Intermediate C-3) and N,N-diisopropylethylamine (3 eq.) were added and stirred at 60 °C for 16 hours. The solution was diluted in ethyl acetate and extracted using sodium bicarbonate and brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain heptadecan-9-yl 8-[(3-{[8-(heptadecan- 9-yloxy)-8-oxooctyl](2-hydroxybutyl)amino}propyl)(24iydroxybutyl)amino]octanoate (Lipid

Chemical Formula: C61H123N2C . MS (ESI): m/z (MH⁺) 979.9374.

'HNMR (400 MHz, CDCI3) 8 4.86 (p, J = 6.3 Hz, 2H), 3.62 (tt, J = 14.9, 7.5 Hz, 2H), 2.81 - 2.42 (m, 10H), 2.28 (t, J = 7.5 Hz. 4H), 1.69 (q, J = 6.9 Hz, 2H). 1.60 (q, J = 7.0 Hz, 4H), 1.55 - 1.37 (m, 16H). 1.29 (d, J = 24.3 Hz. 62H), 0.98 (td. J = 7.4. 1.7 Hz, 6H). 0.88 (t. J = 6.7 Hz, 12H). ¹³C NMR (101 MHz, CDCL) 5 173.57, 74. 13. 68.47, 68. 13. 60.56, 60.20, 54.44, 54.05.

52.54, 52.34, 34.64, 34.13, 31.86, 29.53, 29.50, 29.23, 29.21, 29.19, 29.14, 29.13, 27.88, 27.78,

27.24, 26.31, 25.31, 25.06, 25.04, 22.66, 14.10, 9.99, 9.97.

Example 7; Lipid 7

To a solution of l-({3-[(2-hydroxybutyl)amino]propyl}amino)butan-2-ol (1 eq.) (Intermediate B-l) in ethanol, undecyl 6-bromohexanoate (2.2 eq.) (Intermediate C-4) and N,N-diisopropylethylamine (3 eq.) were added and stirred at 60 °C for 16 hours. The solution was diluted in ethyl acetate and extracted using sodium bicarbonate and brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain undecyl 6-[(2-hydroxybutyl)({3-[(2- hydroxybutyl)[6-oxo-6-(undecyloxy)hexyl]amino]propyl})amino]hexanoate (Lipid 7).

Chemical Formula: C45H91N2C . MS (ESI): m/z (MH⁺) 755.6885.

'H NMR (400 MHz. CDCI3) 54.05 (t. J = 6.7 Hz. 4H), 3.61 (dp, J = 10.4. 5.2 Hz. 2H), 2.77 - 2.57 (m, 4H), 2.48 (tt, J = 14.9, 8.3 Hz, 6H), 2.30 (t, J = 7.4 Hz, 4H), 1.78 - 1.67 (m, 2H), 1.62 (ddt, J = 15.4, 8.7, 4.7 Hz, 10H), 1.56 - 1.38 (m, 8H), 1.29 (d, J = 17.2 Hz, 38H), 1.04 - 0.92 (m, 6H), 0.88 (t, J = 6.7 Hz, 6H).

¹³C NMR (101 MHz. CDCh) 5 173.65, 173.63, 70.37, 68.46, 68.11, 66.05. 64.48, 64.41, 60.53. 60.21, 54.25. 53.89, 52.51. 52.34, 34.30. 34.15, 34.13. 31.88, 29.57. 29.56, 29.50, 29.44, 29.30, 29.24, 28.62, 27.85, 27.77, 26.85, 26.83, 25.99, 25.90, 25.79, 25.68, 24.84, 24.76, 24.74, 23.89, 23.57, 22.65, 15.19, 14.08, 9.96, 9.95.

Example 8; Lipid 8

To a solution of l-({2-[(2-hydroxybutyl)amino]ethyl}amino)butan-2-ol (1 eq.) (Intermediate B-2) in ethanol, 2-hexyldecyl 6-bromohexanoate (2.2 eq.) (Intermediate C-l) and N,N-diisopropylethylamine (3 eq.) were added and stirred at 60 °C for 16 hours. The solution was diluted in ethyl acetate and extracted using sodium bicarbonate and brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain 2-hexyldecyl 6-{[2-({6-[(2- hexyldecyl)oxy]-6-oxohexyl}(2-hydroxybutyl)amino)ethyl](2- hydroxybufy I )amino} hexanoate (Lipid 8).

Chemical Formula: C₅4HIO9N₂06⁺. MS (ESI): m/z (MH⁺) 881.8297.

'H NMR (400 MHz, CDCE) 5 3.97 (d, J= 5.8 Hz, 4H), 3.62 - 3.51 (m, 2H), 2.87 - 2.32 (m, 12H), 2.30 (d, J= 7.5 Hz, 4H), 1.64 (p, J= 7.5 Hz, 6H), 1.55 - 1.36 (m, 8H), 1.34 - 1.20 (m, 52H), 0.96 (t, J= 7.4 Hz, 6H), 0.88 (t, J= 6.6 Hz, 12H).

¹³C NMR (101 MHz, CDCh) 8 173.81, 69.73, 69.26, 67.12, 61.17, 60.31, 55.29. 55.20, 53.54. 52.09, 37.29. 34.29, 31.89, 31.81, 31.25, 29.96. 29.62, 29.56. 29.30, 27.67. 27.61 , 27.03, 27.00, 26.69, 26.65, 26.35, 26.18, 24.90, 22.67, 22.64, 14.10, 10.09, 10.04.

Example 9: Lipid 9

To a solution of l-({2-[(2-hydroxypropyl)amino] ethyl} amino)propan-2-ol (1 eq.) (Intermediate B-4) in ethanol, 2-hexyldecyl 6-bromohexanoate (2.2 eq.) (Intennediate C-l) and N,N-diisopropylethylamine (3 eq.) were added and stirred at 60 °C for 16 hours. The solution was diluted in ethyl acetate and extracted using sodium bicarbonate and brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain 2-hexyldecyl 6-{[2-({6-[(2- hexyldecyl)oxy] -6-oxohexyl } (2-hydroxypropyl)amino)ethyl] (2- hydroxypropyl)amino} hexanoate (Lipid 9).

Chemical Formula: C52H105N2C . MS (ESI): m/z (MH⁺) 853.7981.

'H NMR (400 MHz, CDCE) 8 3.97 (d, J= 5.8 Hz, 4H), 3.87 (tt, J= 6.8, 3.4 Hz, 2H), 2.77 (dd, J= 19.7, 9.2 Hz, 2H), 2.60 (t, J= 8.4 Hz, 4H), 2.52 (d, J= 13.0 Hz, 2H), 2.39 (dd, J = 16.4. 6.5 Hz. 2H), 2.31 (t, J= 7.4 Hz, 4H), 1.64 (p, J = 7.6 Hz, 6H), 1.53 (dt. J= 14.7, 7.5 Hz, 4H), 1.40 - 1. 17 (m, 54H), 1.12 (t, J= 5.5 Hz, 6H), 0.88 (t, J= 6.6 Hz, 12H).

¹³C NMR (101 MHz, CDCh) 8 173.79, 173.77, 67.16, 67.15, 64.59, 64.26, 62.48, 61.80, 55.36, 55.16, 53.23, 52.19, 37.29, 34.24, 31.89, 31.81, 31.25, 29.96, 29.61, 29.56, 29.30, 26.95. 26.91, 26.69. 26.65, 25.95, 25.86, 24.85, 22.67, 22.64, 20.20. 20.13, 14.10.

Example 10: Lipid 10

To a solution of l-({3-[(2-hydroxypropyl)amino]propyl}amino)propan-2-ol (1 eq.) (Intermediate B-3) in ethanol, 2-hexyldecyl 6-bromohexanoate (2.2 eq.) (Intermediate C-l) and N,N-diisopropylethylamine (3 eq.) were added and stirred at 60 °C for 16 hours. The solution was diluted in ethyl acetate and extracted using sodium bicarbonate and brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain 2-hexyldecyl 6-{[3-({6-[(2- hexyldecyl)oxy]-6-oxohexyl}(2-hydroxypropyl)amino)propyl](2- hydroxypropyl)amino}hexanoate (Lipid 10).

Chemical Formula: C₅3HIO7N₂0₆ ⁺. MS (ESI): m/z (MH⁺) 867.8161.

'HNMR (400 MHz, CDCh) 5 3.96 (d, J= 5.8 Hz, 4H), 3.77 - 3.58 (m, 2H), 3.01 - 2.53 (m, 10H), 2.32 (t, J= 7.4 Hz, 4H), 1.83 (h, J= 7.4 Hz, 2H), 1.63 (dt, J= 24.2. 12.1 Hz, 10H), 1.41 - 1.10 (m, 60H), 0.88 (t, J= 6.7 Hz, 12H).

¹³C NMR (101 MHz, CDCh) 8 173.75, 173.71, 127.77, 67.23, 65.05, 63.34, 62.87, 61.76, 61.30, 54.25, 53.57, 52.76, 52.67, 37.27, 34.12, 34.07, 31.89, 31.81, 31.23, 29.95, 29.69, 29.61, 29.55, 29.30. 26.91, 26.74, 26.68, 26.64, 25.23, 24.79, 24.69, 24.66, 22.66, 22.64, 21.49. 20.77, 20.55. 20.08, 14.10.

Example 11 : Lipid 11

To a solution of heptadecan-9-yl 8-[(2-hydroxypropyl)({3-[(2- hydroxypropyl)amino] propyl })amino] octanoate (1 eq.) (Intermediate D-3) in ethanol, undecyl 6-bromohexanoate (1.5 eq.) (Intermediate C-4) and N,N-diisopropylethylamine (3 eq.) were added and stirred for 16 hours at 60 °C. The solution was diluted in ethyl acetate and extracted using sodium bicarbonate and brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain heptadecan-9-yl 8-[(2-hydroxypropyl)({3-[(2-hydroxypropyl)[6-oxo-6- (undecyloxy)hexyl]amino]propyl})amino]octanoate (Lipid 11).

Chemical Formula: C₅IHIO3N₂06⁺.MS (ESI): m/z (MH⁺) 839.7850.

'H NMR (400 MHz. CDCI3) 5 4.86 (s, 1H). 4.05 (t. J= 6.8 Hz. 2H), 3.94 (dq, J = 18.1, 7.1, 6.4 Hz, 2H), 2.84 - 2.56 (m, 10H), 2.31 - 2.27 (m, 4H), 1.88 - 1.74 (m, 2H), 1.62 (d, J= 6.1 Hz, 4H), 1.50 (d, J= 6.3 Hz, 6H), 1.26 (d, J= 5.9 Hz, 54H), 1.18 (t, J= 5.7 Hz, 6H), 0.88 (s, 9H).

¹³C NMR (101 MHz, CDCh) 5 173.64, 173.56, 74.19, 74.17. 64.96, 64.54, 63.43, 63.23, 63.05. 62.87, 62.66. 61.98, 61.61, 60.53, 56.15, 55.27. 54.49, 54.31. 54.12, 53.98.

53.88, 52.64, 52.52, 50.24, 34.59, 34.12, 34.09, 31.89, 31.85, 29.59, 29.57, 29.52, 29.49,

29.32, 29.25, 29.22, 29.14, 29.07, 28.63, 27.19, 27.06, 26.77, 25.98, 25.92, 25.31, 24.98,

24.70, 22.67, 22.65, 21.86. 21.49, 20.55, 20.42, 20.38, 20.03, 14.10.

Example 12: Lipid 12

To a solution of 2-({2-[(2-hydroxyethyl)amino]ethyl}amino)ethan-l-ol (1 eq.) (Intermediate B-6) in ethanol, 2-hexyldecyl 5-bromopentanoate (2.2 eq.) (Intermediate C-2) and N,N-diisopropylethylamine (3 eq.) were added and stirred at 60 °C for 16 hours. The solution was diluted in ethyl acetate and extracted using sodium bicarbonate and brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain 2-hexyldecyl 5-{[2-({5-[(2- hexyldecyl)oxy] -5 -oxopentyl } (2-hy droxy ethy l)amino)ethyl] (2- hydroxyethyl)amino}pentanoate (Lipid 12).

Chemical Formula: C48H₉7N₂O₆ ⁺. MS (ESI): m/z (MH⁺) 797.7340.

'HNMR (400 MHz, CDCls) 8 3.97 (d, J= 5.8 Hz, 4H), 3.71 (t, J= 4.8 Hz, 4H), 2.85 - 2.71 (m, 10H), 2.39 - 2.30 (m, 4H), 1.61 (q, J= 5.6 Hz, 10H), 1.33 - 1.23 (m, 50H), 0.88 (t, J = 6.6 Hz, 12H).

¹³C NMR (101 MHz, CDCh) 6 173.82, 173.50. 70.07, 67.29. 67.12, 66.10, 59.11, 55.59, 54.54, 51.74, 37.28, 34.15, 33.83, 31.89, 31.81, 31.26, 31.22, 29.95, 29.61, 29.56, 29.30, 29.23, 26.69, 26.65, 24.96, 22.66, 22.64, 22.59, 21.82, 15.19, 14.09.

Example 13; Lipid 13

To a solution of 2-({2-[(2-hydroxyethyl)amino]ethyl}amino)ethan-l-ol (1 eq.) (Intermediate B-6) in ethanol, 2-hexyldecyl 6-bromohexanoate (2.2 eq.) (Intermediate C-l) and N,N-diisopropylethylamine (3 eq.) were added and stirred at 60 °C for 16 hours. The solution was diluted in ethyl acetate and extracted using sodium bicarbonate and brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain 2-hexyldecyl 6-{ [2-({6-[(2- hexyldecyl)oxy]-6-oxohexyl}(2-hydroxyethyl)amino)ethyl](2- hydroxyethyl)amino}hexanoate (Lipid 13).

Chemical Formula: C₅OHIOIN₂06⁺. MS (ESI): m/z (MH⁺) 825.7686.

'H NMR (400 MHz. CDCE) 6 3.96 (d, J = 5.8 Hz, 4H), 3.71 (t, J = 4.8 Hz, 4H). 2.84 (s, 2H), 2.80 (t, J= 4.8 Hz, 4H), 2.73 (t, J= 8.1 Hz, 4H), 2.31 (t, J= 7.4 Hz, 4H), 1.61 (dq, J = 24.2, 7.8 Hz, 10H), 1.27 (s, 54H), 0.88 ( 6.6 Hz, 12H). ¹³C NMR (101 MHz, CDCh) 5 173.73. 67.21, 59.05. 55.61, 54.72. 51.72, 37.28. 34.14, 31.89, 31.81, 31.24, 29.96, 29.61 , 29.56, 29.31 , 26.78, 26.69, 26.65, 25.16, 24.73, 22.67, 22.64, 14.10.

Example 14; Lipid 14

To a solution of 2-({3-[(2-hydroxyethyl)amino]propyl}amino)ethan-l-ol (1 eq.) (Intermediate B-5) in ethanol, 2-hexyldecyl 5-bromopentanoate (2.2 eq.) (Intermediate C-2) and N,N-diisopropylethylamine (3 eq.) were added and stirred at 60 °C for 16 hours. The solution was diluted in ethyl acetate and extracted using sodium bicarbonate and brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain 2-hexyldecyl 5-{[3-({5-[(2- hexyldecyl)oxy]-5-oxopentyl}(2-hydroxyethyl)amino)propyl](2- hydroxyethyl)amino}pentanoate (Lipid 14).

Chemical Formula: C₄9H99N₂O6⁺. MS (ESI): m/z (MH⁺) 811.7494.

‘HNMR (400 MHz. CDCh) 5 3.97 (d, J= 5.8 Hz, 4H), 3.77 (t, J= 4.9 Hz, 4H), 2.86 (dt. J= 9.4, 5.8 Hz, 6H), 2.81 - 2.67 (m. 4H), 2.40 - 2.31 (m. 4H), 1.90 (dp, J = 13.2, 6.8 Hz, 2H), 1.63 (dh, J= 8.4, 5.2, 4.7 Hz, 10H), 1.27 (d, J= 2.6 Hz, 50H), 0.88 (t, J= 6.7 Hz, 12H).

¹³C NMR (101 MHz, CDCh) 5 173.49, 129.50, 128.52, 127.60, 67.38, 59.09, 58.83, 58.09, 55.94, 53.64, 52.41, 51.47, 37.28, 33.69, 31.90, 31.82, 31.22, 29.96, 29.62, 29.57, 29.31, 26.70. 26.65, 24.41. 22.67, 22.65, 22.50, 22.35, 14.11.

Example 15; Lipid 15

To a solution of 2-({3-[(2-hydroxyethyl)amino]propyl}amino)ethan-l-ol (1 eq.) (Intermediate B-5) in ethanol, 2-hexyldecyl 6-bromohexanoate (2.2 eq.) (Intermediate C-l) and N,N-diisopropylethylamine (3 eq.) were added and stirred at 60 °C for 16 hours. The solution was diluted in ethyl acetate and extracted using sodium bicarbonate and brine. The organic layers were dried using sodium sulfate, filtered, and evaporated in vacuo. The residue was purified using silica gel chromatography to obtain 2-hexyldecyl 6-{[3-({6-[(2- hexyldecyl)oxy] -6-oxohexyl} (2-hydroxyethyl)amino)propyl] (2- hydroxyethyl)amino}hexanoate (Lipid 15).

Chemical Formula: C51H103N2O6T MS (ESI): m/z (MH⁺) 839.7802.

'HNMR (400 MHz, CDCls) 5 3.96 (d, J= 5.8 Hz, 4H), 3.78 (t, J= 5.0 Hz, 2H), 3.51 - 3.37 (m, 2H), 3.03 - 2.56 (m, 8H), 2.32 (td, J= 1.6, 2.8 Hz, 4H), 1.62 (tq, J= 14.3, 7.2 Hz, 10H), 1.27 (d, J= 6.2 Hz, 58H), 0.88 (t, J = 6.6 Hz, 12H).

¹³C NMR (101 MHz, CDCh) 6 173.94, 173.69, 129.47, 128.50, 70.40, 67.25, 67.09, 66.08, 57.91, 55.80, 53.75, 52.50, 37.28, 34.38, 34.05, 31.89, 31.81, 31.27, 31.23, 29.95, 29.61, 29.56, 29.47, 29.30, 26.69, 26.64, 26.51, 25.82, 24.89, 24.62, 24.52, 22.67, 22.64, 22.12, 15.21, 14.10.

Synthesis of Lipid Nanoparticles

For in vitro studies (transfection efficiencies, particle sizes and surface charge), different lipid nanoparticles (LNPs) were formulated by mixing an ionizable lipid, phospholipid (l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE) or

Distearoylphosphatidylcholine (DSPC)). cholesterol, and l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG) dissolved in ethanol with mRNA buffer solution rapidly by pipette mixing. The lipids were dissolved in ethanol at molar ratios of 40: 10:48:2 (ionizable lipid: phospholipid: cholesterol: DMG-PEG) at an N/P ratio of 8.

For in vivo study and measurement of the polydispersity index (PDI) and mRNA encapsulation efficiency, different LNPs were formulated by mixing an ionizable lipid, DSPC, cholesterol, and DMG-PEG dissolved in ethanol at a molar ratio of 50: 10:38.5: 1.5, respectively. A molar ratio of 6: 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 25 mM citrate buffer (pH 3.0) was mixed with the lipid mixture at a 3: 1 volume ratio using a T-junction. The solution was concentrated using an Ami con filter (MWCO: 100,000 Da) to remove the ethanol and exchange the buffer with PBS.

Experimental Example 1: In-vitro Transfection Efficiencies LNPs with different compositions were tested against Jurkat T cells for mRNA transfection efficiencies. Jurkat cells are representative T lymphocytes which are known to be difficult to transfect.

The cells were seeded in a 96-well plate at a density of 40,000 cells/well. Different formulations containing 100 ng of either FLuc or eGFP mRNA were added to each well and were incubated for 20 hours at 37°C. If FLuc mRNA was used, the luciferase expression was measured by using the Bright-Glo™ luciferase assay (Promega) following the manufacturer’s protocol. The luciferase expression was measured using a luminometer. For quantifying the expression of eGFP mRNA, flow cytometry was used to determine transfection efficacy in human Jurkat T cells. The cells were prepared by diluting them in PBS and were quantified by gating the live cells.

In Jurkat cells, the LNP containing different ionizable lipids such as Lipid 1, Lipid 2, Lipid 3, Lipid 4, Lipid 5. Lipid 6 or Lipid 8 showed an eGFP mRNA transfection efficiency of >~70% (Fig. 1). and the LNP containing different ionizable lipids such as Lipid 1. Lipid 2, Lipid 3, Lipid 4, Lipid 5 or Lipid 6 showed Flue mRNA transfection efficiency measured by luciferase assay ranged between 10³-10⁵ relative light units (RLU) (Fig. 2).

Experimental Example 2: In-vitro Particle Characterization

The LNPs containing different ionizable lipids such as Lipid 1, Lipid 2, Lipid 3, Lipid 4, Lipid 5, Lipid 6, Lipid 7, Lipid 8, Lipid 9, Lipid 10 or Lipid 11 w ere characterized for particle size and surface charge using dynamic light scattering and zeta potential measurements.

The LNPs showed a range of particle sizes of -100-150 nm (Fig. 3(a)). The surface charge of the LNPs ranged from -3 mV to -9 mV (Fig. 3(b)). Since these LNPs contained ionizable lipids, their surface charge was close to neutral when they were measured at physiological pH of 7.4.

The polydispersity index (PDI) of the LNPs (particularly, containing Lipid 1 or Lipid 2) was measured to be about - 0.2. The encapsulation efficiency of mRNA inside the LNP was detennined using QUANT-IT™ Ribogreen® RNA assay (Invitrogen). In particular, the encapsulation efficiency of the LNP containing Lipid 2 was measured to be about 80 %. Experimental Example 3: In-vivo Whole-Body and Organ-Specific Flux Animals

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 w aler and food ad libitum. All procedures involving animal 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.

LNP Administration to Animals mRNA-LNPs diluted in Dulbecco’s PBS were injected i.v. at a dosage of 0.5 mg/kg (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.

Whole-Body and Ex-Vivo Organ Bi olumine scent Imaging of Mice Administered Luciferase mRNA

Balb/c mice were injected with D-Luciferin, Sodium Salt (GoldBio, catalog #: LUCNA) intraperitoneally (i.p.) 10 min prior to 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 prior to 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 with ventral side up and maintained on 3% isoflurane and 97% oxygen. Images were acquired using IVIS Spectrum (PerkinElmer). After whole-body imaging animals were returned to their cage for recovery or w ere euthanized by CO2 administration followed by cervical dislocation. The 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 w as 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).

The mice were imaged 6 hours after the administration of different LNPs at a dosage of 0.5 mg/kg. The total flux was measured using Living image software. After euthanizing the mice and isolating the organs, the liver, spleen, and lymph nodes were measured for luciferase expression (Fig. 4). LNP containing Lipid 1 or Lipid 2 showed atotal flux of ~10⁹ p/sec/cm²/sr. LNP containing Lipid 1 or Lipid 2 showed a liver radiance of ~10⁸ p/sec/cm²/sr, a spleen radiance in the range of 10⁷- 10⁸ p/sec/cm²/sr, and the lymph node radiance in the range of 10⁶- 10⁷ p/sec/cm²/sr.

Claims

1. A compound having the following structure of Formula (I) or a pharmaceutically acceptable salt thereof: wherein:

Ri is a C2-C18 alkyl group or C2-C18 alkenyl group, optionally substituted with R2;

R2 is a C2-C18 alkyl group or C2-C18 alkenyl group;

R3 is a C2-C18 alkyl group:

R4 is a C2-C6 alkyl group;

Rs is a C2-C18 alkyl group:

Re is a C2-C18 alkyl group or C2-C18 alkenyl group, optionally substituted with R7;

R7 is a C2-C18 alkyl group or C2-C18 alkenyl group; and

Rs and R9 are independently hydrogen or a C1-C2 alkyd group.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein:

Ri is a C5-C15 alkyl group or C5-C15 alkenyl group, optionally substituted with R2;

R2 is a C2-C12 alk\ 1 group or C2-C12 alkenyl group;

R3 is a C2-C13 alkyl group;

R4 is a C2-C6 alkyl group;

Rs is a C2-C13 alkyl group:

Re is a C5-C15 alkyl group or C5-C15 alkenyl group, optionally substituted with R7;

R7 is a C2-C 12 alky l group or C2-C12 alkenyl group; and

Rs and R9 are independently hydrogen or a C1-C2 alky l group.

3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein:

Ri is a C9-C11 alky l group, optionally substituted with R2:

R2 is a Ce-Cs alkyl group; R3 is a C4-C7 alkyl group;

R4 is a C2-C6 alkyl group;

R5 is a C4-C7 alkyl group;

Re is a C9-C11 alkyl group, optionally substituted with R7;

R7 is a Ce-Cs alkyl group; and

Rs and R9 are independently hydrogen or a C1-C2 alkyl group.

4. The compound of claim 1, which is selected from the group consisting of the following compounds:

5. A lipid nanoparticle comprising the ionizable lipid compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof.

6. The lipid nanoparticle of claim 5, further comprising: a phospholipid; a sterol; and a PEGylated lipid conjugate.

7. The lipid nanoparticle of claim 6, wherein the phospholipid is selected from the group consisting of: l,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE). 1.2-Dipalmitoyl-sn-glycero-3-

Phosphatidylcholine (DPPC), E2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), and 1, 2- dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG).

8. The lipid nanoparticle of claim 6, wherein the sterol is cholesterol or a derivative thereof.

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

10. The lipid nanoparticle of claim 6, wherein the ionizable lipid compound constitutes about 30-60 mole%, the phospholipid constitutes about 5-15 mole%, the sterol constitutes about 30-55 mole%. and the PEGylated lipid conjugate constitutes about 0.5-5 mole% of the total lipids in the lipid nanoparticle.

11. A composition comprising mRNA formulated in the lipid nanoparticle of claim 5.

12. A method of delivering an mRNA to a subject or cell comprising administering the composition of claim 11 to the subject or cell.