Composition of lipid nanoparticles

Abstract

Provided herein are lipid nanoparticle compositions comprising a cationic lipid, a steroid, a polymer-bound lipid, and no more than 0.5 mole percent of a phospholipid, which can be used to deliver a therapeutic payload (e.g., mRNA, siRNA, DNA) for therapeutic or prophylactic purposes.

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims priority to International Patent Application No. PCT / CN2022 / 113414 filed on August 18, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] Provided herein are lipid nanoparticle (LNP) compositions comprising cationic lipids, steroids, polymer-bound lipids, and no more than 0.5 mole percent of phospholipids, which can be used to deliver therapeutic payloads (e.g., mRNA, siRNA, miRNA, non-coding RNA, DNA, antisense oligonucleotides, gene editing cassettes) for therapeutic or preventive purposes. Background Art

[0004] Therapeutic nucleic acids have great potential to revolutionize vaccination, gene therapy, cell therapy, and the treatment of cancer and genetic diseases. Since the first clinical studies of therapeutic nucleic acids began in the 2000s, the design, modification and delivery of nucleic acid molecules have made significant progress. A variety of nucleic acid-based therapies have been studied, including mRNA, siRNA, plasmid DNA, viral and bacterial vectors, and patient-derived cell therapies, more than a dozen of which have been approved by the U.S. Food and Drug Administration (FDA) for use in humans, and more are in clinical trials. However, nucleic acid therapeutics are still faced with multiple challenges, such as off-target effects, unwanted immune stimulation, and low delivery efficiency. For mRNA-based therapies, the rapid degradation of mRNA by nucleases in serum makes the delivery process more challenging.

[0005] Current LNP delivery systems typically include cationic lipids, phospholipids, cholesterol, and PEG-lipid conjugates. Four lipid component LNP formulations (e.g., with a typical lipid ratio of cationic lipid: cholesterol: DSPC: DSPC: DMG-PEG = 50: 38.5: 10: 1.5) have been shown to be successful in a variety of commercial drug products, but there is still room for improvement. There is still a need to develop simplified lipid nanoparticle compositions without sacrificing the delivery efficiency of nucleic acid payloads. Summary of the invention

[0006] In one embodiment, provided herein is a lipid nanoparticle comprising a reduced amount (eg, compared to conventional four-lipid component lipid nanoparticles) of a phospholipid component.

[0007] In one embodiment, the present invention provides a lipid nanoparticle, wherein the lipid nanoparticle comprises:

[0008] (a) a cationic lipid in an amount of about 40 mol% to about 75 mol% of the total lipids present in the nanoparticle, wherein the cationic lipid comprises (i) at least one tertiary amine or quaternary ammonium moiety, and (ii) at least one C 14 -C 42 an alkyl ester moiety;

[0009] (b) a steroid in an amount from about 22 mol % to about 59.5 mol % of the total lipids present in the nanoparticle;

[0010] (c) polymer-bound lipids in an amount from about 0.5 mol % to about 2.5 mol % of the total lipids present in the nanoparticle; and

[0011] (d) nucleic acids;

[0012] Provided that the lipid nanoparticles contain no more than 0.5 mole percent phospholipids.

[0013] In one embodiment, the cationic lipid has formula (I):

[0014]

[0015] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein G 1 , X 1 , R 1 , R x and R y As defined herein or elsewhere.

[0016] In one embodiment, the cationic lipid has formula (III):

[0017]

[0018] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein G 1 , X 1 , R 1 , R x and R y As defined herein or elsewhere.

[0019] In one embodiment, compared with conventional four lipid component lipid nanoparticles (such as four lipid component lipid nanoparticles wherein the molar ratio of cationic lipid: steroid: phospholipid: polymer-bound lipid is about 50:38.5:10:1.5), three lipid component lipid nanoparticles provided herein (without phospholipid component, or with reduced / insignificant amount of phospholipid component) have increased size. In one embodiment, the size of the three lipid component nanoparticles provided herein is about 110nm to about 130nm.

[0020] Also provided herein are pharmaceutical compositions comprising the lipid nanoparticles provided herein and a pharmaceutically acceptable excipient.

[0021] Also provided herein are methods of using the lipid nanoparticles provided herein or the pharmaceutical compositions provided herein for expressing mRNA in mammalian cells or mammalian tissues, for introducing protein-encoding mRNA into cells, for treating a disease or disorder in a human, or for treating a disease or disorder in a human caused by impaired protein expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 In vivo protein (hEPv) expression 6 hours after intravenous injection of LNPs containing lipid-5 is shown. LNP composition and properties are listed in Table 1 and Table 5. All expression data are normalized to formulation L-0. Error bars are shown to represent standard error of the mean (SEM).

[0023] Figure 2 In vivo protein (hEPO) expression 6 hours after intravenous administration of LNPs containing SM-102 is shown. LNP composition and properties are listed in Table 2 and Table 6. All expression data are normalized to formulation S-0. Error bars are shown to represent SEM.

[0024] Figure 3 In vivo protein (hEPO) expression 6 hours after intravenous administration of LNPs containing C1 or ALC-0315 is shown. LNP composition and properties are listed in Table 3, Table 4, Table 7 and Table 8. All expression data are normalized to formulation A-0 or C1-0. Error bars are shown to represent SEM.

[0025] Figure 4 In vivo protein (hEPO) expression 6 hours after intravenous administration of LNPs containing more than 3 mol% PEG-lipids is shown. All expression data are normalized to formulation L-0 or S-0. Error bars are shown to represent SEM.

[0026] Figure 5A , Figure 5B and Figure 5C Shown is the long-term stability of certain lipid-5 containing LNPs over the course of 4 months stored at room temperature, 2°C-8°C and -20°C, respectively, by evaluating the purity of the encapsulated mRNA using a fragment analysis (FA) assay.

[0027] Figure 5D and Figure 5E Shown are updated long-term stabilities of certain lipid-5-containing LNPs stored for up to 400 days at 2°C-8°C or -20°C, respectively.

[0028] Fig. 6A Shown is in vivo GFP expression 6 hours after intramuscular administration of certain lipid-5-containing LNPs; Figure 6B Shown are IL-6 levels normalized to the amount of GFP expressed.

[0029] Fig. 7A and Figure 7B Shown are changes in body weight of mice monitored over 7 days after intravenous and intramuscular administration of certain lipid-5-containing LNPs, respectively.

[0030] Figure 8 In vivo protein (hEPO) expression 6 hours after intravenous administration of LNPs containing C2 is shown. LNP composition and properties are listed in Tables 9 and 10. All expression data are normalized to formulation C2-0. Error bars are shown to represent SEM.

[0031] Fig. 9A and Fig. 9B Cyro-TEM images of LNPs composed of C1 / cholesterol / DMG-PEG (60% / 38.5% / 1.5%) and LNPs composed of C1 / cholesterol / DMG-PEG (65% / 33% / 2.0%) are shown, respectively. DETAILED DESCRIPTION

[0032] 6.1 General Technology

[0033] The techniques and procedures described or referenced herein include those generally well understood by those skilled in the art and / or commonly employed using routine methods, such as the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Current Protocols in Molecular Biology (Ausubel et al., eds., 2003).

[0034] 6.2 Terminology

[0035] Unless otherwise described, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. For the purpose of interpreting this specification, the following terminology description will be applied, and where appropriate, terms used in the singular will also include the plural form, and vice versa. All patents, applications, published applications and other publications are incorporated by reference in their entirety. If any description of a term set forth conflicts with any document incorporated herein by reference, the term description set forth below shall prevail.

[0036] As used herein and unless otherwise specified, the term "lipid" refers to a group of organic compounds, including but not limited to esters of fatty acids, and is generally characterized by being poorly soluble in water, but soluble in many non-polar organic solvents such as pentane, hexane, dichloromethane, chloroform, ethyl acetate and ether. Although lipids are generally poorly soluble in water, there are certain classes of lipids (e.g., lipids modified by polar groups, such as DMG-PEG2000) that have limited water solubility and can be soluble in water under certain conditions. Known types of lipids include biomolecules such as fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides and phospholipids. Lipids can be divided into at least three categories: (1) "simple lipids", which include fats and oils and waxes; (2) "complex lipids", which include phospholipids and glycolipids (e.g., DMPE-PEG2000); and (3) "derivatized lipids", such as steroids. Further, as used herein, lipids also encompass synthetic lipidoid compounds. The term "lipidoid compound", also referred to as "lipidoid" for short, refers to a lipid-like compound (eg, an amphiphilic compound having lipid-like physical properties).

[0037] The term "lipid nanoparticle" or "LNP" refers to a particle with at least one nanometer (nm) size (e.g., 1 to 1,000 nm), which contains one or more types of lipid molecules. LNP provided herein may further contain at least one non-lipid payload molecule (e.g., one or more nucleic acid molecules). In one embodiment, LNP comprises a non-lipid payload molecule partially or completely encapsulated in the lipid shell. In particular, in one embodiment, wherein the payload is a negatively charged molecule (such as a nucleic acid including RNA and DNA), and the lipid component of LNP comprises at least one cationic lipid. Without being bound by theory, it is expected that cationic lipids can interact with negatively charged payload molecules through electrostatic effects, and promote payload binding and / or encapsulation into LNP during LNP formation. Other lipids that can form a part of LNP as provided herein include but are not limited to neutral lipids and charged lipids, such as steroids, polymer-bound lipids and various zwitterionic lipids. In certain embodiments, LNP according to the present disclosure comprises one or more cationic lipids of formula (I) and (II) as described.

[0038] The term "cationic lipid" refers to a lipid that is positively charged at any pH value or hydrogen ion activity of its environment, or can be positively charged in response to the pH value or hydrogen ion activity of its environment (e.g., the environment of its intended use). Therefore, the term "cationic lipid" encompasses "permanent cationic lipids", "cationizable lipids" and "ionizable lipids". In certain embodiments, the positive charge in the cationic lipid is derived from the presence of a quaternary nitrogen atom. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that is positively charged in the environment of its intended use (e.g., at physiological pH). In certain embodiments, the cationic lipid is an ionizable lipid. In certain embodiments, the cationic lipid is a lipid of one or more formulas (I) or (II) as described herein.

[0039] The term "polymer-bound lipid" refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer-bound lipid is a pegylated lipid (PEG-lipid), wherein the polymer portion comprises polyethylene glycol.

[0040] As used herein and unless otherwise indicated, the term "alkyl" refers to a saturated straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms. The alkyl group does not contain any carbon-carbon double bonds or carbon-carbon triple bonds. In one embodiment, the alkyl group has, for example, one to twenty-four carbon atoms (C1-C 24 Alkyl), four to twenty carbon atoms (C4-C 20 Alkyl), six to sixteen carbon atoms (C6-C 16 alkyl), six to nine carbon atoms (C6-C9 alkyl), one to fifteen carbon atoms (C1-C 15 Alkyl), one to twelve carbon atoms (C1-C 12 alkyl), one to eight carbon atoms (C1-C8 alkyl), one to six carbon atoms (C1-C6 alkyl), two to twelve carbon atoms (C1-C 12 alkyl), 14 to 42 carbon atoms (C 14 -C 42 alkyl), one to six carbon atoms (C1-C6 alkyl), six to forty-two carbon atoms (C6-C 42 Alkyl), six to twenty-four carbon atoms (C6-C 24 Alkyl) or fourteen to twenty-four carbon atoms (C 14 -C 24 Alkyl) and it is connected to the rest of the molecule by a single bond. As non-limiting examples, alkyl includes methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, etc. Unless otherwise indicated, alkyl is optionally substituted.

[0041] As used herein and unless otherwise indicated, the term "alkylene" or "alkylene chain" refers to a straight or branched multivalent (e.g., divalent or trivalent) hydrocarbon chain that connects the rest of the molecule to a radical, which consists only of carbon and hydrogen and is saturated. In one embodiment, an alkylene group has, for example, one to twenty-four carbon atoms (C1-C 24 Alkylene), one to fifteen carbon atoms (C1-C 15 Alkylene), one to twelve carbon atoms (C1-C 12 alkylene), one to eight carbon atoms (C1-C8 alkylene), one to six carbon atoms (C1-C6 alkylene), two to four carbon atoms (C2-C4 alkylene), one to two carbon atoms (C1-C2 alkylene), or two to twelve carbon atoms (C2-C 12 Alkylene). As non-limiting examples, alkylene includes methylene, ethylene, propylene, isopropylene, n-butylene, tert-butylene, etc. The alkylene chain is connected to the rest of the molecule by a single bond and to the radical by a single bond. The connection point of the alkylene chain to the rest of the molecule and to the radical can be through one carbon or any two carbons in the chain. Unless otherwise indicated, the alkylene chain is optionally substituted.

[0042] As used herein and unless otherwise indicated, the term "cycloalkyl" refers to a saturated, non-aromatic, monocyclic or polycyclic hydrocarbon radical consisting only of carbon and hydrogen atoms. Cycloalkyls may include fused or bridged ring systems. In one embodiment, a cycloalkyl has, for example, 3 to 15 ring carbon atoms (C3-C4). 15 cycloalkyl), 3 to 10 ring carbon atoms (C3-C 10 The cycloalkyl radicals of the present invention are cycloalkyl radicals (cycloalkyl radicals) or 3 to 8 ring carbon atoms (C3-C8 cycloalkyl radicals). The cycloalkyl radicals are connected to the rest of the molecule by a single bond. As non-limiting examples, monocyclic cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl radicals. Examples of polycyclic cycloalkyl radicals include adamantyl, norbornyl, decahydronaphthyl, 7,7-dimethyl-bicyclo [2.2.1] heptyl radicals, etc. Unless otherwise indicated, cycloalkyl radicals are optionally substituted.

[0043] As used herein and unless otherwise indicated, the term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic hydrocarbon radical consisting only of carbon and hydrogen atoms and containing one or more carbon-carbon double bonds. Cycloalkenyl groups may include fused or bridged ring systems. In one embodiment, a cycloalkenyl group has, for example, 3 to 15 ring carbon atoms (C3-C 15 cycloalkenyl), 3 to 10 ring carbon atoms (C3-C 10The cycloalkenyl radical is optionally substituted with cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, etc. Unless otherwise indicated, the cycloalkenyl radical is optionally substituted.

[0044] As used herein and unless otherwise specified, the term "aryl" refers to a monocyclic aromatic group and / or a polycyclic monovalent aromatic group containing at least one aromatic hydrocarbon ring. In certain embodiments, an aryl group has 6 to 18 ring carbon atoms (C6-C 18 aryl), 6 to 14 ring carbon atoms (C6-C 14 aryl) or 6 to 10 ring carbon atoms (C6-C 10 The term "aryl" also refers to a bicyclic, tricyclic or other polycyclic hydrocarbon ring, wherein at least one ring is an aromatic ring, and the other rings may be saturated, partially unsaturated or aromatic rings, such as dihydronaphthyl, indenyl, dihydroindenyl or tetrahydronaphthyl (tetrahydronaphthyl / tetralinyl). Unless otherwise indicated, aryl is optionally substituted.

[0045] When a group described herein is referred to as being "substituted", it may be substituted with one or more any suitable substituents. Illustrative examples of substituents include, but are not limited to, those found in the exemplary compounds and embodiments provided herein, as well as: halogen atoms, such as F, Cl, Br or I; cyano; oxo (=O); hydroxyl (-OH); alkyl; alkenyl; alkynyl; cycloalkyl; aryl; -(C=O)OR'; -O(C=O)R'; -C(=O)R'; -OR'; -S(O) x R';-S-SR';-C(=O)SR';-SC(=O)R';-NR'R';-NR'C(=O)R';-C(=O)NR'R';-NR'C(=O)NR'R';-OC(=O)NR'R';-NR'C(=O)OR';-NR'S(O) x NR'R';-NR'S(O) x R'; and -S(O) x NR'R', wherein: R' is independently H, C1-C 15 alkyl or cycloalkyl, and x is 0, 1 or 2. In one embodiment, the substituent is C1-C 12In other embodiments, the substituent is a cycloalkyl. In other embodiments, the substituent is a halo, such as a fluoro group. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group (-OR'). In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amino group (-NR'R').

[0046] As used herein and unless otherwise indicated, the term "optional" or "optionally" (e.g., optionally substituted) means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur. For example, "optionally substituted alkyl" means that the alkyl may or may not be substituted, and that the description includes both substituted alkyls and alkyls without substitution.

[0047] As used herein and unless otherwise indicated, the term "pharmaceutically acceptable salt" includes both acid addition salts and base addition salts.

[0048] As non-limiting examples, pharmaceutically acceptable acid addition salts include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, acid), dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc.

[0049] As non-limiting examples, pharmaceutically acceptable base addition salts include salts prepared by adding an inorganic base or an organic base to a free acid compound. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. In one embodiment, the inorganic salt is an ammonium salt, a sodium salt, a potassium salt, a calcium salt, and a magnesium salt. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, denanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, phenethylbenzylamine, benzathine penicillin, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. In one embodiment, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0050] The compounds provided herein may contain one or more asymmetric centers, and thus may produce enantiomers, diastereomers, and other stereoisomeric forms, which may be defined as (R)- or (S)-, or as (D)- or (L)- for amino acids, according to absolute stereochemistry. Unless otherwise indicated, the compounds provided herein are intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for preparing / separating individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high performance liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other geometrically asymmetric centers, and unless otherwise indicated, the compounds are intended to include E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0051] As used herein and unless otherwise indicated, the term "isomer" refers to different compounds having the same molecular formula. "Stereoisomers" are isomers that differ only in the arrangement of their atoms in space. "Atropisomers" are stereoisomers resulting from hindered rotation about a single bond. "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A mixture of a pair of enantiomers in any ratio may be referred to as a "racemic" mixture. "Diastereomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other.

[0052] "Stereoisomers" may also include E and Z isomers or mixtures thereof, as well as cis and trans isomers or mixtures thereof. In certain embodiments, the compounds described herein are isolated as E or Z isomers. In other embodiments, the compounds described herein are mixtures of E and Z isomers.

[0053] "Tautomers" refers to isomeric forms of a compound that are in equilibrium with each other. The concentrations of the isomeric forms will depend on the environment in which the compound is located, and may vary depending on, for example, whether the compound is a solid or in an organic or aqueous solution.

[0054] It should also be noted that the compounds described herein may contain unnatural proportions of atomic isotopes at one or more atoms. For example, the compounds may be radiolabeled with a radioactive isotope, such as tritium ( 3 H), carbon-14 ( 14 C), carbon-11 ( 11 C) Fluorine-18 ( 18 C) Sulfur-35( 35 S), iodine-125 ( 125 I), or may be isotopically enriched, such as deuterium ( 2 H), carbon-13 ( 13 C) or nitrogen-15( 15 N). As used herein, an "isotopologue" is an isotopically enriched compound. The term "isotopically enriched" refers to an atom whose isotopic composition is different from the natural isotopic composition of the atom. "Isotopically enriched" may also refer to a compound containing at least one atom whose isotopic composition is different from the natural isotopic composition of the atom. The term "isotopic composition" refers to the amount of each isotope present in a given atom. Radiolabeled and isotopically enriched compounds can be used as therapeutic agents, such as cancer therapeutics; research reagents, such as binding assay reagents; and diagnostic agents, such as in vivo imaging agents. All isotopic variants of the compounds described herein, whether or not radioactive, are intended to be included within the scope of the embodiments provided herein. In one embodiment, isotopologues of the compounds described herein are provided, for example, isotopologues are enriched in deuterium, carbon-13, and / or nitrogen-15. As used herein, "deuterated" refers to a compound in which at least one hydrogen (H) has been replaced by deuterium (as D or 2 H) substitution, that is, the compound is enriched in deuterium at at least one position.

[0055] It should be noted that if there is a discrepancy between a depicted structure and the name of the structure, the depicted structure shall prevail.

[0056] As used herein and unless otherwise indicated, the term "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the U.S. Food and Drug Administration for acceptable use in humans or livestock.

[0057] The term "composition" is intended to encompass a product containing the specified ingredients (eg, mRNA molecules provided herein), optionally in the specified amounts.

[0058] As used interchangeably herein, the term "polynucleotide" or "nucleic acid" refers to a nucleotide polymer of any length, and includes, for example, DNA and RNA. Nucleic acids can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or analogs thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Nucleic acids can contain modified nucleotides, such as methylated nucleotides and their analogs. Nucleic acids can be in single-stranded or double-stranded form. As used herein and unless otherwise indicated, "nucleic acid" also includes nucleic acid mimetics, such as locked nucleic acids (LNA), peptide nucleic acids (PNA) and morpholino nucleic acids. As used herein, "oligonucleotide" refers to a short synthetic polynucleotide, which is generally but not necessarily less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Unless otherwise indicated, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of the nascent RNA transcript is called the transcription direction; the sequence region on the DNA chain that has the same sequence as the RNA transcript and is located 5' relative to the 5' end of the RNA transcript is called the "upstream sequence"; the sequence region on the DNA chain that has the same sequence as the RNA transcript and is located 3' relative to the 3' end of the RNA transcript is called the "downstream sequence".

[0059] As used herein, the term "non-naturally occurring" is intended to mean that the nucleic acid molecule does not exist in nature when used with reference to a nucleic acid molecule as described herein. Non-naturally occurring nucleic acids encoding viral peptides or proteins contain at least one genetic alteration or chemical modification that is not usually present in naturally occurring strains of the virus (including wild-type strains of the virus). Genetic alterations include, for example, modifications of expressible nucleic acid sequences that introduce heterologous peptides or polypeptides encoding the virus, other nucleic acid additions, nucleic acid deletions, nucleic acid substitutions, and / or other functional disruptions to the genetic material of the virus. Such modifications include, for example, modifications of coding regions and functional fragments thereof of heterologous, homologous, or heterologous and homologous polypeptides of viral species. Additional modifications include, for example, modifications of non-coding regulatory regions, wherein the modifications change the expression of a gene or operon. Additional modifications also include, for example, incorporating nucleic acid sequences into vectors such as plasmids or artificial chromosomes. Chemical modifications include, for example, one or more functional nucleotide analogs as described herein.

[0060] "Isolated nucleic acid" refers to nucleic acids, such as RNA, DNA or nucleic acid mixtures, that are substantially separated from other genomic DNA sequences and proteins or complexes (such as ribosomes and polymerases) that naturally accompany native sequences. "Isolated" nucleic acid molecules are nucleic acid molecules that are separated from other nucleic acid molecules that are present in the natural source of nucleic acid molecules. In addition, when manufactured by recombinant technology, "isolated" nucleic acid molecules, such as mRNA molecules, may be substantially free of other cell materials or culture media, or when chemically synthesized, they may be substantially free of chemical precursors or other chemicals. In a specific embodiment, one or more nucleic acid molecules encoding antigens as described herein are isolated or purified. The term includes nucleic acid sequences removed from their naturally occurring environment, and includes recombinant or cloned DNA or RNA isolates and chemically synthesized analogs or analogs synthesized by heterologous systems. Substantially pure molecules may include isolated forms of molecules.

[0061] The term "coding nucleic acid" or its grammatical equivalents when used to refer to nucleic acid molecules include: (a) nucleic acid molecules that can be transcribed to produce mRNA and then translated into peptides and / or polypeptides when in a natural state or by methods well known to those skilled in the art; and (b) mRNA molecules themselves. The antisense strand is the complementary sequence of such nucleic acid molecules, and the coding sequence can be inferred therefrom. The term "coding region" refers to the portion of a coding nucleic acid sequence that is translated into a peptide or polypeptide. The term "untranslated region" or "UTR" refers to the portion of a coding nucleic acid that is not translated into a peptide or polypeptide. Depending on the orientation of the UTR relative to the coding region of the nucleic acid molecule, the UTR is called a 5'-UTR if it is located at the 5' end of the coding region, and the UTR is called a 3'-UTR if it is located at the 3' end of the coding region.

[0062] As used herein, the term "mRNA" refers to a messenger RNA molecule comprising one or more open reading frames (ORFs), which can be translated by a cell or organism having the mRNA to produce one or more peptides or protein products. The region containing one or more ORFs is referred to as the coding region of the mRNA molecule. In certain embodiments, the mRNA molecule also comprises one or more untranslated regions (UTRs).

[0063] In certain embodiments, mRNA is a monocistronic mRNA comprising only one ORF. In certain embodiments, monocistronic mRNA encodes a peptide or protein comprising at least one epitope of a selected antigen (e.g., a pathogenic antigen or a tumor-associated antigen). In other embodiments, mRNA is a polycistronic mRNA comprising two or more ORFs. In certain embodiments, polycistronic mRNA encodes two or more peptides or proteins that may be identical or different from each other. In certain embodiments, each peptide or protein encoded by polycistronic mRNA comprises at least one epitope of a selected antigen. In certain embodiments, different peptides or proteins encoded by polycistronic mRNA each comprise at least one epitope of a different antigen. In any one of the embodiments described herein, at least one epitope may be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 epitopes of an antigen.

[0064] The term "nucleobase" encompasses purines and pyrimidines, including the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural or synthetic analogs or derivatives thereof.

[0065] As used herein, the term "functional nucleotide analogue" refers to a modified version of a classical nucleotide A, G, C, U or T that (a) retains the base pairing properties of the corresponding classical nucleotide and (b) contains at least one chemical modification of the (i) nucleobase, (ii) sugar group, (iii) phosphate group or (iv) any combination of (i) to (iii) of the corresponding natural nucleotide. As used herein, base pairing encompasses not only the classical Watson-Crick adenine-thymine, adenine-uracil or guanine-cytosine base pairs, but also base pairs formed between a classical nucleotide and a functional nucleotide analogue or between a pair of functional nucleotide analogues, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonds to be formed between a modified nucleobase and a classical nucleobase or between two complementary modified nucleobase structures. For example, a functional analogue of guanosine (G) retains the ability to base pair with cytosine (C) or a functional analogue of cytosine. An example of such non-classical base pairing is base pairing between modified nucleotides inosine and adenine, cytosine or uracil. As described herein, functional nucleotide analogs can be naturally occurring or non-naturally occurring. Therefore, nucleic acid molecules containing functional nucleotide analogs can have at least one modified nucleobase, sugar group and / or internucleoside linkage. Exemplary chemical modifications to the nucleobase, sugar group or internucleoside linkage of nucleic acid molecules are provided herein.

[0066] As used herein, the terms "translation enhancer element", "TEE" and "translation enhancer" refer to a region in a nucleic acid molecule that is used to promote the translation of a coding sequence of a nucleic acid into a protein or peptide product, such as via cap-dependent or cap-independent translation into a protein or peptide product. TEEs are typically located in the UTR region of a nucleic acid molecule (e.g., mRNA) and enhance the translation level of a coding sequence located upstream or downstream. For example, a TEE in the 5'-UTR of a nucleic acid molecule may be located between the promoter and the start codon of the nucleic acid molecule. Various TEE sequences are known in the art (Wellensiek et al. Genome-wide profiling of human cap-independent translation-enhancing elements, Nature Methods, August 2013; 10(8):747-750; Chappell et al. PNAS June 29, 2004 101(26)9590-9594). Some TEEs are known to be conserved in multiple species (Pánek et al. Nucleic Acids Research, Vol. 41, No. 16, Sept. 1, 2013, pp. 7625-7634).

[0067] As used herein, the term "siRNA" refers to "small interfering" or "short interfering RNA". siRNA is an RNA duplex of nucleotides that can target a target gene. "RNA duplex" refers to a structure formed by complementary pairing between two regions of an RNA molecule. siRNA "targets" a gene because the nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the targeted gene. In one embodiment, the length of the duplex of the siRNA is less than 30 nucleotides. In one embodiment, the length of the duplex of the siRNA is between 20 and 25 nucleotides. In one embodiment, the length of the duplex of the siRNA can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 18, 16, 14, 12 or 10 nucleotides. The RNA duplex portion of the siRNA can be a part of a hairpin structure. In addition to the duplex portion, the hairpin structure can also contain a loop portion located between the two sequences that form the duplex. The length of the loop can vary. In one embodiment, the length of the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides. The hairpin structure may also contain a 3' or 5' overhang. In one embodiment, the overhang is a 3' or 5' overhang of 0, 1, 2, 3, 4 or 5 nucleotides in length. The siRNA may be encoded by a nucleic acid sequence, and the nucleic acid sequence may also include a promoter. The nucleic acid sequence may also include a polyadenylation signal. In one embodiment, the polyadenylation signal is a synthetic minimum polyadenylation signal. In one embodiment, siRNA is used for "gene silencing". "Gene silencing" refers to inhibiting gene expression. Gene silencing may be mediated by the process of affecting transcription and / or by the process of affecting post-transcriptional mechanisms. In one embodiment, gene silencing occurs when siRNA initiates the degradation of the mRNA of the target gene in a sequence-specific manner via RNA interference. In one embodiment, siRNA interferes with the expression of a specific gene with a complementary nucleotide sequence by degrading mRNA after transcription, thereby preventing translation. In one embodiment, gene silencing may be allele-specific, which refers to the specific silencing of one allele of a gene. In one embodiment, siRNA is used to "knock down" a gene. "Knock down" refers to a gene silencing technique in which the expression of a target gene is reduced compared to the gene expression before the introduction of the siRNA, which can result in inhibition of the production of the target gene product. The term "reduced" is used herein to indicate a 1%-100% reduction in target gene expression. For example, expression can be reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, or 99%. siRNA can be used to guide the knockdown of gene expression.

[0068] The terms "polypeptide" and "protein" are used interchangeably herein and refer to polymers having more than fifty (50) amino acid residues linked by covalent peptide bonds. That is, the description of polypeptides applies equally to the description of proteins, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally occurring amino acids (e.g., amino acid analogs). As used herein, the terms encompass amino acid chains of any length, including full-length proteins (e.g., antigens).

[0069] In the case of peptides or polypeptides, as used herein, the term "derivative" refers to a peptide or polypeptide comprising an amino acid sequence of a viral peptide or protein or a fragment of a viral peptide or protein, which has been changed by introducing an amino acid residue substitution, deletion or addition. As used herein, the term "derivative" also refers to a viral peptide or protein, or a fragment of a viral peptide or protein, which has been chemically modified, for example, by making any type of molecule covalently attached to a polypeptide. For example, but not limited to, a viral peptide or protein or a fragment of a viral peptide or protein can be chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, by known protective groups / blocking group derivatization, proteolytic cleavage, chemical cleavage, preparation, tunicamycin metabolic synthesis, connection with a cell ligand or other protein, etc. Derivatives are modified in a manner different from naturally occurring or starting peptides or polypeptides in terms of the type or position of the connected molecule. Derivatives also include the absence of one or more chemical groups naturally present in viral peptides or proteins. In addition, the derivatives of viral peptides or proteins or fragments of viral peptides or proteins can contain one or more non-classical amino acids. In a specific embodiment, the derivative is a functional derivative of the native or unmodified peptide or polypeptide from which the derivative is derived.

[0070] The term "functional derivative" refers to a derivative that retains one or more functions or activities of the naturally occurring or starting peptide or polypeptide from which the derivative is derived. For example, a functional derivative of a coronavirus S protein may retain the ability to bind to one or more of its receptors on a host cell. For example, a functional derivative of a coronavirus N protein may retain the ability to bind to RNA or package the viral genome.

[0071] The term "consistency" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by alignment and comparison of sequences. "Amino acid sequence identity percentage (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and, if necessary, introducing spaces (achieving maximum sequence identity percentage) and not treating any conservative substitutions as part of sequence identity. Alignment for purposes of determining amino acid sequence identity percentage can be achieved in a variety of ways within the technical scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNAStar, Inc.) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment within the full length of the compared sequence.

[0072] "Modification" of an amino acid residue / position refers to a change in the primary amino acid sequence as compared to the starting amino acid sequence, wherein the change is caused by a sequence change involving the amino acid residue / position. For example, typical modifications include replacing a residue with another amino acid (e.g., conservative or non-conservative substitutions), inserting one or more (e.g., usually less than 5, 4 or 3) amino acids next to the residue / position, and / or deleting the residue / position.

[0073] In the case of a peptide or polypeptide, as used herein, the term "fragment" refers to a peptide or polypeptide comprising an amino acid sequence that is less than the full length. Such fragments may, for example, come from truncation of the amino terminus, truncation of the carboxyl terminus, and / or internal deletions of residues in the amino acid sequence. Fragments may, for example, be produced by selective RNA splicing or by in vivo protease activity. In certain embodiments, a fragment refers to at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 30 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 110 consecutive amino acid residues, at least 120 consecutive amino acid residues, at least 130 consecutive amino acid residues, at least 140 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 160 consecutive amino acid residues, at least 170 consecutive amino acid residues, at least 180 consecutive amino acid residues, at least 190 consecutive amino acid residues, at least 200 consecutive amino acid residues, at least 210 consecutive amino acid residues, at least 220 consecutive amino acid residues, at least 230 consecutive amino acid residues, at least 240 consecutive amino acid residues, at least 250 consecutive amino acid residues, at least 260 consecutive amino acid residues, at least 270 consecutive amino acid residues, at least 280 consecutive amino acid residues, at least 290 consecutive amino acid residues, at least 300 consecutive amino acid residues, at least 310 consecutive In some embodiments, the fragment of a polypeptide has an amino acid sequence of at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900 or at least 950 consecutive amino acid residues. In a specific embodiment, the fragment of a polypeptide retains at least one, at least two, at least three or more functions of the polypeptide.

[0074] As used herein in the context of a peptide or polypeptide (e.g., a protein), the term "immunogenic fragment" refers to a fragment of a peptide or polypeptide that retains the ability of the peptide or polypeptide to elicit an immune response (including an innate immune response and / or an adaptive immune response) when in contact with a mammalian immune system. In one embodiment, an immunogenic fragment of a peptide or polypeptide may be an epitope.

[0075] The term "antigen" refers to a substance that can be recognized by a subject's immune system (including an adaptive immune system) and can trigger an immune response (including an antigen-specific immune response) after contacting the subject with the antigen. In certain embodiments, the antigen is a protein associated with a diseased cell (such as a cell infected by a pathogen) or a neoplastic cell (e.g., a tumor-associated antigen (TAA)).

[0076] "Epitope" is a site on the surface of an antigen molecule that binds to a single antibody molecule, such as a local area on the surface of an antigen that can bind to one or more antigen binding regions of an antibody and has antigenic or immunogenic activity that can trigger an immune response in an animal, such as a mammal (e.g., a human). An epitope with immunogenic activity is a part of a polypeptide that triggers an antibody response in an animal. An epitope with antigenic activity is a part of a polypeptide to which an antibody binds, as determined by any method known in the art (including, for example, by immunoassay). Antigenic epitopes are not necessarily immunogenic. Epitopes are usually composed of chemically active surface groups of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics and specific charge characteristics. Antibody epitopes can be linear epitopes or conformational epitopes. Linear epitopes are formed by continuous amino acid sequences in proteins. Conformational epitopes are formed by amino acids that are discontinuous in the protein sequence but aggregate together when the protein is folded into its three-dimensional structure. When the three-dimensional structure of a protein is in an altered conformation, such as after activation or binding of another protein or ligand, an inductive epitope is formed. In certain embodiments, an epitope is a three-dimensional surface feature of a polypeptide. In other embodiments, an epitope is a linear feature of a polypeptide. Typically, an antigen has several or many different epitopes and can react with many different antibodies.

[0077] As used herein, the term "genetic vaccine" refers to a therapeutic or preventive composition comprising at least one nucleic acid molecule, the at least one nucleic acid molecule encoding an antigen associated with a target disease (e.g., an infectious disease or a neoplastic disease). Administration of a vaccine to a subject ("vaccination") allows the production of encoded peptides or proteins, thereby inducing an immune response to the target disease in the subject. In certain embodiments, the immune response includes an adaptive immune response, such as the production of antibodies to the encoded antigen, and / or the activation and proliferation of immune cells that can specifically eliminate diseased cells expressing the antigen. In certain embodiments, the immune response also includes an innate immune response. According to the present disclosure, the vaccine can be administered to a subject before or after the onset of clinical symptoms of the target disease. In one embodiment, vaccination of healthy or asymptomatic subjects makes the vaccinated subject immune or less sensitive to the development of the target disease. In one embodiment, vaccination of subjects showing symptoms of the disease improves the disease condition of the vaccinated subject or treats the disease.

[0078] The terms "innate immune response" and "innate immunity" are recognized in the art and refer to non-specific defense mechanisms initiated by the body's immune system when pathogen-associated molecular patterns are recognized, which involve different forms of cellular activity, including cytokine production and cell death through various pathways. As used herein, the innate immune response includes, but is not limited to, increased production of inflammatory cytokines (e.g., type I interferon or IL-10 production); activation of the NFκB pathway; increased proliferation, maturation, differentiation and / or survival of immune cells, and in some cases induction of apoptosis. Activation of innate immunity can be detected using methods known in the art, such as measuring (NF)-κB activation.

[0079] The terms "adaptive immune response" and "adaptive immunity" are recognized in the art and refer to antigen-specific defense mechanisms initiated by the body's immune system when it recognizes a specific antigen, including humoral responses and cell-mediated responses. As used herein, an adaptive immune response includes a cellular response triggered and / or enhanced by a vaccine composition, such as a genetic composition described herein. In one embodiment, the vaccine composition comprises an antigen that is a target of an antigen-specific adaptive immune response. In other embodiments, the vaccine composition, upon administration, allows the production of an antigen in an immunized subject that is a target of an antigen-specific adaptive immune response. Activation of an adaptive immune response can be detected using methods known in the art, such as measuring the production of antigen-specific antibodies or the level of antigen-specific cell-mediated cytotoxicity.

[0080] The term "antibody" is intended to include polypeptide products of B cells within the polypeptide class of immunoglobulins, which can bind to specific molecular antigens and are composed of two pairs of identical polypeptide chains, each pair having a heavy chain (about 50-70 kDa) and a light chain (about 25 kDa), each amino terminal portion of each chain comprising a variable region of about 100 to about 130 or more amino acids, and each carboxyl terminal portion of each chain comprising a constant region. See, for example, Antibody Engineering (Borrebaeck, ed., 2nd ed., 1995); and Kuby, Immunology (3rd ed., 1997). In a specific embodiment, a specific molecular antigen can be bound by an antibody provided herein, including a polypeptide, a fragment thereof or an epitope. Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intracellular antibodies, anti-idiotype (anti-Id) antibodies, and functional fragments of any of the above, which refers to a portion of the antibody heavy chain or light chain polypeptide that retains part or all of the binding activity of the antibody as the source of the fragment. Non-limiting examples of functional fragments include single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as antigen binding domains or molecules containing antigen binding sites (e.g., one or more CDRs of an antibody). Such antibody fragments can be found in, for example, Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22: 189-224; Plückthun and Skerra, 1989, Meth. Enzymol. 178: 497-515; and Day, Advanced Immunochemistry (2nd ed., 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules.

[0081] The term "administer" or "administration" refers to the operation of injecting or otherwise physically delivering a substance (e.g., a lipid nanoparticle composition described herein) present in vitro into a patient's body, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When treating a disease, disorder, illness, or symptom thereof, the administration of the substance is usually performed after the onset of the disease, disorder, illness, or symptom thereof. When preventing a disease, disorder, illness, or symptom thereof, the administration of the substance is usually performed before the onset of the disease, disorder, illness, or symptom thereof.

[0082] "Long-term" administration, as opposed to acute mode, refers to administration of one or more agents in a continuous mode (e.g., for a period of time, such as days, weeks, months or years), thereby maintaining the initial therapeutic effect (activity) over a longer period of time. "Intermittent" administration refers to treatment that is not continuous without interruption, but rather is cyclical in nature.

[0083] As used herein, the term "targeted delivery" or verb form "targeting" refers to a process in which the delivered agent (such as the therapeutic payload molecule in the lipid nanoparticle composition as described herein) reaches a specific organ, tissue, cell and / or intracellular compartment (referred to as the target location) compared to delivery to any other organ, tissue, cell or intracellular compartment (referred to as a non-target location). Targeted delivery can be detected using methods known in the art, such as by comparing the concentration of the delivered agent in the target cell population with the concentration of the delivered agent at the non-target cell population after systemic administration. In certain embodiments, targeted delivery makes the concentration at the target location at least 2 times higher than the concentration at the non-target location.

[0084] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms; eliminate symptoms and / or potential causes; prevent the occurrence of symptoms and / or their potential causes; and / or improve or remedy damage caused by a disease, disorder or condition, including, for example, an infection and neoplasm formation or associated therewith. In one embodiment, an effective amount is a therapeutically effective amount or a prophylactically effective amount.

[0085] As used herein, the term "therapeutically effective amount" refers to an amount of an agent (e.g., a vaccine composition) sufficient to reduce and / or improve the severity and / or duration of a given disease, disorder or condition, and / or its associated symptoms (e.g., infectious diseases, such as those caused by viral infection, or neoplastic diseases, such as cancer). The "therapeutically effective amount" of a substance / molecule / agent disclosed herein (e.g., a lipid nanoparticle composition described herein) may vary according to many factors, such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule / agent to elicit a desired response in the individual. A therapeutically effective amount includes an amount in which the therapeutically beneficial effects of a substance / molecule / agent outweigh any toxic or deleterious effects thereof. In certain embodiments, the term "therapeutically effective amount" refers to an amount of a lipid nanoparticle composition as described herein or a therapeutic agent or prophylactic agent (e.g., therapeutic mRNA) contained therein that is effective to "treat" a disease, disorder, or condition of a subject or mammal.

[0086] A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., prevent a disease, disorder, illness, or related symptom (e.g., an infectious disease, such as an infectious disease caused by a viral infection, or a neoplastic disease, such as cancer), delay its onset (or recurrence), or reduce the likelihood of its onset (or recurrence). Typically, but not necessarily, since a prophylactic dose is used for a subject before a disease, disorder, or illness, or at an early stage thereof, the prophylactic effective amount may be less than the therapeutically effective amount. A complete therapeutic or prophylactic effect may not occur by administering a single dose, but may occur only after a series of doses are administered. Therefore, a therapeutically or prophylactically effective amount may be administered in one or more administrations.

[0087] As used herein and unless otherwise indicated, the terms "treat," "treating," and "treatment" refer to alleviating in whole or in part a condition, disease, or disorder, or one or more symptoms associated with a condition, disease, or disorder, or slowing or stopping the further progression or worsening of those symptoms, or reducing or eradicating the cause of the condition, disease, or disorder itself.

[0088] As used herein and unless otherwise indicated, the terms "prevent," "preventing," and "prevention" refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms (e.g., an infectious disease, such as caused by a viral infection, or a neoplastic disease, such as cancer).

[0089] As used herein and unless otherwise indicated, the terms "manage", "managing" and "management" refer to the beneficial effects that a subject obtains from a therapy (e.g., a prophylactic or therapeutic agent) that does not result in a cure of the disease. In certain embodiments, one or more therapies (e.g., a prophylactic or therapeutic agent, such as a lipid nanoparticle composition described herein) are administered to a subject to "manage" an infectious or neoplastic disease, one or more symptoms thereof, thereby preventing progression or worsening of the disease.

[0090] The term "prophylactic agent" refers to any agent that can completely or partially inhibit the development, recurrence, onset, or spread of a disease and / or its associated symptoms in a subject.

[0091] The term "therapeutic agent" refers to any agent useful in treating, preventing or alleviating a disease, disorder or condition, including any agent useful in treating, preventing or alleviating one or more symptoms of a disease, disorder or condition and / or its associated symptoms.

[0092] The term "therapy" refers to any regimen, method and / or agent that can be used to prevent, control, treat and / or improve a disease, disorder or illness. In certain embodiments, the term "therapy" refers to biological therapy, supportive therapy and / or other therapies known to those skilled in the art, such as medical personnel, that can be used to prevent, control, treat and / or improve a disease, disorder or illness.

[0093] As used herein, a "prophylactically effective serum titer" is a serum titer of antibodies in a subject (e.g., a human) that completely or partially inhibits the development, recurrence, onset or spread of a disease, disorder or condition and / or symptoms associated therewith in the subject.

[0094] The term "side effect" encompasses unwanted and / or adverse effects of a therapy (e.g., a preventive or therapeutic agent). Unwanted effects are not necessarily adverse. Adverse effects of a therapy (e.g., a preventive or therapeutic agent) may be harmful, uncomfortable, or risky. Examples of side effects include diarrhea, cough, gastroenteritis, wheezing, nausea, vomiting, anorexia, abdominal cramps, fever, pain, weight loss, dehydration, alopecia, dyspnea, insomnia, dizziness, mucositis, nerve and muscle effects, fatigue, dry mouth, loss of appetite, rash or swelling at the site of administration, flu-like symptoms such as fever, chills, and fatigue, digestive tract problems, and allergic reactions. Other undesirable effects experienced by patients are numerous and known in the art. There are many effects described in Physician's Desk Reference (68th edition, 2014).

[0095] The terms "subject" and "patient" are used interchangeably. As used herein, in certain embodiments, the subject is a mammal, such as a non-primate (e.g., cattle, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans). In a specific embodiment, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) suffering from an infectious disease or a neoplastic disease. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing an infectious disease or a neoplastic disease.

[0096] By "substantially all" is meant at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

[0097] As used herein and unless otherwise indicated, the term "about" or "approximately" means an acceptable error of a particular value determined by one of ordinary skill in the art, which depends in part on the manner in which the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the terms "about" and "approximately" mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, within 0.5%, within 0.05%, or lower of a given value or range.

[0098] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0099] All publications, patent applications, accession numbers and other references cited in this specification are incorporated herein by reference in their entirety, and the extent of their citation is as if each individual publication or patent application is specifically and individually indicated to be incorporated by reference. The publications discussed herein are provided only for their disclosures prior to the filing date of the present application. Nothing herein should be construed as admitting that the present invention is not entitled to predate such publications by virtue of prior invention. In addition, the publication date provided may be different from the actual publication date, which may require independent confirmation.

[0100] A number of embodiments of the present invention have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the present invention. Therefore, the descriptions in the experimental section and examples are intended to illustrate rather than limit the scope of the invention described in the claims.

[0101] 6.3 Lipid Nanoparticle (LNP) Composition

[0102] Phospholipids are generally considered as auxiliary lipids or structural lipids in LNPs and play an important role in conventional LNP formulations. Phospholipids are believed to impart structural stability and promote in vivo gene delivery efficiency by mediating membrane fusion. Three commercial products based on LNPs ( and mRNA-1273) contain DSPC as an essential component (Eygeris et al. Chemistry of Lipid Nanoparticles for RNA Delivery. Acc. Chem. Res. 2022, 55, 2-12). However, as provided herein, it is shown that phospholipids are not essential in LNP formulations as generally believed.

[0103] In one embodiment, the present invention provides a lipid nanoparticle comprising a reduced amount of phospholipid components (e.g., compared to conventional four-lipid component lipid nanoparticles). In one embodiment, the LNP comprises no more than 0.5 molar percent of phospholipids.

[0104] In one embodiment, the present invention provides a lipid nanoparticle, wherein the lipid nanoparticle comprises:

[0105] (a) a cationic lipid in an amount of about 40 mol% to about 75 mol% of the total lipids present in the nanoparticle, wherein the cationic lipid comprises (i) at least one tertiary amine or quaternary ammonium moiety, and (ii) at least one C 14 -C 42 an alkyl ester moiety;

[0106] (b) a steroid in an amount from about 22 mol % to about 59.5 mol % of the total lipids present in the nanoparticle;

[0107] (c) polymer-bound lipids in an amount from about 0.5 mol % to about 2.5 mol % of the total lipids present in the nanoparticle; and

[0108] (d) nucleic acids;

[0109] Provided that the lipid nanoparticles contain no more than 0.5 mole percent phospholipids.

[0110] In one embodiment, the amount of cationic lipid is about 40mol% to about 75mol% of the total lipid present in the nanoparticle. In one embodiment, the amount of cationic lipid is about 40mol% to about 55mol%. In one embodiment, the amount of cationic lipid is about 45mol% to about 60mol%. In one embodiment, the amount of cationic lipid is about 50mol% to about 65mol%. In one embodiment, the amount of cationic lipid is about 55mol% to about 70mol%. In one embodiment, the amount of cationic lipid is about 60mol% to about 75mol%. In one embodiment, the amount of cationic lipid is about 40mol% to about 50mol%. In one embodiment, the amount of cationic lipid is about 45mol% to about 55mol%. In one embodiment, the amount of cationic lipid is about 50mol% to about 60mol%. In one embodiment, the amount of cationic lipid is about 55mol% to about 65mol%. In one embodiment, the amount of cationic lipid is about 60mol% to about 70mol%. In one embodiment, the amount of cationic lipid is about 65mol% to about 75mol%. In one embodiment, the amount of cationic lipid is about 40mol% to about 45mol%. In one embodiment, the amount of cationic lipid is about 45mol% to about 50mol%. In one embodiment, the amount of cationic lipid is about 50mol% to about 55mol%. In one embodiment, the amount of cationic lipid is about 55mol% to about 60mol%. In one embodiment, the amount of cationic lipid is about 60mol% to about 65mol%. In one embodiment, the amount of cationic lipid is about 65mol% to about 70mol%. In one embodiment, the amount of cationic lipid is about 70mol% to about 75mol%.

[0111] In one embodiment, the amount of cationic lipid is about 40 mol%, about 42.5 mol%, about 45 mol%, about 47.5 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 55.5 mol%, about 56 mol%, about 56.5 mol%, about 57 mol%, about 57.5 mol%, about 58 mol%, about 58.5 mol%, about 59 mol%, about 59.5 ... About 60 mol%, about 60.5 mol%, about 61 mol%, about 61.5 mol%, about 62 mol%, about 62.5 mol%, about 63 mol%, about 63.5 mol%, about 64 mol%, about 64.5 mol%, about 65 mol%, about 65.5 mol%, about 66 mol%, about 66.5 mol%, about 67 mol%, about 67.5 mol%, about 68 mol%, about 68.5 mol%, about 69 mol%, about 70 mol%, about 72.5 mol% or about 75 mol%.

[0112] In one embodiment, the amount of cationic lipid is about 42.5mol% of the total lipid present in the nanoparticle. In one embodiment, the amount of cationic lipid is about 45mol%. In one embodiment, the amount of cationic lipid is about 47.5mol%. In one embodiment, the amount of cationic lipid is about 50mol%. In one embodiment, the amount of cationic lipid is about 52.5mol%. In one embodiment, the amount of cationic lipid is about 55mol%. In one embodiment, the amount of cationic lipid is about 57.5mol%. In one embodiment, the amount of cationic lipid is about 60mol%. In one embodiment, the amount of cationic lipid is about 62.5mol%. In one embodiment, the amount of cationic lipid is about 65mol%. In one embodiment, the amount of cationic lipid is about 67.5mol%. In one embodiment, the amount of cationic lipid is about 70mol%.

[0113] In one embodiment, the amount of polymer-bound lipid is about 0.5mol% to about 2.5mol% of the total lipid present in the nanoparticle. In one embodiment, the amount of polymer-bound lipid is about 0.5mol% to about 0.75mol%, about 0.75mol% to about 1mol%, about 0.5mol% to about 1mol%, about 1mol% to about 1.25mol%, about 1.25mol% to about 1.5mol%, about 1mol% to about 1.5mol%, about 1mol% to about 1.5mol%, about 1.5mol% to about 1.75mol%, about 1.75mol% to about 2mol%, about 1mol% to about 2mol%, about 2mol% to about 2.25mol%, about 2.25mol% to about 2.5mol% or about 2mol% to about 2.5mol%. In one embodiment, the amount of polymer-bound lipid is about 0.5mol% to about 2mol%. In one embodiment, the amount of polymer-bound lipid is about 1mol% to about 2.5mol%. In one embodiment, the amount of polymer-bound lipid is about 0.5mol% to about 1.5mol%. In one embodiment, the amount of polymer-bound lipid is about 1mol% to about 2mol%. In one embodiment, the amount of polymer-bound lipid is about 1.5mol% to about 2.5mol%. In one embodiment, the amount of polymer-bound lipid is about 0.5mol% to about 1mol%. In one embodiment, the amount of polymer-bound lipid is about 1mol% to about 1.5mol%. In one embodiment, the amount of polymer-bound lipid is about 1.5mol% to about 2mol%. In one embodiment, the amount of polymer-bound lipid is about 2mol% to about 2.5mol%.

[0114] In one embodiment, the amount of polymer-bound lipid is about 0.5 mol%, about 0.6 mol%, about 0.7 mol%, about 0.8 mol%, about 0.9 mol%, about 1 mol%, about 1.1 mol%, about 1.2 mol%, about 1.25 mol%, about 1.3 mol%, about 1.35 mol%, about 1.4 mol%, about 1.45 mol%, about 1.5 mol%, about 1.55 mol%, about 1.6 mol%, about 1.65 mol%, about 1.7 mol%, about 1.75 mol%, about 1.8 mol%, about 1.85 mol%, about 1.9 mol%, about 1.95 mol%, about 2 mol%, about 2.1 mol%, about 2.2 mol%, about 2.3 mol%, about 2.4 mol%, or about 2.5 mol% of the total lipid present in the nanoparticle.

[0115] In one embodiment, the amount of polymer-bound lipid is about 1 mol% of the total lipid present in the nanoparticle. In one embodiment, the amount of polymer-bound lipid is about 1.25 mol%. In one embodiment, the amount of polymer-bound lipid is about 1.5 mol%. In one embodiment, the amount of polymer-bound lipid is about 1.75 mol%. In one embodiment, the amount of polymer-bound lipid is about 2 mol%. In one embodiment, the amount of polymer-bound lipid is about 2.25 mol%. In one embodiment, the amount of polymer-bound lipid is about 2.5 mol%.

[0116] In one embodiment, the amount of steroid is about 22mol% to about 59.5mol% of the total lipid present in the nanoparticle. In one embodiment, the amount of steroid is about 27mol% to about 54.5mol%. In one embodiment, the amount of steroid is about 32mol% to about 49.5mol%. In one embodiment, the amount of steroid is about 30mol% to about 50mol%. In one embodiment, the amount of steroid is about 33mol% to about 44mol%. In one embodiment, the amount of steroid is about 33mol% to about 38mol%. In one embodiment, the amount of steroid is about 38mol% to about 48mol%. In one embodiment, the amount of steroid is about 38mol% to about 44mol%. In one embodiment, the amount of steroid is about 44mol% to about 49mol%. In one embodiment, the amount of steroid is the remainder of the total lipid (except cationic lipid and polymer-bound lipid) present in the nanoparticle.

[0117] In one embodiment, the amount of steroid is about 22 mol % to about 23.5 mol %, about 23.5 mol % to about 29 mol %, about 29 mol % to about 33 mol %, about 33 mol % to about 36.5 mol %, about 36.5 mol % to about 38 mol %, about 38 mol % to about 42 mol %, about 42 mol % to about 44 mol %, about 33 mol % to about 44 mol %, about 44 mol % to about 49 mol %, about 49 mol % to about 54 mol %, or about 54 mol % to about 59.5 mol % of the total lipid present in the nanoparticle.

[0118] In one embodiment, the amount of steroid is about 33 mol% of the total lipid present in the nanoparticle. In one embodiment, the amount of steroid is about 38 mol%. In one embodiment, the amount of steroid is about 38.5 mol%. In one embodiment, the amount of steroid is about 39 mol%. In one embodiment, the amount of steroid is about 44 mol%. In one embodiment, the amount of steroid is about 48 mol%. In one embodiment, the amount of steroid is about 48.5 mol%. In one embodiment, the amount of steroid is about 49 mol%.

[0119] In one embodiment, the amount of cationic lipid is about 45 mol% to about 70 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0120] In one embodiment, the amount of cationic lipid is about 45 mol% to about 70 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0121] In one embodiment, the amount of cationic lipid is about 50 mol% to about 65 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1.5 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0122] In one embodiment, the amount of cationic lipid is about 45 mol% to about 65 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0123] In one embodiment, the amount of cationic lipid is about 45 mol% to about 65 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0124] In one embodiment, the amount of cationic lipid is about 50 mol% to about 65 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 0.5 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0125] In one embodiment, the amount of cationic lipid is about 50 mol% to about 65 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0126] In one embodiment, the amount of cationic lipid is about 50 mol% to about 65 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 1.5 mol% of the total lipid present in the nanoparticle.

[0127] In one embodiment, the amount of cationic lipid is about 50 mol% to about 65 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1.5 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0128] In one embodiment, the amount of cationic lipid is about 50 mol% to about 65 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 2 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0129] In one embodiment, the amount of cationic lipid is about 50 mol% to about 60 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 0.8 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0130] In one embodiment, the amount of cationic lipid is about 50 mol% to about 60 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0131] In one embodiment, the amount of cationic lipid is about 50 mol% to about 60 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1.5 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0132] In one embodiment, the amount of cationic lipid is about 50 mol% to about 60 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 2 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0133] In one embodiment, the amount of cationic lipid is about 55 mol% to about 65 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0134] In one embodiment, the amount of cationic lipid is about 55 mol% to about 65 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0135] In one embodiment, the amount of cationic lipid is about 55 mol% to about 65 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 0.5 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0136] In one embodiment, the amount of cationic lipid is about 55 mol% to about 65 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 0.8 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0137] In one embodiment, the amount of cationic lipid is about 60 mol% to about 65 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0138] In one embodiment, the amount of cationic lipid is about 60 mol% to about 65 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1.5 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0139] In one embodiment, the amount of cationic lipid is about 45 mol% to about 60 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0140] In one embodiment, the amount of cationic lipid is about 45 mol% to about 60 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0141] In one embodiment, the amount of cationic lipid is about 45 mol% to about 55 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0142] In one embodiment, the amount of cationic lipid is about 45 mol% to about 50 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0143] In one embodiment, the amount of cationic lipid is about 50 mol% to about 55 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0144] In one embodiment, the amount of cationic lipid is about 50 mol% to about 55 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0145] In one embodiment, the amount of cationic lipid is about 50 mol% to about 55 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1.5 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0146] In one embodiment, the amount of cationic lipid is about 55 mol% to about 60 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0147] In one embodiment, the amount of cationic lipid is about 55 mol% to about 60 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0148] In one embodiment, the amount of cationic lipid is about 55 mol% to about 60 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 0.5 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0149] In one embodiment, the amount of cationic lipid is about 55 mol% to about 60 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 0.8 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0150] In one embodiment, the amount of cationic lipid is about 60 mol% to about 70 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0151] In one embodiment, the amount of cationic lipid is about 40 mol% to about 55 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0152] In one embodiment, the amount of cationic lipid is about 40 mol% to about 55 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0153] In one embodiment, the amount of cationic lipid is about 67.5 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 2 mol% of the total lipid present in the nanoparticle.

[0154] In one embodiment, the amount of cationic lipid is about 65 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% of the total lipid present in the nanoparticle.

[0155] In one embodiment, the amount of cationic lipid is about 65 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1.5 mol% of the total lipid present in the nanoparticle.

[0156] In one embodiment, the amount of cationic lipid is about 65 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 2 mol% of the total lipid present in the nanoparticle.

[0157] In one embodiment, the amount of cationic lipid is about 65 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 2.5 mol% of the total lipid present in the nanoparticle.

[0158] In one embodiment, the amount of cationic lipid is about 60 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% of the total lipid present in the nanoparticle.

[0159] In one embodiment, the amount of cationic lipid is about 60 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1.5 mol% of the total lipid present in the nanoparticle.

[0160] In one embodiment, the amount of cationic lipid is about 60 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 2 mol% of the total lipid present in the nanoparticle.

[0161] In one embodiment, the amount of cationic lipid is about 60 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 2.5 mol% of the total lipid present in the nanoparticle.

[0162] In one embodiment, the amount of cationic lipid is about 55 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% of the total lipid present in the nanoparticle.

[0163] In one embodiment, the amount of cationic lipid is about 55 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1.5 mol% of the total lipid present in the nanoparticle.

[0164] In one embodiment, the amount of cationic lipid is about 55 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 2 mol% of the total lipid present in the nanoparticle.

[0165] In one embodiment, the amount of cationic lipid is about 55 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 2.5 mol% of the total lipid present in the nanoparticle.

[0166] In one embodiment, the amount of cationic lipid is about 50 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1 mol% of the total lipid present in the nanoparticle.

[0167] In one embodiment, the amount of cationic lipid is about 50 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 1.5 mol% of the total lipid present in the nanoparticle.

[0168] In one embodiment, the amount of cationic lipid is about 50 mol% of the total lipid present in the nanoparticle and the amount of polymer-bound lipid is about 2 mol% of the total lipid present in the nanoparticle.

[0169] In one embodiment, the lipid nanoparticles having an amount of a cationic lipid and an amount of a polymer-conjugated lipid provided herein have an increased nanoparticle size as described herein or elsewhere (e.g., from about 100 nm to about 140 nm, or from about 110 nm to about 130 nm) or as shown in the Examples.

[0170] In one embodiment, the lipid component of the nanoparticle composition includes one or more polymer-bound lipids, such as PEGylated lipids (PEG lipids). Without being bound by theory, it is expected that the polymer-bound lipid component in the nanoparticle composition can improve colloidal stability, reduce protein absorption of the nanoparticles and / or increase the blood circulation time of the nanoparticles.

[0171] In one embodiment, the polymer-bound lipid is a pegylated lipid, such as a pegylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropyl carbamate such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

[0172] In one embodiment, the polymer-bound lipid is a polyethylene glycol (PEG)-bound lipid. In one embodiment, the polymer-bound lipid is a PEG-diacylglycerol (PEG-DAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, a PEG-dimyristyloxypropyl (PEG-DMA) conjugate, a PEG-distearyloxypropyl (PEG-DSA) conjugate, or a mixture thereof. In one embodiment, the polymer-bound lipid is PEG-DAG. In one embodiment, the polymer-bound lipid is PEG-DAA. In one embodiment, the polymer-bound lipid is PEG-DMA. In one embodiment, the polymer-bound lipid is PEG-DSA. In one embodiment, the polymer-bound lipid is PEG-DMG. In one embodiment, the polymer-bound lipid is PEG-DMPE. In one embodiment, the polymer-bound lipid is DMG-PEG2000. In one embodiment, the polymer-bound lipid is DMPE-PEG2000.

[0173] In one embodiment, the polymer-bound lipid is a polyethylene glycol (PEG)-bound lipid, wherein the molecular weight of PEG is about 500 g / mol, about 750 g / mol, about 1000 g / mol, about 1500 g / mol, about 2000 g / mol, about 2500 g / mol, about 3000 g / mol, about 3500 g / mol, about 4000 g / mol, about 4500 g / mol, about 5000 g / mol or about 10000 g / mol.

[0174] In one embodiment, the polymer-bound lipid is selected from the group consisting of: DMG-PEG500, DMG-PEG1000, DMG-PEG1500, DMG-PEG2000, DMG-PEG2500, DMG-PEG3000, DMG-PEG5000, and DMG-PEG10000. In one embodiment, the polymer-bound lipid is selected from the group consisting of: DMPE-PEG500, DMPE-PEG1000, DMPE-PEG1500, DMPE-PEG2000, DMPE-PEG2500, DMPE-PEG3000, DMPE-PEG5000, and DMPE-PEG10000.

[0175] In one embodiment, the steroid is a glucocorticoid, a mineralocorticoid, clobetasol, cholesterol, or a cholesterol derivative. In one embodiment, the steroid is cholesterol. In one embodiment, the steroid is a cholesterol derivative. In one embodiment, the steroid is a glucocorticoid. In one embodiment, the steroid is a mineralocorticoid. In one embodiment, the steroid is clobetasol. In one embodiment, the steroid is a mixture of a glucocorticoid, a mineralocorticoid, clobetasol, cholesterol, or a cholesterol derivative.

[0176] In one embodiment, the polymer-bound lipid is a PEG-bound lipid, and the steroid is cholesterol or a cholesterol derivative. In one embodiment, the polymer-bound lipid is DMG-PEG2000, and the steroid is cholesterol or a cholesterol derivative. In one embodiment, the polymer-bound lipid is DMPE-PEG2000, and the steroid is cholesterol or a cholesterol derivative.

[0177] In one embodiment, the cationic lipid has a pKa of at least 6. In one embodiment, the cationic lipid has a pKa of at least 7. In one embodiment, the cationic lipid has a pKa of at least 8. In one embodiment, the cationic lipid has a pKa of at least 9. In one embodiment, the cationic lipid has a pKa of at least 9.5. In one embodiment, the cationic lipid has a pKa of at least 10. In one embodiment, the cationic lipid has a pKa of at least 11. In one embodiment, the cationic lipid has a pKa of at least 12.

[0178] In one embodiment, the cationic lipid comprises only one ester moiety, which is C 14 -C 42 In one embodiment, the ester moiety is C 14 -C 37 In one embodiment, the ester moiety is C14 -C 32 In one embodiment, the ester moiety is C 14 -C 27 In one embodiment, the ester moiety is C 14 -C 22 In one embodiment, the ester moiety is C 14 -C 17 In one embodiment, the ester moiety is C 15 In one embodiment, the ester moiety is C 16 In one embodiment, the ester moiety is C 17 Alkyl ester moiety.

[0179] In one embodiment, the cationic lipid comprises C 14 -C 42 In one embodiment, the cationic lipid comprises C 14 -C 42 and a second alkyl ester moiety. In one embodiment, the cationic lipid comprises two C 14 -C 42 In one embodiment, two C 14 -C 42 The alkyl ester moieties are identical. In one embodiment, the two C 14 -C 42 In one embodiment, the cationic lipid comprises two C 15 In one embodiment, the cationic lipid comprises two C 16 In one embodiment, the cationic lipid comprises C 10 The alkyl ester part and C 17 Alkyl ester moiety.

[0180] In one embodiment, at least one ester moiety of the cationic lipid is -C(=O)OR 1 or -OC(=O)R 1 , where R 1 It is a branched chain C 14 -C 42 In one embodiment, R 1 It is a branched chain C 14 -C 19 In one embodiment, R 1 It is a branched chain C 20 -C 25 In one embodiment, R 1 It is a branched chain C 26-C 31 In one embodiment, R 1 It is a branched chain C 32 -C 37 In one embodiment, R 1 It is a branched chain C 38 -C 42 In one embodiment, R 1 It is a branched chain C 15 In one embodiment, R 1 It is a branched chain C 16 In one embodiment, R 1 It is a branched chain C 17 Alkyl ester moiety.

[0181] In one embodiment, the second ester moiety of the cationic lipid is -C(=O)OR 2 or -OC(=O)R 2 , where R 2 Is a straight chain or branched C6-C 42 In one embodiment, R 2 Is a straight chain or branched C6-C 11 In one embodiment, R 2 Is it a straight chain or branched chain C 12 -C 17 In one embodiment, R 2 Is it a straight chain or branched chain C 18 -C 23 In one embodiment, R 2 Is it a straight chain or branched chain C 24 -C 29 In one embodiment, R 2 Is it a straight chain or branched chain C 30 -C 35 In one embodiment, R 2 Is it a straight chain or branched chain C 36 -C 42 alkyl.

[0182] In one embodiment, the second ester moiety of the cationic lipid is -C(=O)OR 2 or -OC(=O)R 2 , where R 2 It is a straight chain C6-C 42 In one embodiment, R 2 It is a straight chain C6-C 12 In one embodiment, R 2 It is a straight chain C 13 -C 18In one embodiment, R 2 It is a straight chain C 19 -C 25 In one embodiment, R 2 It is a straight chain C 26 -C 32 In one embodiment, R 2 It is a straight chain C 32 -C 42 In one embodiment, R 2 is a straight chain C9 alkyl group. 2 It is a straight chain C 10 In one embodiment, R 2 It is a straight chain C 11 alkyl.

[0183] In one embodiment, the second ester moiety of the cationic lipid is -C(=O)OR 2 or -OC(=O)R 2 , where R 2 It is a branched C6-C 12 In one embodiment, R 2 It is a branched chain C 13 -C 24 In one embodiment, R 2 It is a branched chain C 25 -C 42 In one embodiment, R 2 It is a branched chain C 15 In one embodiment, R 2 It is a branched chain C 16 In one embodiment, R 2 It is a branched chain C 17 alkyl.

[0184] In one embodiment, the cationic lipid comprises C 14 -C 42 In one embodiment, the cationic lipid comprises C 14 -C 42 In one embodiment, the second alkyl ester moiety and the third alkyl ester moiety are independently -C(=O)OR 2 or -OC(=O)R 2 , where each R 2 Independently straight chain C7-C 11 In one embodiment, the second alkyl ester moiety and the third alkyl ester moiety are part of a part, where each L2 are independently -OC(=O)R 2 , and each R 2 Independently straight chain C7-C 11 In one embodiment, the second alkyl ester moiety and the third alkyl ester moiety are part of a part, where each L 2 are independently -C(=O)OR 2 , and each R 2 Independently straight chain C7-C 11 alkyl.

[0185] In one embodiment, the cationic lipid comprises only one tertiary amine. In one embodiment, the cationic lipid comprises two tertiary amines. In one embodiment, the tertiary amine is protonated. In one embodiment, the tertiary amine is positively charged. In one embodiment, the tertiary amine is present in the form of a quaternary ammonium salt. In one embodiment, the tertiary amine is protonated when the pH is about 5 to about 9. In one embodiment, the tertiary amine is protonated at pH 5, pH 5.5, pH 6, pH 6.5, pH 7, pH 7.5, pH 8, pH 8.5 or pH 9.

[0186] In one embodiment, the cationic lipid comprises at least one tertiary amine and at least one C 14 -C 42 In one embodiment, the cationic lipid comprises a tertiary amine and two C 14 -C 42 In one embodiment, the cationic lipid comprises two tertiary amines and two C 14 -C 42 In one embodiment, the cationic lipid comprises a tertiary amine, a branched C 14 -C 42 The alkyl ester moiety and a linear C6-C 42 In one embodiment, the cationic lipid comprises two tertiary amines, a C 14 -C 42 Alkyl ester part, straight chain C7-C 11 The alkyl second ester portion and the linear C7-C 11 Alkyl tertiary ester moiety.

[0187] In one embodiment, the cationic lipid comprises part, where G 1 It is C2-C 12 Alkylene; X 1 is -C(=O)O- or -OC(=O)-; and R 1 It is a branched chain C 14 -C42 In one embodiment, G 1 is C2-C5 alkylene. 1 is C6-C9 alkylene. 1 It is C 10 -C 12 Alkylene.

[0188] In one embodiment, G 1 It is C2-C 12 Alkylene; X 1 is -C(=O)O- or -OC(=O)-; and R 1 It is a branched chain C 14 -C 19 In one embodiment, R 1 It is a branched chain C 20 -C 25 In one embodiment, R 1 It is a branched chain C 26 -C 31 In one embodiment, R 1 It is a branched chain C 32 -C 37 In one embodiment, R 1 It is a branched chain C 38 -C 42 alkyl.

[0189] In one embodiment, the cationic lipid has formula (I):

[0190]

[0191] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0192] G 1 It is C2-C 12 Alkylene;

[0193] X 1 is -C(=O)O- or -OC(=O)-;

[0194] R 1 It is a branched chain C 14 -C 42 alkyl;

[0195] R y is C1-C6 alkyl or -G 2 -X 2 -R 2 ;

[0196] G 2 It is C2-C12 Alkylene;

[0197] X 2 is -C(=O)O- or -OC(=O)-;

[0198] R 2 Is a straight chain or branched C6-C 42 alkyl;

[0199] R x is C1-C6 alkyl or -G 3 -R 3 ;

[0200] G 3 It is C2-C 12 Alkylene;

[0201] R 3 Yes-N(R 4 )R 5 OR 6 ;

[0202] R 4 It is C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 Aryl;

[0203] R 5 It is C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 Aryl;

[0204] or R 4 and R 5 Together with the nitrogen to which they are attached they form a ring portion;

[0205] R 6 is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 aryl; and

[0206] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, alkylene and cyclic moiety is independently optionally substituted.

[0207] In one embodiment, the cationic lipid has formula (II):

[0208]

[0209] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0210] In one embodiment, the cationic lipid has formula (II-A):

[0211]

[0212] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0213] G 1 is C5-C7 alkylene;

[0214] R 1 It is a branched chain C 14 -C 19 alkyl;

[0215] G 2 is C5-C7 alkylene;

[0216] R 2 It is a straight chain C7-C 13 alkyl;

[0217] G 3 It is a C2-C4 alkylene group.

[0218] In one embodiment, the cationic lipid has formula (II-A-1):

[0219]

[0220] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0221] In one embodiment, the cationic lipid has formula (II-B):

[0222]

[0223] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: G 1 is C5-C7 alkylene;

[0224] R 1 It is a branched chain C 14 -C 19 alkyl;

[0225] G 2 is C5-C7 alkylene;

[0226] R 2 It is a branched chain C 14 -C 19 alkyl;

[0227] G 3 It is a C2-C4 alkylene group.

[0228] In one embodiment, the cationic lipid has formula (II-B-1):

[0229] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0230] In one embodiment, the cationic lipid has formula (II-C):

[0231] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: G 1 is C5-C7 alkylene;

[0232] R 1 It is a branched chain C 14 -C 19 alkyl;

[0233] G 2 is C5-C7 alkylene;

[0234] R 2 It is a branched chain C 14 -C 19 alkyl;

[0235] G 3 is C2-C4 alkylene;

[0236] R 4 is a C3-C8 cycloalkyl group; and

[0237] R 5 It is a C2-C4 alkyl group substituted by a hydroxy group.

[0238] In one embodiment, the cationic lipid has formula (II-C-1):

[0239] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0240] In one embodiment, the cationic lipid has formula (III):

[0241]

[0242] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0243] G 1 It is C2-C 12 Alkylene;

[0244] X 1 is -C(=O)O- or -OC(=O)-;

[0245] R 1 It is a branched chain C 14 -C 42 alkyl;

[0246] G2 It is C2-C 12 Alkylene;

[0247] Each L 2 Independently for -X 2 -R 2 ;

[0248] Each X 2 are independently -C(=O)O- or -OC(=O)-;

[0249] Each R 2 are independently straight chain or branched C6-C 42 alkyl;

[0250] m is 1 or 2;

[0251] G 3 It is C2-C 12 Alkylene;

[0252] R 4 It is C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 Aryl;

[0253] R 5 It is C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 aryl; and

[0254] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, alkylene and cyclic moiety is independently optionally substituted.

[0255] In one embodiment, m is 1. In one embodiment, m is 2.

[0256] In one embodiment, the cationic lipid has formula (IV):

[0257]

[0258] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0259] y1 is an integer from 0 to 9.

[0260] In one embodiment, y1 is an integer from 2 to 6. In one embodiment, y1 is 2. In one embodiment, y1 is 3. In one embodiment, y1 is 4. In one embodiment, y1 is 5. In one embodiment, y1 is 6.

[0261] In one embodiment, each L 2Independently -OC(=O)R 2 In one embodiment, each L 2 are independently -OC(=O)R 2 , and each R 2 Independently straight chain C7-C 11 In one embodiment, each L 2 are independently -C(=O)OR 2 In one embodiment, each L 2 are independently -C(=O)OR 2 , and each R 2 Independently straight chain C7-C 11 alkyl.

[0262] In one embodiment, the cationic lipid has formula (IV-A):

[0263]

[0264] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0265] G 1 is C5-C7 alkylene;

[0266] R 1 It is a branched chain C 14 -C 19 alkyl;

[0267] Each R 2 Independently straight chain C7-C 13 alkyl;

[0268] G 3 is C2-C4 alkylene;

[0269] R 4 is a C3-C8 cycloalkyl group;

[0270] R 5 is a C2-C4 alkyl group substituted with a hydroxy group; and

[0271] y1 is an integer from 2 to 6.

[0272] In one embodiment, the cationic lipid has formula (IV-A-1):

[0273]

[0274] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0275] In one embodiment, G 1 is C2-C5 alkylene.1 is C5-C7 alkylene. 1 is C6-C9 alkylene. 1 It is C 10 -C 12 In one embodiment, G 1 is C2 alkylene. In one embodiment, G 1 is C3 alkylene. In one embodiment, G 1 is C4 alkylene. In one embodiment, G 1 is C5 alkylene. In one embodiment, G 1 is C6 alkylene. In one embodiment, G 1 is C7 alkylene. In one embodiment, G 1 is C8 alkylene. In one embodiment, G 1 is C9 alkylene. In one embodiment, G 1 It is C 10 In one embodiment, G 1 It is C 11 In one embodiment, G 1 It is C 12 Alkylene.

[0276] In one embodiment, X 1 is -C(=O)O-. In one embodiment, X 1 is -OC(=O)-. Unless otherwise specified, the left side is toward G 1 , and the right side faces R 1 .

[0277] In one embodiment, R 1 It is a branched chain C 14 -C 24 In one embodiment, R 1 It is a branched chain C 14 -C 19 In one embodiment, R 1 It is a branched chain C 20 -C 25 In one embodiment, R 1 It is a branched chain C 26 -C 31 In one embodiment, R 1 It is a branched chain C 32 -C 37 In one embodiment, R 1 It is a branched chain C 38 -C 42In one embodiment, R 1 Yes-R 7 -CH(R 8 )(R 9 ), where R 7 is C0-C5 alkylene, and R 8 and R 9 Independently for C2-C 10 In one embodiment, R 1 Yes-R 7 -CH(R 8 )(R 9 ), where R 7 is C0-C1 alkylene, and R 8 and R 9 are independently C4-C8 alkyl.

[0278] In one embodiment, R 1 It is a branched chain C 14 In one embodiment, R 1 It is a branched chain C 15 In one embodiment, R 1 It is a branched chain C 16 In one embodiment, R 1 It is a branched chain C 17 In one embodiment, R 1 It is a branched chain C 18 In one embodiment, R 1 It is a branched chain C 19 In one embodiment, R 1 It is a branched chain C 20 In one embodiment, R 1 It is a branched chain C 22 In one embodiment, R 1 It is a branched chain C 24 In one embodiment, R 1 It is a branched chain C 26 In one embodiment, R 1 It is a branched chain C 28 In one embodiment, R 1 It is a branched chain C 30 In one embodiment, R 1 It is a branched chain C 32 In one embodiment, R 1 It is a branched chain C 34 In one embodiment, R 1 It is a branched chain C 36 In one embodiment, R 1 It is a branched chain C38 In one embodiment, R 1 It is a branched chain C 40 In one embodiment, R 1 It is a branched chain C 42 alkyl.

[0279] In one embodiment, R y is C1-C6 alkyl. In one embodiment, R y In one embodiment, R y In one embodiment, R y is a C3 alkyl group. In one embodiment, R y is a C4 alkyl group. In one embodiment, R y is a C5 alkyl group. y It is a C6 alkyl group.

[0280] In one embodiment, R y Yes-G 2 -X 2 -R 2 .

[0281] In one embodiment, G 2 It is C2-C 12 In one embodiment, G 2 is C2-C5 alkylene. 2 is C5-C7 alkylene. 2 is C6-C9 alkylene. 2 It is C 10 -C 12 In one embodiment, G 2 is C2 alkylene. In one embodiment, G 2 is C3 alkylene. In one embodiment, G 2 is C4 alkylene. In one embodiment, G 2 is C5 alkylene. In one embodiment, G 2 is C6 alkylene. In one embodiment, G 2 is C7 alkylene. In one embodiment, G 2 is C8 alkylene. In one embodiment, G 2 is C9 alkylene. In one embodiment, G 2 It is C 10 In one embodiment, G 2 It is C 11 In one embodiment, G 2 It is C12 Alkylene.

[0282] In one embodiment, G 2 yes In one embodiment, G 2 yes In one embodiment, G 2 yes In one embodiment, G 2 yes In one embodiment, G 2 yes In one embodiment, G 2 yes In one embodiment, G 2 yes

[0283] In one embodiment, X 2 is -C(=O)O-. In one embodiment, X 2 is -OC(=O)-. Unless otherwise specified, the left side is toward G 2 , and the right side faces R 2 .

[0284] In one embodiment, R 2 Is a straight chain or branched C6-C 11 In one embodiment, R 2 Is it a straight chain or branched chain C 12 -C 17 In one embodiment, R 2 Is it a straight chain or branched chain C 18 -C 23 In one embodiment, R 2 Is it a straight chain or branched chain C 24 -C 29 In one embodiment, R 2 Is it a straight chain or branched chain C 30 -C 35 In one embodiment, R 2 Is it a straight chain or branched chain C 36 -C 42 alkyl.

[0285] In one embodiment, R 2 It is a straight chain C6-C 24 In one embodiment, R 2 It is a straight chain C7-C 13 In one embodiment, R 2 It is a straight chain C7-C 11 In one embodiment, R2 is a straight chain C8 alkyl group. 2 is a straight chain C9 alkyl group. 2 It is a straight chain C 10 In one embodiment, R 2 It is a straight chain C 11 In one embodiment, R 2 It is a straight chain C 12 In one embodiment, R 2 It is a straight chain C 13 In one embodiment, R 2 It is a straight chain C 14 In one embodiment, R 2 It is a straight chain C 15 In one embodiment, R 2 It is a straight chain C 16 alkyl.

[0286] In one embodiment, R 2 It is a branched chain C 14 -C 24 In one embodiment, R 2 It is a branched chain C 14 -C 19 In one embodiment, R 2 Yes-R 7 -CH(R 8 )(R 9 ), where R 7 is C0-C5 alkylene, and R 8 and R 9 Independently for C2-C 10 In one embodiment, R 2 Yes-R 7 -CH(R 8 )(R 9 ), where R 7 is C0-C1 alkylene, and R 8 and R 9 are independently C4-C8 alkyl.

[0287] In one embodiment, R 2 is a branched C6 alkyl group. 2 is a branched C8 alkyl group. 2 It is a branched chain C 10 In one embodiment, R 2 It is a branched chain C 12 In one embodiment, R 2 It is a branched chain C14 In one embodiment, R 2 It is a branched chain C 15 In one embodiment, R 2 It is a branched chain C 16 In one embodiment, R 2 It is a branched chain C 17 In one embodiment, R 2 It is a branched chain C 18 In one embodiment, R 2 It is a branched chain C 19 In one embodiment, R 2 It is a branched chain C 20 In one embodiment, R 2 It is a branched chain C 22 In one embodiment, R 2 It is a branched chain C 24 In one embodiment, R 2 It is a branched chain C 26 In one embodiment, R 2 It is a branched chain C 28 In one embodiment, R 2 It is a branched chain C 30 In one embodiment, R 2 It is a branched chain C 32 In one embodiment, R 2 It is a branched chain C 34 In one embodiment, R 2 It is a branched chain C 36 In one embodiment, R 2 It is a branched chain C 38 In one embodiment, R 2 It is a branched chain C 40 In one embodiment, R 2 It is a branched chain C 42 alkyl.

[0288] In one embodiment, R 1 , R 2 Each is independently one of the following structures:

[0289]

[0290] In one embodiment, R x is C1-C6 alkyl. In one embodiment, R x In one embodiment, R x In one embodiment, R xis a C3 alkyl group. In one embodiment, R x is a C4 alkyl group. In one embodiment, R x is a C5 alkyl group. x It is a C6 alkyl group.

[0291] In one embodiment, R x Yes-G 3 -R 3 .

[0292] In one embodiment, G 3 It is C2-C 12 In one embodiment, G 3 is C2-C4 alkylene. In one embodiment, G 3 is C2-C5 alkylene. 3 is C6-C9 alkylene. 3 It is C 10 -C 12 In one embodiment, G 3 is C2 alkylene. In one embodiment, G 3 is C3 alkylene. In one embodiment, G 3 is C4 alkylene. In one embodiment, G 3 is C5 alkylene. In one embodiment, G 3 is C6 alkylene. In one embodiment, G 3 is C7 alkylene. In one embodiment, G 3 is C8 alkylene. In one embodiment, G 3 is C9 alkylene. In one embodiment, G 3 It is C 10 In one embodiment, G 3 It is C 11 In one embodiment, G 3 It is C 12 Alkylene.

[0293] In one embodiment, R 3 Yes-N(R 4 )R 5 .

[0294] In one embodiment, R 4 It is C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 Aryl.

[0295] In one embodiment, R4 It is C1-C 12 In one embodiment, R 4 is C1-C8 alkyl. 4 is C1-C6 alkyl. In one embodiment, R 4 is C1-C4 alkyl. In one embodiment, R 4 In one embodiment, R 4 In one embodiment, R 4 In one embodiment, R 4 In one embodiment, R 4 In one embodiment, R 4 In one embodiment, R 4 In one embodiment, R 4 In one embodiment, R 4 It's Zheng Renji.

[0296] In one embodiment, R 4 is a C3-C8 cycloalkyl group. 4 In one embodiment, R 4 In one embodiment, R 4 In one embodiment, R 4 In one embodiment, R 4 In one embodiment, R 4 It's cyclooctyl.

[0297] In one embodiment, R 4 is C3-C8 cycloalkenyl. In one embodiment, R 4 In one embodiment, R 4 In one embodiment, R 4 In one embodiment, R 4 In one embodiment, R 4 In one embodiment, R 4 It is cyclooctenyl.

[0298] In one embodiment, R 4 It is C6-C 10 In one embodiment, R 4 It is phenyl.

[0299] In one embodiment, R 4 Not replaced.

[0300] In one embodiment, R 4 Substituted by one or more substituents selected from the group consisting of: oxo, -OR g 、-NR g C(=O)R h 、-C(=O)NR g R h 、-C(=O)R h 、-OC(=O)R h 、-C(=O)OR h AND-OR i -OH, where:

[0301] R g is independently H or C1-C6 alkyl at each occurrence;

[0302] R h is independently C1-C6 alkyl at each occurrence; and

[0303] R i is independently C1-C6 alkylene at each occurrence.

[0304] In one embodiment, R 4 is substituted with one or more hydroxyl groups. 4 is substituted with one hydroxyl group. 4 is substituted with one or more hydroxyl groups and one or more oxo groups. 4 Substituted with one hydroxyl group and one oxo group.

[0305] In one embodiment, R 5 It is C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 Aryl.

[0306] In one embodiment, R 5 It is C1-C 12 In one embodiment, R 5 is C1-C8 alkyl. 5 is C1-C6 alkyl. In one embodiment, R 5 is C1-C4 alkyl. In one embodiment, R 5 is C2-C4 alkyl. In one embodiment, R 5 In one embodiment, R 5 In one embodiment, R 5 In one embodiment, R 5In one embodiment, R 5 In one embodiment, R 5 In one embodiment, R 5 In one embodiment, R 5 In one embodiment, R 5 It's Zheng Renji.

[0307] In one embodiment, R 5 is a C3-C8 cycloalkyl group. 5 In one embodiment, R 5 In one embodiment, R 5 In one embodiment, R 5 In one embodiment, R 5 In one embodiment, R 5 It's cyclooctyl.

[0308] In one embodiment, R 5 is C3-C8 cycloalkenyl. In one embodiment, R 5 In one embodiment, R 5 In one embodiment, R 5 In one embodiment, R 5 In one embodiment, R 5 In one embodiment, R 4 It is cyclooctenyl.

[0309] In one embodiment, R 5 It is C6-C 10 In one embodiment, R 5 It is phenyl.

[0310] In one embodiment, R 4 and R 5 Together with the nitrogen to which they are attached they form a cyclic moiety.

[0311] In one embodiment, the cyclic portion (composed of R 4 and R 5In one embodiment, the cyclic moiety is a heterocycloalkyl. In one embodiment, the cyclic moiety is a 4- to 8-membered heterocycloalkyl. In one embodiment, the cyclic moiety is a 4-membered heterocycloalkyl. In one embodiment, the cyclic moiety is a 5-membered heterocycloalkyl. In one embodiment, the cyclic moiety is a 6-membered heterocycloalkyl. In one embodiment, the cyclic moiety is a 7-membered heterocycloalkyl. In one embodiment, the cyclic moiety is an 8-membered heterocycloalkyl.

[0312] In one embodiment, the cyclic portion (composed of R 4 and R 5 In one embodiment, the cyclic moiety is azetidin-1-yl. In one embodiment, the cyclic moiety is pyrrolidin-1-yl. In one embodiment, the cyclic moiety is piperidin-1-yl. In one embodiment, the cyclic moiety is azepan-1-yl. In one embodiment, the cyclic moiety is azocyclooctan-1-yl. In one embodiment, the cyclic moiety is morpholinyl. In one embodiment, the cyclic moiety is piperazin-1-yl. The point of attachment in these groups is with G 3 .

[0313] As described herein and unless otherwise indicated, R 5 The substitution pattern also applies to R 4 and R 5 Together with the nitrogen to which they are attached they form a ring portion.

[0314] In one embodiment, R 5 Not replaced.

[0315] In one embodiment, R 5 Substituted by one or more substituents selected from the group consisting of: oxo, -OR g 、-NR g C(=O)R h 、-C(=O)NR g R h 、-C(=O)R h 、-OC(=O)R h 、-C(=O)OR h AND-OR i -OH, where:

[0316] R g is independently H or C1-C6 alkyl at each occurrence;

[0317] R h is independently C1-C6 alkyl at each occurrence; and

[0318] R iis independently C1-C6 alkylene at each occurrence.

[0319] In one embodiment, R 5 is substituted with one or more hydroxyl groups. 5 is substituted with a hydroxyl group.

[0320] In one embodiment, R 5 is substituted with one or more hydroxyl groups and one or more oxo groups. 5 is substituted with one hydroxyl group and one oxo group. 5 is a C2-C4 alkyl group substituted with a hydroxyl group. 5 It is -CH2CH2OH.

[0321] In one embodiment, R 3 Yes-OR 6 .

[0322] In one embodiment, R 6 is hydrogen (i.e., R 3 is -OH). In one embodiment, R 6 It is C1-C 12 In one embodiment, R 6 is C1-C8 alkyl. 6 is C1-C6 alkyl. In one embodiment, R 6 is C1-C4 alkyl. In one embodiment, R 6 In one embodiment, R 6 In one embodiment, R 6 is a C3-C8 cycloalkyl group. 6 is C3-C8 cycloalkenyl. In one embodiment, R 6 It is C6-C 10 In one embodiment, R 6 In one embodiment, R 6 In one embodiment, R 6 was replaced.

[0323] In one embodiment, the cationic lipid is

[0324]

[0325] It should be understood that any embodiment of the compound provided herein as stated above, and any specific substituent and / or variable of the compound provided herein as stated above can be independently combined with other embodiments and / or substituents and / or variables of the compound to form the above embodiments not specifically stated. In addition, in the case of listing the substituent and / or variable list of any specific group or variable, it should be understood that each individual substituent and / or variable can be deleted from a specific embodiment and / or technical solution, and the remaining substituent and / or variable list will be considered to be within the scope of the embodiment provided herein.

[0326] It is understood that in the present specification, combinations of substituents and / or variables in the various formulae depicted are permissible only if such contributions result in stable compounds.

[0327] In one embodiment, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In one embodiment, the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the phospholipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC). In one embodiment, the phospholipid is 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC). In one embodiment, the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In one embodiment, the phospholipid is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). In one embodiment, the phospholipid is 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC). In one embodiment, the phospholipid is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC).

[0328] In one embodiment, the lipid nanoparticle comprises no more than 0.5mol% of phospholipids. In one embodiment, the lipid nanoparticle comprises no more than 0.01mol% of phospholipids, no more than 0.05mol% of phospholipids, no more than 0.1mol% of phospholipids, no more than 0.15mol% of phospholipids, no more than 0.2mol% of phospholipids, no more than 0.25mol% of phospholipids, no more than 0.3mol% of phospholipids, no more than 0.35mol% of phospholipids, no more than 0.4mol% of phospholipids, no more than 0.45mol% of phospholipids or no more than 0.5mol% of phospholipids. In one embodiment, the lipid nanoparticle does not comprise phospholipids.

[0329] In one embodiment, the lipid nanoparticle has a lipid: nucleic acid mass ratio of about 9: 1 to about 20: 1. In one embodiment, the lipid nanoparticle has a lipid: nucleic acid mass ratio of about 9: 1 to about 15: 1. In one embodiment, the lipid nanoparticle has a lipid: nucleic acid mass ratio of about 9: 1 to about 12: 1. In one embodiment, the lipid: nucleic acid mass ratio is about 9: 1. In one embodiment, the lipid: nucleic acid mass ratio is about 10: 1. In one embodiment, the lipid: nucleic acid mass ratio is about 11: 1. In one embodiment, the lipid: nucleic acid mass ratio is about 12: 1. In one embodiment, the lipid: nucleic acid mass ratio is about 13: 1. In one embodiment, the lipid: nucleic acid mass ratio is about 14: 1. In one embodiment, the lipid: nucleic acid mass ratio is about 15: 1. In one embodiment, the lipid: nucleic acid mass ratio is about 16: 1. In one embodiment, the lipid: nucleic acid mass ratio is about 17: 1. In one embodiment, the lipid: nucleic acid mass ratio is about 18: 1. In one embodiment, the lipid: nucleic acid mass ratio is about 19: 1. In one embodiment, the lipid:nucleic acid mass ratio is about 20:1.

[0330] In one embodiment, the size and encapsulation efficiency of the LNP provided herein are comparable to the size and encapsulation efficiency of conventional LNP formulations. In one embodiment, the LNP provided herein has improved in vitro and / or in vivo mRNA delivery efficiency, as evaluated by protein expression levels, compared to conventional LNP formulations.

[0331] In one embodiment, LNP is a vesicle comprising one or more lipid bilayers. In one embodiment, LNP comprises two or more concentric bilayers separated by an aqueous compartment. The lipid bilayers may be functionalized and / or cross-linked to each other. The lipid bilayers may include one or more ligands, proteins or channels.

[0332] In one embodiment, the nanoparticles comprise one or more nucleic acid molecules as a therapeutic payload.

[0333] In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is messenger RNA (mRNA), small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), small guide RNA (sgRNA), Cas9 RNA or a mixture thereof. In one embodiment, the nucleic acid is mRNA. In one embodiment, the nucleic acid is DNA. In one embodiment, the nucleic acid is plasmid DNA. In one embodiment, the nucleic acid is supercoiled DNA. In one embodiment, the nucleic acid is linear DNA.

[0334] In one embodiment, the mRNA is an mRNA encoding an antigen or a fragment or epitope thereof. In one embodiment, the mRNA is an mRNA encoding a pathogenic antigen. In one embodiment, the mRNA is an mRNA encoding a tumor-associated antigen. In one embodiment, the mRNA is an mRNA encoding a tumor-specific antigen.

[0335] In one embodiment, mRNA is a monocistronic mRNA. In one embodiment, mRNA is a polycistronic mRNA. In one embodiment, mRNA is a polycistronic mRNA comprising two or more open reading frames (ORFs). In one embodiment, polycistronic mRNA encodes two identical peptides or proteins. In one embodiment, polycistronic mRNA encodes two different peptides or proteins.

[0336] In one embodiment, each peptide or protein encoded by the polycistronic mRNA comprises at least one epitope of a selected antigen. In one embodiment, the different peptides or proteins encoded by the polycistronic mRNA each comprise at least one epitope of a different antigen.

[0337] In one embodiment, the at least one epitope can be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 epitopes of an antigen.

[0338] In one embodiment, the nucleic acid is a small interfering RNA (siRNA).

[0339] In one embodiment, upon administration of a nanoparticle composition comprising siRNA to a subject in need thereof, the siRNA selectively silences a gene associated with a particular disease, disorder, or condition.

[0340] In one embodiment, the nucleic acid is an immunomodulatory siRNA.

[0341] In one embodiment, the siRNA comprises a sequence that is complementary to an mRNA sequence encoding a target protein product.

[0342] In one embodiment, the nucleic acid molecule contains only canonical nucleotides selected from A (adenosine), G (guanosine), C (cytosine), U (uridine) and T (thymidine). In one embodiment, the nucleic acid comprises at least one functional nucleotide analog. In one embodiment, the functional nucleotide analog contains a non-classical nucleobase.

[0343] In one embodiment, the nucleic acid is chemically modified. In one embodiment, the nucleic acid comprises at least one chemical modification to a nucleobase. Exemplary modifications to a nucleobase include, but are not limited to, one or more substitutions or modifications, including, but not limited to, alkyl, aryl, halo, oxo, hydroxyl, alkoxy, and / or thio substitutions; one or more fused or open rings, oxidations, and / or reductions. In one embodiment, the nucleic acid comprises at least one chemical modification to a sugar group. In one embodiment, the nucleic acid comprises at least one chemical modification to a nucleoside linkage.

[0344] In one aspect, nucleic acid molecules as described herein are formulated for in vitro and in vivo delivery. In particular, in one embodiment, nucleic acid molecules are formulated into lipid nanoparticles. In one embodiment, lipid nanoparticles encapsulate nucleic acid molecules in lipid shells. In one embodiment, lipid shells protect nucleic acid molecules from degradation. In one embodiment, lipid nanoparticles also promote the transportation of sealed nucleic acid molecules to intracellular compartments and / or mechanisms to exert expected treatment or preventive functions. In certain embodiments, when present in lipid nanoparticles, nucleic acids resist nuclease degradation in aqueous solution. Lipid nanoparticles comprising nucleic acids and methods for making the same are known in the art, such as those disclosed in, for example, U.S. Patent Publication No. 2004 / 0142025, U.S. Patent Publication No. 2007 / 0042031, PCT Publication No. WO 2017 / 004143, PCT Publication No. WO 2015 / 199952, PCT Publication No. WO 2013 / 016058, and PCT Publication No. WO 2013 / 086373, the complete disclosure of each of which is incorporated herein by reference in its entirety for all purposes.

[0345] In one embodiment, the LNP compositions provided herein have a maximum dimension of 1 μm or less (e.g., ≤1 μm, ≤900 nm, ≤800 nm, ≤700 nm, ≤600 nm, ≤500 nm, ≤400 nm, ≤300 nm, ≤200 nm, ≤175 nm, ≤150 nm, ≤125 nm, ≤100 nm, ≤75 nm, ≤50 nm, or less), such as when measured by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or another method.

[0346] In one embodiment, lipid nanoparticles are provided herein, wherein the size of the nanoparticle is about 40nm to about 150nm. In one embodiment, the size of the nanoparticle is about 50nm to about 100nm. In one embodiment, the size of the nanoparticle is about 40nm to about 120nm. In one embodiment, the size of the nanoparticle is about 40nm to about 100nm. In one embodiment, the size of the nanoparticle is about 50nm to about 150nm. In one embodiment, the size of the nanoparticle is about 60nm to about 150nm. In one embodiment, the size of the nanoparticle is about 60nm to about 120nm. In one embodiment, the size of the nanoparticle is about 70nm to about 120nm. In one embodiment, the size of the nanoparticle is about 80nm to about 120nm.

[0347] As used herein and unless otherwise specified, the size of nanoparticles refers to the average size of the nanoparticle population or multiple nanoparticles. In one embodiment, compared with a reference preparation, the size of the three lipid component lipid nanoparticles provided herein (without a phospholipid component, or with a reduced / insignificant amount of phospholipid component) is increased; wherein the reference preparation is different from the lipid nanoparticles in that: (i) the reference preparation comprises phospholipids, and (ii) the molar ratio of cationic lipids: steroids: phospholipids: polymer-bound lipids is about 50:38.5:10:1.5. In one embodiment, the three lipid component nanoparticles provided herein have at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% size increase. In one embodiment, compared with a reference preparation, the size of the nanoparticles increases by at least about 50%. In one embodiment, the three lipid component nanoparticles provided herein have a size increase of no more than about 90%, no more than about 100% or no more than about 110%. In one embodiment, the size (of the three lipid component nanoparticles provided herein) is about 90nm to about 150nm. In one embodiment, the size is about 100nm to about 140nm. In one embodiment, the size is about 110nm to about 130nm. In one embodiment, the size is about 100nm to about 110nm. In one embodiment, the size is about 110nm to about 120nm. In one embodiment, the size is about 120nm to about 130nm.

[0348] In one embodiment, the size of the nanoparticles is about 40nm. In one embodiment, the size of the nanoparticles is about 50nm. In one embodiment, the size of the nanoparticles is about 55nm. In one embodiment, the size of the nanoparticles is about 60nm. In one embodiment, the size of the nanoparticles is about 65nm. In one embodiment, the size of the nanoparticles is about 70nm. In one embodiment, the size of the nanoparticles is about 75nm. In one embodiment, the size of the nanoparticles is about 80nm. In one embodiment, the size of the nanoparticles is about 85nm. In one embodiment, the size of the nanoparticles is about 90nm. In one embodiment, the size of the nanoparticles is about 95nm. In one embodiment, the size of the nanoparticles is about 100nm. In one embodiment, the size of the nanoparticles is about 105nm. In one embodiment, the size of the nanoparticles is about 110nm. In one embodiment, the size of the nanoparticles is about 115nm. In one embodiment, the size of the nanoparticles is about 120nm. In one embodiment, the size of the nanoparticles is about 125nm. In one embodiment, the size of the nanoparticles is about 125nm. In one embodiment, the size of the nanoparticles is about 130nm. In one embodiment, the size of the nanoparticles is about 135nm. In one embodiment, the size of the nanoparticles is about 140nm. In one embodiment, the size of the nanoparticles is about 145nm. In one embodiment, the size of the nanoparticles is about 150nm. In one embodiment, the size of the nanoparticles is about 155nm.

[0349] In one embodiment, the lipid nanoparticles provided herein have a nucleic acid encapsulation efficiency of at least about 80%. In one embodiment, the lipid nanoparticles provided herein have an mRNA encapsulation efficiency of at least about 80%. In one embodiment, the lipid nanoparticles provided herein have a DNA encapsulation efficiency of at least about 80%. In one embodiment, the lipid nanoparticles provided herein have a siRNA encapsulation efficiency of at least about 80%.

[0350] In one embodiment, the lipid nanoparticles provided herein have a nucleic acid encapsulation efficiency of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, or at least about 96%.

[0351] In one embodiment, the lipid nanoparticles provided herein have an mRNA encapsulation efficiency of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, or at least about 96%.

[0352] In one embodiment, the nanoparticles have a nucleic acid encapsulation efficiency of at least about 90%. In one embodiment, the nanoparticles have an mRNA encapsulation efficiency of at least about 90%. In one embodiment, the nanoparticles have a DNA encapsulation efficiency of at least about 90%. In one embodiment, the nanoparticles have a siRNA encapsulation efficiency of at least about 90%.

[0353] In one embodiment, the nucleic acid encodes RNA or protein, wherein the amount of RNA or protein expressed by the nucleic acid in the nanoparticle in a mammalian cell or a mammalian tissue is greater than the amount of RNA or protein expressed by the nucleic acid in a reference preparation; wherein the reference preparation differs from the lipid nanoparticle in that (i) the reference preparation comprises a phospholipid, and (ii) the molar ratio of cationic lipid:steroid:phospholipid:polymer-bound lipid is about 50:38.5:10:1.5.

[0354] In one embodiment, the amount of phospholipids in the reference preparation is greater than 5 mol% of the total lipids present in the reference preparation. In one embodiment, the amount of phospholipids in the reference preparation is greater than 10 mol% of the total lipids present in the reference preparation. In one embodiment, the amount of phospholipids in the reference preparation is greater than 15 mol% of the total lipids present in the reference preparation. In one embodiment, the amount of phospholipids in the reference preparation is greater than 20 mol% of the total lipids present in the reference preparation. In one embodiment, the amount of phospholipids in the reference preparation is greater than 25 mol% of the total lipids present in the reference preparation. In one embodiment, the amount of phospholipids in the reference preparation is greater than 30 mol% of the total lipids present in the reference preparation. In one embodiment, the amount of phospholipids in the reference preparation is greater than 35 mol% of the total lipids present in the reference preparation. In one embodiment, the amount of phospholipids in the reference preparation is greater than 40 mol% of the total lipids present in the reference preparation. In one embodiment, the amount of phospholipids in the reference preparation is greater than 45 mol% of the total lipids present in the reference preparation. In one embodiment, the amount of phospholipids in the reference preparation is greater than 50 mol% of the total lipids present in the reference preparation.

[0355] In one embodiment, the amount of phospholipids in the reference preparation is about 10 mol % of the total lipids present in the reference preparation.

[0356] In one embodiment, the reference formulation comprises DSPC.In one embodiment, the reference formulation comprises DOPE.

[0357] In one embodiment, the amount of RNA or protein expressed by the nucleic acid in the nanoparticles provided herein is at least about 50% greater than the amount of RNA or protein expressed by the nucleic acid in a reference preparation.

[0358] In one embodiment, the amount of RNA or protein expressed by the nucleic acids in the nanoparticles provided herein is at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% greater than the amount of RNA or protein expressed by the nucleic acids in a reference preparation.

[0359] In one embodiment, the amount of RNA or protein expressed by the nucleic acid in the nano-particle provided herein is at least about 100% more than the amount of RNA or protein expressed by the nucleic acid in the reference preparation. In one embodiment, the amount of RNA or protein expressed by the nucleic acid in the nano-particle provided herein is at least about 150% more than the amount of RNA or protein expressed by the nucleic acid in the reference preparation. In one embodiment, the amount of RNA or protein expressed by the nucleic acid in the nano-particle provided herein is at least about 200% more than the amount of RNA or protein expressed by the nucleic acid in the reference preparation. In one embodiment, the amount of RNA or protein expressed by the nucleic acid in the nano-particle provided herein is at least about 250% more than the amount of RNA or protein expressed by the nucleic acid in the reference preparation. In one embodiment, the amount of RNA or protein expressed by the nucleic acid in the nano-particle provided herein is at least about 300% more than the amount of RNA or protein expressed by the nucleic acid in the reference preparation.

[0360] In one embodiment, the amount of RNA or protein expressed by the nucleic acids in the nanoparticles provided herein is at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, or at least about 350% more than the amount of RNA or protein expressed by the nucleic acids in the reference preparation.

[0361] In one embodiment, provided herein is a pharmaceutical composition comprising the lipid nanoparticles provided herein and a pharmaceutically acceptable excipient. In one embodiment, the excipient is approved for human and for veterinary use.

[0362] In one embodiment, one or more excipients may constitute greater than 50% of the total mass or volume of the pharmaceutical composition comprising the nanoparticle composition. For example, one or more excipients may constitute 50%, 60%, 70%, 80%, 90% or more of the pharmaceutical composition.

[0363] In one embodiment, a pharmaceutical composition may comprise between 0.1% and 100% (wt / wt) of one or more nanoparticle compositions.

[0364] In one embodiment, the pharmaceutical composition comprises 100% (wt / wt) of the nanoparticle composition. In one embodiment, the pharmaceutical composition comprises about 75% (wt / wt) of the nanoparticle composition. In one embodiment, the pharmaceutical composition comprises about 50% (wt / wt) of the nanoparticle composition. In one embodiment, the pharmaceutical composition comprises about 25% (wt / wt) of the nanoparticle composition. In one embodiment, the pharmaceutical composition comprises 1% (wt / wt) of the nanoparticle composition. In one embodiment, the pharmaceutical composition comprises 0.1% (wt / wt) of the nanoparticle composition.

[0365] In one embodiment, provided herein is a method for expressing mRNA in a mammalian cell or a mammalian tissue, the method comprising delivering a lipid nanoparticle provided herein or a pharmaceutical composition provided herein to a mammalian cell or a mammalian tissue, wherein the nucleic acid is mRNA; and wherein the delivered mRNA is expressed in the mammalian cell or in the mammalian tissue.

[0366] In one embodiment, provided herein is a method for introducing mRNA encoding a protein into a cell, the method comprising contacting the cell with a lipid nanoparticle provided herein or a pharmaceutical composition provided herein.

[0367] In one embodiment, the cell is a prokaryotic cell. In one embodiment, the cell is a eukaryotic cell. In one embodiment, the cell is in a mammal. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is in a human.

[0368] In one embodiment, the cell is in the liver. In one embodiment, the cell is in the lung. In one embodiment, the cell is in the spleen. In one embodiment, the cell is in the colon. In one embodiment, the cell is in the kidney.

[0369] In one embodiment, the cell is an immune cell. Examples of immune cells include, but are not limited to, T cells, B cells, natural killer cells, neutrophils, monocytes, and macrophages.

[0370] In one embodiment, the cell is a genetically engineered cell.

[0371] In one embodiment, the cell is a stem cell. Examples of stem cells include, but are not limited to, pluripotent stem cells, mesenchymal stem cells, neural stem cells, and hematopoietic stem cells.

[0372] In one embodiment, the cell is a cancer cell.

[0373] In one embodiment, provided herein is a method for treating a disease or disorder in a human, the method comprising administering to the human a therapeutically effective amount of a lipid nanoparticle provided herein or a pharmaceutical composition provided herein.

[0374] In one embodiment, a method for treating a disease or condition caused by impaired expression of a protein in a human is provided herein, the method comprising administering to a human a therapeutically effective amount of a lipid nanoparticle provided herein or a pharmaceutical composition provided herein, wherein the nucleic acid is an mRNA encoding a protein. In one embodiment, the expression level of the protein increases by at least 10%, at least 20%, at least 40%, at least 60%, at least 80%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 750% or at least 1000%.

[0375] In one embodiment, a method for treating a disease or condition caused by overexpression of a protein in a human is provided herein, the method comprising administering to a human a therapeutically effective amount of a lipid nanoparticle provided herein or a pharmaceutical composition provided herein, wherein the nucleic acid is an siRNA that reduces the expression level of the protein. In one embodiment, the expression level of the protein is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%.

[0376] In one embodiment, the disease or condition is a viral infection. In one embodiment, the disease or condition is a liver disease. In one embodiment, the disease or condition is cancer.

[0377] In one embodiment, administration is intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, subcutaneous, or intradermal. In one embodiment, administration is intravenous. In one embodiment, administration is intramuscular. In one embodiment, administration is oral.

[0378] In one embodiment, the present invention provides a method for preventing a disease in a subject by administering to the subject a vaccine comprising a therapeutically effective amount of a lipid nanoparticle provided herein or a pharmaceutical composition provided herein, wherein the nucleic acid encodes an antigen associated with the disease, thereby triggering an immune response against the target disease in the subject. In one embodiment, the immune response is an adaptive immune response or an innate immune response or both.

[0379] In one embodiment, the vaccine is administered before clinical symptoms of the target disease appear, thereby making the vaccinated person immune or less susceptible to the development of the target disease. In one embodiment, the vaccine is administered after clinical symptoms appear, thereby alleviating symptoms and / or preventing recurrence.

[0380] In one embodiment, the antigen is a virus. In one embodiment, the antigen is a bacterium. In one embodiment, the antigen is a fungus. In one embodiment, the antigen is a parasite.

[0381] In one embodiment, the pathogen is a coronavirus (e.g., SARS, SARS-Cov-2, MERS), influenza virus, measles virus, human papillomavirus (HPV), rabies virus, meningitis virus, pertussis virus, tetanus virus, plague virus, hepatitis virus, and tuberculosis virus.

[0382] In one embodiment, the therapeutic payload comprises a nucleic acid sequence (e.g., mRNA) encoding a pathogenic protein or an antigenic fragment or epitope thereof that is specific to a pathogen. After administration to a vaccinated subject, the vaccine allows the encoded pathogenic protein (or an antigenic fragment or epitope thereof) to be expressed, thereby inducing immunity against the pathogen in the subject.

[0383] In one embodiment, provided herein is a method for preventing an infectious disease in a subject by administering to the subject a vaccine comprising a therapeutically effective amount of a lipid nanoparticle provided herein or a pharmaceutical composition provided herein, wherein the nucleic acid encodes a polypeptide associated with the infectious disease, thereby triggering an immune response against the target disease in the human.

[0384] In one embodiment, the infectious disease is a viral infection. In one embodiment, the infectious disease is a bacterial infection.

[0385] In one embodiment, the nucleic acid is mRNA encoding a polypeptide present in the antigen.

[0386] 6.3.1 Therapeutic Payload

[0387] Nanoparticle compositions as described herein may include one or more therapeutic and / or prophylactic agents. These therapeutic and / or prophylactic agents are sometimes referred to as "therapeutic payloads" or "payloads" in this disclosure. In some embodiments, the therapeutic payload can be administered in vivo or in vitro using nanoparticles as a delivery vehicle.

[0388] In one embodiment, the therapeutic payload is a nucleic acid as provided herein. In one embodiment, the nanoparticle composition comprises a therapeutic payload in addition to a nucleic acid as provided herein. In one embodiment, the nanoparticle composition comprises a therapeutic payload in place of a nucleic acid as provided herein.

[0389] In some embodiments, the nanoparticle composition comprises as a therapeutic payload: a small molecule compound (e.g., a small molecule drug), such as an anticancer agent (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), an anti-tumor agent (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside, arabinoside), anthracyclines, alkylating agents, platinum compounds, antimetabolites and nucleoside analogs such as methotrexate and purine and pyrimidine analogs), anti-infectives, local anesthetics (such as dibucaine and chlorpromazine), beta-adrenergic blocking agents (such as propranolol, timolol and labetalol), antihypertensives (such as clonidine and hydralazine), antidepressants (such as imipramine, amitriptyline and doxepin), anticonvulsants (such as phenytoin), antihistamines

[0013] The invention also includes antihistamines, antiseptics, and topical agents (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterials (e.g., gentamycin, ciprofloxacin, and cefoxitin), antifungals (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, anesthetics, and imaging agents.

[0390] In some embodiments, the therapeutic payload comprises a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and / or another therapeutic and / or prophylactic agent. Cytotoxins or cytotoxic agents include any agent that may be harmful to cells. Examples include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, such as maytansinol, rachelmycin (CC-1065), and analogs or homologs thereof. Radioactive ions include, but are not limited to, iodine (eg, iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium.

[0391] In other embodiments, the therapeutic payload of the nanoparticle compositions of the invention can include, but are not limited to, therapeutic and / or prophylactic agents, such as antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, razithromycin (CC-1065), melphalan, carmustine (BSNU), lomustine (

[0013] In some embodiments, the present invention includes but is not limited to: anti-infective agents such as streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines such as daunomycin (formerly daunomycin) and doxorubicin, antibiotics such as dactinomycin (formerly actinomycin), bleomycin, milramycin, and anthramycin (AMC), and anti-mitotic agents such as vincristine, vinblastine, paclitaxel, and maytansine compounds.

[0392] In some embodiments, the nanoparticle composition comprises biomolecules such as peptides and polypeptides as therapeutic payloads. The biomolecules forming part of the nanoparticle composition of the present invention can be of natural origin or synthetic. For example, in some embodiments, the therapeutic payload of the nanoparticle composition of the present invention can include, but is not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), factor VIR, luteinizing hormone releasing hormone (LHRH) analogs, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, cholera vaccine, and peptides and polypeptides.

[0393] 6.3.1.1 Additional description of nucleic acids

[0394] In some embodiments, the nanoparticle compositions of the present invention include one or more nucleic acid molecules (e.g., DNA or RNA molecules) as therapeutic payloads. Exemplary forms of nucleic acid molecules that can be included in the nanoparticle compositions of the present invention as therapeutic payloads include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA), ribonucleic acid (RNA), including messenger mRNA (mRNA), hybrids thereof, RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces the formation of triple helices, aptamers, vectors, and the like. In certain embodiments, the therapeutic payload comprises RNA. RNA molecules that can be included in the nanoparticle compositions of the present invention as therapeutic payloads include, but are not limited to, shortmers, agomirs, antagomirs, antisenses, ribozymes, small interfering RNAs (siRNAs), asymmetric interfering RNAs (aiRNAs), microRNAs (miRNAs), Dicer-substrate RNAs (dsRNAs), small hairpin RNAs (shRNAs), transfer RNAs (tRNAs), messenger RNAs (mRNAs), and other forms of RNA molecules known in the art. In certain embodiments, the RNA is mRNA.

[0395] In one embodiment, the nanoparticle composition of the present invention comprises one or more nucleic acid molecules (e.g., DNA or RNA molecules) as a therapeutic payload. Exemplary forms of nucleic acid molecules that can be included in the nanoparticle composition of the present invention as a therapeutic payload include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA), ribonucleic acid (RNA), including messenger mRNA (mRNA), its hybrids, RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces the formation of triple helices, aptamers, vectors, etc. In certain embodiments, the therapeutic payload comprises RNA. RNA molecules that can be included in the nanoparticle composition of the present invention as a therapeutic payload include, but are not limited to, shortmers, agomirs, antagomirs, antisenses, ribozymes, small interfering RNAs (siRNAs), asymmetric interfering RNAs (aiRNAs), microRNAs (miRNAs), Dicer-substrate RNAs (dsRNAs), small hairpin RNAs (shRNAs), transfer RNAs (tRNAs), messenger RNAs (mRNAs), and other forms of RNA molecules known in the art. In specific embodiments, the RNA is mRNA.

[0396] In other embodiments, the nanoparticle composition comprises siRNA molecules as therapeutic payloads. In particular, in one embodiment, the siRNA molecules can selectively interfere with and downregulate the expression of target genes. For example, in one embodiment, after administering a nanoparticle composition comprising siRNA to a subject in need, the siRNA payload selectively silences genes associated with a particular disease, disorder or condition. In one embodiment, the siRNA molecule comprises a sequence complementary to an mRNA sequence encoding a target protein product. In one embodiment, the siRNA molecule is an immunomodulatory siRNA.

[0397] In one embodiment, the nanoparticle composition comprises a shRNA molecule or a vector encoding an shRNA molecule as a therapeutic payload. In particular, in one embodiment, the therapeutic payload produces shRNA in the target cell after being administered to the target cell. Constructs and mechanisms associated with shRNA are well known in the relevant art.

[0398] In one embodiment, nanoparticle compositions include mRNA molecules as therapeutic payloads. In particular, in one embodiment, mRNA molecules encode target polypeptides, including any natural or non-naturally occurring or otherwise modified polypeptides. The polypeptides encoded by the mRNA can have any size and can have any secondary structure or activity. In one embodiment, the polypeptides encoded by the mRNA payload can have a therapeutic effect when expressed in a cell.

[0399] In some embodiments, the nucleic acid molecules of the present disclosure include mRNA molecules. In a specific embodiment, the nucleic acid molecule includes at least one coding region (e.g., open reading frame (ORF)) encoding a target peptide or polypeptide. In one embodiment, the nucleic acid molecule also includes at least one untranslated region (UTR). In a specific embodiment, the untranslated region (UTR) is located upstream (5' end) of the coding region and is referred to herein as 5'-UTR. In a specific embodiment, the untranslated region (UTR) is located downstream (3' end) of the coding region and is referred to herein as 3'-UTR. In a specific embodiment, the nucleic acid molecule includes both 5'-UTR and 3'-UTR. In one embodiment, 5'-UTR includes 5'-cap structure. In one embodiment, the nucleic acid molecule includes a Kozak sequence (e.g., in 5'-UTR). In one embodiment, the nucleic acid molecule includes a poly-A region (e.g., in 3'-UTR). In one embodiment, the nucleic acid molecule includes a polyadenylation signal (e.g., in 3'-UTR). In one embodiment, the nucleic acid molecule includes a stabilizing region (e.g., in 3'-UTR). In one embodiment, the nucleic acid molecule includes a secondary structure. In one embodiment, the secondary structure is a stem-loop. In one embodiment, the nucleic acid molecule comprises a stem-loop sequence (e.g., in a 5'-UTR and / or a 3'-UTR). In one embodiment, the nucleic acid molecule comprises one or more intronic regions that can be excised during splicing. In a specific embodiment, the nucleic acid molecule comprises one or more regions selected from a 5'-UTR and a coding region. In a specific embodiment, the nucleic acid molecule comprises one or more regions selected from a coding region and a 3'-UTR. In a specific embodiment, the nucleic acid molecule comprises one or more regions selected from a 5'-UTR, a coding region, and a 3'-UTR.

[0400] Coding region

[0401] In one embodiment, nucleic acid molecules of the present disclosure include at least one coding region. In one embodiment, the coding region is an open reading frame (ORF) encoding a single peptide or protein. In one embodiment, the coding region includes at least two ORFs, each ORF encoding a peptide or protein. In those embodiments where the coding region includes more than one ORF, the encoded peptide and / or protein may be identical or different from each other. In one embodiment, a plurality of ORFs in the coding region are separated by non-coding sequences. In a specific embodiment, the non-coding sequence separating two ORFs includes an internal ribosome entry site (IRES).

[0402] Without being bound by theory, it is expected that an internal ribosome entry site (IRES) can be used as a sole ribosome binding site, or as one of a plurality of ribosome binding sites of an mRNA. An mRNA molecule comprising more than one functional ribosome binding site can encode several peptides or polypeptides that are independently translated by ribosomes (e.g., polycistronic mRNA). Thus, in one embodiment, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises one or more internal ribosome entry sites (IRES). Examples of IRES sequences that can be used in conjunction with the present disclosure include, but are not limited to, those from picornaviruses (e.g., FMDV), pestiviruses (CFFV), polioviruses (PV), encephalomyocarditis virus (ECMV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), swine fever virus (CSFV), murine leukemia virus (MLV), simian immunodeficiency virus (SIV), or cricket paralysis virus (CrPV).

[0403] In various embodiments, the nucleic acid molecules of the present disclosure encode at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 peptides or proteins. The peptides and proteins encoded by the nucleic acid molecules may be the same or different. In one embodiment, the nucleic acid molecules of the present disclosure encode dipeptides (e.g., carnosine and anserine). In one embodiment, the nucleic acid molecules encode tripeptides. In one embodiment, the nucleic acid molecules encode tetrapeptides. In one embodiment, the nucleic acid molecules encode pentapeptides. In one embodiment, the nucleic acid molecules encode hexapeptides. In one embodiment, the nucleic acid molecules encode heptapeptides. In one embodiment, the nucleic acid molecules encode octapeptides. In one embodiment, the nucleic acid molecules encode nonapeptides. In one embodiment, the nucleic acid molecules encode decapeptides. In one embodiment, the nucleic acid molecules encode peptides or polypeptides having at least about 15 amino acids. In one embodiment, the nucleic acid molecules encode peptides or polypeptides having at least about 50 amino acids. In one embodiment, the nucleic acid molecules encode peptides or polypeptides having at least about 100 amino acids. In one embodiment, the nucleic acid molecules encode peptides or polypeptides having at least about 150 amino acids. In one embodiment, the nucleic acid molecules encode peptides or polypeptides having at least about 300 amino acids. In one embodiment, the nucleic acid molecule encodes a peptide or polypeptide having at least about 500 amino acids. In one embodiment, the nucleic acid molecule encodes a peptide or polypeptide having at least about 1000 amino acids.

[0404] In one embodiment, the length of the nucleic acid molecule of the present disclosure is at least about 30 nucleotides (nt). In one embodiment, the length of the nucleic acid molecule is at least about 35nt. In one embodiment, the length of the nucleic acid molecule is at least about 40nt. In one embodiment, the length of the nucleic acid molecule is at least about 45nt. In one embodiment, the length of the nucleic acid molecule is at least about 50nt. In one embodiment, the length of the nucleic acid molecule is at least about 55nt. In one embodiment, the length of the nucleic acid molecule is at least about 60nt. In one embodiment, the length of the nucleic acid molecule is at least about 65nt. In one embodiment, the length of the nucleic acid molecule is at least about 70nt. In one embodiment, the length of the nucleic acid molecule is at least about 75nt. In one embodiment, the length of the nucleic acid molecule is at least about 80nt. In one embodiment, the length of the nucleic acid molecule is at least about 85nt. In one embodiment, the length of the nucleic acid molecule is at least about 90nt. In one embodiment, the length of the nucleic acid molecule is at least about 95nt. In one embodiment, the length of the nucleic acid molecule is at least about 100nt. In one embodiment, the length of the nucleic acid molecule is at least about 120nt. In one embodiment, the length of the nucleic acid molecule is at least about 140nt. In one embodiment, the length of the nucleic acid molecule is at least about 160nt. In one embodiment, the length of the nucleic acid molecule is at least about 180nt. In one embodiment, the length of the nucleic acid molecule is at least about 200nt. In one embodiment, the length of the nucleic acid molecule is at least about 250nt. In one embodiment, the length of the nucleic acid molecule is at least about 300nt. In one embodiment, the length of the nucleic acid molecule is at least about 400nt. In one embodiment, the length of the nucleic acid molecule is at least about 500nt. In one embodiment, the length of the nucleic acid molecule is at least about 600nt. In one embodiment, the length of the nucleic acid molecule is at least about 700nt. In one embodiment, the length of the nucleic acid molecule is at least about 800nt. In one embodiment, the length of the nucleic acid molecule is at least about 900nt. In one embodiment, the length of the nucleic acid molecule is at least about 1000nt. In one embodiment, the length of the nucleic acid molecule is at least about 1100nt. In one embodiment, the length of the nucleic acid molecule is at least about 1200nt. In one embodiment, the length of the nucleic acid molecule is at least about 1300nt. In one embodiment, the length of the nucleic acid molecule is at least about 1400nt. In one embodiment, the length of the nucleic acid molecule is at least about 1500nt. In one embodiment, the length of the nucleic acid molecule is at least about 1600nt. In one embodiment, the length of the nucleic acid molecule is at least about 1700 nt. In one embodiment, the length of the nucleic acid molecule is at least about 1800 nt. In one embodiment, the length of the nucleic acid molecule is at least about 1900 nt.In one embodiment, the length of the nucleic acid molecule is at least about 2000nt. In one embodiment, the length of the nucleic acid molecule is at least about 2500nt. In one embodiment, the length of the nucleic acid molecule is at least about 3000nt. In one embodiment, the length of the nucleic acid molecule is at least about 3500nt. In one embodiment, the length of the nucleic acid molecule is at least about 4000nt. In one embodiment, the length of the nucleic acid molecule is at least about 4500nt. In one embodiment, the length of the nucleic acid molecule is at least about 5000nt.

[0405] In a specific embodiment, the therapeutic payload comprises a vaccine composition as described herein (e.g., a gene vaccine). In one embodiment, the therapeutic payload comprises a compound capable of triggering immunity to one or more target diseases or diseases. In one embodiment, the target disease is associated with or caused by a pathogen infection, such as a coronavirus (e.g., 2019-nCoV), influenza virus, measles virus, human papillomavirus (HPV), rabies virus, meningitis virus, pertussis virus, tetanus virus, plague virus, hepatitis virus, and tuberculosis virus. In one embodiment, the therapeutic payload comprises a nucleic acid sequence (e.g., mRNA) encoding a pathogenic protein or its antigenic fragment or epitope specific to the pathogen. After the vaccine is administered to a vaccinated subject, the encoded pathogenic protein (or its antigenic fragment or epitope) is allowed to be expressed, thereby inducing immunity to the pathogen in the subject.

[0406] In one embodiment, the target disease is associated with or caused by the neoplastic growth of cells (such as cancer). In one embodiment, the therapeutic payload comprises a nucleic acid sequence (e.g., mRNA) encoding a tumor-associated antigen (TAA) or an antigenic fragment or epitope thereof that is specific to cancer. Upon administration to a vaccinated subject, the vaccine allows expression of the encoded TAA (or its antigenic fragment or epitope), thereby inducing immunity in the subject against neoplastic cells expressing the TAA.

[0407] 5'-cap structure

[0408] Without being bound by theory, it is expected that the 5'-cap structure of the polynucleotide participates in nuclear export and increases polynucleotide stability, and binds to mRNA cap binding protein (CBP), which is responsible for polynucleotide stability in cells and causes translational competence by forming mature circular mRNA species through the association of CBP with poly-A binding protein. The 5'-cap structure further contributes to the removal of 5'-proximal introns during mRNA splicing. Therefore, in one embodiment, the nucleic acid molecule of the present disclosure comprises a 5'-cap structure.

[0409] Nucleic acid molecules can be capped at the 5' end by the endogenous transcriptional machinery of the cell, thereby generating a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue of the polynucleotide and the sense nucleotide transcribed at the 5' end. This 5'-guanylate cap can then be methylated to produce an N7-methyl-guanylate residue. The ribose of the nucleotides transcribed at the terminal and / or anteterminal of the 5' end of the polynucleotide can also be optionally 2'-O-methylated. 5'-decapping via hydrolysis and cleavage of the guanylate cap structure can target nucleic acid molecules, such as mRNA molecules, for degradation.

[0410] In one embodiment, the nucleic acid molecules of the present disclosure comprise one or more changes to a native 5'-cap structure produced by an endogenous process. Without being bound by theory, modifications to the 5'-cap may increase the stability of the polynucleotide, increase the half-life of the polynucleotide, and may increase the translation efficiency of the polynucleotide.

[0411] Exemplary changes to the natural 5'-cap structure include the production of a non-hydrolyzable cap structure to prevent decapping and thereby increase the half-life of the polynucleotide. In one embodiment, because the hydrolysis of the cap structure requires the cleavage of the 5'-ppp-5' phosphodiester linkage, in one embodiment, modified nucleotides may be used during the capping reaction. For example, in one embodiment, the vaccinia capping enzyme from New England Biolabs (Ipswich, Mass.) may be used for α-thioguanosine nucleotides to produce thiophosphate linkages in the 5'-ppp-5' cap according to the manufacturer's instructions. Additional modified guanosine nucleotides such as α-methylphosphonic acid and selenophosphate nucleotides may be used.

[0412] Additional exemplary changes to the native 5'-cap structure also include modifications at the 2'- and / or 3'-position of the capped guanosine triphosphate (GTP), replacement of the sugar ring oxygen with a methylene moiety (CH2) (generating a carbocycle), modifications at the triphosphate bridge portion of the cap structure, or modifications at the nucleobase (G) portion.

[0413] Additional exemplary changes to the natural 5'-cap structure include, but are not limited to, 2'-O-methylation of the ribose of the 5'-terminus of the polynucleotide and / or the 5'-terminal nucleotide on the sugar 2'-hydroxyl group (as described above). A plurality of different 5'-cap structures can be used to generate the 5'-cap of a polynucleotide (such as an mRNA molecule). Additional exemplary 5'-cap structures that can be used in conjunction with the present disclosure also include those described in International Patent Publications: WO2008127688, WO2008016473, and WO 2011015347, the entire contents of which are incorporated herein by reference.

[0414] In various embodiments, the 5'-terminal cap may include a cap analog. Cap analogs, also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, have a chemical structure that is different from a natural (ie, endogenous, wild-type, or physiological) 5'-cap and retains cap function. Cap analogs can be synthesized and / or attached to a polynucleotide chemically (ie, non-enzymatically) or enzymatically.

[0415] For example, the anti-reverse cap analog (ARCA) cap contains two guanosines linked by a 5'-5'-triphosphate group, one of which contains an N7-methyl group and a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m 7 G-3'mppp-G, which can be equivalently referred to as 3'O-Me-m7G(5')ppp(5')G). The 3'-O atom of another unchanged guanosine is attached to the 5'-terminal nucleotide of the capped polynucleotide (e.g., mRNA). The N7- and 3'-O-methylated guanosine provides the terminal portion of the capped polynucleotide (e.g., mRNA). Another exemplary cap structure is mCAP, which is similar to ARCA, but has a 2'-O-methyl group on the guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G).

[0416] In one embodiment, the cap analog can be a dinucleotide cap analog. As a non-limiting example, a dinucleotide cap analog can be modified with a boranophosphate or a phophoroselenoate at different phosphate positions, such as the dinucleotide cap analogs described in U.S. Pat. No. 8,519,110, the entire contents of which are incorporated herein by reference in their entirety.

[0417] In one embodiment, the cap analog can be a N7-(4-chlorophenoxyethyl) substituted dinucleotide cap analog known in the art and / or described herein. Non-limiting examples of N7-(4-chlorophenoxyethyl) substituted dinucleotide cap analogs include N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and N7-(4-chlorophenoxyethyl)-m3'-OG(5')ppp(5')G cap analogs (see, e.g., Kore et al. Bioorganic & Medicinal Chemistry 2013 21:4570-4574 for various cap analogs and methods for synthesizing cap analogs; the entire contents of the document are incorporated herein by reference). In other embodiments, the cap analog that can be used in conjunction with the nucleic acid molecules of the present disclosure is a 4-chloro / bromophenoxyethyl analog.

[0418] In various embodiments, the cap analog may include a guanosine analog. Useful guanosine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0419] Without being bound by theory, it is expected that although cap analogs allow concomitant capping of polynucleotides in in vitro transcription reactions, up to 20% of transcripts remain uncapped. This situation, as well as the structural differences between the cap analogs and the natural 5'-cap structure of polynucleotides produced by the endogenous transcription machinery of the cell, may lead to reduced translation capacity and reduced cellular stability.

[0420] Therefore, in one embodiment, the nucleic acid molecules of the present disclosure may also be capped after transcription using an enzyme to produce a more authentic 5'-cap structure. As used herein, the phrase "more authentic" refers to a feature that closely reflects or mimics an endogenous or wild-type feature in structure or function. That is, a "more authentic" feature better represents endogenous, wild-type, natural or physiological cell function and / or structure than a synthetic feature or analog of the prior art, or it outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more aspects. A non-limiting example of a more authentic 5'-cap structure that can be used in conjunction with the nucleic acid molecules of the present disclosure is a structure that has enhanced binding to a cap-binding protein, increased half-life, reduced sensitivity to 5'-endonucleases, and / or reduced 5'-decapping, compared to synthetic 5'-cap structures known in the art (or compared to wild-type, natural or physiological 5'-cap structures). For example, in one embodiment, a recombinant vaccinia virus capping enzyme and a recombinant 2'-O-methyltransferase can generate a classic 5'-5'-triphosphate linkage between the 5'-terminal nucleotide of a polynucleotide and a guanosine cap nucleotide, wherein the cap guanosine contains an N7-methylation and the 5'-terminal nucleotide of the polynucleotide contains a 2'-O-methyl group. This structure is referred to as a cap 1 structure. Compared to other 5' cap analog structures known in the art, for example, this cap results in higher translational capacity, cellular stability, and reduced activation of cellular proinflammatory cytokines. Other exemplary cap structures include 7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), 7mG(5')-ppp(5')NlmpN2mp (cap 2), and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up (cap 4).

[0421] Without being bound by theory, it is contemplated that the nucleic acid molecules of the present disclosure can be capped after transcription, and because this method is relatively efficient, nearly 100% of the nucleic acid molecules can be capped.

[0422] Untranslated region (UTR)

[0423] In one embodiment, the nucleic acid molecules of the present disclosure include one or more untranslated regions (UTRs). In one embodiment, the UTR is located upstream of the coding region in the nucleic acid molecule and is referred to as the 5'-UTR. In one embodiment, the UTR is located downstream of the coding region in the nucleic acid molecule and is referred to as the 3'-UTR. The sequence of the UTR may be homologous or heterologous to the sequence of the coding region found in the nucleic acid molecule. Multiple UTRs may be included in the nucleic acid molecule and may have the same or different sequences and / or gene origins. According to the present disclosure, any portion of the UTR in the nucleic acid molecule (including without any portion) may be codon optimized, and any portion may contain one or more different structures or chemical modifications independently before and / or after codon optimization.

[0424] In one embodiment, nucleic acid molecules (e.g., mRNA) of the present disclosure comprise UTRs and coding regions that are homologous to each other. In other embodiments, nucleic acid molecules (e.g., mRNA) of the present disclosure comprise UTRs and coding regions that are heterologous to each other. In one embodiment, to monitor the activity of UTR sequences, nucleic acid molecules comprising coding sequences of UTRs and detectable probes may be administered in vitro (e.g., in cell or tissue culture) or in vivo (e.g., to a subject), and the effects of UTR sequences (e.g., regulating expression levels, cellular localization of the encoded product, or half-life of the encoded product) may be measured using methods known in the art.

[0425] In one embodiment, the UTR of a nucleic acid molecule (e.g., mRNA) of the present disclosure includes at least one translation enhancer element (TEE), which acts to increase the amount of a polypeptide or protein produced by the nucleic acid molecule. In one embodiment, TEE is located in the 5'-UTR of a nucleic acid molecule. In other embodiments, TEE is located at the 3'-UTR of a nucleic acid molecule. In other embodiments, at least two TEEs are located at the 5'-UTR and 3'-UTR of a nucleic acid molecule, respectively. In one embodiment, a nucleic acid molecule (e.g., mRNA) of the present disclosure may include one or more copies of a TEE sequence or include more than one different TEE sequence. In one embodiment, different TEE sequences present in a nucleic acid molecule of the present disclosure may be homologous or heterologous relative to each other.

[0426] Various TEE sequences are known in the art and can be used in conjunction with the present disclosure. For example, in one embodiment, the TEE can be an internal ribosome entry site (IRES), HCV-IRES, or an IRES element. Chappell et al. Proc. Natl. Acad. Sci. USA 101:9590-9594, 2004; Zhou et al. Proc. Natl. Acad. Sci. 102:6273-6278, 2005. Additional internal ribosome entry sites (IRES) that can be used in conjunction with the present disclosure include, but are not limited to, U.S. Patent No. 7,468,275, U.S. Patent Publication No. 2007 / 0048776, and U.S. Patent Publication No. 2011 / 0124100, and International Patent Publication No. WO2007 / 025008 and International Patent Publication No. WO2001 / 055369, the contents of each of which are incorporated herein by reference in their entirety. In one embodiment, the TEE can be the TEE described in Supplementary Table 1 and Supplementary Table 2 of Wellensiek et al. Genome-wide profiling of human cap-independent translation-enhancing elements, Nature Methods, 2013 Aug; 10(8):747-750; the contents of which are incorporated herein by reference in their entirety.

[0427] Additional exemplary TEEs that may be used in conjunction with the present disclosure include, but are not limited to, U.S. Patent No. 6,310,197, U.S. Patent No. 6,849,405, U.S. Patent No. 7,456,273, U.S. Patent No. 7,183,395, U.S. Patent Publication No. 2009 / 0226470, U.S. Patent Publication No. 2013 / 0177581, U.S. Patent Publication No. 2007 / 0048776, U.S. Patent Publication No. 2011 / 0124100, U.S. Patent Publication No. 2013 / 0177581, U.S. Patent Publication No. 2013 / 0177582, U.S. Patent Publication No. 2013 / 0177583, U.S. Patent Publication No. 2013 / 0177584, U.S. Patent Publication No. 2013 / 0177585, U.S. Patent Publication No. 2013 / 0177586, U.S. Patent Publication No. 2013 / 0177587 009 / 0093049, International Patent Publication No. WO2009 / 075886, International Patent Publication No. WO2012 / 009644 and International Patent Publication No. WO1999 / 024595, International Patent Publication No. WO2007 / 025008, International Patent Publication No. WO2001 / 055371, European Patent No. 2610341, European Patent No. 2610340, the contents of each of which are incorporated herein by reference in their entirety.

[0428] In various embodiments, nucleic acid molecules (e.g., mRNA) disclosed herein include at least one UTR, which includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences. In one embodiment, the TEE sequence in the UTR of nucleic acid molecules is a copy of the same TEE sequence. In other embodiments, at least two TEE sequences in the UTR of nucleic acid molecules have different TEE sequences. In one embodiment, a plurality of different TEE sequences are arranged in the UTR region of nucleic acid molecules with one or more repetitive patterns. For the purpose of illustration only, the repetition pattern can be, for example, ABABAB, AABBAABBAAB, ABCABCABC, etc., wherein in these exemplary patterns, each capital letter (A, B or C) represents a different TEE sequence. In one embodiment, in the UTR of a nucleic acid molecule, at least two TEE sequences are continuous with each other (i.e., there is no spacer sequence therebetween). In other embodiments, at least two TEE sequences are separated by a spacer sequence. In one embodiment, UTR may include a TEE sequence-spacer sequence module, which is repeated in UTR at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times or more than 9 times. In any embodiment described in this paragraph, UTR can be 5'-UTR, 3'-UTR or 5'-UTR and 3'-UTR of a nucleic acid molecule.

[0429] In one embodiment, the UTR of nucleic acid molecules (e.g., mRNA) disclosed herein comprises at least one translation inhibition element, which acts to reduce the amount of polypeptide or protein produced by the nucleic acid molecules. In one embodiment, the UTR of nucleic acid molecules comprises one or more miR sequences or fragments thereof (e.g., miR seed sequences (seed sequence)) identified by one or more microRNAs. In one embodiment, the UTR of nucleic acid molecules comprises one or more stem-loop structures that lower the translation activity of nucleic acid molecules. Other mechanisms for suppressing the translation activity associated with nucleic acid molecules are known in the art. In any embodiment described in this section, UTR can be 5'-UTR, 3'-UTR or both 5'-UTR and 3'-UTR of nucleic acid molecules.

[0430] Polyadenylation (Poly-A) region

[0431] During natural RNA processing, long chains of adenosine nucleotides (poly-A regions) are often added to messenger RNA (mRNA) molecules to increase the stability of the molecule. Immediately after transcription, the 3'-end of the transcript is cleaved to release the 3'-hydroxyl. Next, poly-A polymerase adds a series of adenosine nucleotides to the RNA. The process is called polyadenylation, and poly-A regions between 100 and 250 residues in length are added. Without being bound by theory, it is expected that poly-A regions can confer a number of advantages to the nucleic acid molecules of the present disclosure.

[0432] Therefore, in one embodiment, the nucleic acid molecules (e.g., mRNA) of the present disclosure comprise a polyadenylation signal. In one embodiment, the nucleic acid molecules (e.g., mRNA) of the present disclosure comprise one or more polyadenylation (poly-A) regions. In one embodiment, the poly-A region is entirely composed of adenine nucleotides or functional analogs thereof. In one embodiment, the nucleic acid molecule comprises at least one poly-A region at its 3' end. In one embodiment, the nucleic acid molecule comprises at least one poly-A region at its 5' end. In one embodiment, the nucleic acid molecule comprises at least one poly-A region at its 5' end and at least one poly-A region at its 3' end.

[0433] According to the present disclosure, in different embodiments, the poly-A region may have different lengths. In particular, in one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 30 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 35 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 40 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 45 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 50 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 55 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 60 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 65 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 70 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 75 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 80 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 85 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 90 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 95 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 100 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 110 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 120 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 130 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 140 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 150 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 160 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 170 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 180 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 190 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 200 nucleotides.In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 225 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 250 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 275 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 300 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 350 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 400 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 450 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 500 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 600 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 700 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 800 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 900 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 1000 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 1100 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 1200 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 1300 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 1400 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 1500 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 1600 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 1700 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 1800 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 1900 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 2000 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 2250 nucleotides. In one embodiment, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 2500 nucleotides.In one embodiment, the poly-A region of the nucleic acid molecule of the present disclosure is at least 2750 nucleotides in length. In one embodiment, the poly-A region of the nucleic acid molecule of the present disclosure is at least 3000 nucleotides in length.

[0434] In one embodiment, the length of the poly-A region in the nucleic acid molecule can be selected based on the total length of the nucleic acid molecule or a portion thereof, such as the length of the coding region of the nucleic acid molecule or the length of the open reading frame, etc. For example, in one embodiment, the poly-A region accounts for about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the total length of the nucleic acid molecule containing the poly-A region.

[0435] Without being bound by theory, it is expected that certain RNA binding proteins can bind to the poly-A region located at the 3' end of the mRNA molecule. These poly-A binding proteins (PABPs) can regulate mRNA expression, such as interacting with the translation initiation machinery in the cell and / or protecting the 3'-poly-A tail from degradation. Therefore, in one embodiment, the nucleic acid molecules (e.g., mRNA) of the present disclosure comprise at least one binding site for a poly-A binding protein (PABP). In other embodiments, the nucleic acid molecules are conjugated or complexed with PABP before being loaded into a delivery vehicle (e.g., lipid nanoparticle).

[0436] In one embodiment, the nucleic acid molecules (e.g., mRNA) of the present disclosure comprise poly-AG quadruplexes. G-quadruplexes are circular arrays of four hydrogen-bonded guanosine nucleotides that can be formed by G-rich sequences in DNA and RNA. In this embodiment, the G-quadruplexes are incorporated at the ends of the poly-A region. The stability, protein yield, and other parameters of the resulting polynucleotides (e.g., mRNA) can be determined, including the half-life at different time points. It has been found that the protein yield of the poly-AG quadruplex structure is equal to at least 75% of the protein yield observed using only a poly-A region containing 120 nucleotides.

[0437] In one embodiment, the nucleic acid molecules (e.g., mRNA) of the present disclosure may include a poly-A region and may be stabilized by adding a 3'-stabilizing region. In one embodiment, the 3'-stabilizing region that can be used to stabilize nucleic acid molecules (e.g., mRNA) includes a poly-A or poly-AG quadruplex structure as described in International Patent Publication No. WO2013 / 103659, the contents of which are incorporated herein by reference in their entirety.

[0438] In other embodiments, the 3'-stabilizing region that can be used in conjunction with the nucleic acid molecules of the present disclosure includes chain terminating nucleosides such as, but not limited to, 3'-deoxyadenosine (cordycepin); 3'-deoxyuridine; 3'-deoxycytosine; 3'-deoxyguanosine; 3'-deoxythymidine; 2',3'-dideoxynucleosides, such as 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymidine; 2'-deoxynucleosides; or O-methyl nucleosides; 3'-deoxynucleosides; 2',3'-dideoxynucleosides; 3'-O-methyl nucleosides; 3'-O-ethyl nucleosides; 3'-arabinoside, as well as other alternative nucleosides known in the art and / or described herein.

[0439] Secondary structure

[0440] Without being bound by theory, it is expected that the stem-loop structure can guide RNA folding, protect the structural stability of nucleic acid molecules (e.g., mRNA), provide recognition sites for RNA binding proteins, and serve as substrates for enzymatic reactions. For example, the incorporation of miR sequences and / or TEE sequences will change the shape of the stem-loop region, thereby increasing and / or decreasing translation (Kedde et al. A Pumilio-induced RNA structure switch in p27-3'UTR controls miR-221 and miR-222 accessibility. Nat Cell Biol., 2010 Oct; 12(10): 1014-20, the contents of which are incorporated herein by reference in their entirety).

[0441] Therefore, in one embodiment, nucleic acid molecules as described herein (e.g., mRNA) or parts thereof may present a stem-loop structure, such as, but not limited to, histone stem-loops. In one embodiment, the stem-loop structure is formed by a stem-loop sequence having a length of about 25 or about 26 nucleotides, such as, but not limited to, the structure described in International Patent Publication No. WO2013 / 103659, the contents of which are incorporated herein by reference in their entirety. Additional examples of stem-loop sequences include sequences described in International Patent Publication No. WO2012 / 019780 and International Patent Publication No. WO201502667, the contents of which are incorporated herein by reference. In one embodiment, the stem-loop sequence includes TEE as described herein. In one embodiment, the stem-loop sequence includes a miR sequence as described herein. In a specific embodiment, the stem-loop sequence may include a miR-122 seed sequence. In a specific embodiment, the nucleic acid molecule includes two stem-loop sequences described in International Patent Publication No. WO2021204175, the entire contents of which are incorporated herein by reference.

[0442] In one embodiment, nucleic acid molecules (e.g., mRNA) of the present disclosure include stem-loop sequences located upstream of the coding region (at the 5' end) in the nucleic acid molecule. In one embodiment, the stem-loop sequences are located in the 5'-UTR of the nucleic acid molecule. In one embodiment, nucleic acid molecules (e.g., mRNA) of the present disclosure include stem-loop sequences located downstream of the coding region (at the 3' end) in the nucleic acid molecule. In one embodiment, the stem-loop sequences are located in the 3'-UTR of the nucleic acid molecule. In some cases, nucleic acid molecules may contain more than one stem-loop sequences. In some embodiments, nucleic acid molecules include at least one stem-loop sequence in the 5'-UTR and at least one stem-loop sequence in the 3'-UTR.

[0443] In one embodiment, the nucleic acid molecule comprising a stem-loop structure further comprises a stabilizing region. In one embodiment, the stabilizing region comprises at least one chain terminating nucleoside, which acts to slow degradation and thereby increase the half-life of the nucleic acid molecule. Exemplary chain terminating nucleosides that can be used in conjunction with the nucleic acid molecules of the present disclosure include, but are not limited to, 3'-deoxyadenosine (cordycepin); 3'-deoxyuridine; 3'-deoxycytosine; 3'-deoxyguanosine; 3'-deoxythymidine; 2',3'-dideoxynucleosides, such as 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymidine; 2'-deoxynucleosides; or O-methyl nucleosides; 3'-deoxynucleosides; 2',3'-dideoxynucleosides; 3'-O-methyl nucleosides; 3'-O-ethyl nucleosides; 3'-arabinoside, as well as other alternative nucleosides known in the art and / or described herein. In other embodiments, the stem-loop structure can be stabilized by altering the 3 '-region of the polynucleotide, which alterations can prevent and / or inhibit the addition of oligo(U) (International Patent Publication No. WO2013 / 103659, which is herein incorporated by reference in its entirety).

[0444] In one embodiment, the nucleic acid molecules of the present disclosure include at least one stem-loop sequence and a poly-A region or a polyadenylation signal. Non-limiting examples of polynucleotide sequences including at least one stem-loop sequence and a poly-A region or a polyadenylation signal include International Patent Publication No. WO2013 / 120497, International Patent Publication No. WO2013 / 120629, International Patent Publication No. WO2013 / 120500, International Patent Publication No. WO2013 / 120627, International Patent Publication No. WO2013 / 120498, International Patent Publication No. WO2013 / 120626, International Patent Publication No. WO2013 / 120499 and International Patent Publication No. WO2013 / 120628, the contents of each of which are incorporated herein by reference in their entirety.

[0445] In one embodiment, a nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal may encode a pathogen antigen or a fragment thereof, such as the polynucleotide sequences described in International Patent Publication No. WO2013 / 120499 and International Patent Publication No. WO2013 / 120628, the contents of each of which are incorporated herein by reference in their entirety.

[0446] In one embodiment, a nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal may encode a therapeutic protein, such as the polynucleotide sequences described in International Patent Publication No. WO2013 / 120497 and International Patent Publication No. WO2013 / 120629, the contents of each of which are incorporated herein by reference in their entirety.

[0447] In one embodiment, a nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal may encode a tumor antigen or a fragment thereof, such as the polynucleotide sequences described in International Patent Publication No. WO2013 / 120500 and International Patent Publication No. WO2013 / 120627, the contents of each of which are incorporated herein by reference in their entirety.

[0448] In one embodiment, a nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal may encode a sensitizing antigen or an autoimmune self-antigen, such as the polynucleotide sequences described in International Patent Publication No. WO2013 / 120498 and International Patent Publication No. WO2013 / 120626, the contents of each of which are incorporated herein by reference in their entirety.

[0449] Functional nucleotide analogs

[0450] In one embodiment, the payload nucleic acid molecules described herein contain only canonical nucleotides selected from A (adenosine), G (guanosine), C (cytosine), U (uridine) and T (thymidine). Without being bound by theory, it is expected that certain functional nucleotide analogs can confer useful properties on nucleic acid molecules. In the context of the present disclosure, examples of such useful properties include, but are not limited to, increased stability of nucleic acid molecules, reduced immunogenicity of nucleic acid molecules in inducing innate immune responses, increased production of proteins encoded by nucleic acid molecules, increased intracellular delivery and / or retention of nucleic acid molecules, and / or reduced cytotoxicity of nucleic acid molecules, etc.

[0451] Therefore, in one embodiment, the effective load nucleic acid molecule comprises at least one functional nucleotide analog as described herein. In one embodiment, the functional nucleotide analog contains at least one chemical modification for a nucleobase, a sugar group and / or a phosphate group. Therefore, the effective load nucleic acid molecule comprising at least one functional nucleotide analog contains at least one chemical modification for a nucleobase, a sugar group and / or an internucleoside linkage. Exemplary chemical modifications to the nucleobase, sugar group or internucleoside linkage of a nucleic acid molecule are provided herein.

[0452] As described herein, nucleotides ranging from 0% to 100% of all nucleotides in a payload nucleic acid molecule can be functional nucleotide analogs as described herein. For example, in various embodiments, about 1% to about 20%, about 1% to about 25%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 95%, about 10% to about 20%, about 10% to about 25%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 25%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 25%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70 In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein, and the nucleotides of the present invention are functional nucleotide analogs as described herein. In any one of these embodiments, the functional nucleotide analogs can be present in any position of the nucleic acid molecule, including the 5'-end, the 3'-end and / or one or more internal positions. In one embodiment, a single nucleic acid molecule can contain different sugar modifications, different nucleobase modifications, and / or different types of internucleoside linkages (eg, backbone structures).

[0453] As described herein, a range from 0% to 100% of all nucleotides of one type (e.g., all purine-containing nucleotides as a type, or all pyrimidine-containing nucleotides as a type, or all A, G, C, T, or U as a type) in a payload nucleic acid molecule can be functional nucleotide analogs as described herein. For example, in various embodiments, about 1% to about 20%, about 1% to about 25%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 95%, about 10% to about 20%, about 10% to about 25%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 25%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 25%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 10% to about 80%, In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein. In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein. In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein. In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein. In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein. In any one of these embodiments, the functional nucleotide analogs can be present in any position of the nucleic acid molecule, including the 5'-end, the 3'-end and / or one or more internal positions. In one embodiment, a single nucleic acid molecule can contain different sugar modifications, different nucleobase modifications, and / or different types of internucleoside linkages (eg, backbone structures).

[0454] Nucleobase modification

[0455] In one embodiment, the functional nucleotide analog contains a non-classical nucleobase. In one embodiment, the classical nucleobases in the nucleotide (e.g., adenine, guanine, uracil, thymine and cytosine) can be modified or replaced to provide one or more functional analogs of the nucleotide. Exemplary modifications of nucleobases include, but are not limited to, one or more substitutions or modifications, including, but not limited to, alkyl, aryl, halo, oxo, hydroxyl, alkoxy and / or thio substitutions; one or more fused rings or ring opening, oxidation and / or reduction.

[0456] In one embodiment, the non-classical nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (ψ), pyridin-4-ketoribonucleoside, 5-azauracil, 6-azauracil, 2-thio-5-azauracil, 2-thiouracil (s 2 U), 4-thio-uracil (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil (ho 5 U), 5-aminoallyl-uracil, 5-halouracil (e.g., 5-iodouracil or 5-bromouracil), 3-methyluracil (m 3 U), 5-methoxyuracil (mo 5 U), uracil 5-oxyacetic acid (cmo 5 U), uracil 5-oxyacetate (mcmo 5 U), 5-carboxymethyl-uracil (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uracil (chm 5 U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uracil (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouracil (mcm 5 s 2 U), 5-aminomethyl-2-thiouracil (nm 5 s 2 U), 5-methylaminomethyluracil (mnm 5 U), 5-methylaminomethyl-2-thiouracil (mnm 5 s 2 U), 5-methylaminomethyl-2-selenouracil (mnm 5 s 2 U), 5-carbamoylmethyluracil (ncm 5 U), 5-carboxymethylaminomethyl-uracil (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouracil (cmnm 5 s 2 U), 5-propynyl-uracil, 1-propynyl-pseudouracil, 5-taurine methyl-uracil (τm 5 U), 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thio-uracil (τm 5 5s 2 U), 1-taurine methyl-4-thio-pseudouridine, 5-methyl-uracil (m 5 U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m1 ψ), 1-ethyl-pseudouridine (Et 1 ψ), 5-methyl-2-thiouracil (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouracil (D), dihydropseudouridine, 5,6-dihydrouracil, 5-methyl-dihydrouracil (m 5 D), 2-thio-dihydrouracil, 2-thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl) uracil (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl) pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uracil (m 5 U), 5-(isopentenylaminomethyl)-2-thio-uracil (m 5 s 2 U), 5,2'-O-dimethyl-uridine (m 5 Um), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um) and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uracil, deoxythymidine, 5-(2-methoxycarbonylvinyl)-uracil, 5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thio-uracil, 5-carboxymethyl-2-thio-uracil, 5-cyanomethyl-uracil, 5-methoxy-2-thio-uracil and 5-[3-(1-E-propenylamino)]uracil.

[0457] In one embodiment, the non-classical nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosines include 5-azacytosine, 6-azacytosine, pseudoisocytidine, 3-methylcytosine (m3C), N4-acetylcytosine (ac4C), 5-formylcytosine (f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C), 5-halo-cytosine (e.g., 5-iodo-cytosine), 5-hydroxymethyl-cytosine (hm5C), 1-methyl-pseudoisocytidine, pyrrolocytosine, pyrrolopseudoisocytidine, 2-thiocytosine (s2C), 2-thio-5-methylcytosine, 4-thiopseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5 -aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytosine, 2-methoxy-5-methyl-cytosine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), 5,2'-O-dimethyl-cytidine (m5Cm), N4-ethyl Acyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (fSCm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytosine, 5-hydroxy-cytosine, 5-(3-azidopropyl)-cytosine and 5-(2-azidoethyl)-cytosine.

[0458] In one embodiment, the non-classical nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having an alternative adenine include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2 ,6-diaminopurine, 1-methyl-adenine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenine (m6A), 2-methylthio-N6-methyl-adenine (ms2m6A), N6-isopentenyl-adenine (i6A), 2-methylthio-N6-isopentenyl-adenine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycylcarbamoyl N6-methyl-N6-threonylcarbamoyl-adenine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A), N6,N6-dimethyl-adenine (m62A), N6-hydroxynorvalylcarbamoyl-adenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenine (ms2hn6A), N6-acetyl-adenine (ac6A) , 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2-amino-N6-methyl-purine, 1-thio-adenine, 8-azido-adenine, N6-(19-amino-pentaoxanonadecanyl)-adenine, 2,8-dimethyl-adenine, N6-formyl-adenine and N6-hydroxymethyl-adenine.

[0459] In one embodiment, the non-classical nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methyl wyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxy wybutosine (o2yW), hydroxy wybutosine (OHyW), undermodified hydroxy wybutosine (OHyW*), 7-deaza- Guanine, queuosine (Q), epoxyqueuosine (oQ), galactosylqueuosine (galQ), mannosylqueuosine (manQ), 7-cyano-7-deaza-guanine (preQO), 7-aminomethyl-7-deaza-guanine (preQ1), archaeosine (G+), 7-deaza-8-aza-guanine, 6-thio-guanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl guanine (m7G), 6-thio-7-methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1-methyl-guanine (m1G), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine (m22G), N2,7-dimethyl-guanine (m2,7G), N2,N2,7-dimethyl-guanine (m2,2,7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1-methyl-6-thio-guanine, N2-methyl-6- Thio-guanine, N2,N2-dimethyl-6-thio-guanine, N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 1-thio-guanine and O-6-methyl-guanine.

[0460] In one embodiment, the non-classical nucleobase of the functional nucleotide analogue can independently be a purine, a pyrimidine, a purine analogue or a pyrimidine analogue. For example, in one embodiment, the non-classical nucleobase can be a modified adenine, cytosine, guanine, uracil or hypoxanthine. In other embodiments, non-classical nucleobases may also include, for example, naturally occurring and synthetic derivatives of bases, including pyrazolo[3,4-d]pyrimidine; 5-methylcytosine (5-me-C); 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine and 2-thiocytosine; 5-propynyluracil and cytosine; 6-azouracil, cytosine and thymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halo (e.g., 8-bromo), 8-amino, 8-thiol , 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines; 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine; deazaguanine, 7-deazaguanine, 3-deazaguanine; deazaadenine, 7-deazaadenine, 3-deazaadenine; pyrazolo[3,4-d]pyrimidine; imidazo[1,5-a]1,3,5-triazinone; 9-deazapurine; imidazo[4,5-d]pyrazine; thiazolo[4,5-d]pyrimidine; pyrazin-2-one; 1,2,4-triazine; pyridazine; or 1,3,5-triazine.

[0461] Sugar modification

[0462] In one embodiment, the functional nucleotide analog contains a non-classical sugar group. In various embodiments, the non-classical sugar group can be a 5- or 6-carbon sugar (such as pentose, ribose, arabinose, xylose, glucose, galactose or its deoxy derivatives) with one or more substitutions, such as halogen, hydroxyl, thiol, alkyl, alkoxy, alkenyloxy, alkynyloxy, cycloalkyl, aminoalkoxy, alkoxyalkoxy, hydroxyalkoxy, amino, azido, aryl, aminoalkyl, aminoalkenyl, aminoalkynyl, etc.

[0463] In general, RNA molecules contain a ribose sugar group that is an oxygen-containing 5-membered ring. Exemplary non-limiting alternative nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or an alkylene group, such as methylene or ethylene); addition of double bonds (e.g., replacement of ribose with a cyclopentenyl or cyclohexenyl group); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6-membered or 7-membered ring with additional carbon or heteroatoms, such as for anhydrohexitol, altritol, mannitol, cyclohexane, cyclohexane, etc.); hexenyl and N-morpholinyl (which also have a phosphoramidate backbone); polycyclic forms (e.g., tricyclic and "unlocked" forms, such as glycol nucleic acids (GNAs) (e.g., R-GNA or S-GNA, in which ribose is replaced by a glycol unit linked to a phosphodiester linkage), threose nucleic acids (TNA, in which ribose is replaced by an α-L-threofuranosyl-(3'→2')) and peptide nucleic acids (PNA, in which 2-amino-ethyl-glycine linkages replace ribose and the phosphodiester backbone)).

[0464] In one embodiment, the sugar group contains one or more carbons having a stereochemical configuration opposite to the corresponding carbon in ribose. Therefore, the nucleic acid molecule may include nucleotides containing, for example, arabinose or L-ribose as sugar. In one embodiment, the nucleic acid molecule includes at least one nucleoside wherein the sugar is L-ribose, 2'-O-methyl ribose, 2'-fluororibose, arabinose, hexitol, LNA or PNA.

[0465] Modification of internucleoside linkages

[0466] In one embodiment, the disclosed payload nucleic acid molecules may contain one or more modified internucleoside linkages (eg, phosphate backbones). The backbone phosphate groups may be altered by replacing one or more oxygen atoms with different substituents.

[0467] In one embodiment, functional nucleotide analogs can include another internucleoside linkage replacement unchanged phosphate moiety as described herein.The example of alternative phosphate groups includes but is not limited to phosphorothioate, selenophosphate, borane phosphate, borane phosphate, hydrogen phosphonate, phosphoramidate, diamino phosphoroester, alkyl or aryl phosphonate and phosphotriester.Two non-connected oxygens of dithiophosphate are all replaced by sulfur.It is also possible to change the phosphate linker by replacing the connection oxygen with nitrogen (phosphoramidate of bridge), sulfur (phosphorothioate of bridge) and carbon (methylene phosphonate of bridge).

[0468] Alternative nucleosides and nucleotides may include one or more non-bridging oxygens replaced by a borane moiety (BH3), sulfur (thio), methyl, ethyl and / or methoxy. As a non-limiting example, two non-bridging oxygens at the same position (e.g., alpha (α), beta (β) or gamma (γ) position) may be replaced by sulfur (thio) and methoxy. One or more oxygen atoms at the position of a replacement phosphate moiety (e.g., α-thiophosphate) may impart RNA and DNA stability (such as stability against exonucleases and endonucleases) via non-natural thiophosphate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and therefore have a longer half-life in a cellular environment.

[0469] Other internucleoside linkages that can be used in accordance with the present disclosure are described herein, including internucleoside linkages that do not contain a phosphorus atom.

[0470] Additional examples of nucleic acid molecules (e.g., mRNA), related compositions, formulations, and / or methods that can be used in conjunction with the present disclosure include WO2002 / 098443, WO2003 / 051401, WO2008 / 052770, WO2009127230, WO2006122828, WO2008 / 083949, WO2010088927, WO2010 / 037539, WO2004 / 004743, WO2005 / 016 376. WO2006 / 024518, WO2007 / 095976, WO2008 / 014979, WO2008 / 077592, WO2009 / 030481, WO2009 / 095226, W O2011069586, WO2011026641, WO2011 / 144358, WO2012019780, WO2012013326, WO2012089338, WO201211351 3. WO2012116811, WO2012116810, WO2013113502, WO2013113501, WO2013113736, WO2013143698, WO2013143 699. WO2013143700, WO2013 / 120626, WO2013120627, WO2013120628, WO2013120629, WO2013174409, WO2014 Those described in WO2015 / 027917, WO2015 / 024669, WO2015 / 024668, WO2015 / 024667, WO2015 / 024665, WO2015 / 024666, WO2015 / 024664, WO2015101415, WO2015101414, WO2015024667, WO2015062738, WO2015101416, the contents of each of which are incorporated herein in their entirety.

[0471] 6.3.2 Preparation

[0472] The lipid nanoparticle compositions described herein may include at least one lipid component and one or more additional components, such as a therapeutic and / or prophylactic agent (e.g., a therapeutic nucleic acid as described herein). Nanoparticle compositions may be designed for one or more specific applications or targets. The composition of the nanoparticle composition may be selected based on a specific application or target, and / or based on the efficacy, toxicity, cost, ease of use, availability or other characteristics of one or more components. Similarly, the specific formulation of the nanoparticle composition may be selected for a specific application or target, based on the efficacy and toxicity of a specific combination of, for example, each component.

[0473] In one embodiment, the therapeutic and / or prophylactic agent encapsulated in the nanoparticle can be delivered to a host cell in vitro, for example by contacting the host cell with a nanoparticle composition, or delivered to a host cell in vivo, for example by administering the nanoparticle composition to a subject containing a host cell. In one embodiment, after delivery, the therapeutic nucleic acid molecule encapsulated in the nanoparticle can be expressed via the host cell endogenous transcription and translation machinery.

[0474] In one embodiment, the nanoparticle composition comprises a non-lipid component comprising a therapeutic agent. In one embodiment, the therapeutic agent is a nucleic acid molecule. In one embodiment, the therapeutic agent is an mRNA molecule.

[0475] In one embodiment, after the nucleic acid containing the nanoparticle composition is delivered to the host cell, the nucleic acid is expressed via the host cell endogenous transcription and / or translation machinery to form RNA and / or protein. In one embodiment, the expression level of the nucleic acid formulated in the LNP is enhanced compared to the nucleic acid formulated in the reference LNP composition.

[0476] Therefore, the nanoparticle compositions described herein can be used in methods for expressing mRNA in host cells or tissues of host subjects, wherein the method comprises formulating mRNA in a nanoparticle composition and delivering the nanoparticle composition to the host cell or host subject; and wherein the delivered mRNA is expressed in the host cell or the host subject. In one embodiment, the host cell is a mammalian cell (such as a cell derived from a human or non-human vertebrate). In one embodiment, the host subject is a mammal (such as a human or non-human vertebrate). In one embodiment, the nanoparticle composition can be delivered by contacting the nanoparticle composition with the host cell in vitro. In one embodiment, the nanoparticle composition can be delivered by administering the nanoparticle composition to the host subject in vivo.

[0477] In some embodiments, the ratio of therapeutic agent to lipid (i.e., N / P, where N represents the number of moles of cationic lipid and P represents the number of moles of phosphate present as part of the nucleic acid backbone) in the nanoparticle composition is in the range of 9: 1 to 20: 1, such as in the range of 10: 1 to 15: 1. Exemplary N / P ranges include about 9: 1, about 10: 1, about 11: 1, about 12: 1, about 13: 1, about 14: 1, about 15: 1, about 16: 1, about 17: 1, about 18: 1, about 19: 1, and about 20: 1.

[0478] In one embodiment, lipid nanoparticles for targeted delivery of therapeutic payloads are provided herein. Nanoparticle compositions can be designed for one or more specific applications or targets. For example, nanoparticle compositions can be designed for delivering therapeutic and / or preventive agents, such as RNA, to specific cells, tissues, organs or systems or groups thereof in mammals. The physicochemical properties of nanoparticle compositions can be changed to increase selectivity for specific body targets. For example, the particle size can be adjusted based on the window size of different organs. The therapeutic and / or preventive agents contained in the nanoparticle compositions can also be selected based on one or more desired delivery targets. For example, therapeutic and / or preventive agents can be selected for specific indications, illnesses, diseases or conditions and / or for delivery to specific cells, tissues, organs or systems or groups thereof (e.g., local or specific delivery). In certain embodiments, nanoparticle compositions can include mRNA encoding a target polypeptide, which can be translated intracellularly to produce a target polypeptide. Such compositions can be designed to be specifically delivered to specific organs. In one embodiment, the composition can be designed to be specifically delivered to the liver of a mammal. In one embodiment, the composition can be designed to be specifically delivered to the lungs of a mammal. In one embodiment, the composition can be designed to be delivered specifically to a target site, such as a site of inflammation, cancer, or infection.

[0479] The amount of therapeutic and / or preventive agents in the nanoparticle composition can depend on the size, composition, target and / or application required, or other characteristics of the nanoparticle composition, and the characteristics of the therapeutic and / or preventive agents. For example, the amount of RNA that can be used in the nanoparticle composition can depend on the size, sequence and other characteristics of the RNA. The relative amount of therapeutic and / or preventive agents and other components (such as lipids) in the nanoparticle composition can also vary. In one embodiment, the wt / wt ratio of lipid components and therapeutic and / or preventive agents in the nanoparticle composition can be about 5:1 to about 60:1, such as about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 22:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1 and 60:1. For example, the wt / wt ratio of the lipid component to the therapeutic and / or prophylactic agent can be about 10: 1 to about 40: 1. In certain embodiments, the wt / wt ratio is about 20: 1. The amount of the therapeutic and / or prophylactic agent in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., UV-visible spectroscopy).

[0480] In one embodiment, the nanoparticle composition comprises one or more RNAs, and the one or more RNAs, lipids, and amounts thereof can be selected to provide a specific N:P ratio.

[0481] Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of nanoparticle compositions. Zeta potential can be measured using dynamic light scattering or potentiometric methods (e.g., potentiometric titration). Dynamic light scattering can also be used to determine particle size. Instruments such as Zetasizer Nano ZS (Malvem Instruments Ltd, Malvem, Worcestershire, UK) can also be used to measure multiple features of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

[0482] In various embodiments, the average size of the nanoparticle composition can be between tens of nanometers and hundreds of nanometers. For example, the average size can be about 40nm to about 150nm, such as about 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm or 150nm. In one embodiment, the lipid nanoparticle composition comprises a plurality of nanoparticles, and the average size of a plurality of nanoparticles is about 40nm to about 150nm. In one embodiment, the average size of a plurality of particles is about 50nm to about 100nm. In one embodiment, the average size of a plurality of particles is about 95nm.

[0483] In one embodiment, compared with the average size of conventional four lipid component lipid nanoparticle compositions (such as four lipid component lipid nanoparticles with a molar ratio of cationic lipid: steroid: phospholipid: polymer-bound lipid of about 50:38.5:10:1.5), the average size of the three lipid component lipid nanoparticle compositions provided herein (without phospholipid components, or with a reduced / insignificant amount of phospholipid components) has an increased size. In one embodiment, the average size of the three lipid component nanoparticle compositions provided herein has an increase in size of at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80%. In one embodiment, the average size of the three lipid component nanoparticle compositions provided herein has an increase in size of no more than about 90%, no more than about 100% or no more than about 110%. In one embodiment, the average size of the three lipid component nanoparticle compositions provided herein is about 90nm to about 150nm. In one embodiment, the average size of (three lipid component nanoparticle compositions provided herein) is about 100nm to about 140nm. In one embodiment, the average size is about 110nm to about 130nm. In one embodiment, the average size is about 100nm to about 110nm. In one embodiment, the average size is about 110nm to about 120nm. In one embodiment, the average size is about 120nm to about 130nm. In one embodiment, the average size is about 90nm. In one embodiment, the average size is about 95nm. In one embodiment, the average size is about 100nm. In one embodiment, the average size is about 105nm. In one embodiment, the average size is about 110nm. In one embodiment, the average size is about 115nm. In one embodiment, the average size is about 120nm. In one embodiment, the average size is about 125nm. In one embodiment, the average size is about 130nm. In one embodiment, the average size is about 135nm. In one embodiment, the average size is about 140nm. In one embodiment, the average size is about 145nm. In one embodiment, the average size is about 150nm. In one embodiment, the average size is about 155nm.

[0484] The nanoparticle composition can be relatively homogeneous. The polydispersity index can be used to indicate the uniformity of the nanoparticle composition, such as the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In one embodiment, the polydispersity index of the nanoparticle composition can be about 0.10 to about 0.20. In one embodiment, the lipid nanoparticle composition comprises a plurality of nanoparticles, and the polydispersity index (PDI) of the nanoparticle composition is about 0 to about 0.25. In one embodiment, the PDI of the nanoparticle composition is less than 0.1.

[0485] The zeta potential of a nanoparticle composition can be used to indicate the zeta potential of the composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low positive or negative charges are generally desirable because higher charged materials can interact undesirably with cells, tissues, and other components in the body. In one embodiment, the zeta potential of the nanoparticle composition can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0486] The encapsulation efficiency of therapeutic and / or prophylactic agents describes the amount of therapeutic and / or prophylactic agents encapsulated or otherwise associated with nanoparticle compositions after preparation relative to the initial amount provided. Encapsulation efficiency is desirably high (e.g., close to 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or prophylactic agents in a solution containing nanoparticle compositions before and after the nanoparticle compositions are destroyed with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agents (e.g., RNA) in a solution. For nanoparticle compositions described herein, the encapsulation efficiency of therapeutic and / or prophylactic agents can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0487] The nanoparticle composition may optionally include one or more coatings. For example, the nanoparticle composition may be formulated into a capsule, film, or tablet having a coating. The capsule, film, or tablet containing the composition described herein may have any useful size, tensile strength, hardness, or density.

[0488] 6.3.3 Pharmaceutical Compositions

[0489] According to the present disclosure, the nanoparticle composition can be formulated as a pharmaceutical composition in whole or in part. The pharmaceutical composition can include one or more nanoparticle compositions. For example, the pharmaceutical composition can include one or more nanoparticle compositions, and the one or more nanoparticle compositions include one or more different therapeutic agents and / or preventive agents. The pharmaceutical composition may also include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as those described herein. General guidelines for the preparation and manufacture of pharmaceutical compositions and agents can be found in, for example, Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and auxiliary ingredients can be used in any pharmaceutical composition, unless any conventional excipient or auxiliary ingredient is incompatible with one or more components of the nanoparticle composition. If the combination of the excipient or auxiliary ingredient with the components of the nanoparticle composition will result in any undesirable biological effect or other harmful effect, the excipient or auxiliary ingredient is incompatible with the components of the nanoparticle composition.

[0490] In one embodiment, one or more excipients or adjuvants may constitute greater than 50% of the total mass or volume of the pharmaceutical composition comprising the nanoparticle composition. For example, one or more excipients or adjuvants may constitute 50%, 60%, 70%, 80%, 90% or more of the pharmaceutical composition. In one embodiment, the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure. In one embodiment, the excipient is approved for human and for veterinary use. In one embodiment, the excipient is approved by the U.S. Food and Drug Administration. In one embodiment, the excipient is pharmaceutical grade. In one embodiment, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia and / or the International Pharmacopoeia.

[0491] The relative amounts of one or more nanoparticle compositions, one or more pharmaceutically acceptable excipients, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated and further on the route of administration of the composition. For example, a pharmaceutical composition may include between 0.1% and 100% (wt / wt) of one or more nanoparticle compositions.

[0492] In some embodiments, nanoparticle compositions and / or pharmaceutical compositions of the disclosure are refrigerated or frozen for storage and transport (for example, stored at 4°C or lower, such as between about -150°C and about 0°C or between about -80°C and about -20°C (for example, about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C)). In certain embodiments, the present disclosure is also directed to methods of maintaining or increasing the stability of lipid nanoparticle compositions and / or pharmaceutical compositions by storing the nanoparticle compositions and / or pharmaceutical compositions at 4°C or lower, such as between about -150°C and about 0°C, or between about -80°C and about -20°C (e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C). For example, the nanoparticle compositions and / or pharmaceutical compositions disclosed herein are stable for about at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months at a temperature of, for example, 4°C or less (e.g., between about 4°C and -20°C). In one embodiment, the formulation is stable for at least 4 weeks at about 4°C. In certain embodiments, the pharmaceutical compositions of the present disclosure comprise a nanoparticle composition disclosed herein and a pharmaceutically acceptable carrier selected from one or more of the following: Tris, acetate (e.g., sodium acetate), citrate (e.g., sodium citrate), saline, PBS, and sucrose. In certain embodiments, the pH of the pharmaceutical composition of the present disclosure is between about 7 and 8 (e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or between 7.5 and 8, or between 7 and 7.8). For example, the pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein, Tris, saline, and sucrose, and has a pH of about 7.5-8, which is suitable for storage and / or transportation at, for example, about -20°C. For example, the pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein and PBS, and has a pH of about 7-7.8, which is suitable for storage and / or transportation at, for example, about 4°C or lower.In the context of the present disclosure, "stability", "stabilized" and "stable" refer to the resistance of the nanoparticle compositions and / or pharmaceutical compositions disclosed herein to chemical or physical changes (e.g., degradation, particle size changes, aggregation, changes in encapsulation, etc.) under given manufacturing, preparation, transportation, storage and / or use conditions, for example, when stress is applied, such as shear force, freeze / thaw stress, etc.

[0493] Nanoparticle compositions and / or pharmaceutical compositions comprising one or more nanoparticle compositions can be administered to any patient or subject, including patients or subjects who can benefit from the therapeutic effect provided by delivering therapeutic and / or prophylactic agents to one or more specific cells, tissues, organs or systems or groups thereof, such as the renal system. Although the description of nanoparticle compositions and pharmaceutical compositions comprising nanoparticle compositions provided herein is mainly directed to compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other mammal. Improvements to compositions suitable for administration to humans in order to make the compositions suitable for administration to various animals are well known, and veterinary pharmacologists with ordinary skills can design and / or perform such improvements only through ordinary experiments (if any). It is contemplated that subjects to whom the compositions are administered include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats.

[0494] Pharmaceutical compositions comprising one or more nanoparticle compositions can be prepared by any method known or later developed in the art of pharmacology. In general, such preparation methods include combining the active ingredient with an excipient and / or one or more other auxiliary ingredients, and then, if desired or necessary, dividing, shaping and / or packaging the product into desired single or multiple dosage units.

[0495] Pharmaceutical compositions according to the present disclosure can be prepared, packaged and / or sold in bulk, as a single unit dose and / or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient (e.g., a nanoparticle composition). The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to the subject and / or a convenient portion of such a dose, such as half or one-third of such a dose.

[0496] Pharmaceutical compositions can be prepared into various forms suitable for various routes and methods of administration. For example, pharmaceutical compositions can be prepared into liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders and granules), dosage forms for topical and / or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and patches), suspensions, powders and other forms.

[0497] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups and / or elixirs. In addition to the active ingredient, the liquid dosage form may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan fatty acid esters, and mixtures thereof. In addition to inert diluents, oral compositions may also contain additional therapeutic and / or prophylactic agents, additional agents, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and / or flavoring agents. In certain embodiments for parenteral administration, the composition is mixed with a solubilizer, such as Cremophor TM , alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers and / or combinations thereof.

[0498] Injectable preparations, such as sterile injectable aqueous or oily suspensions, can be prepared according to known techniques using suitable dispersants, wetting agents and / or suspending agents. Sterile injectable preparations can be sterile injectable solutions, suspensions and / or emulsions in nontoxic parenterally acceptable diluents and / or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, USP and isotonic sodium chloride solution. Sterile fixed oils are generally used as solvents or suspending media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid can be used to prepare injections.

[0499] The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter and / or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0500] The present disclosure provides methods for delivering therapeutic and / or prophylactic agents to mammalian cells or organs, producing target polypeptides in mammalian cells, and treating diseases or disorders in mammals in need thereof, the methods comprising administering a nanoparticle composition comprising a therapeutic and / or prophylactic agent to a mammal and / or contacting mammalian cells with the nanoparticle composition.

[0501] 7. Examples

[0502] The examples in this section are offered by way of illustration and not limitation.

[0503] Example 1: Preparation of lipid nanoparticle formulations

[0504] LNP preparations were prepared by mixing lipid and mRNA stock solutions at a flow rate of 1:3 through a T-connector, with a total flow rate of 12 mL / min. Lipid stock solutions were prepared in ethanol with a total lipid concentration of 12.5 mM. mRNA stock solutions were prepared in 20 mM pH 4.0 citrate buffer with a final concentration of 0.125 mg / mL. The size and distribution of all preparations were determined by dynamic light scattering (DLS) measurements. According to the supplier's instructions for the kit, the encapsulation efficiency (EE%) of each preparation was determined by Ribogreen determination. HPLC was used to determine the concentration of the preparation by measuring the UV absorption at 260 nm and comparing the standard concentration linear curve.

[0505] Example 2: Physical properties of lipid nanoparticle formulations

[0506] Lipid nanoparticle compositions comprising mRNA and variable lipid compositions are listed below in Tables 1 to 4 and 9. Physical characterizations of the LNP compositions are listed below in Tables 5 to 8 and 10.

[0507] The structures and names of the cationic lipids tested are listed below:

[0508]

[0509]

[0510] Table 1. LNPs containing lipid-5

[0511]

[0512]

[0513] Table 2. LNPs containing SM-102

[0514]

[0515]

[0516] Table 3. LNPs containing ALC-0315

[0517]

[0518] Table 4. LNPs containing lipid C1

[0519]

[0520] Table 5. Physicochemical characterization of LNPs containing lipid-5

[0521]

[0522]

[0523] Table 6. Physicochemical characterization of LNPs containing SM-102

[0524]

[0525]

[0526] Table 7. Physicochemical Characterization of LNPs Containing ALC-0315

[0527] Preparation ID Size(nm) PDI EE% A-0 65.2 0.061 88.9 A-55 / 1.5 87.81 0.091 85.90 A-55 / 2.0 77.36 0.036 86.17 A-55 / 2.5 75.68 0.006 87.78 A-60 / 1.0 126.2 0.027 87.82 A-60 / 1.5 103.7 0.008 86.50 A-60 / 2.0 93.14 0.014 85.94 A-60 / 2.5 84.05 0.022 83.99 A-65 / 1.0 148.9 0.019 86.4 A-65 / 1.5 129.1 0.019 81.8 A-65 / 2.0 93.1 0.014 85.9

[0528] Table 8. Physicochemical characterization of LNPs containing C1

[0529]

[0530] Table 9. LNPs containing lipid C2

[0531]

[0532] Table 10. Physicochemical characterization of LNPs containing C2

[0533] Preparation ID Size(nm) PDI EE% C2-0 80.7 0.03 96.4 C2-35 / 1.5 148.5 0.27 77.9 C2-37.5 / 1.5 130.2 0.27 85.3 C2-40 / 1.0 124.2 0.01 91.5 C2-40 / 1.5 95.0 0.06 91.9 C2-40 / 2.0 78.2 0.09 94.3 C2-40 / 2.5 71.6 0.06 92.4 C2-40 / 3.0 60.6 0.07 92.8 C2-45 / 0.75 160.2 0.11 77.5 C2-45 / 1.0 130.4 0.02 93.4 C2-45 / 1.5 103.5 0.06 92.5 C2-45 / 2.0 88.8 0.04 91.7 C2-45 / 2.5 79.6 0.04 91.6 C2-45 / 3.0 68.1 0.06 92.3 C2-45 / 3.5 61.2 0.07 89.3 C2-47.5 / 0.75 164.9 0.09 82.0 C2-47.5 / 1.0 135.7 0.05 90.5 C2-47.5 / 1.5 110.9 0.11 93.4 C2-47.5 / 2.0 95.6 0.06 93.2 C2-47.5 / 2.5 81.51 0.02 91.5 C2-47.5 / 3.0 72.5 0.09 90.1 C2-50 / 0.5 188.7 0.05 77.9 C2-50 / 0.75 168.8 0.08 85.0 C2-50 / 1.0 142.1 0.02 94.6 C2-50 / 1.5 124.3 0.01 93.0 C2-50 / 2.0 104.5 0.02 92.0 C2-50 / 2.5 81.1 0.02 94.7 C2-55 / 1.0 156.1 0.00 94.4 C2-55 / 1.5 129.1 0.11 92.4 C2-55 / 2.0 114.2 0.01 91.6 C2-55 / 2.5 91.4 0.03 93.0 C2-60 / 0.5 235.8 0.02 82.1 C2-60 / 1.0 173.6 0.00 91.8 C2-60 / 1.5 140.1 0.01 90.8 C2-60 / 2.0 118.9 0.03 76.3 C2-60 / 2.5 99.7 0.04 84.0 C2-65 / 0.5 286.3 0.03 75.0 C2-65 / 1.0 211.6 0.03 82.7 C2-65 / 1.5 160.0 0.01 82.1 C2-65 / 2.0 133.0 0.07 69.0 C2-65 / 2.5 111.6 0.01 53.6

[0534] Cyro-TEM images of LNPs composed of C1 / cholesterol / DMG-PEG (60% / 38.5% / 1.5%) and LNPs composed of C1 / cholesterol / DMG-PEG (65% / 33% / 2.0%) are provided in Fig. 9A and Fig. 9B Use Ultra-4 is concentrated 20 times (volume change) by centrifugation by 3 component LNP sample.Sample (3 μ l) is deposited on the holey carbon grid (Quantifoil R1.2 / 1.3) of glow discharge and is vitrified using Vitrobot Mark IV (Thermo Fisher Scientific) instrument respectively.Cryo-EM imaging is carried out on the Talos F200C equipped with Ceta 4k x4k camera operated under 200kV accelerating voltage.The result shows that the 3 component LNP based on C1 shows the uniform morphology of the solid sphere lacking aqueous core, which is different from multi-lamellar solid nanoparticle or conventional liposome.

[0535] Example 3: In vivo protein expression

[0536] The lipid nanoparticles with different compositions (listed in Tables 1-4) encapsulating human erythropoietin (hEPO) mRNA were systemically administered to female ICR mice aged 6 to 8 weeks at 0.5 mg / kg per week by tail vein injection, and the mouse blood was sampled at a specific time point (e.g., 6 hours) after administration. For the same preparation, three to five mice were included for administration. After the last sampling time point, the mice were euthanized by overdose CO2. Serum was separated from whole blood by centrifugation at 5000g for 10 minutes at 4°C, rapidly frozen and stored at -80°C for analysis. In order to evaluate hEPO expression, ELISA was performed using a commercially available kit (DEP00, R&D systems;) according to the manufacturer's instructions.

[0537] GFP encoding mRNA was encapsulated in lipid-5 containing LNPs with different lipid compositions. LNPs were systemically administered to 8 to 10 week old female Balb / c mice at a dose of 0.1 mg / kg by intramuscular injection. Muscles around the injection point were collected 6 hours after intramuscular administration and then cell lysis was performed with cell lysis buffer. Total protein in the samples was detected using the Pierce BCA protein assay kit. GFP and IL-6 in muscle were determined by ELISA assay using the GFP SimpleStem ELISA kit and the mouse IL-6 ELISA kit, respectively.

[0538] Figure 1Normalized hEPO expression level when showing the three lipid LNPs comprising lipid-5 used in vivo.Lipid-5 formulation group also shows impressive hEPO expression in vivo.Use conventional four lipid LNPs (L-0) as reference group to normalize hEPO expression level.In lipid-5 formulation group, it is found that 25 different LNP formulations have higher hEPO expression level in vivo than conventional formulations (L-0).In these formulations, the amount of cationic lipid (lipid-5) is in the range of about 40mol% to about 70mol%, and the amount of PEG-lipid is in the range of about 0.5mol% to about 3.0mol%.In addition, it is also observed that compared with L-0 control, 7 of these formulations make the gene expression level in vivo increase by more than 2 times.Data also show that the three lipid LNP formulations containing lipid-5 less than 40mol% or more than 70mol% and PEG-lipid greater than 3mol% have relatively low in vivo activity.

[0539] Figure 2 Normalized hEPO expression level when being shown in vivo using three lipid LNPs comprising SM-102.When compared with reference preparation (S-0), SM-102 preparation group also shows obvious benefit in terms of hEPO expression in vivo.In SM-102 preparation group, it is found that 20 kinds of LNP preparations tested have higher hEPO expression level in vivo than conventional preparation (S-0).In these preparations, the amount of cationic lipid (SM-102) is in the range of about 40mol% to about 70mol%, and the amount of PEG-lipid is in the range of about 0.5mol% to about 3mol%.It is also observed that, compared with S-0 control, 10 kinds in these preparations improve gene expression level in vivo by more than 2 times.In addition, when compared with reference preparation (S-0), one in the preparation shows hEPO expression level in vivo exceeding three times.Data also show that the three lipid LNP preparations containing SM-102 less than 40mol% or more than 70mol% and PEG-lipid greater than 3mol% have relatively low in vivo activity.

[0540] Based on the results, LNPs comprising cationic lipids in amounts ranging from about 40 mol% to about 70 mol% and PEG-lipids in amounts less than 3 mol% appear to be a general area where tri-lipid LNP formulations have superior in vivo activity over conventional tetra-lipid LNP formulations.

[0541] Figure 3Normalized hEPO expression levels when three lipid LNPs containing cationic lipids ALC-0315 or C1 are shown in vivo. In the ALC-0315 group, ten different LNP compositions (Table 3 and Table 7) have been prepared and characterized. All 10 LNP preparations containing ALC-0315 were found to have higher hEPO expression levels in vivo than conventional preparations (A-0). It was also observed that 5 of these preparations increased the gene expression level in vivo by more than 2 times compared with the A-0 control. In addition, 6 LNP preparations containing C1 (Table 4 and Table 8) have been prepared and characterized. These LNP preparations show higher in vivo activity than conventional preparations (C1-0).

[0542] In U.S. Pat. No. 11,191,849, it was mentioned that increasing the PEG-lipid ratio to 3.3% in a PL-free (phospholipid-free) formulation slightly affected the efficiency of the LNP in terms of ApoB silencing activity. However, when using tri-lipid LNPs containing more than 3 mol% of PEG-lipid, the data presented herein show a very different situation in terms of mRNA delivery. Figure 4 Normalized hEPO expression levels when three lipid LNPs containing more than 3 mol% PEG-lipids are shown when administered in vivo. All formulations containing more than 3 mol% PEG-lipids show very low in vivo hEPO expression compared to other three lipid LNPs containing lower levels of PEG-lipids. The hEPO expression levels of all three lipid LNPs containing more than 3 mol% PEG-lipids are less than 50% of the hEPO expression levels of conventional formulations.

[0543] Table 11 and Figure 8 Normalized hEPO expression level when showing the three lipid LNPs comprising C2 used in vivo. The three lipid LNPs containing C2 show good hEPO expression activity in vivo. It is found that the LNP preparations of seventeen kinds of tests have higher hEPO expression levels than conventional preparations (C2-0). The cationic lipids in the preparations are in the range of about 40% to about 55%, and the PEG-lipids are in the range of about 1.0% to about 2.5%.

[0544] Table 11. hEPO expression of LNPs containing C2

[0545]

[0546] Example 4: Fragment Analysis

[0547] like Figure 5A , Figure 5B and Figure 5CAs shown, fragment analysis (FA) was performed to test the long-term stability of LNP formulations at different temperatures. mRNA was isolated from LNP using the RNeasy micro kit (50). Fragment analysis was performed on an Agilant 5280 analyzer.

[0548] LNP can protect RNA from degradation by ubiquitous RNase. Three kinds of LNPs containing lipid-5 that encapsulate mRNA encoding GFP were used in fragment analysis. These preparations were stored for more than four months at room temperature (rt), 2°C-8°C or -20°C. At room temperature, mRNA purity decreased over time in all three preparations. No significant differences were found between conventional LNP preparations and three lipid LNP preparations. When these LNPs were stored at 2°C-8°C or -20°C, RNA purity was not significantly reduced even after storage for four months in all three preparations tested. These results show that the three lipid LNP preparations have similar or identical characteristics in protecting the RNA encapsulated from degradation.

[0549] Figure 5D and Figure 5E The updated long-term stability of lipid-5-containing LNPs stored for up to 400 days at 2° C.-8° C. or -20° C., respectively, is shown. No significant decrease in mRNA purity in lipid-5-containing LNPs was found over a year under both storage conditions.

[0550] Example 5: Intramuscular Administration

[0551] The intramuscular administration of two LNPs containing lipid 5 (L-50 / 1.5, L-60 / 1.5) was investigated by comparing the GFP expression level with the conventional formulation (L-0). Fig. 6A and Figure 6B As shown, in the group of intramuscular administration of three lipid LNPs (L-50 / 1.5, L-60 / 1.5), GFP expression level increased by about 2 times compared with conventional preparations (L-0). Data show that the three lipid LNP preparations have significant advantages in gene expression whether by intramuscular injection or intravenous injection. Detection of cytokine interleukin-6 (IL-6) to evaluate the effect of LNP administration on immunostimulation. When IL-6 production was normalized for GFP expression, it was found that under the same GFP expression level, the three lipid LNP preparations induced relatively less IL-6 production in vivo, indicating that the three lipid LNP preparations (L-50 / 1.5 and L-60 / 1.5) have relatively less side effects compared with conventional four lipid LNP preparations (L-0).

[0552] Example 6: Weight Change

[0553] After administration, some side effects, such as weight changes, can be induced in mice due to the immune stimulation of LNPs. Different LNP compositions containing lipid-5 have been used to study the level and duration of side effects after intravenous or intramuscular administration. All LNP formulations encapsulate mRNA encoding luciferase. After administration of LNPs, changes in mouse body weight were tracked over the course of 7 days ( Fig. 7A and Figure 7B ). For intravenous administration, the tri-lipid LNP formulation group showed lower maximum weight loss and faster weight recovery. The mouse groups injected intravenously with the tri-lipid LNP formulation (L-50 / 1.5 and L-60 / 1.5) recovered to 100% body weight within two days, while the mouse group injected intravenously with the conventional tetra-lipid LNP formulation (L-0) took more than four days to recover. The L-50 / 1.5 group showed a relatively faster weight gain, indicating that the side effects were minimal after intravenous administration of LNP.

[0554] For intramuscular injection, all three groups lost more than 5% of body weight rapidly within one day of administration, indicating that intramuscular administration has more serious side effects than intravenous administration in this system. Similarly, the mice group treated with three lipid LNP formulations (L-50 / 1.5 and L-60 / 1.5) recovered the required time of their body weight after intramuscular administration less than the group treated with conventional four lipid LNP formulations (L-0). L-50 / 1.5 and L-60 / 1.5 groups need 7 days to fully recover to their original body weight, while the body weight in the L-0 group only changes slightly. The mice in the L-50 / 1.5 group gain weight faster than the other two groups, indicating that the side effects caused by this preparation are relatively few.

Claims

1. A lipid nanoparticle, comprising: (a) a cationic lipid in an amount of about 40 mol% to about 75 mol% of the total lipids present in the nanoparticle, wherein the cationic lipid comprises (i) at least one tertiary amine or quaternary ammonium moiety, and (ii) at least one C 14 -C 42 an alkyl ester moiety; (b) a steroid in an amount from about 22 mol % to about 59.5 mol % of the total lipids present in the nanoparticle; (c) polymer-bound lipids in an amount from about 0.5 mol % to about 2.5 mol % of the total lipids present in the nanoparticle; and (d) nucleic acids; Provided that the lipid nanoparticles contain no more than 0.5 mole percent phospholipids.

2. The lipid nanoparticle of claim 1, wherein the amount of the cationic lipid is about 55 mol % to about 65 mol % of the total lipid present in the nanoparticle.

3. The lipid nanoparticle of claim 2, wherein the amount of the cationic lipid is about 60 mol % of the total lipid present in the nanoparticle.

4. The lipid nanoparticle of any one of claims 1 to 3, wherein the amount of the polymer-bound lipid is about 1 mol % to about 2 mol % of the total lipid present in the nanoparticle.

5. The lipid nanoparticle of claim 4, wherein the amount of the polymer-bound lipid is about 1.5 mol % of the total lipid present in the nanoparticle.

6. The lipid nanoparticle of any one of claims 1 to 5, wherein the amount of the steroid is about 33 mol % to about 44 mol % of the total lipid present in the nanoparticle.

7. The lipid nanoparticle of claim 1, wherein the amount of the cationic lipid is about 45 mol % to about 70 mol % of the total lipid present in the nanoparticle, and wherein the amount of the polymer-bound lipid is about 1 mol % to about 2.5 mol % of the total lipid present in the nanoparticle.

8. The lipid nanoparticle of claim 1, wherein the amount of the cationic lipid is about 45 mol % to about 65 mol % of the total lipid present in the nanoparticle, and wherein the amount of the polymer-bound lipid is about 1 mol % to about 2.5 mol % of the total lipid present in the nanoparticle.

9. The lipid nanoparticle of claim 1, wherein the amount of the cationic lipid is about 45 mol % to about 65 mol % of the total lipid present in the nanoparticle, and wherein the amount of the polymer-bound lipid is about 1 mol % to about 2 mol % of the total lipid present in the nanoparticle.

10. The lipid nanoparticle of claim 1, wherein the amount of the cationic lipid is about 50 mol % to about 65 mol % of the total lipid present in the nanoparticle, and wherein the amount of the polymer-bound lipid is about 0.5 mol % to about 2.5 mol % of the total lipid present in the nanoparticle.

11. The lipid nanoparticle of claim 1, wherein the amount of the cationic lipid is about 50 mol % to about 60 mol % of the total lipid present in the nanoparticle, and wherein the amount of the polymer-bound lipid is about 0.8 mol % to about 2 mol % of the total lipid present in the nanoparticle.

12. The lipid nanoparticle of claim 1, wherein the amount of the cationic lipid is about 50 mol % to about 60 mol % of the total lipid present in the nanoparticle, and wherein the amount of the polymer-bound lipid is about 1 mol % to about 2 mol % of the total lipid present in the nanoparticle.

13. The lipid nanoparticle of claim 1, wherein the amount of the cationic lipid is about 55 mol % to about 65 mol % of the total lipid present in the nanoparticle, and wherein the amount of the polymer-bound lipid is about 1 mol % to about 2.5 mol % of the total lipid present in the nanoparticle.

14. The lipid nanoparticle of claim 1, wherein the amount of the cationic lipid is about 60 mol % to about 65 mol % of the total lipid present in the nanoparticle, and wherein the amount of the polymer-bound lipid is about 1 mol % to about 2 mol % of the total lipid present in the nanoparticle.

15. The lipid nanoparticle of claim 1, wherein the amount of the cationic lipid is about 60 mol % to about 65 mol % of the total lipid present in the nanoparticle, and wherein the amount of the polymer-bound lipid is about 1.5 mol % to about 2.5 mol % of the total lipid present in the nanoparticle.

16. The lipid nanoparticle of claim 1, wherein the amount of the cationic lipid is about 40 mol % to about 55 mol % of the total lipid present in the nanoparticle, and the amount of the polymer-bound lipid is about 1 mol % to about 2.5 mol % of the total lipid present in the nanoparticle.

17. The lipid nanoparticle of any one of claims 1 to 16, wherein the polymer-bound lipid is a polyethylene glycol (PEG)-bound lipid.

18. The lipid nanoparticle of claim 17, wherein the polymer-bound lipid is a PEG-diacylglycerol (PEG-DAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, a PEG-dimyristyloxypropyl (PEG-DMA) conjugate, a PEG-distearyloxypropyl (PEG-DSA) conjugate, or a mixture thereof.

19. The lipid nanoparticle of claim 17, wherein the polymer-bound lipid is DMG-PEG2000 or DMPE-PEG2000.

20. The lipid nanoparticle of any one of claims 1 to 19, wherein the steroid is a glucocorticoid, a mineralocorticoid, clobetasol, cholesterol or a cholesterol derivative.

21. The lipid nanoparticle of any one of claims 1 to 20, wherein the cationic lipid has a pKa of at least 9.

22. The lipid nanoparticle of any one of claims 1 to 21, wherein the cationic lipid comprises only one ester moiety, the ester moiety being C 14 -C 42 Alkyl ester moiety.

23. The lipid nanoparticle of any one of claims 1 to 21, wherein the cationic lipid comprises the C 14 -C 42 An alkyl ester moiety and at least a second ester moiety.

24. The lipid nanoparticle of any one of claims 1 to 23, wherein at least one ester portion of the cationic lipid is -C(=O)OR 1 or -OC(=O)R 1 , where R 1 It is a branched chain C 14 -C 42 alkyl.

25. The lipid nanoparticle of claim 23 or 24, wherein the second ester portion of the cationic lipid is -C(=O)OR 2 or -OC(=O)R 2 , where R 2 Is a straight chain or branched C6-C 42 alkyl.

26. The lipid nanoparticle of any one of claims 1 to 21, wherein the cationic lipid comprises the C 14 -C 42 An alkyl ester moiety and at least a second ester moiety and a third ester moiety.

27. The lipid nanoparticle of claim 26, wherein the second alkyl ester moiety and the third alkyl ester moiety are independently -C(=O)OR 2 or -OC(=O)R 2 , where each R 2 Independently straight chain C7-C 11 alkyl.

28. The lipid nanoparticle of any one of claims 1 to 27, wherein the cationic lipid comprises only one tertiary amine.

29. The lipid nanoparticle of any one of claims 1 to 27, wherein the cationic lipid comprises two tertiary amines.

30. The lipid nanoparticle of any one of claims 1 to 29, wherein the cationic lipid comprises part, where G 1 It is C2-C 12 Alkylene; X 1 is -C(=O)O- or -OC(=O)-; and R 1 It is a branched chain C 14 -C 42 alkyl.

31. The lipid nanoparticle of any one of claims 1 to 30, wherein the cationic lipid has formula (I): or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, in: G 1 It is C2-C 12 Alkylene; X 1 is -C(=O)O- or -OC(=O)-; R 1 It is a branched chain C 14 -C 42 alkyl; R y is C1-C6 alkyl or -G 2 -X 2 -R 2 ; G 2 It is C2-C 12 Alkylene; X 2 is -C(=O)O- or -OC(=O)-; R 2 Is a straight chain or branched C6-C 42 alkyl; R x is C1-C6 alkyl or -G 3 -R 3 ; G 3 It is C2-C 12 Alkylene; R 3 is -N(R 4 )R 5 or -OR 6 ; R 4 It is C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 Aryl; R 5 It is C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 Aryl; or R 4 and R 5 Together with the nitrogen to which they are attached they form a ring portion; R 6 is hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 aryl; and wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, alkylene and cyclic moiety is independently optionally substituted.

32. The lipid nanoparticle of claim 31, wherein the cationic lipid has formula (II): or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

33. The lipid nanoparticle of claim 31 or 32, wherein R 1 It is a branched chain C 14 -C 24 alkyl.

34. The lipid nanoparticle of any one of claims 31 to 33, wherein R 2 It is a straight chain C6-C 24 alkyl.

35. The lipid nanoparticle of any one of claims 31 to 33, wherein R 2 It is a branched chain C 14 -C 24 alkyl.

36. The lipid nanoparticle of any one of claims 31 to 35, wherein R 3 It is -OH.

37. The lipid nanoparticle of any one of claims 31 to 35, wherein R 3 Yes-N(R 4 )R 5 .

38. The lipid nanoparticle of claim 32, wherein the cationic lipid has formula (II-A): or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, in: G 1 is C5-C7 alkylene; R 1 It is a branched chain C 14 -C 19 alkyl; G 2 is C5-C7 alkylene; R 2 It is a straight chain C7-C 13 alkyl; G 3 It is a C2-C4 alkylene group.

39. The lipid nanoparticle of claim 38, wherein the cationic lipid has formula (II-A-1): or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

40. The lipid nanoparticle of claim 32, wherein the cationic lipid has formula (II-B): or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, in: G 1 is C5-C7 alkylene; R 1 It is a branched chain C 14 -C 19 alkyl; G 2 is C5-C7 alkylene; R 2 It is a branched chain C 14 -C 19 alkyl; G 3 It is a C2-C4 alkylene group.

41. The lipid nanoparticle of claim 40, wherein the cationic lipid has formula (II-B-1): or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

42. The lipid nanoparticle of claim 32, wherein the cationic lipid has formula (II-C): or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, in: G 1 is C5-C7 alkylene; R 1 It is a branched chain C 14 -C 19 alkyl; G 2 is C5-C7 alkylene; R 2 It is a branched chain C 14 -C 19 alkyl; G 3 is C2-C4 alkylene; R 4 is a C3-C8 cycloalkyl group; and R 5 It is a C2-C4 alkyl group substituted by a hydroxy group.

43. The lipid nanoparticle of claim 42, wherein the cationic lipid has formula (II-C-1): or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

44. The lipid nanoparticle of claim 31, wherein the cationic lipid is 45. The lipid nanoparticle of any one of claims 1 to 30, wherein the cationic lipid has formula (III): or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, in: G 1 It is C2-C 12 Alkylene; X 1 is -C(=O)O- or -OC(=O)-; R 1 It is a branched chain C 14 -C 42 alkyl; G 2 It is C2-C 12 Alkylene; Each L 2 Independently for -X 2 -R 2 ; Each X 2 are independently -C(=O)O- or -OC(=O)-; Each R 2 are independently straight chain or branched C6-C 42 alkyl; m is 1 or 2; G 3 It is C2-C 12 Alkylene; R 4 It is C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 Aryl; R 5 It is C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 Aryl; and wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, alkylene and cyclic moiety is independently optionally substituted.

46. ​​The lipid nanoparticle of claim 45, wherein the cationic lipid has formula (IV): or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, in: y1 is an integer from 0 to 9.

47. The lipid nanoparticle of claim 45 or 46, wherein R 1 It is a branched chain C 14 -C 24 alkyl.

48. The lipid nanoparticle of any one of claims 45 to 47, wherein each L 2 are independently -OC(=O)R 2 , and each R 2 Independently straight chain C7-C 11 alkyl.

49. The lipid nanoparticle of any one of claims 45 to 48, wherein R 5 It is -CH2CH2OH.

50. The lipid nanoparticle of claim 46, wherein the cationic lipid has formula (IV-A): or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, in: G 1 is C5-C7 alkylene; R 1 It is a branched chain C 14 -C 19 alkyl; Each R 2 Independently straight chain C7-C 13 alkyl; G 3 is C2-C4 alkylene; R 4 is a C3-C8 cycloalkyl group; R 5 is a C2-C4 alkyl group substituted with a hydroxy group; and y1 is an integer from 2 to 6.

51. The lipid nanoparticle of claim 47, wherein the cationic lipid has formula (IV-A-1): or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

52. The lipid nanoparticle of claim 45, wherein the cationic lipid is 53. The lipid nanoparticle of any one of claims 1 to 52, wherein the lipid nanoparticle does not comprise a phospholipid.

54. The lipid nanoparticle of any one of claims 1 to 53, wherein the lipid nanoparticle has a lipid:nucleic acid mass ratio of about 9:1 to about 20:

1.

55. The lipid nanoparticle of any one of claims 1 to 54, wherein the nucleic acid is mRNA.

56. The lipid nanoparticle of claim 55, wherein the mRNA is an mRNA encoding an antigen or a fragment or epitope thereof.

57. The lipid nanoparticle of claim 56, wherein the mRNA is a monocistronic mRNA.

58. The lipid nanoparticle of claim 56, wherein the mRNA is a polycistronic mRNA.

59. The lipid nanoparticle of any one of claims 56 to 58, wherein the antigen is a pathogenic antigen.

60. The lipid nanoparticle of any one of claims 56 to 58, wherein the antigen is a tumor-associated antigen.

61. The lipid nanoparticle of any one of claims 1 to 60, wherein the nucleic acid is a small interfering RNA (siRNA).

62. The lipid nanoparticle of any one of claims 1 to 61, wherein the nucleic acid is chemically modified.

63. A lipid nanoparticle as described in any one of claims 1 to 62, wherein the size of the nanoparticle is increased compared to a reference formulation; wherein the reference formulation differs from the lipid nanoparticle in that: (i) the reference formulation comprises a phospholipid, and (ii) the molar ratio of the cationic lipid: the steroid: the phospholipid: the polymer-bound lipid is approximately 50:38.5:10:1.

5.

64. The lipid nanoparticle of claim 63, wherein the size of the nanoparticle is increased by at least about 50% compared to the reference formulation.

65. The lipid nanoparticle of any one of claims 1 to 62, wherein the size of the nanoparticle is about 100 nm to about 140 nm.

66. The lipid nanoparticle of claim 65, wherein the size of the nanoparticle is about 110 nm to about 130 nm.

67. The lipid nanoparticle of any one of claims 1 to 66, having an encapsulation efficiency of at least about 80% of the nucleic acid.

68. The lipid nanoparticle of claim 67, having an encapsulation efficiency of at least about 90% of the nucleic acid.

69. A lipid nanoparticle as described in any one of claims 1 to 68, wherein the nucleic acid encodes an RNA or a protein; and wherein the amount of RNA or protein expressed by the nucleic acid in the nanoparticle in a mammalian cell or a mammalian tissue is greater than the amount of RNA or protein expressed by the nucleic acid in a reference preparation; wherein the reference preparation differs from the lipid nanoparticle in that (i) the reference preparation comprises a phospholipid, and (ii) the molar ratio of the cationic lipid: the steroid: the phospholipid: the polymer-bound lipid is about 50:38.5:10:1.

5.

70. The lipid nanoparticle of claim 69, wherein the amount of RNA or protein expressed by the nucleic acid in the nanoparticle is at least about 50% greater than the amount of RNA or protein expressed by the nucleic acid in a reference preparation.

71. The lipid nanoparticle of claim 70, wherein the amount of RNA or protein expressed by the nucleic acid in the nanoparticle is at least about 100% greater than the amount of RNA or protein expressed by the nucleic acid in a reference preparation.

72. A pharmaceutical composition comprising the lipid nanoparticles of any one of claims 1 to 71 and a pharmaceutically acceptable excipient.

73. A method for expressing mRNA in a mammalian cell or a mammalian tissue, the method comprising delivering the lipid nanoparticle of any one of claims 1 to 71 or the pharmaceutical composition of claim 72 to the mammalian cell or the tissue of the mammal, wherein the nucleic acid is mRNA; and wherein the delivered mRNA is expressed in the mammalian cell or in the tissue of the mammal.

74. A method for introducing mRNA encoding a protein into a cell, the method comprising contacting the cell with the lipid nanoparticle of any one of claims 1 to 71 or the pharmaceutical composition of claim 72.

75. The method of claim 74, wherein the cell is in a mammal.

76. A method for treating a disease or condition in a human, the method comprising administering to the human a therapeutically effective amount of the lipid nanoparticle of any one of claims 1 to 71 or the pharmaceutical composition of claim 72.

77. A method for treating a disease or condition caused by impaired expression of a protein in a human, the method comprising administering to the human a therapeutically effective amount of the lipid nanoparticle of any one of claims 1 to 71 or the pharmaceutical composition of claim 72, wherein the nucleic acid is an mRNA encoding the protein.

78. The method of claim 76 or 77, wherein the disease or condition is a viral infection, a liver disease or condition, or cancer.

79. The method of any one of claims 76 to 78, wherein the administration is intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, subcutaneous, or intradermal.

80. A method for preventing a disease or condition in a subject, the method comprising administering to the subject a vaccine comprising a therapeutically effective amount of a lipid nanoparticle as described in any one of claims 1 to 71 or a pharmaceutical composition as described in claim 72, wherein a nucleic acid encodes an antigen associated with the disease or condition, thereby triggering an immune response against the disease or condition in the subject.

81. The method of claim 80, wherein the disease or condition is a viral infection, a liver disease or condition, or cancer.

82. The method of claim 80 or 81, wherein the antigen is a virus.

83. The method of any one of claims 80 to 82, wherein the nucleic acid is mRNA.

84. The method of claim 83, wherein the mRNA encodes a pathogenic protein or an antigenic fragment or epitope thereof that is specific to a pathogen.

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