WO2024019770A1

WO2024019770A1 - Methods of making ionizable lipids and lipid nanoparticles for mrna delivery

Info

Publication number: WO2024019770A1
Application number: PCT/US2023/010671
Authority: WO
Inventors: Joo-Youp LEE; Vishnu SRIRAM
Original assignee: University Of Cincinnati
Priority date: 2022-07-20
Filing date: 2023-01-12
Publication date: 2024-01-25
Also published as: CN117881428A; CA3226487A1

Abstract

Provided herein are an ionizable lipid compound, a lipid nanoparticle including the ionizable lipid compound, a composition including 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

METHODS OF MAKING IONIZABLE LIPIDS AND LIPID NANOPARTICLES FOR

MRNA DELIVERY

FIELD

BACKGROUND Messenger RNA (mRNA) has been presented as a new category of therapeutic agent to prevent and treat various diseases. There has been a need for lipid nanoparticles which may effectively and stably deliver mRNA to a target cell or subject.

SUMMARY The present disclosure provides novel ionizable lipid compounds, which may be used for the delivery of mRNA. In one embodiment, the present disclosure provides a compound having the following structure (I):

Here, in one embodiment Ri may be

In addition, R2 and R3 may be each independently a C10-C24 alkyl group

which optionally may contain one, two or three double bonds.

In some embodiment, the compound having the structure (I) may be selected from the following compounds.

14

In another embodiment, the present disclosure provides a compound having the following structure (II):

Here, in one embodiment, R4 may be

In addition, in one embodiment, R₅ and R₆ may be each independently a C10-C24 alkyl group which optionally may contain one, two or three double bonds. In some embodiment, the compound having the structure (II) may be selected from the

In another embodiment, the present disclosure provides a compound having the following structure (III):

Here, in one embodiment, R7 may be or

In addition, in one

embodiment, R8 and R9 may be each independently a C10-C24 alkyl group which optionally may contain one, two or three double bonds.

In one embodiment, the compound having structure (III) may be selected from the

The present disclosure also provides a lipid nanoparticle comprising the compound having structure (I), (II) or (III), or the specific compounds disclosed above.

In addition, the present disclosure provides a composition comprising an mRNA formulated in the lipid nanoparticle which comprises the compound having structure (I), (II) or (III), or the specific compounds disclosed above.

Moreover, the present disclosure provides a method of delivering an mRNA to a subject or cell comprising administering the composition comprising an mRNA formulated in the lipid nanoparticle which comprises the compound having structure (I), (II) or (III), or the specific compounds disclosed above to the subject or cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the synthesis of Im lipid.

FIG. 2 is a NMR of Product 1.

FIG. 3 is a NMR of Product 2.

FIG. 4 is an ESI-MS of Im lipid. Theoretical mass: 782.59. Observed mass: 782.59.

FIG. 5 is a pair of graphs showing (a) Particle size and (b) surface charge of different LNPs.

FIG. 6 is a graph showing the in vitro cell uptake and transfection efficiency of different LNP formulations in HeLa cells after 20-hour incubation using 100 ng Cy5 -labeled eGFP mRNA. Cellular uptake and transfection efficiencies are relative to the negative control (i.e., no treatment).

FIG. 7 is a graph showing the in vitro cell uptake and transfection efficiency of different LNP formulations in Jurkat cells after 19-hour incubation using 100 ng Cy5 -labeled eGFP mRNA.

FIG. 8 is a synthesis route of Product 4 ([3-Alanine, N-(2-hydroxyethyl)-N-[3-(9- octadecenyloxy)-3-oxopropyl]-, 9-octadecenyl ester, (Z,Z)- (9CI)).

FIG. 9 is a 1H NMR of Product 3 ((9Z)-9-Octadecen-l-yl 2-propenoate).

FIG. 10 is a 1H NMR of Product 4 (P-Alanine, N-(2-hydroxyethyl)-N-[3-(9- octadecenyloxy)-3-oxopropyl]-, 9-octadecenyl ester, (Z,Z)- (9CI)).

FIG. 11 is a synthesis route of Product 5 (2AEOAP2 lipid).

FIG. 12 is a synthesis route of Product 6 (2AEOAP4 lipid). FIG. 13 is a synthesis route of Product 7 (2AEOAD2 lipid).

FIG. 14 is a synthesis route of Product 8 (2AEOAD3 lipid).

FIG. 15 is a synthesis route of linoleyl alcohol (LA) tail.

FIG. 16 is a 1H NMR of Product 9 (2-Propenoic acid, 9,12-octadecadienyl ester, (Z,Z)- (9CI)).

FIG. 17 is a 1H NMR of Product 10 (LA tail).

FIG. 18 is a synthesis route of Product 11 (2AELAP2 lipid).

FIG. 19 is a synthesis route of Product 12 (2AELAP4 lipid).

FIG. 20 is a synthesis route of Product 13 (2AELAD2 lipid).

FIG. 21 is a synthesis route of Product 14 (2AELAD3 lipid).

FIG. 22 is a pair of graphs showing (a) particle size and (b) surface charge of the different LNPs. Here, 5 LNPs was prepared by using Product 5. 6 LNPs was prepared by using Product 6. 7 LNPs was prepared by using Product 7. 8 LNPs was prepared by using Product 8. 9 LNPs was prepared by using Product 9. 10 LNPs was prepared by using Product 10. 11 LNPs was prepared by using Product 11. 12 LNPs was prepared by using Product 12. 13 LNPs was prepared by using Product 13. 14 LNPs was prepared by using Product 14.

FIG. 23 is a graph showing in vitro cell uptake and transfection efficiency of different LNP formulations in Jurkat cells after 21 -hour incubation using 100 ng Cy5 -labeled eGFP mRNA. Negative control represents cells with no treatment. LipoMM represents mRNA complexed with Lipofectamine MessengerMax. Cellular uptake and transfection efficiencies are relative to the negative control (i.e., no treatment). Here, 5 LNPs was prepared by using Product 5. 6 LNPs was prepared by using Product 6. 7 LNPs was prepared by using Product 7. 8 LNPs was prepared by using Product 8. 9 LNPs was prepared by using Product 9. 10 LNPs was prepared by using Product 10. 11 LNPs was prepared by using Product 11. 12 LNPs was prepared by using Product 12. 13 LNPs was prepared by using Product 13. 14 LNPs was prepared by using Product 14.

A of FIG. 24 is a schematic of synthesis of ring lipids. B of FIG. 24 shows structures of four different ring lipids (i.e., Product 24(B)(a), Product 24(B)(b), Product 24(B)(c), and Product 24(B)(d)).

A of FIG. 25 is a schematic of the synthesis of piperidine-based lipids. B of FIG. 25 shows structures of piperidine -based lipids (i.e., Product 25(B)(a) and Product 25(B)(b)). DEFINITIONS

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this disclosure is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

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 this disclosure belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply.

As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise.

As used herein, the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc., without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of’ and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc., and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of’ denotes the recited feature(s), element(s), method step(s), etc., and any additional feature(s), element(s), method step(s), etc., that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of’ and/or “consisting essentially of’ embodiments, which may alternatively be claimed or described using such language.

As used herein, the term “C10-C24 alkyl group” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated or unsaturated (i.e., contains one or more double and/or triple bonds), having from ten to twenty four carbon atoms (C10-C24 alkyl), and optionally substituted with methyl, ethyl, n-propyl, 1- methylethyl(isopropyl), n-butyl, n-pentyl, l,l-dimethylethyl(t-butyl), 3 -methylhexyl, 2- methylhexyl, ethenyl, prop-l-enyl, but-l-enyl, pent-l-enyl, penta- 1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.

As used herein, the term “messenger ribonucleic acid (mRNA)” refers to a singlestranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein.

As used herein, the term “an mRNA formulated in a lipid nanoparticle” refers to a structure where mRNA is 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.

As used herein, the term “pharmaceutically acceptable carrier” refers to any substance or vehicle 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, a lipid particle, etc.

As used herein, the term “ionizable lipid” has its ordinary meaning in the art and may refer to a lipid comprising one or more charged moieties. In some embodiments, an ionizable lipid may be positively charged, in which case it can be referred to as “cationic lipid”.

As used herein, the term “lipid nanoparticle” refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm). In some embodiments, lipid nanoparticles are included in a formulation that can be used to deliver an mRNA to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). In some embodiments, the lipid nanoparticle has a mean diameter of 50-200 nm. In some embodiments, the lipid nanoparticle comprises ionizable lipids, PEG-lipids, structural lipids, e.g., cholesterol, and neutral lipids, e.g., phospholipids. In one embodiment, the lipid nanoparticle comprises (i) at least one ionizable lipid compound having structure (I), (II) or (III), or the specific compounds disclosed above, (ii) at least one phospholipid selected from DOPE, DSPC, DPPC and DPyPE, (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.

An “effective amount” of the composition (e.g. mRNA) is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size, and extent of modified nucleosides) and other components of the therapeutic composition. For instance, an effective amount of the vaccine (e.g., mRNA) provides an induced or boosted immune response as a function of antigen production in the cell, preferably more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or a peptide antigen. Increased antigen production may be demonstrated by increased cell transfection (the percentage of cells transfected with the RNA, e.g., mRNA, vaccine), increased protein translation from the polynucleotide, decreased nucleic acid degradation (as demonstrated, for example, by increased duration of protein translation from a modified polynucleotide), or altered antigen specific immune response of the host cell.

The terms "subject," "patient," "individual," and the like are used interchangeably herein, and refer to any animal, any mammalian subject, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

DETAILED DESCRIPTION

1. The 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 (I):

In one embodiment, Ri is

In one embodiment, R2 and R3 are each independently a C10-C24 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R2 and R3 are each independently a C15-C21 alkyl group which optionally contains one, two or three double bonds, In another embodiment, R2 and R3 are each independently a C16-C20 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R2 and R3 are each independently a C17-C19 alkyl group which optionally contains one, two or three double bonds. In some embodiment, R2 and R3 may be the same or different from each other. In one embodiment, R2 and R3 are each independently a CIO alkyl group which optionally contains one, two or three double bonds. In another embodiment, R2 and R3 are each independently a Cl 1 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R2 and R3 are each independently a C12 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R2 and R3 are each independently a C13 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R2 and R3 are each independently a C14 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R2 and R3 are each independently a C15 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R2 and R3 are each independently a C16 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R2 and R3 are each independently a C17 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R2 and R3 are each independently a Cl 8 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R2 and R3 are each independently a C19 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R2 and R3 are each independently a C20 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R2 and R3 are each independently a C21 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R2 and R3 are each independently a C22 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R2 and R3 are each independently a C23 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R2 and R3 are each independently a C24 alkyl group which optionally contains one, two or three double bonds.

In some embodiment, the compound having structure (I) may comprise the following compounds.

Here, in one embodiment, R4 is

In one embodiment, R5 and R₆ are each independently a C10-C24 alkyl group which optionally contains one, two or three double bonds.

In another embodiment, R5 and R₆ are each independently a C15-C21 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R5 and R₆ are each independently a C16-C20 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R5 and R₆ are each independently a C17-C19 alkyl group which optionally contains one, two or three double bonds. In some embodiment, R5 and R₆ may be the same or different from each other. In one embodiment, R5 and R₆ are each independently a CIO alkyl group which optionally contains one, two or three double bonds. In another embodiment, R₅ and R₆ are each independently a C 11 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R5 and R₆ are each independently a C12 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R5 and R₆ are each independently a Cl 3 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R5 and R₆ are each independently a C14 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R5 and R₆ are each independently a C15 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R5 and R₆ are each independently a C16 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R5 and R₆ are each independently a C17 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R5 and R₆ are each independently a Cl 8 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R5 and R₆ are each independently a C19 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R5 and R₆ are each independently a C20 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R5 and R₆ are each independently a C21 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R5 and R₆ are each independently a C22 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R5 and R₆ are each independently a C23 alkyl group which optionally contains one, two or three double bonds. In another embodiment, R5 and R₆ are each independently a C24 alkyl group which optionally contains one, two or three double bonds.

In some embodiment, the compound having structure (II) may comprise the following compounds.

Here, in one embodiment, R7 is In another embodiment, Rs and R9

are each independently a C10-C24 alkyl group which optionally contains one, two or three double bonds.

In another embodiment, Rs and R9 are each independently a C15-C21 alkyl group which optionally contains one, two or three double bonds. In another embodiment, Rs and R9 are each independently a C16-C20 alkyl group which optionally contains one, two or three double bonds.

In another embodiment, Rs and R9 are each independently a C17-C19 alkyl group which optionally contains one, two or three double bonds. In some embodiment, Rs and R9 may be the same or different from each other. In one embodiment, Rs and R9 are each independently a CIO alkyl group which optionally contains one, two or three double bonds. In another embodiment, Rs and R9 are each independently a C 11 alkyl group which optionally contains one, two or three double bonds. In another embodiment, Rs and R9 are each independently a C12 alkyl group which optionally contains one, two or three double bonds. In another embodiment, Rs and R9 are each independently a C 13 alkyl group which optionally contains one, two or three double bonds. In another embodiment, Rs and R9 are each independently a C14 alkyl group which optionally contains one, two or three double bonds. In another embodiment, Rs and R9 are each independently a C15 alkyl group which optionally contains one, two or three double bonds. In another embodiment, Rs and R9 are each independently a C16 alkyl group which optionally contains one, two or three double bonds. In another embodiment, Rs and R9 are each independently a C17 alkyl group which optionally contains one, two or three double bonds. In another embodiment, Rs and R9 are each independently a Cl 8 alkyl group which optionally contains one, two or three double bonds. In another embodiment, Rs and R9 are each independently a C19 alkyl group which optionally contains one, two or three double bonds. In another embodiment, Rs and R9 are each independently a C20 alkyl group which optionally contains one, two or three double bonds. In another embodiment, Rs and R9 are each independently a C21 alkyl group which optionally contains one, two or three double bonds. In another embodiment, Rs and R9 are each independently a C22 alkyl group which optionally contains one, two or three double bonds. In another embodiment, Rs and R9 are each independently a C23 alkyl group which optionally contains one, two or three double bonds. In another embodiment, Rs and R9 are each independently a C24 alkyl group which optionally contains one, two or three double bonds.

In some embodiment, the compound having structure (III) may comprise the following compounds.

2. The lipid nanoparticle

The present disclosure also provides a lipid nanoparticle comprising the compound having structure (I), (II) or (III), or the specific compounds disclosed above. 2.1. Neutral lipids (Phospholipids) In some embodiments, the lipid component of a lipid nanoparticle composition can include one or more neutral lipids such as phospholipids including one or more (poly) unsaturated lipids. Without being bound by the theory, it is contemplated that phospholipids may assemble into one or more lipid bilayers structures.

Exemplary phospholipids that can form part of the present lipid nanoparticle composition include but are not limited to 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2 -dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1 -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), 1-hexadecyl- sn-glycero-3 -phosphocholine (Cl 6 Lyso PC), 1, 2 -dilinolenoyl-sn-glycero-3 -phosphocholine, 1 ,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, 1 ,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- diarachidonoyl-sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl -phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 carboxylate (DOPE- mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearioyl-2-oleoylphosphatidyethanol amine (SOPE), 1,2-dielaidoyl-sn- glycero-3-phophoethanolamine (transDOPE), and sphingomyelin (SM).

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

In certain embodiments, a lipid nanoparticle composition includes at least one phospholipid selected from DOPE, DSPC, DPPC and DPyPE. In certain embodiments, a lipid nanoparticle composition includes DOPE. In one embodiment, the lipid nanoparticle includes 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 composition can include one or more polymer conjugated lipids, such as PEGylated lipids (PEG-lipids). Without being bound by the theory, it is contemplated that a polymer conjugated lipid component in a nanoparticle composition can improve of colloidal stability and/or reduce protein absorption of the nanoparticles.

Exemplary cationic lipids that can be used in connection with the present disclosure include but are not limited to PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG-lipid may be 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 includes from about 0.5% to about 5% on a molar basis of the PEG-lipids 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 2%, or about 3% on a molar basis.

2.3. Structural Lipids

In some embodiments, the lipid component of a lipid nanoparticle composition can 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 include but are not limited to cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In certain embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof. In one embodiment, the lipid nanoparticles provided herein comprise a steroid or steroid analogue. In one embodiment, the steroid or steroid analogue is cholesterol.

In one embodiment, the lipid nanoparticle includes from about 30% to about 55% on a molar basis of the structural lipids e.g., from about 40 to about 55%, from about 45 to about 55%, from about 45 to about 50%, or about 45%, about 47%, or about 48% on a molar basis.

2.4. Ionizable lipids

In some embodiments, the lipid component of a lipid nanoparticle composition can include one or more ionizable lipids. In one embodiment, the lipid nanoparticle comprises (i) at least one ionizable lipid compound having structure (I), (II) or (III), or the specific compounds disclosed above. 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 include but are not limited to 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

In one embodiment, the lipid nanoparticle includes from about 30% to about 60% on a molar basis of the ionizable lipids e.g., from about 35 to about 55%, from about 35 to about 50%, from about 35 to about 45%, or about 35%, about 40%, or about 45% on a molar basis.

In one embodiment, the lipid nanoparticle comprises (i) at least one ionizable lipid compound having structure (I), (II) or (III), or the specific compounds disclosed above, (ii) at least one phospholipid selected from DOPE, DSPC, DPPC and DPyPE, (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 comprises (i) at least one ionizable lipid compound having structure (I), (II) or (III), or the specific compounds disclosed above, (ii) at least one phospholipid selected from DOPE, DSPC, DPPC and DPyPE, (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 35-45% ionizable lipid : 5-15% phospholipid : 0.5-5% PEG-lipid : 45-55% structural lipid.

In one embodiment, the lipid nanoparticle comprises (i) at least one ionizable lipid compound having structure (I), (II) or (III), or the specific compounds disclosed above, (ii) at least one phospholipid selected from DOPE, DSPC, DPPC and DPyPE, (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 40% ionizable lipid : 10% phospholipid : 2% PEG-lipid : 48% 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 50-200 nm.

3. The 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 structure (I), (II) or (III), or the specific compounds disclosed above. In one embodiment, in the above composition, mRNA is 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 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. The 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 structure (I), (II) or (III), or the specific compounds disclosed above to the subject or cell.

In some embodiment, the cell can be a mammalian cell, such as, but not limited to, a human cell. In another embodiment, the cell can 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 is administered by one or more of a variety of routes, including parenteral (e.g., subcutaneous, intracutaneous, intravenous, intraperitoneal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, 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 is 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 a non-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 have 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 subject 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 times 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 μg, administered for example as a single dose or as two 5 pg doses. In some embodiments, the effective amount is a total dose of 10 μg-300 μg. For example, the effective amount may be a total dose of 10 μg, 20 μg, 25 pg, 30 pg, 35 pg, 40 μg, 45 pg, 50 μg, 55 pg, 60 μg, 65 pg, 70 μg, 75 pg, 80 μg, 85 pg, 90 μg, 95 pg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg or 200 μg, 210 pg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290 μg or 300 μg. In some embodiments, the effective amount is a total dose of 10 μg-300 μg. In some embodiments, the effective amount is a total dose of 30 μg- 100 μg or 50 μ.g-200 ng.

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 are encompassed by the present disclosure. For example, the composition comprising mRNA formulated in the lipid nanoparticle may be administered three or four times.

EXAMPLE

The ionizable lipid compound and lipid nanoparticles were synthesized additionally using DOPE, DSPC, DPPC, DPyPE, cholesterol, DMG-PEG, DSPE-PEG, etc. and tested as follows.

1 -bromotetradecane and potassium carbonate was stirred overnight with 2- mercaptoethanol in acetonitrile. The reaction solution was extracted using ethyl acetate to yield Product Iwithout purification. 1H NMR (400 MHz, CDC13) 5 3.71 (t, J = 6.0 Hz, 2H), 2.73 (t, J = 6.0 Hz, 2H), 2.60 - 2.45 (m, 2H), 2.32 (s, 1H), 1.58 (t, J = 7.6 Hz, 2H), 1.26 (s, 22H), 0.88 (t, J = 6.7 Hz, 3H).

The Product 1 was stirred with triethylamine in dichloromethane and a solution of acryloyl chloride was added dropwise to the solution. After reacting overnight, the solution was extracted using dichloromethane to yield Product 2. 1H NMR (400 MHz, CDC13) 5 6.43 (dd, J = 17.4, 1.4 Hz, 1H), 6.13 (dd, J = 17.3, 10.5 Hz, 1H), 5.85 (dd, J = 10.4, 1.4 Hz, 1H), 4.31 (s, 2H), 2.78 (t, J = 7.0 Hz, 2H), 2,57 (t, J = 7.5 Hz, 2H), 1,59 (t, J = 7.5 Hz, 2H), 1,26 (s, 22H), 0.88 (t, J = 6.7 Hz, 3H). The Product 2 was reacted neat with l-(3-Aminopropyl)imidazole at 70°C for 48 hours. The crude product was used without further purification. ESI-MS of Im lipid [M+H]+. Theoretical mass: 782.59. Observed mass: 782.59.

Six different lipid nanoparticles were formulated by varying the molar percentage of the two ionizable lipids of Im lipid and DLin-MC3-DMA (MC3). The six formulations included 100% Im/0% MC3, 90% Im/10% MC3, 75% Im/25% MC3, 50% Im/ 50% MC3, 0% Im/100% MC3 and 25% Im/75% MC3. The lipids including the ionizable lipids, DOPE, cholesterol, and DMG-PEG were dissolved in ethanol at a concentration of 2 mg/ml and were mixed at a molar ratio of 40: 10:48:2, respectively. A molar ratio of 8: 1 was used between the amine group of the ionizable lipid and the phosphate group of the mRNA (Trilink Biotechnologies, L-7701). mRNA diluted in 5 mM citrate buffer (pH 5.0) was mixed with the lipid mixture at a 3:1 volume ratio. After incubating the sample for 30 minutes, the solution was concentrated using an Amicon filter (MWCO: 30,000 Da) to remove the ethanol. The encapsulation efficiency of mRNA was determined using QuantiFluor® RNA system (Promega). The particle size and surface charge of the LNPs were determined using Nanobrook Omni.

Flow cytometry was used to determine in vitro cellular uptake and transfection efficacy in both human Jurkat T cells and HeLa cells. The cells were seeded in a 96-well plate at a density of 40,000 cells/well. Different formulations containing 100 ng of mRNA were added to each well and were incubated for 18-20 hours at 37°C. The cells were washed (for suspension cells) or trypsinized (for adherent cells) and centrifuged at 300 x g. After the cells were diluted in PBS, flow cytometry analysis was performed using the FL-1 and FL-4 channels to quantify the amounts of cellular uptake and transfection efficiency, respectively.

The LNPs were characterized for particle size and surface charge using dynamic light scattering and zeta potential measurements (Fig. 5). LNPs showed a range of particle sizes of -120-175 nm. The surface charge of the LNPs ranged from +10 mV to -10 mV. Since these LNPs contained ionizable lipids, their surface charge was close to neutral when they were measured at physiological pH of 7.4.

LNPs with different compositions were tested against HeLa and Jurkat cells for both cellular uptake and mRNA transfection efficiencies (Figs. 6 and 7). The HeLa cells are relatively easy to transfect with transfecting agents and thus were chosen as a representative cell line. Human Jurkat cells are representative T lymphocytes which are known to be difficult to transfect. The LNPs were tested for cellular uptake using Cy5-labeled eGFP mRNA. Cy5 fluorescent dye was used to determine cellular uptake while eGFP fluorescence was used to determine the transfection efficiency of the mRNA encapsulated in the different LNP formulations. After 20-hour incubation, fluorescence was measured by flow cytometry. From all the different LNP formulations, -100% cellular uptake was obtained in HeLa cells. The positive control with Lipofectamine MessengerMax showed a cellular uptake efficacy of -82%. In Jurkat cells, the cellular uptake was -55% for the positive control, Lipofectamine MessengerMax. LNPs showed mixed results for the cellular uptake efficiencies in terms of different ionizable lipid ratios. Even the positive control with -55% cellular uptake showed -4% transfection efficiency.

Synthesis of Product 3 ( -9-Octadecen-l-yl 2-propenoate)

To a solution of oleyl alcohol (1 eq.) stirred with triethylamine (TEA) (2 eq.) in dichloromethane (DCM), acryloyl chloride (1.5 eq.) was added dropwise. The reaction was stirred overnight. The solution was extracted using DCM and the solvent was dried off to yield a pure sample of Product 3. 1H NMR (400 MHz, CDC13) 8 6.48 - 6.33 (m, 1H), 6.14 (d, J = 10.4 Hz, 1H), 5.81 (dd, J = 10.4, 1.5 Hz, 1H), 5,50 - 5.24 (m, 2H), 4,15 (t, J = 6.7 Hz, 2H), 2.01 (d, J = 6.2 Hz, 3H), 1.67 (s, 2H), 1.30 (dt, J = 17.4, 10.5 Hz, 22H), 0.88 (t, J = 6.6 Hz, 3H).

Synthesis of Product 4 (3- Alanine, N-(2-hvdroxyethyl)-N-r3-(9-octadecenyloxy)-3-oxopropyl]-, 9-octadecenyl ester, (Z,Z)- (9CD)

(9Z)-9-Octadecen-l-yl 2-propenoate (Product 3) (1 eq.) was stirred with 2-aminoethanol (0.6 eq.) neat at 70 °C for 48 hours. After monitoring the reaction using thin layer chromatography (TLC), water was added and the product was extracted using DCM. The solvent was dried off to yield Product 4.

Synthesis of Product 5 (2AEOAP2 lipid)

Product 4 (1 eq.) was dissolved in a solution of DCM and dimethylformamide (DMF). 1- Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (1.8 eq.) and 4-Dimethylaminopyridine (DMAP) (0.4 eq.) were added to the solution. 1 -Methylpiperidine -2-carboxylic acid hydrochloride (1.5 eq.) was added to the stirring solution. After stirring overnight, the solvent was reduced using a rotary evaporator. The Product 5 was used without further purification. Synthesis of Product 6 (2AEOAP4 lipid)

Product 4 (1 eq.) was dissolved in a mixed solution of DCM and DMF. 1 -Ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) (1.8 eq.) and 4-Dimethylaminopyridine (DMAP) (0.4 eq.) were added to the solution. l-Methyl-4-piperidinecarboxylic acid (1.5 eq.) was added to the stirring solution. After stirring overnight, the solvent was evaporated using a rotary evaporator. Product 6 was purified and isolated using column chromatography. Chemical Formula: C51H95N2O6+. MS (ESI): m/z (MH+) 831.7164

Synthesis of Product 7 (2AEOAD2 lipid)

Product 4 (1 eq.) was dissolved in a mixed solution of DCM and DMF. 1 -Ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) (1.8 eq.) and 4-Dimethylaminopyridine (DMAP) (0.4 eq.) were added to the solution. 3 -(Dimethylamino) propionic acid hydrochloride (1.5 eq.) was added to the stirring solution. After stirring overnight, the solvent was evaporated using a rotary evaporator. The Product 7 was used without further purification.

Synthesis of Product 8 (2AEOAD3 lipid)

Product 4 (1 eq.) was dissolved in a mixed solution of DCM and DMF. 1 -Ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) (1.8 eq.) and 4-Dimethylaminopyridine (DMAP) (0.4 eq.) were added to the solution. 4-Dimethylaminobutyric acid hydrochloride (1.5 eq.) was added to the stirring solution. After stirring overnight, the solvent was evaporated using a rotary evaporator. The Product 8 was used without further purification.

Synthesis of Product 9 (2-Propenoic acid, 9,12-octadecadienyl ester, (Z,Z)- (9CI))

To a solution of linoleyl alcohol (1 eq.) stirred with triethylamine (TEA) (2 eq.) in DCM, acryloyl chloride (1.5 eq.) was added dropwise. The reaction was stirred overnight. The solution was extracted using DCM and the solvent was dried off to yield a pure sample of Product 9 (2-Propenoic acid, 9,12-octadecadienyl ester, (Z,Z)-(9CI)). 1H NMR (400 MHz, CDC13) 5 6.40 (dd, J = 17.3, 1.5 Hz, 1H), 6.12 (dd, J = 17.3, 10.4 Hz, 1H), 5.81 (dd, J = 10.4, 1.5 Hz, 1H), 5.46 - 5.25 (m, 4H), 4.15 (t, J = 6.7 Hz, 2H), 2.77 (t, J = 6.5 Hz, 2H), 2.05 (q, J = 6.8 Hz, 4H), 1.67 (p, J = 6.8 Hz, 2H), 1.31 (qt, J = 13.7, 5.4 Hz, 16H), 0.89 (t, J = 6.7 Hz, 3H).

Synthesis of Product 10 (LA tail) 2-Propenoic acid, 9,12-octadecadienyl ester, (Z,Z)-(9CI) (Product 9) (1 eq.) was stirred with 2 -aminoethanol (0.6 eq.) neat at 70 °C for 48 hours. After monitoring the reaction using TLC, water was added and the product was extracted using DCM. The solvent was dried off to yield product (8).

Synthesis of Product 11 (2AELAP2 lipid)

Product 10 (1 eq.) was dissolved in a mixed solution of DCM and DMF. 1 -Ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) (1.8 eq.) and 4-Dimethylaminopyridine (DMAP) (0.4 eq.) were added to the solution. 1 -Methylpiperidine -2-carboxylic acid hydrochloride (1.5 eq.) was added to the stirring solution. After stirring overnight, the solvent was evaporated using a rotary evaporator. Product 11 was used without further purification.

Synthesis of Product 12 (2AELAP4 lipid)

Product 10 (1 eq.) was dissolved in a solution of DCM and DMF. 1 -Ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) (1.8 eq.) and 4-Dimethylaminopyridine (DMAP) (0.4 eq.) were added to the solution, l-methyl-4-piperidinecarboxylic acid (1.5 eq.) was added to the stirring solution. After stirring overnight, the solvent was evaporated using a rotary evaporator. Product 12 was purified and isolated using column chromatography. Chemical Formula: C51H91N2O6+. MS (ESI): m/z (MH+) 827.6849.

Synthesis of Product 13 (2AELAD2 lipid)

Product 10 (1 eq.) was dissolved in a mixed solution of DCM and DMF. 1 -Ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) (1.8 eq.) and 4-Dimethylaminopyridine (DMAP) (0.4 eq.) were added to the solution. 3 -(Dimethylamino) propionic acid hydrochloride (1.5 eq.) was added to the stirring solution. After stirring overnight, the solvent was evaporated using a rotary evaporator. Product 13 was used without further purification.

Synthesis of Product 14 (2AELAD3 lipid)

Product 10 (1 eq.) was dissolved in a mixed solution of DCM and DMF. 1 -Ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) (1.8 eq.) and 4-Dimethylaminopyridine (DMAP) (0.4 eq.) were added to the solution. 4-Dimethylaminobutyric acid hydrochloride (1.5 eq.) was added to the stirring solution. After stirring overnight, the solvent was evaporated using a rotary evaporator. Product 14 was used without further purification. Preparation of lipid nanoparticles including the ionizable lipid of Products 5 to 8 and 11 to 14

The lipids including the ionizable lipids (Products 5 to 8 and 11 to 14), DOPE, cholesterol, and DMG-PEG were dissolved in ethanol at a concentration of 2 mg/ml and mixed at a molar ratio of 40, 10, 48, and 2, respectively. A molar ratio of 8: 1 was used between the amine group of the ionizable lipid and the phosphate group of the mRNA. mRNA diluted in 6.25 mM citrate buffer (pH 5.0) was mixed with the lipid mixture at a 3:1 volume ratio. After incubating the sample for 30 minutes, the solution was concentrated using an Amicon fdter (MWCO: 30,000 Da) to remove the ethanol. The particle sizes and surface charges of the LNPs were determined using Nanobrook Omni.

Particle size and surface charge

The LNPs were characterized for particle size and surface charge using dynamic light scattering and zeta potential measurements (Fig. 22). LNPs showed a range of particle size of from - 100 to -200 nm. The surface charge of the LNPs ranged from -10 mV to -20 mV. Since these LNPs contained ionizable lipids, their surface charge was close to neutral when they were measured at physiological pH of 7.4.

In vitro cellular uptake and transfection efficiencies

LNPs with different compositions were tested against Jurkat cells for both cellular uptake and mRNA transfection efficiencies (Fig. 23). Human Jurkat cells are representative T lymphocytes which are known to be difficult to transfect. The LNPs were tested for cellular uptake using Cy5 -labeled eGFP mRNA. Cy5 fluorescent dye was used to determine the cellular uptake while eGFP fluorescence was used to determine the transfection efficiency of the mRNA encapsulated in the different LNP formulations. The cells were seeded in a 96-well plate at a density of 40,000 cells/well. Different LNP formulations containing 100 ng of mRNA were added to each well and were incubated for 21 hours at 37°C. The cells were washed and centrifuged at 300 x g. After the cells were diluted in PBS, flow cytometry analysis was performed quantify the amounts of cellular uptake and transfection efficiency. The cellular uptake varied from -2% to -90%. MC3 LNPs, used as a positive control, showed a cell uptake of 90% and a transfection efficiency -92%. LNPs showed mixed results for the cellular uptake efficiencies in terms of different ionizable lipid ratios. The transfection efficiencies for the different LNPs varied -10% to a maximum of -75%. The LNPs with the maximum transfection efficiencies were identified to be 5 LNPs (including Product 5), 6 LNPs (including Product 6), and 12 LNPs (including Product 12).

To a solution of cyclohexane-l,3,5-triol in dichloromethane, N, N'-dicyclohexyl carbodiimide and 4-dimethylaminopyridine will be added. A solution of 1 -methylpiperidine -2- carboxylic acid hydrochloride or l-methyl-4-piperidinecarboxylic acid will be added and stirred overnight. The reaction mixture will be monitored using thin layer chromatography to make sure the reactant is completely consumed. The reaction will be extracted using ethyl acetate and the desired Product 15 will be separated using column chromatography. After confirming the structure using NMR and MS, the next reaction would be performed. To synthesize Product 16, Product 15 will be stirred withN, N'-dicyclohexyl carbodiimide and 4-dimethylaminopyridine. Oleic acid or linoleic acid will be added and the reaction will be stirred overnight. The reaction mixture will be extracted and the desired product will be separated using column chromatography. Products 24(B)(a), Products 24(B)(b), Products 24(B)(c) and Products 24(B)(d) will be synthesized as described in FIG. 24. LNPs will be prepared using each of Products 24(B)(a), Products 24(B)(b), Products 24(B)(c) and Products 24(B)(d) and tested in the similar manner as described above.

To synthesize Product 17, linoleyl alcohol will be reacted with methanesulfonyl chloride in the presence of triethylamine (TEA) in dichloromethane (DCM). The reaction mixture will be extracted, and Product 17 will be confirmed using NMR and MS. To a solution of Product 17 in diethyl ether, magnesium bromide ethyl etherate will be added. The reaction mixture will be extracted, and Product 18 will be confirmed using NMR and MS. Magnesium turnings will be added to a dry flask, and then diethyl ether will be added. Then Product 18 will be dropwise added, and an exothermic reaction will be noticed. Iodine may be added to initiate the reaction. After the reaction is completed, ethyl formate will be dropwise added. The reaction mixture will be extracted, and the product will be separated by column chromatography. The reaction system will be degassed using nitrogen and reflux condensation will be used. Product 19 will be stirred with N, N'-dicyclohexyl carbodiimide, 4-dimethylaminopyridine, and l-Methylpiperidine-2- carboxylic acid hydrochloride, l-methyl-4-piperidinecarboxylic acid or IH-Imidazol-l-ylacetic acid will be added to produce Product 20. Products 25(B)(a) and Product 25(B)(b) will be synthesized as described in FIG. 25. LNPs will be prepared using each of Products 25(B)(a) and Product 25(B)(b) and tested in the similar manner as described above. Preparation of lipid nanoparticles using an ethanol dilution method

Different lipid nanoparticles (LNPs) were formulated by incorporating different ionizable lipids. DLin-MC3-DMA (MC3) LNPs were prepared as a control. The lipids including the ionizable lipids, DOPE, cholesterol, and DMG-PEG were dissolved in ethanol at a concentration of 2 mg/ml. Other helper lipids such as l,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), l,2-Dipalmitoyl-sn-glycero-3-Phosphatidylcholine (DPPC), 1,2-diphytanoyl-sn- glycero-3-phosphatidylethanolamine (DPyPE) could be used in place of DOPE. DSPE-PEG could be used in place of DMG-PEG. The composition of ionizable lipid varied from 40-60%, DOPE ranged from 10-20%, cholesterol ranged from 30-50% and DMG-PEG ranged from 1-5%, A molar ratio of ranging from 5:1 to 15:1 was used between the amine group of the ionizable lipid and the phosphate group of the mRNA. mRNA diluted in 5 mM citrate buffer (pH 5.0) was mixed with the lipid mixture at a 3: 1 volume ratio. After incubating the sample for 30 minutes, the solution was concentrated using an Amicon fdter (MWCO: 30,000 Da) to remove the ethanol. The encapsulation efficiency of mRNA was determined using QuantiFluor® RNA system (Promega). Particle size and surface charge of the LNPs were determined using Nanobrook Omni.

Claims

1. A compound having the following structure (I):

R2 and R3 are each independently a C10-C24 alkyl group which optionally contains one, two or three double bonds.

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

3. A compound having the following structure (II):

wherein R4 is and

R₅ and R₆ are each independently a C10-C24 alkyl group which optionally contains one, two or three double bonds.

4. The compound of claim 3 which is selected from the group consisting of the following compounds :

5. A compound having the following structure (III):

wherein R7 is

R8 and R9 are each independently a C10-C24 alkyl group which optionally contains one, two or three double bonds.

6. The compound of claim 5 which is selected from the group consisting of the following compounds :

7. A lipid nanoparticle comprising the compound of claim 1.

8. A lipid nanoparticle comprising the compound of claim 2.

9. A lipid nanoparticle comprising the compound of claim 3.

10. A lipid nanoparticle comprising the compound of claim 4.

11. A lipid nanoparticle comprising the compound of claim 5.

12. A lipid nanoparticle comprising the compound of claim 6.

13. A composition comprising an mRNA formulated in the lipid nanoparticle of claim

7.

14. A composition comprising an mRNA formulated in the lipid nanoparticle of claim

8.

15. A composition comprising an mRNA formulated in the lipid nanoparticle of claim

9.

16. A composition comprising an mRNA formulated in the lipid nanoparticle of claim

10.

17. A composition comprising an mRNA formulated in the lipid nanoparticle of claim

11.

18. A composition comprising an mRNA formulated in the lipid nanoparticle of claim 12.

19. A method of delivering an mRNA to a subject or cell comprising administering the composition of claim 13 to the subject or cell.

20. A method of delivering an mRNA to a subject or cell comprising administering the composition of claim 14 to the subject or cell.

21. A method of delivering an mRNA to a subject or cell comprising administering the composition of claim 15 to the subject or cell.

22. A method of delivering an mRNA to a subject or cell comprising administering the composition of claim 16 to the subject or cell.

23. A method of delivering an mRNA to a subject or cell comprising administering the composition of claim 17 to the subject or cell.

24. A method of delivering an mRNA to a subject or cell comprising administering the composition of claim 18 to the subject or cell.