Lipid nanoparticle composition

Abstract

Provided herein are lipid nanoparticle compositions comprising a cationic lipid, a steroid, a polymer-conjugated lipid, and 0.5 mole percent or less of a phospholipid, which may be used to deliver a therapeutic payload (e.g., mRNA, siRNA, DNA) for therapeutic or prophylactic purposes.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Patent Application No. PCT / CN2022 / 113414, filed August 18, 2022, the contents of which are incorporated herein by reference.

[0002] 2.Technical Field Provided herein are lipid nanoparticle (LNP) compositions comprising a cationic lipid, a steroid, a polymer-conjugated lipid, and 0.5 mole percent or less of a phospholipid that can be used to deliver a therapeutic payload (e.g., mRNA, siRNA, miRNA, non-coding RNA, DNA, antisense oligonucleotides, gene editing cassettes) for therapeutic or prophylactic purposes. [Background technology]

[0003] 3.Background technology 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 on therapeutic nucleic acids began in the 2000s, significant progress has been made in the design, modification, and delivery of nucleic acid molecules. Various nucleic acid-based therapies, including mRNA, siRNA, plasmid DNA, viral and bacterial vectors, and patient-derived cell therapies, have been investigated, and more than a dozen of these have been approved by the U.S. Food and Drug Administration (FDA) for human use, with many more in clinical trials. However, nucleic acid therapeutics still face multiple challenges, including off-target effects, unwanted immune stimulation, and low delivery efficiency. In the case of mRNA-based therapies, rapid degradation of mRNA by nucleases in serum makes the delivery process more difficult.

[0004] Current LNP delivery systems typically contain cationic lipids, phospholipids, cholesterol, and PEG-lipid conjugates. Four-lipid LNP formulations (e.g., with a typical lipid ratio of cationic lipid:cholesterol:DSPC:DMG-PEG=50:38.5:10:1.5) have proven successful in several marketed pharmaceuticals, but still leave room for improvement. There remains a need to develop simplified lipid nanoparticle compositions without sacrificing the delivery efficiency of nucleic acid payloads. Summary of the Invention [Problem to be solved by the invention]

[0005] 4. Summary of the Invention In one embodiment, provided herein are lipid nanoparticles that comprise a reduced amount of phospholipid components (eg, compared to conventional four-lipid component lipid nanoparticles).

[0006] In one embodiment, as used herein, (a) a cationic lipid in an amount of about 40 mol% to about 75 mol% of the total lipid present in the nanoparticle, the cationic lipid comprising: (i) at least one tertiary amine or quaternary ammonium moiety; and (ii) at least one C 14 ~C 42 the cationic lipid comprising an alkyl ester moiety; and (b) a steroid in an amount of about 22 mol% to about 59.5 mol% of the total lipid present in the nanoparticles; and (c) a polymer-conjugated lipid in an amount of about 0.5 mol % to about 2.5 mol % of the total lipid present in the nanoparticle; (d) nucleic acid, Lipid nanoparticles are provided, with the proviso that the lipid nanoparticles comprise 0.5 mole percent or less of phospholipids.

[0007] In one embodiment, the cationic lipid has formula (I): [ka] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein G 1 , X 1 , R 1 , R x and R y is as defined herein or elsewhere.

[0008] In one embodiment, the cationic lipid has formula (III): [ka] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein G 1 , X 1 , R 1 , R x and R y is as defined herein or elsewhere.

[0009] In one embodiment, the three-lipid component lipid nanoparticles provided herein (containing no or reduced / insignificant amounts of phospholipid component) have an increased size compared to conventional four-lipid component lipid nanoparticles, e.g., four-lipid component lipid nanoparticles having a molar ratio of cationic lipid:steroid:phospholipid:polymer-conjugated lipid of about 50:38.5:10:1.5. In one embodiment, the three-lipid component nanoparticles provided herein have a size of about 110 nm to about 130 nm.

[0010] Also provided herein are pharmaceutical compositions comprising the lipid nanoparticles provided herein and a pharmaceutically acceptable excipient.

[0011] Also provided herein are methods of using the lipid nanoparticles provided herein or the pharmaceutical compositions provided herein to express mRNA in mammalian cells or mammalian tissues, to introduce mRNA encoding a protein into a cell, to treat a disease or disorder in a human, or to treat a disease or disorder in a human caused by impaired expression of a protein. [Brief explanation of the drawings]

[0012] 5. Brief description of the drawings [Figure 1] This figure illustrates in vivo protein (hEPO) expression levels 6 hours after intravenous administration of LNPs containing lipid-5. The composition and properties of the LNPs are listed in Tables 1 and 5. All expression data are normalized to formulation L-0. Error bars are shown to represent the standard error of the mean (SEM). [Figure 2] Figure 1 illustrates in vivo protein (hEPO) expression levels 6 hours after intravenous administration of LNPs containing SM-102. The composition and properties of the LNPs are listed in Tables 2 and 6. All expression data are normalized to formulation S-0. Error bars are shown to represent SEM. [Figure 3] Figure 1 illustrates in vivo protein (hEPO) expression levels 6 hours after intravenous administration of LNPs containing C1 or ALC-0315. The composition and properties of the LNPs are listed in Tables 3, 4, 7, and 8. All expression data are normalized to formulation A-0 or C1-0. Error bars are shown to represent SEM. [Figure 4] Figure 1 illustrates in vivo protein (hEPO) expression levels 6 hours after intravenous administration of LNPs containing greater than 3 mol% PEG-lipid. All expression data are normalized to formulation L-0 or S-0. Error bars represent SEM. [Figure 5A] 1 shows the long-term stability of certain lipid-5-containing LNPs stored at room temperature over the course of four months by assessing the purity of the encapsulated mRNA using a fragment analysis (FA) assay. [Figure 5B] Figure 1 shows the long-term stability of certain lipid-5-containing LNPs stored at 2-8 °C over the course of 4 months, using a fragment analysis (FA) assay to assess the purity of the encapsulated mRNA. [Figure 5C]1 shows the long-term stability of certain lipid-5-containing LNPs stored at −20° C. over the course of 4 months by assessing the purity of the encapsulated mRNA using a fragment analysis (FA) assay. [Figure 5D] Figure 1 shows the updated long-term stability of certain lipid-5-containing LNPs stored at 2-8 °C for up to 400 days. [Figure 5E] 1 shows the updated long-term stability of certain lipid-5-containing LNPs stored at −20° C. for up to 400 days. [Figure 6A] 1 shows the in vivo GFP expression level 6 hours after intramuscular administration of certain lipid-5-containing LNPs. [Figure 6B] IL-6 levels normalized by the amount of GFP expressed are shown. [Figure 7A] 1 shows changes in mouse body weight monitored over 7 days after intravenous administration of certain lipid-5-containing LNPs. [Figure 7B] 1 shows changes in mouse body weight monitored over 7 days after intramuscular administration of certain lipid-5-containing LNPs. [Figure 8] Figure 1 illustrates in vivo protein (hEPO) expression levels 6 hours after intravenous administration of LNPs containing C2. The composition and properties of the LNPs are listed in Tables 9 and 10. All expression data are normalized to formulation C2-0. Error bars are shown to represent SEM. [Figure 9] A and B show cryo-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%), respectively. DETAILED DESCRIPTION OF THE INVENTION

[0013] 6. MODE FOR CARRYING OUT THE INVENTION 6.1 General techniques The techniques and procedures described or referenced herein generally include those that are generally well understood and / or commonly employed by those of skill in the art using conventional methodology, such as the widely utilized methodology described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001), Current Protocols in Molecular Biology (Ausubel et al. eds., 2003).

[0014] 6.2 Terminology Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. For the purpose of interpreting this specification, the following explanations of terms shall apply, and whenever appropriate, terms used in the singular shall also include the plural, and vice versa. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. If the explanation of any term described contradicts any document incorporated by reference herein, the explanation of the definition set forth below shall prevail.

[0015] 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, 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 diethyl ether. Lipids are generally poorly soluble in water, although certain categories of lipids (e.g., lipids modified with polar groups, e.g., DMG-PEG2000) exist that have limited water solubility and can be dissolved in water under certain conditions. Known types of lipids include fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, and biomolecules such as phospholipids. Lipids can be classified into at least three classes: (1) "simple lipids," including fats and oils and waxes; (2) "complex lipids," including phospholipids and glycolipids (e.g., DMPE-PEG2000); and (3) "derived lipids," such as steroids. Furthermore, as used herein, lipid also encompasses synthetic lipidoid compounds. The term "lipidoid compound" (also simply "lipidoid") refers to a lipid-like compound (e.g., an amphiphilic compound with lipid-like physical properties).

[0016] The term "lipid nanoparticle" or "LNP" refers to a particle having at least one dimension in the nanometer (nm) scale (e.g., 1-1,000 nm) containing one or more lipid molecules. The LNPs provided herein may further contain at least one non-lipid payload molecule (e.g., one or more nucleic acid molecules). In one embodiment, the LNP comprises a non-lipid payload molecule either partially or completely encapsulated inside the lipid shell. In particular, in one embodiment, the payload is a negatively charged molecule, such as a nucleic acid, including RNA and DNA, and the lipid component of the LNP comprises at least one cationic lipid. Without being bound by theory, it is contemplated that the cationic lipid can interact with the negatively charged payload molecule through electrostatic effects, facilitating the incorporation and / or encapsulation of the payload into the LNP during LNP formation. Other lipids that may form part of the LNPs provided herein include, but are not limited to, neutral and charged lipids such as steroids, polymer-conjugated lipids, and various zwitterionic lipids. In certain embodiments, LNPs according to the present disclosure comprise one or more cationic lipids of formulas (I) and (II) as described.

[0017] The term "cationic lipid" refers to a lipid that is either positively charged at any pH value or hydrogen ion activity in its environment, or that can become positively charged in response to the pH value or hydrogen ion activity in its environment (e.g., its intended use environment). Thus, the term "cationic lipid" encompasses "permanently cationic lipids," "cationizable lipids," and "ionizable lipids." In certain embodiments, the positive charge in cationic lipids results from the presence of a quaternary nitrogen atom. In certain embodiments, cationic lipids include zwitterionic lipids that are positively charged in their intended use environment (e.g., at physiological pH). In certain embodiments, the cationic lipid is an ionizable lipid. In certain embodiments, the cationic lipid is one or more lipids of formula (I) or (II) described herein.

[0018] The term "polymer-conjugated lipid" refers to a molecule that includes both a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a pegylated lipid (PEG lipid), in which the polymer portion includes polyethylene glycol.

[0019] As used herein, and unless otherwise specified, the term "alkyl" refers to a saturated, straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms. An alkyl does not contain any carbon-carbon double or triple bonds. In one embodiment, an alkyl group is, for example, an alkyl group having 1 to 24 carbon atoms (C1 to C6). 24 alkyl), 4 to 20 carbon atoms (C4 to C 20 alkyl), 6 to 16 carbon atoms (C6 to C 16 alkyl), 6 to 9 carbon atoms (C6 to C9 alkyl), 1 to 15 carbon atoms (C1 to C 15 alkyl), 1 to 12 carbon atoms (C1 to C 12 alkyl), 1 to 8 carbon atoms (C1 to C8 alkyl), 1 to 6 carbon atoms (C1 to C6 alkyl), 2 to 12 carbon atoms (C1 to C 12 alkyl), 14 to 42 carbon atoms (C 14 ~C 42 alkyl), 1 to 6 carbon atoms (C1 to C6 alkyl), 6 to 42 carbon atoms (C6 to C 42 alkyl), 6 to 24 carbon atoms (C6 to C 24 alkyl), or 14 to 24 carbon atoms (C 14 ~C 24 alkyl), which is attached to the rest of the molecule by a single bond. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, and the like. Unless otherwise specified, alkyl groups are optionally substituted.

[0020] As used herein, and unless otherwise specified, the term "alkylene" or "alkylene chain" refers to a linear or branched polyvalent (e.g., divalent or trivalent) hydrocarbon chain that connects the remainder of the molecule to a radical group, consists solely of carbon and hydrogen, and is saturated. In one embodiment, alkylene is, for example, an alkylene having 1 to 24 carbon atoms (C1 to C6). 24 Alkylene, 1 to 15 carbon atoms (C1 to C 15 Alkylene, 1 to 12 carbon atoms (C1 to C 12 alkylene), 1 to 8 carbon atoms (C1-C8 alkylene), 1 to 6 carbon atoms (C1-C6 alkylene), 2 to 4 carbon atoms (C2-C4 alkylene), 1 to 2 carbon atoms (C1-C2 alkylene), or 2 to 12 carbon atoms (C2-C 12 Non-limiting examples of alkylene groups include methylene, ethylene, propylene, iso-propylene, n-butylene, t-butylene, and the like. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless otherwise specified, an alkylene chain is optionally substituted.

[0021] As used herein, and unless otherwise specified, the term "cycloalkyl" refers to a non-aromatic monocyclic or polycyclic hydrocarbon radical, consisting solely of carbon and hydrogen atoms and which is saturated. Cycloalkyl groups can include fused or bridged ring systems. In one embodiment, cycloalkyl refers to a group having, for example, 3 to 15 ring carbon atoms (C3 to C4). 15 Cycloalkyl), 3 to 10 ring carbon atoms (C3 to C 10Cycloalkyls have from 1 to 8 ring carbon atoms (C3-C8 cycloalkyl). Cycloalkyls are attached to the rest of the molecule by a single bond. Non-limiting examples of monocyclic cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic cycloalkyl radicals include adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwise specified, cycloalkyl groups are optionally substituted.

[0022] As used herein, and unless otherwise specified, the term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms and containing one or more carbon-carbon double bonds. Cycloalkenyls can include fused or bridged ring systems. In one embodiment, cycloalkenyls are rings of, for example, 3 to 15 ring carbon atoms (C3 to C4). 15 Cycloalkenyl), 3 to 10 ring carbon atoms (C3 to C 10 Cycloalkenyls have from 1 to 8 ring carbon atoms (C-C cycloalkenyl), or from 3 to 8 ring carbon atoms (C-C cycloalkenyl). Cycloalkenyls are attached to the rest of the molecule by a single bond. Non-limiting examples of cycloalkenyl radicals include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like. Unless otherwise specified, cycloalkenyl groups are optionally substituted.

[0023] 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 is an aromatic group having 6 to 18 ring carbon atoms (C6 to C8). 18 aryl), 6 to 14 ring carbon atoms (C6 to C 14 aryl), or 6 to 10 ring carbon atoms (C6 to C 10aryl). Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, pyrenyl, biphenyl, and terphenyl. The term "aryl" also refers to bicyclic, tricyclic, or other polycyclic hydrocarbon rings in which at least one of the rings is aromatic and the others can be saturated, partially unsaturated, or aromatic, such as dihydronaphthyl, indenyl, indanyl, or tetrahydronaphthyl (tetralinyl). Unless otherwise specified, aryl groups are optionally substituted.

[0024] When groups described herein are said to be "substituted," they can be substituted with any suitable substituent(s). Illustrative examples of substituents include those found in the exemplary compounds and embodiments provided herein, as well as halogen atoms such as F, CI, 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 and the like. Examples include, but are not limited to, NR'R', where R', in each occurrence, 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 another embodiment, the substituent is an alkyl group. In another embodiment, the substituent is a cycloalkyl group. In another embodiment, the substituent is a halo group, such as fluoro. In another embodiment, the substituent is an oxo group. In another embodiment, the substituent is a hydroxyl group. In another embodiment, the substituent is an alkoxy group (-OR'). In another embodiment, the substituent is a carboxyl group. In another embodiment, the substituent is an amino group (-NR'R').

[0025] As used herein, and unless otherwise specified, the term "optional" or "optionally" (e.g., optionally substituted) means that the subsequently described circumstance event may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, "optionally substituted alkyl" means that the alkyl radical may or may not be substituted, and that the description includes both substituted alkyl radicals and alkyl radicals with no substitution.

[0026] As used herein, and unless otherwise specified, the term "pharmaceutically acceptable salts" includes both acid and base addition salts.

[0027] By way of non-limiting example, pharmaceutically acceptable acid addition salts include those with 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, dodecylsulfuric 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, glycerin, glycerol ... These include glutamic acid, glutaric acid, 2-oxo-glutaric 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, and undecylenic acid.

[0028] Non-limiting examples of pharmaceutically acceptable base addition salts include salts prepared from the addition of an inorganic base or an organic base to a free acid compound. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. In one embodiment, the inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. 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, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, pipericine, N-ethylpiperidine, polyamine resins, etc. In one embodiment, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0029] The compounds provided herein may contain one or more asymmetric centers and may therefore give rise to enantiomers, diastereomers, and other stereoisomeric forms, which may be defined in terms of absolute stereochemistry as (R)- or (S)-, or for amino acids, as (D)- or (L)-. Unless otherwise specified, 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 the preparation / isolation of individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of the racemate (or racemate of a salt or derivative), using, for example, chiral high-performance liquid chromatography (HPLC). When a compound described herein contains an olefinic double bond or other center of geometric asymmetry, and unless otherwise specified, the compound is intended to include both E and Z geometric isomers. Likewise, all tautomeric forms are intended to be included.

[0030] As used herein, and unless otherwise specified, the term "isomer" refers to different compounds that have the same molecular formula. "Stereoisomers" are isomers that differ only in the way the atoms are arranged in space. "Atropisomers" are stereoisomers due to restricted 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 proportions may be known as a "racemic" mixture. "Diastereoisomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other.

[0031] "Stereoisomer" can 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 either the E or Z isomer. In other embodiments, the compounds described herein are a mixture of E and Z isomers.

[0032] "Tautomers" refer to isomeric forms of a compound that are in equilibrium with each other. The concentrations of isomeric forms depend on the environment in which the compound is found, and can vary depending on, for example, whether the compound is a solid or in an organic or aqueous solution.

[0033] It should also be noted that the compounds described herein may contain unnatural proportions of atomic isotopes at one or more of the atoms. For example, the compounds may contain, for example, tritium ( 3 H), carbon-14( 14 C), carbon-11( 11 C), fluorine-18( 18 C), sulfur-35( 35 S), iodine-125( 125 It may be radiolabeled with a radioisotope such as I) or deuterium ( 2 H), carbon-13( 13 C), or nitrogen-15( 15The compound may be enriched in an isotope, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 112, 113, 120, 121, 134, 140, 141, 152, 153, 164, 170, 171, 182, 191, 192, 193, 194, 195, 196, 197, As used herein, "deuterated" means that at least one hydrogen (H) has been replaced with a deuterium (D or 2 H), meaning that the compound is enriched with deuterium at at least one position.

[0034] Note that if there is a discrepancy between the illustrated structure and the name for that structure, the illustrated structure shall prevail.

[0035] As used herein, and unless otherwise specified, the term "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, lubricant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the U.S. Food and Drug Administration (FDA) as acceptable for use in humans or veterinary medicine.

[0036] The term "composition" is intended to encompass a product containing specified components (eg, mRNA molecules provided herein), optionally in specified amounts.

[0037] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to polymers of nucleotides of any length, including, for example, DNA and RNA. Nucleic acids can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Nucleic acids may contain modified nucleotides, such as methylated nucleotides and their analogs. Nucleic acids can be in either single-stranded or double-stranded form. As used herein, and unless otherwise specified, "nucleic acid" also includes nucleic acid mimetics such as locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholinos. As used herein, "oligonucleotide" refers to short synthetic polynucleotides, generally, although 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 specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The 5' to 3' addition direction of the nascent RNA transcript is referred to as the transcription direction, and the region of the sequence on the DNA strand that is 5' to the 5' end of the RNA transcript and has the same sequence as the RNA transcript is referred to as the "upstream sequence," and the region of the sequence on the DNA strand that is 3' to the 3' end of the RNA transcript and has the same sequence as the RNA transcript is referred to as the "downstream sequence."

[0038] As used herein, the term "non-naturally occurring" when used with respect to a nucleic acid molecule described herein is intended to mean that the nucleic acid molecule is not found in nature. Non-naturally occurring nucleic acids encoding viral peptides or proteins contain at least one genetic alteration or chemical modification not normally found in naturally occurring viral strains, including wild-type viral strains. Genetic alterations include, for example, modifications that introduce expressible nucleic acid sequences encoding peptides or polypeptides heterologous to the virus, other nucleic acid additions, nucleic acid deletions, nucleic acid substitutions, and / or other functional disruptions of the viral genetic material. Such modifications include, for example, modifications in coding regions and functional fragments of polypeptides heterologous, homologous, or both heterologous and homologous to the viral species. Additional modifications include, for example, modifications in non-coding regulatory regions that alter gene or operon expression. Additional modifications also include, for example, the incorporation of nucleic acid sequences into vectors, such as plasmids or artificial chromosomes. Chemical modifications include, for example, one or more functional nucleotide analogs described herein.

[0039] An "isolated nucleic acid" is a nucleic acid, e.g., RNA, DNA, or mixture of nucleic acids, that has been substantially separated from other genomic DNA sequences and proteins or complexes, such as ribosomes and polymerases, that naturally accompany the naturally occurring sequence. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the nucleic acid molecule's natural source. Furthermore, an "isolated" nucleic acid molecule, such as an mRNA molecule, can be substantially free of other cellular material or culture media when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In specific embodiments, one or more nucleic acid molecules encoding the antigens described herein are isolated or purified. This term encompasses nucleic acid sequences that have been removed from their naturally occurring environment, including recombinant or cloned DNA or RNA isolates, and chemically synthesized analogs or analogs biologically synthesized by heterologous systems. A substantially pure molecule can include isolated forms of the molecule.

[0040] When used in reference to a nucleic acid molecule, the term "encoding nucleic acid" or its grammatical equivalents encompasses (a) a nucleic acid molecule in its natural state, or a nucleic acid molecule that, when manipulated by methods well known to those of skill in the art, can be transcribed to produce mRNA, which is then translated into peptides and / or polypeptides, and (b) the mRNA molecule itself. An antisense strand is the complementary sequence of such a nucleic acid molecule, from which a coding sequence can be derived. 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 a portion of a coding nucleic acid that is not translated into a peptide or polypeptide. Depending on the orientation of a UTR relative to the coding region of a nucleic acid molecule, a UTR is referred to as a 5'-UTR if it is located at the 5' end of the coding region, or a 3'-UTR if it is located at the 3' end of the coding region.

[0041] As used herein, the term "mRNA" refers to a message RNA molecule that contains one or more open reading frames (ORFs) that can be translated by a cell or organism that contains the mRNA to produce one or more peptide or protein products. The region containing one or more ORFs is referred to as the coding region of an mRNA molecule. In certain embodiments, an mRNA molecule further contains one or more untranslated regions (UTRs).

[0042] In certain embodiments, the mRNA is a monocistronic mRNA containing only one ORF. In certain embodiments, the monocistronic mRNA encodes a peptide or protein containing at least one epitope of a selected antigen (e.g., a pathogenic antigen or a tumor-associated antigen). In other embodiments, the mRNA is a multicistronic mRNA containing two or more ORFs. In certain embodiments, the multicistronic mRNA encodes two or more peptides or proteins, which can be the same or different from one another. In certain embodiments, each peptide or protein encoded by the multicistronic mRNA contains at least one epitope of a selected antigen. In certain embodiments, different peptides or proteins encoded by the multicistronic mRNA each contain at least one epitope of a different antigen. In any of the embodiments described herein, the at least one epitope can be at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten epitopes of an antigen.

[0043] The term "nucleobase" encompasses purines and pyrimidines, including the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and their natural or synthetic analogues or derivatives.

[0044] As used herein, the term "functional nucleotide analog" refers to a modified version of the standard nucleotides A, G, C, U, or T that (a) retains the base-pairing properties of the corresponding standard nucleotide and (b) contains at least one chemical modification to (i) the nucleobase, (ii) the sugar group, (iii) the phosphate group, or (iv) any combination of (i)-(iii) of the corresponding natural nucleotide. As used herein, base pairing encompasses not only standard Watson-Crick adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between a standard nucleotide and a functional nucleotide analog or between a pair of functional nucleotide analogs, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between a modified nucleobase and a standard nucleobase or between two complementary nucleobase structures. For example, a functional analog of guanosine (G) retains the ability to base pair with cytosine (C) or a functional analog of cytosine. An example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. As described herein, functional nucleotide analogs can be either naturally occurring or non-naturally occurring. Thus, 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.

[0045] As used herein, the terms "translation enhancer element," "TEE," and "translation enhancer" refer to a region in a nucleic acid molecule that functions to promote translation of a coding sequence of the nucleic acid into a protein or peptide product, e.g., via cap-dependent or cap-independent translation. TEEs are typically located in the UTR region of a nucleic acid molecule (e.g., mRNA) and enhance the level of translation of a coding sequence located either upstream or downstream. For example, a TEE in the 5' UTR of a nucleic acid molecule can be located between the promoter and 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, 2013 Aug;10(8):747-750; Chappell et al., PNAS June 29, 2004 101(26)9590-9594). Some TEEs are known to be conserved across multiple species (Panek et al. Nucleic Acids Research, Volume 41, Issue 16, 1 September 2013, Pages 7625-7634).

[0046] As used herein, the term "siRNA" refers to "small interfering RNA" or "short interfering RNA." siRNA is an RNA duplex of nucleotides that can be targeted to a gene of interest. An "RNA duplex" refers to the structure formed by complementary pairing between two regions of an RNA molecule. An siRNA is "targeted" to a gene when 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 siRNA duplex is less than 30 nucleotides. In one embodiment, the length of the siRNA duplex is 20-25 nucleotides. In one embodiment, the siRNA duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 18, 16, 14, 12, or 10 nucleotides long. The RNA duplex portion of the siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure can contain a loop portion located between the two sequences that form the duplex. The length of the loop can vary. In one embodiment, the loop is 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in length. The hairpin structure may also contain a 3' or 5' overhang portion. In one embodiment, the overhang is 0, 1, 2, 3, 4, or 5 nucleotides in length. The siRNA may be encoded by a nucleic acid sequence, which may also include a promoter. The nucleic acid sequence may also include a polyadenylation signal. In one embodiment, the polyadenylation signal is a synthetic minimal polyadenylation signal. In one embodiment, the siRNA is used for "gene silencing." "Gene silencing" refers to the suppression of gene expression. Gene silencing can be mediated through processes that affect transcription and / or processes that affect post-transcriptional mechanisms. In one embodiment, gene silencing occurs when the siRNA initiates degradation of the mRNA of the gene of interest in a sequence-specific manner via RNA interference. In one embodiment, siRNAs disrupt the expression of specific genes that have complementary nucleotide sequences by degrading the mRNA after transcription and 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. "Knockdown" 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 lead to inhibition of production of the target gene product. The term "reduced" is used herein to indicate that target gene expression is reduced by 1 to 100%. For example, expression can be reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 97, or 99%. Knockdown of gene expression can be achieved using siRNA.

[0047] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of more than 50 amino acid residues linked by covalent peptide bonds. That is, a description of a polypeptide applies equally to a description of a protein, and vice versa. The term applies 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 term encompasses amino acid chains of any length, including full-length proteins (e.g., antigens).

[0048] In the context of a peptide or polypeptide, the term "derivative" as used herein refers to a peptide or polypeptide containing the amino acid sequence of a viral peptide or protein, or a fragment of a viral peptide or protein, altered by the introduction of substitutions, deletions, or additions of amino acid residues. As used herein, the term "derivative" also refers to a viral peptide or protein, or a fragment of a viral peptide or protein, that has been chemically modified, for example, by covalently attaching any type of molecule to the 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 by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, chemical cleavage, formulation, metabolic synthesis of tunicamycin, linkage to cellular ligands or other proteins, etc. A derivative is modified in a manner that differs from the native or starting peptide or polypeptide in either the type or location of the attached molecule. A derivative further includes the deletion of one or more chemical groups naturally present on the viral peptide or protein. Furthermore, derivatives of viral peptides or proteins or fragments of viral peptides or proteins may contain one or more non-classical amino acids. In specific embodiments, the derivatives are functional derivatives of the native or unmodified peptide or polypeptide from which they are derived.

[0049] The term "functional derivative" refers to a derivative that retains one or more functions or activities of the native or starting peptide or polypeptide from which it 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 viral genomes.

[0050] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of one in 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 algorithms needed to achieve maximum alignment over the entire length of the sequences being compared.

[0051] A "modification" of an amino acid residue / position refers to an alteration in the primary amino acid sequence compared to the starting amino acid sequence, where the alteration results from a change in the sequence containing that amino acid residue / position. For example, typical modifications include substitution of the residue with another amino acid (e.g., a conservative or non-conservative substitution), insertion of one or more (usually less than 5, 4, or 3) amino acids adjacent to that residue / position, and / or deletion of that residue / position.

[0052] The term "fragment," as used herein in the context of a peptide or polypeptide, refers to a peptide or polypeptide comprising an amino acid sequence that is shorter than the full-length amino acid sequence. Such fragments can result, for example, from amino-terminal truncation, carboxy-terminal truncation, and / or internal deletion of a residue(s) from the amino acid sequence. Fragments can result, for example, from alternative RNA splicing or in vivo protease activity. In certain embodiments, a fragment comprises at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 10 ...0 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 100 contiguous amino acid residues, at A fragment of a polypeptide refers to a polypeptide comprising an amino acid sequence of at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous 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 contiguous amino acid residues. In specific embodiments, a fragment of a polypeptide retains at least one, at least two, at least three, or more functions of the polypeptide.

[0053] The term "immunogenic fragment," as used herein in the context of a peptide or polypeptide (e.g., a protein), refers to a fragment of a peptide or polypeptide that retains the ability of the peptide or polypeptide to elicit an immune response upon contact with a mammalian immune system, including the innate and / or adaptive immune response. In one embodiment, an immunogenic fragment of a peptide or polypeptide may be an epitope.

[0054] The term "antigen" refers to a substance that can be recognized by a subject's immune system (including the adaptive immune system) and that can induce an immune response (including an antigen-specific immune response) after the subject comes into contact with the antigen. In certain embodiments, the antigen is a protein associated with a diseased cell, such as a cell infected with a pathogen or a tumor cell (e.g., a tumor-associated antigen (TAA)).

[0055] An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds, such as a localized region on the surface of an antigen that has antigenic or immunogenic activity that can bind to one or more antigen-binding regions of an antibody and elicit an immune response in an animal, such as a mammal (e.g., a human). An epitope with immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. An epitope with antigenic activity is a portion of a polypeptide to which an antibody binds, as determined by any method known in the art, including, for example, immunoassays. An antigenic epitope is not necessarily immunogenic. Epitopes often consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. Antibody epitopes can be linear or conformational epitopes. Linear epitopes are formed by a consecutive sequence of amino acids in a protein. Conformational epitopes are formed by amino acids that are discontinuous in the protein sequence but are grouped together when the protein folds into its three-dimensional structure. Induced epitopes are formed when the three-dimensional structure of a protein is in a changed conformation, such as after activation or binding of another protein or ligand. In certain embodiments, epitopes are three-dimensional surface features of a polypeptide. In other embodiments, epitopes are linear features of a polypeptide. Generally, an antigen has several or many different epitopes and can react with many different antibodies.

[0056] As used herein, the term "genetic vaccine" refers to a therapeutic or prophylactic composition comprising 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 the vaccine to a subject ("vaccination") allows for the production of the encoded peptide or protein, thereby eliciting an immune response in the subject against the target disease. In certain embodiments, the immune response comprises an adaptive immune response, such as the production of antibodies against the encoded antigen and / or the activation and proliferation of immune cells capable of specifically eliminating diseased cells expressing the antigen. In certain embodiments, the immune response further comprises an innate immune response. In accordance with the present disclosure, a vaccine can be administered to a subject either before or after the onset of clinical symptoms of the target disease. In one embodiment, vaccination of a healthy or asymptomatic subject immunizes or reduces the susceptibility of the vaccinated subject to the development of the target disease. In one embodiment, vaccination of a subject exhibiting disease symptoms ameliorates the disease state or treats the disease in the vaccinated subject.

[0057] The terms "innate immune response" and "innate immunity" are art-recognized and refer to nonspecific defense mechanisms initiated by the body's immune system upon recognition of pathogen-associated molecular patterns, involving different forms of cellular activity, including cytokine production and cell death via various pathways. As used herein, an 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 cellular apoptosis. Activation of innate immunity can be detected using methods known in the art, such as measuring (NF)-κB activation.

[0058] The terms "adaptive immune response" and "adaptive immunity" are art-recognized and refer to antigen-specific defense mechanisms initiated by the body's immune system upon recognition of a specific antigen, including both humoral and cell-mediated responses. As used herein, an adaptive immune response includes a cellular response elicited and / or augmented by a vaccine composition, such as the genetic compositions described herein. In one embodiment, the vaccine composition comprises an antigen that is the target of the antigen-specific adaptive immune response. In other embodiments, the vaccine composition, upon administration, enables the immunized subject to produce the antigen that is the target of the antigen-specific adaptive immune response. Activation of the adaptive immune response can be detected using methods known in the art, such as measuring the level of antigen-specific antibody production or antigen-specific cell-mediated cytotoxicity.

[0059] The term "antibody" is intended to include polypeptide products of B cells within the immunoglobulin class of polypeptides that can bind to a specific molecular antigen and are composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), with the amino-terminal portion of each chain containing a variable region of about 100 to about 130 amino acids or more, and the carboxy-terminal portion of each chain containing a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2nd ed. 1995) and Kuby, Immunology (3rd ed. 1997). In certain embodiments, specific molecular antigens can be bound by antibodies provided herein, including polypeptides, fragments thereof, or epitopes. Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments of any of the above, where a functional fragment refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments include single-chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and molecules containing immunologically active portions of immunoglobulin molecules, such as antigen-binding domains or antigen-binding sites (e.g., one or more CDRs of an antibody).Such antibody fragments can be found, for example, in 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, Pluckthun and Skerra, 1989, Meth. Enzymol. Enzymol. 178:497-515, and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, and IgA), or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0060] The term "administering" or "administration" refers to the act of injecting or otherwise physically delivering an exogenous substance (e.g., a lipid nanoparticle composition described herein) to a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When a disease, disorder, condition, or symptom thereof is being treated, administration of the substance typically occurs after the onset of the disease, disorder, condition, or symptom thereof. When a disease, disorder, condition, or symptom thereof is being prevented, administration of the substance typically occurs before the onset of the disease, disorder, condition, or symptom thereof.

[0061] "Chronic" administration refers to the administration of an agent(s) in a continuous manner (e.g., over a period of days, weeks, months, or years), as opposed to an acute manner, such that the initial therapeutic effect (activity) is maintained over an extended period of time. "Intermittent" administration is treatment that is cyclic in nature, rather than continuous without interruption.

[0062] As used herein, the term "targeted delivery" or the verb form "target" refers to a process that facilitates a delivered agent (such as a therapeutic payload molecule in a lipid nanoparticle composition described herein) reaching a particular organ, tissue, cell, and / or intracellular compartment (referred to as a targeted location) more than any other organ, tissue, cell, or intracellular compartment (referred to as a non-targeted location). Targeted delivery can be detected using methods known in the art, for example, by comparing the concentration of the delivered agent in a targeted cell population after systemic administration with the concentration of the delivered agent in a non-targeted cell population. In certain embodiments, targeted delivery results in at least a two-fold higher concentration in the targeted location compared to non-targeted locations.

[0063] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate symptoms and / or their underlying causes, prevent the occurrence of symptoms and / or their underlying causes, and / or ameliorate or repair damage caused by or associated with a disease, disorder, or condition, including, for example, infection and tumors. In one embodiment, the effective amount is a therapeutically effective amount or a prophylactically effective amount.

[0064] 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 ameliorate the severity and / or duration of a given disease, disorder, or condition, and / or its associated symptoms (e.g., an infectious disease, such as that caused by a viral infection, or a neoplastic disease, such as cancer). A "therapeutically effective amount" of a substance / molecule / agent of the present disclosure (e.g., a lipid nanoparticle composition described herein) may vary according to factors such as the individual's condition, age, sex, and weight, as well as the ability of the substance / molecule / agent to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which the therapeutically beneficial effects outweigh any toxic or adverse effects of the substance / molecule / agent. In certain embodiments, the term "therapeutically effective amount" refers to an amount of a lipid nanoparticle composition described herein or a therapeutic or prophylactic agent (e.g., a therapeutic mRNA) contained therein that is effective to "treat" a disease, disorder, or condition in a subject or mammal.

[0065] A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, achieves the intended prophylactic effect, e.g., prevents, delays, or reduces the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom(s) (e.g., an infectious disease such as caused by a viral infection, or a neoplastic disease such as cancer). Typically, but not necessarily, a prophylactically effective amount may be less than a therapeutically effective amount, since a prophylactic dose is used in a subject prior to or at an early stage of a disease, disorder, or condition. A complete therapeutic or prophylactic effect does not necessarily occur by administration of a single dose, but may occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations.

[0066] As used herein, and unless otherwise specified, the terms "treat," "treating," and "treatment" refer to the total or partial alleviation of a disorder, disease, or condition, or the total or partial alleviation of one or more symptoms associated with a disorder, disease, or condition, or the slowing or halting of further progression or worsening of these symptoms, or the amelioration or eradication of the cause(s) of the disorder, disease, or condition itself.

[0067] As used herein, and unless otherwise specified, the terms "prevent," "preventing," and "prevention" refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom(s) (e.g., an infectious disease, such as caused by a viral infection, or a neoplastic disease, such as cancer).

[0068] As used herein, and unless otherwise specified, the terms "manage," "managing," and "management" refer to the beneficial effects a subject derives from a therapy (e.g., a prophylactic or therapeutic agent) that does not result in a cure of the disease. In certain embodiments, a subject is administered one or more therapies (e.g., a prophylactic or therapeutic agent, such as a lipid nanoparticle composition described herein) to "manage" an infectious or neoplastic disease, one or more symptoms thereof, so as to prevent progression or worsening of the disease.

[0069] The term "prophylactic agent" refers to any agent that can completely or partially inhibit the onset, recurrence, development, or spread of a disease and / or its associated symptoms in a subject.

[0070] The term "therapeutic agent" refers to any agent that can be used to treat, prevent, or alleviate a disease, disorder, or condition, including treating, preventing, or alleviating one or more symptoms of the disease, disorder, or condition and / or symptoms associated therewith.

[0071] The term "therapy" refers to any protocol, method, and / or agent that can be used in the prevention, management, treatment, and / or amelioration of a disease, disorder, or condition. In certain embodiments, the terms "therapies" and "therapy" refer to biological, supportive, and / or other therapies known to those of skill in the art, such as health care professionals, that are useful in the prevention, management, treatment, and / or amelioration of a disease, disorder, or condition.

[0072] 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 onset, recurrence, manifestation, or spread of a disease, disorder, or condition, and / or symptoms associated therewith, in the subject.

[0073] The term "side effect" encompasses unwanted and / or adverse effects of a therapy (e.g., a prophylactic or therapeutic agent). Unwanted effects are not necessarily harmful. Side effects from a therapy (e.g., a prophylactic or therapeutic agent) can be harmful, uncomfortable, or dangerous. Examples of side effects include diarrhea, cough, gastroenteritis, wheezing, nausea, vomiting, anorexia, abdominal cramps, fever, pain, weight loss, dehydration, hair loss, difficulty breathing, 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, fatigue, digestive problems, and allergic reactions. Additional unwanted effects experienced by patients are numerous and known in the art. Many are described in the Physician's Desk Reference (68th ed. 2014).

[0074] The terms "subject" and "patient" may be used interchangeably. As used herein, in certain embodiments, a subject is a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans). In certain embodiments, a subject is a human. In one embodiment, a subject is a mammal (e.g., a human) with an infectious disease or a neoplastic disease. In another embodiment, a subject is a mammal (e.g., a human) at risk of developing an infectious disease or a neoplastic disease.

[0075] "Substantially all" refers to 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%.

[0076] As used herein, and unless otherwise indicated, the term "about" or "approximately" refers to the tolerance of error for a particular value as determined by one of ordinary skill in the art, which depends on how 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 term "about" or "approximately" means within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.05%, or less of a given value or range.

[0077] As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0078] All publications, patent applications, accession numbers, and other references cited herein are incorporated by reference in their entirety as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0079] Several embodiments of the present invention have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, the description of the experimental section and examples is intended to be illustrative, but not limiting, of the scope of the invention as set forth in the claims.

[0080] 6.3 Lipid Nanoparticle (LNP) Composition Phospholipids, generally recognized as helper or structural lipids in LNPs, play an important role in conventional LNP formulations. Phospholipids are thought to confer structural stability and promote gene delivery efficiency in vivo by mediating membrane fusion. Three commercially available LNP-based products (Onpattro®, Comirnaty®, and mRNA-1273) all contain DSPC as a basic component (Eygeris et al. Chemistry of Lipid Nanoparticles for RNA Delivery. Acc. Chem. Res. 2022, 55, 2-12). However, as defined herein, it has been shown that phospholipids are not as essential as commonly believed in LNP formulations.

[0081] In one embodiment, provided herein are lipid nanoparticles comprising a reduced amount of phospholipid components (e.g., compared to conventional four-lipid component lipid nanoparticles). In one embodiment, the LNP comprises 0.5 mole percent or less of phospholipids.

[0082] In one embodiment, as used herein, (a) a cationic lipid in an amount of about 40 mol% to about 75 mol% of the total lipid present in the nanoparticle, the cationic lipid comprising: (i) at least one tertiary amine or quaternary ammonium moiety; and (ii) at least one C 14 ~C 42 the cationic lipid comprising an alkyl ester moiety; and (b) a steroid in an amount of about 22 mol% to about 59.5 mol% of the total lipid present in the nanoparticles; and (c) a polymer-conjugated lipid in an amount of about 0.5 mol % to about 2.5 mol % of the total lipid present in the nanoparticle; (d) nucleic acid, Lipid nanoparticles are provided, with the proviso that the lipid nanoparticles comprise 0.5 mole percent or less of phospholipids.

[0083] In one embodiment, the amount of cationic lipid is about 40 mol% to about 75 mol% of the total lipid present in the nanoparticle. In one embodiment, the amount of cationic lipid is about 40 mol% to about 55 mol%. In one embodiment, the amount of cationic lipid is about 45 mol% to about 60 mol%. In one embodiment, the amount of cationic lipid is about 50 mol% to about 65 mol%. In one embodiment, the amount of cationic lipid is about 55 mol% to about 70 mol%. In one embodiment, the amount of cationic lipid is about 60 mol% to about 75 mol%. In one embodiment, the amount of cationic lipid is about 40 mol% to about 50 mol%. In one embodiment, the amount of cationic lipid is about 45 mol% to about 55 mol%. In one embodiment, the amount of cationic lipid is about 50 mol% to about 60 mol%. In one embodiment, the amount of cationic lipid is about 55 mol% to about 65 mol%. In one embodiment, the amount of cationic lipid is about 60 mol% to about 70 mol%. In one embodiment, the amount of cationic lipid is about 65 mol% to about 75 mol%. In one embodiment, the amount of cationic lipid is about 40 mol% to about 45 mol%. In one embodiment, the amount of cationic lipid is about 45 mol% to about 50 mol%. In one embodiment, the amount of cationic lipid is about 50 mol% to about 55 mol%. In one embodiment, the amount of cationic lipid is about 55 mol% to about 60 mol%. In one embodiment, the amount of cationic lipid is about 60 mol% to about 65 mol%. In one embodiment, the amount of cationic lipid is about 65 mol% to about 70 mol%. In one embodiment, the amount of cationic lipid is about 70 mol% to about 75 mol%.

[0084] 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 mol%, 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%.

[0085] In one embodiment, the amount of cationic lipid is about 42.5 mol% of the total lipid present in the nanoparticle. In one embodiment, the amount of cationic lipid is about 45 mol%. In one embodiment, the amount of cationic lipid is about 47.5 mol%. In one embodiment, the amount of cationic lipid is about 50 mol%. In one embodiment, the amount of cationic lipid is about 52.5 mol%. In one embodiment, the amount of cationic lipid is about 55 mol%. In one embodiment, the amount of cationic lipid is about 57.5 mol%. In one embodiment, the amount of cationic lipid is about 60 mol%. In one embodiment, the amount of cationic lipid is about 62.5 mol%. In one embodiment, the amount of cationic lipid is about 65 mol%. In one embodiment, the amount of cationic lipid is about 67.5 mol%. In one embodiment, the amount of cationic lipid is about 70 mol%.

[0086] In one embodiment, the amount of polymer-conjugated lipid is about 0.5 mol% to about 2.5 mol% of the total lipid present in the nanoparticle. In one embodiment, the amount of polymer-conjugated lipid is about 0.5 mol% to about 0.75 mol%, about 0.75 mol% to about 1 mol%, about 0.5 mol% to about 1 mol%, about 1 mol% to about 1.25 mol%, about 1.25 mol% to about 1.5 mol%, about 1 mol% to about 1.5 mol%, about 1.5 mol% to about 1.75 mol%, about 1.75 mol% to about 2 mol%, about 1 mol% to about 2 mol%, about 2 mol% to about 2.25 mol%, about 2.25 mol% to about 2.5 mol%, or about 2 mol% to about 2.5 mol% of the total lipid present in the nanoparticle. In one embodiment, the amount of polymer-conjugated lipid is about 0.5 mol% to about 2 mol%. In one embodiment, the amount of polymer-conjugated lipid is about 1 mol% to about 2.5 mol%. In one embodiment, the amount of polymer-conjugated lipid is about 0.5 mol% to about 1.5 mol%. In one embodiment, the amount of polymer-conjugated lipid is about 1 mol% to about 2 mol%. In one embodiment, the amount of polymer-conjugated lipid is about 1.5 mol% to about 2.5 mol%. In one embodiment, the amount of polymer-conjugated lipid is about 0.5 mol% to about 1 mol%. In one embodiment, the amount of polymer-conjugated lipid is about 1 mol% to about 1.5 mol%. In one embodiment, the amount of polymer-conjugated lipid is about 1.5 mol% to about 2 mol%. In one embodiment, the amount of polymer-conjugated lipid is about 2 mol% to about 2.5 mol%.

[0087] In one embodiment, the amount of polymer-conjugated 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.

[0088] In one embodiment, the amount of polymer-conjugated lipid is about 1 mol% of the total lipid present in the nanoparticle. In one embodiment, the amount of polymer-conjugated lipid is about 1.25 mol%. In one embodiment, the amount of polymer-conjugated lipid is about 1.5 mol%. In one embodiment, the amount of polymer-conjugated lipid is about 1.75 mol%. In one embodiment, the amount of polymer-conjugated lipid is about 2 mol%. In one embodiment, the amount of polymer-conjugated lipid is about 2.25 mol%. In one embodiment, the amount of polymer-conjugated lipid is about 2.5 mol%.

[0089] In one embodiment, the amount of steroid is about 22 mol% to about 59.5 mol% of the total lipid present in the nanoparticle. In one embodiment, the amount of steroid is about 27 mol% to about 54.5 mol%. In one embodiment, the amount of steroid is about 32 mol% to about 49.5 mol%. In one embodiment, the amount of steroid is about 30 mol% to about 50 mol%. In one embodiment, the amount of steroid is about 33 mol% to about 44 mol%. In one embodiment, the amount of steroid is about 33 mol% to about 38 mol%. In one embodiment, the amount of steroid is about 38 mol% to about 48 mol%. In one embodiment, the amount of steroid is about 38 mol% to about 44 mol%. In one embodiment, the amount of steroid is about 44 mol% to about 49 mol%. In one embodiment, the amount of steroid is the remainder of the total lipid present in the nanoparticle (excluding the cationic lipid and the polymer-conjugated lipid).

[0090] 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 nanoparticles.

[0091] 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%.

[0092] 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-conjugated lipid is about 1 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0093] 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-conjugated lipid is about 1 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0094] 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-conjugated lipid is about 1.5 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0095] 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-conjugated lipid is about 1 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0096] 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-conjugated lipid is about 1 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0097] 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-conjugated lipid is about 0.5 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0098] 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-conjugated lipid is about 1 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0099] 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-conjugated lipid is about 1 mol% to about 1.5 mol% of the total lipid present in the nanoparticle.

[0100] 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-conjugated lipid is about 1.5 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0101] 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-conjugated lipid is about 2 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0102] 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-conjugated lipid is about 0.8 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0103] 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-conjugated lipid is about 1 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0104] 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-conjugated lipid is about 1.5 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0105] 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-conjugated lipid is about 2 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0106] 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-conjugated lipid is about 1 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0107] 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-conjugated lipid is about 1 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0108] 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-conjugated lipid is about 0.5 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0109] 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-conjugated lipid is about 0.8 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0110] 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-conjugated lipid is about 1 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0111] 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-conjugated lipid is about 1.5 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0112] 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-conjugated lipid is about 1 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0113] 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-conjugated lipid is about 1 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0114] 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-conjugated lipid is about 1 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0115] 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-conjugated lipid is about 1 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0116] 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-conjugated lipid is about 1 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0117] 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-conjugated lipid is about 1 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0118] 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-conjugated lipid is about 1.5 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0119] 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-conjugated 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 55 mol% to about 60 mol% of the total lipid present in the nanoparticle, and the amount of polymer-conjugated 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 55 mol% to about 60 mol% of the total lipid present in the nanoparticle, and the amount of polymer-conjugated lipid is about 0.5 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 55 mol% to about 60 mol% of the total lipid present in the nanoparticle, and the amount of polymer-conjugated lipid is about 0.8 mol% to about 2 mol% of the total lipid present in the nanoparticle.

[0123] 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-conjugated lipid is about 1 mol% to about 2.5 mol% of the total lipid present in the nanoparticle.

[0124] 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-conjugated lipid is about 1 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 40 mol% to about 55 mol% of the total lipid present in the nanoparticle, and the amount of polymer-conjugated 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 67.5 mol % of the total lipid present in the nanoparticle, and the amount of polymer-conjugated lipid is about 2 mol % of the total lipid present in the nanoparticle.

[0127] 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-conjugated lipid is about 1 mol % of the total lipid present in the nanoparticle.

[0128] 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-conjugated lipid is about 1.5 mol % of the total lipid present in the nanoparticle.

[0129] 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-conjugated lipid is about 2 mol % of the total lipid present in the nanoparticle.

[0130] 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-conjugated lipid is about 2.5 mol % of the total lipid present in the nanoparticle.

[0131] 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-conjugated lipid is about 1 mol % of the total lipid present in the nanoparticle.

[0132] 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-conjugated lipid is about 1.5 mol % of the total lipid present in the nanoparticle.

[0133] 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-conjugated lipid is about 2 mol % of the total lipid present in the nanoparticle.

[0134] 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-conjugated lipid is about 2.5 mol % of the total lipid present in the nanoparticle.

[0135] 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-conjugated lipid is about 1 mol % of the total lipid present in the nanoparticle.

[0136] 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-conjugated lipid is about 1.5 mol % of the total lipid present in the nanoparticle.

[0137] 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-conjugated lipid is about 2 mol % of the total lipid present in the nanoparticle.

[0138] 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-conjugated lipid is about 2.5 mol % of the total lipid present in the nanoparticle.

[0139] 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-conjugated lipid is about 1 mol % of the total lipid present in the nanoparticle.

[0140] 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-conjugated lipid is about 1.5 mol % of the total lipid present in the nanoparticle.

[0141] 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-conjugated lipid is about 2 mol % of the total lipid present in the nanoparticle.

[0142] In one embodiment, lipid nanoparticles having an amount of cationic lipid and an amount of polymer-conjugated lipid provided herein have an increased nanoparticle size (e.g., about 100 nm to about 140 nm, or about 110 nm to about 130 nm) as described herein or elsewhere, or as illustrated in the Examples.

[0143] In one embodiment, the lipid component of the nanoparticle composition comprises one or more polymer-conjugated lipids, such as PEGylated lipids (PEG lipids). Without being bound by theory, it is contemplated that the polymer-conjugated 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.

[0144] In one embodiment, the polymer-conjugated lipid is a PEGylated lipid, e.g., 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)butanedioate (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.

[0145] In one embodiment, the polymer-conjugated lipid is a polyethylene glycol (PEG)-conjugated lipid. In one embodiment, the polymer-conjugated 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-conjugated lipid is a PEG-DAG. In one embodiment, the polymer-conjugated lipid is a PEG-DAA. In one embodiment, the polymer-conjugated lipid is a PEG-DMA. In one embodiment, the polymer-conjugated lipid is a PEG-DSA. In one embodiment, the polymer-conjugated lipid is a PEG-DMG. In one embodiment, the polymer-conjugated lipid is a PEG-DMPE. In one embodiment, the polymer-conjugated lipid is a DMG-PEG2000. In one embodiment, the polymer-conjugated lipid is a DMPE-PEG2000.

[0146] In one embodiment, the polymer-conjugated lipid is a polyethylene glycol (PEG)-conjugated 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.

[0147] In one embodiment, the polymer-conjugated 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-conjugated 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.

[0148] 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.

[0149] In one embodiment, the polymer-conjugated lipid is a PEG-conjugated lipid, and the steroid is cholesterol or a cholesterol derivative. In one embodiment, the polymer-conjugated lipid is DMG-PEG2000, and the steroid is cholesterol or a cholesterol derivative. In one embodiment, the polymer-conjugated lipid is DMPE-PEG2000, and the steroid is cholesterol or a cholesterol derivative.

[0150] 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.

[0151] In one embodiment, the cationic lipid is C 14 ~C 42 In one embodiment, the ester moiety is a C alkyl ester moiety. 14 ~C 37 In one embodiment, the ester moiety is C 14 ~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 Contains an alkyl ester moiety.

[0152] In one embodiment, the cationic lipid is C 14 ~C 42 In one embodiment, the cationic lipid comprises an alkyl ester moiety and at least a second ester moiety. 14 ~C 42In one embodiment, the cationic lipid comprises two C alkyl ester moieties. 14 ~C 42 In one embodiment, the alkyl ester moiety is selected from two C 14 ~C 42 The alkyl ester moieties are identical. In one embodiment, two C 14 ~C 42 The alkyl ester moieties are different. In one embodiment, the cationic lipid has two C 15 In one embodiment, the cationic lipid comprises two C alkyl ester moieties. 16 In one embodiment, the cationic lipid comprises a C 10 Alkyl ester moiety and C 17 Contains an alkyl ester moiety.

[0153] 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 is branch C 14 ~C 42 In one embodiment, R 1 is branch C 14 ~C 19 In one embodiment, R 1 is branch C 20 ~C 25 In one embodiment, R 1 is branch C 26 ~C 31 In one embodiment, R 1 is branch C 32 ~C 37 In one embodiment, R 1 is branch C 38 ~C 42 In one embodiment, R 1 is branch C 15 In one embodiment, R 1 is branch C 16 In one embodiment, R 1 is branch C17 It is an alkyl ester moiety.

[0154] 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 or branched C6-C 42 In one embodiment, R 2 is a straight or branched C6-C 11 In one embodiment, R 2 is a linear or branched C 12 ~C 17 In one embodiment, R 2 is a linear or branched C 18 ~C 23 In one embodiment, R 2 is a linear or branched C 24 ~C 29 In one embodiment, R 2 is a linear or branched C 30 ~C 35 In one embodiment, R 2 is a linear or branched C 36 ~C 42 It is alkyl.

[0155] 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 C6-C 42 In one embodiment, R 2 is a straight chain C6-C 12 In one embodiment, R 2 is a linear C 13 ~C 18 In one embodiment, R 2 is a linear C 19 ~C 25 In one embodiment, R 2 is a linear C 26 ~C 32 In one embodiment, R2 is a linear C 32 ~C 42 In one embodiment, R 2 is a straight chain C alkyl. In one embodiment, R 2 is a linear C 10 In one embodiment, R 2 is a linear C 11 It is alkyl.

[0156] In one embodiment, the second ester moiety of the cationic lipid is —C(═O)OR 2 or -OC(=O)R 2 where R 2 Branch C6~C 12 In one embodiment, R 2 is branch C 13 ~C 24 In one embodiment, R 2 is branch C 25 ~C 42 In one embodiment, R 2 is branch C 15 In one embodiment, R 2 is branch C 16 In one embodiment, R 2 is branch C 17 It is alkyl.

[0157] In one embodiment, the cationic lipid is C 14 ~C 42 In one embodiment, the cationic lipid comprises an alkyl ester moiety and at least a second ester moiety and a third ester moiety. 14 ~C 42 In one embodiment, the alkyl ester moiety comprises an alkyl ester moiety, a second alkyl ester moiety, and a third ester moiety. 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 are independently straight chain C7 to C 11In one embodiment, the second alkyl ester moiety and the third alkyl ester moiety are [ka] is part of the moiety, wherein each L 2 are independently -OC(=O)R 2 and each R 2 are independently straight chain C7 to C 11 In one embodiment, the second alkyl ester moiety and the third alkyl ester moiety are [ka] is part of the moiety, wherein each L 2 are independently -C(=O)OR 2 and each R 2 are independently straight chain C7 to C 11 It is alkyl.

[0158] In one embodiment, the cationic lipid contains only one tertiary amine. In one embodiment, the cationic lipid contains 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 at a pH of 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.

[0159] 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 one tertiary amine and two C alkyl ester moieties. 14 ~C 42 In one embodiment, the cationic lipid comprises two tertiary amines and two C alkyl ester moieties. 14 ~C 42In one embodiment, the cationic lipid comprises one tertiary amine, one branched C 14 ~C 42 Alkyl ester moiety and one straight chain C6-C 42 In one embodiment, the cationic lipid comprises two tertiary amines, one C alkyl ester moiety. 14 ~C 42 Alkyl ester moiety, straight chain C7~C 11 Alkyl second ester moiety and straight chain C7-C 11 It contains an alkyl third ester moiety.

[0160] In one embodiment, the cationic lipid is [ka] wherein G 1 is C2~C 12 is alkylene, and X 1 is -C(=O)O- or -OC(=O)-, and R 1 is branch C 14 ~C 42 In one embodiment, G is an alkyl group. 1 is a C2-C5 alkylene. 1 is a C6-C9 alkylene. 1 is C 10 ~C 12 It is alkylene.

[0161] In one embodiment, G 1 is C2~C 12 is alkylene, and X 1 is -C(=O)O- or -OC(=O)-, and R 1 is branch C 14 ~C 19 In one embodiment, R 1 is branch C 20 ~C 25 In one embodiment, R 1 is branch C 26 ~C 31 In one embodiment, R1 is branch C 32 ~C 37 In one embodiment, R 1 is branch C 38 ~C 42 It is alkyl.

[0162] In one embodiment, the cationic lipid has formula (I): [ka] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: G 1 But C2~C 12 is alkylene, X 1 is -C(=O)O- or -OC(=O)-, R 1 But branch C 14 ~C 42 is alkyl, R y is C1-C6 alkyl or -G 2 -X 2 -R 2 and G 2 But C2~C 12 is alkylene, X 2 is -C(=O)O- or -OC(=O)-, R 2 but linear or branched C6-C 42 is alkyl, R x is C1-C6 alkyl or -G 3 -R 3 and G 3 But C2~C 12 is alkylene, R 3 But -N(R 4 )R 5 -OR 6 and R 4 However, C1~C 12Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C 10 is aryl, R 5 However, C1~C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C 10 is aryl, or Or R 4 and R 5 together with the nitrogen to which they are attached form a cyclic moiety, R 6 But hydrogen, C1~C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C 10 is aryl, wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, alkylene, and cyclic moiety is independently optionally substituted.

[0163] In one embodiment, the cationic lipid has formula (II): [ka] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0164] In one embodiment, the cationic lipid has formula (II-A): [ka] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: G 1 is a C5-C7 alkylene; R 1 But branch C 14 ~C 19 is alkyl, G 2 is a C5-C7 alkylene; R 2 However, straight chain C7~C 13 is alkyl, G 3 is a C2-C4 alkylene.

[0165] In one embodiment, the cationic lipid has the formula (II-A-1): [ka] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0166] In one embodiment, the cationic lipid has formula (II-B): [ka] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: G 1 is a C5-C7 alkylene; R 1 But branch C 14 ~C 19 is alkyl, G 2 is a C5-C7 alkylene; R 2 But branch C 14 ~C 19 is alkyl, G 3 is a C2-C4 alkylene.

[0167] In one embodiment, the cationic lipid has the formula (II-B-1): [ka] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0168] In one embodiment, the cationic lipid has formula (II-C): [ka] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: G 1 is a C5-C7 alkylene; R 1 But branch C 14 ~C 19 is alkyl, G 2 is a C5-C7 alkylene; R 2 But branch C 14 ~C 19 is alkyl, G 3 is a C2-C4 alkylene, R 4 is a C3-C8 cycloalkyl; R 5 is a C2-C4 alkyl substituted with hydroxy.

[0169] In one embodiment, the cationic lipid has the formula (II-C-1): [ka] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0170] In one embodiment, the cationic lipid has formula (III): [ka] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: G 1 But C2~C 12 is alkylene, X 1 is -C(=O)O- or -OC(=O)-, R 1 But branch C 14 ~C 42 is alkyl, G 2 But C2~C 12 is alkylene, Each L2 independently, -X 2 -R 2 and each X 2 are independently —C(═O)O— or —OC(═O)—; Each R 2 are independently linear or branched C6-C 42 is alkyl, m is 1 or 2; G 3 But C2~C 12 is alkylene, R 4 However, C1~C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C 10 is aryl, R 5 However, C1~C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C 10 is aryl, wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, alkylene, and cyclic moiety is independently optionally substituted.

[0171] In one embodiment, m is 1. In one embodiment, m is 2.

[0172] In one embodiment, the cationic lipid has formula (IV): [ka] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: y1 is an integer from 0 to 9.

[0173] 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.

[0174] In one embodiment, each L 2 are independently -OC(=O)R 2 In one embodiment, each L 2 are independently -OC(=O)R 2 and each R 2 are independently straight chain C7 to 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 are independently straight chain C7 to C 11 It is alkyl.

[0175] In one embodiment, the cationic lipid has formula (IV-A): [ka] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: G 1 is a C5-C7 alkylene; R 1 But branch C 14 ~C 19 is alkyl, Each R 2 are independent, straight chain C7~C 13 is alkyl, G 3 is a C2-C4 alkylene, R 4 is a C3-C8 cycloalkyl; R 5 is a C2-C4 alkyl substituted with hydroxy; y1 is an integer from 2 to 6.

[0176] In one embodiment, the cationic lipid has the formula (IV-A-1): [ka] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0177] In one embodiment, G 1 is a C2-C5 alkylene. 1 is a C5-C7 alkylene. 1 is a C6-C9 alkylene. 1 is C 10 ~C 12 In one embodiment, G is an alkylene. 1 is a C alkylene. In one embodiment, G 1 is a C alkylene. In one embodiment, G 1 is a C4 alkylene. In one embodiment, G 1 is a C5 alkylene. In one embodiment, G 1 is a C alkylene. In one embodiment, G 1 is a C7 alkylene. In one embodiment, G 1 is a C alkylene. In one embodiment, G 1 is a C alkylene. In one embodiment, G 1 is C 10 In one embodiment, G is an alkylene. 1 is C 11 In one embodiment, G is an alkylene. 1 is C 12 It is alkylene.

[0178] In one embodiment, X 1 is —C(═O)O—. In one embodiment, X 1 is -OC(=O)-. Unless otherwise specified, the left side is G 1 On the right side is R 1 It is the latter.

[0179] In one embodiment, R 1 is branch C 14 ~C 24 In one embodiment, R 1 is branch C 14 ~C 19In one embodiment, R 1 is branch C 20 ~C 25 In one embodiment, R 1 is branch C 26 ~C 31 In one embodiment, R 1 is branch C 32 ~C 37 In one embodiment, R 1 is branch C 38 ~C 42 In one embodiment, R 1 -R 7 -CH(R 8 )(R 9 ) where R 7 is a C0-C5 alkylene, and R 8 and R 9 are independently C2 to C 10 In one embodiment, R 1 -R 7 -CH(R 8 )(R 9 ) where R 7 is a C0-C1 alkylene, and R 8 and R 9 are independently C4 to C8 alkyl.

[0180] In one embodiment, R 1 is branch C 14 In one embodiment, R 1 is branch C 15 In one embodiment, R 1 is branch C 16 In one embodiment, R 1 is branch C 17 In one embodiment, R 1 is branch C 18 In one embodiment, R 1 is branch C 19 In one embodiment, R 1 is branch C 20 In one embodiment, R 1 is branch C22 In one embodiment, R 1 is branch C 24 In one embodiment, R 1 is branch C 26 In one embodiment, R 1 is branch C 28 In one embodiment, R 1 is branch C 30 In one embodiment, R 1 is branch C 32 In one embodiment, R 1 is branch C 34 In one embodiment, R 1 is branch C 36 In one embodiment, R 1 is branch C 38 In one embodiment, R 1 is branch C 40 In one embodiment, R 1 is branch C 42 It is alkyl.

[0181] In one embodiment, R y is C1-C6 alkyl. In one embodiment, R y is methyl. In one embodiment, R y is ethyl. In one embodiment, R y is C alkyl. In one embodiment, R y is C4 alkyl. In one embodiment, R y is C alkyl. In one embodiment, R y is a C6 alkyl.

[0182] In one embodiment, R y -G 2 -X 2 -R 2 is.

[0183] In one embodiment, G 2 is C2~C 12 In one embodiment, G is an alkylene. 2is a C2-C5 alkylene. 2 is a C5-C7 alkylene. 2 is a C6-C9 alkylene. 2 is C 10 ~C 12 In one embodiment, G is an alkylene. 2 is a C alkylene. In one embodiment, G 2 is a C alkylene. In one embodiment, G 2 is a C4 alkylene. In one embodiment, G 2 is a C5 alkylene. In one embodiment, G 2 is a C alkylene. In one embodiment, G 2 is a C7 alkylene. In one embodiment, G 2 is a C alkylene. In one embodiment, G 2 is a C alkylene. In one embodiment, G 2 is C 10 In one embodiment, G is an alkylene. 2 is C 11 In one embodiment, G is an alkylene. 2 is C 12 It is alkylene.

[0184] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] In one embodiment, G 2 teeth, [ka] is.

[0185] In one embodiment, X 2 is —C(═O)O—. In one embodiment, X 2 is -OC(=O)-. Unless otherwise specified, the left side is G 2 On the right side is R 2 It is the latter.

[0186] In one embodiment, R 2 is a straight or branched C6-C 11 In one embodiment, R 2 is a linear or branched C 12 ~C 17 In one embodiment, R 2 is a linear or branched C 18 ~C 23 In one embodiment, R 2 is a linear or branched C 24 ~C 29 In one embodiment, R 2 is a linear or branched C 30 ~C 35 In one embodiment, R 2 is a linear or branched C 36 ~C 42 It is alkyl.

[0187] In one embodiment, R 2 is a straight chain C6-C 24 In one embodiment, R2 is a straight chain C7~C 13 In one embodiment, R 2 is a straight chain C7~C 11 In one embodiment, R 2 is a straight chain C alkyl. In one embodiment, R 2 is a straight chain C alkyl. In one embodiment, R 2 is a linear C 10 In one embodiment, R 2 is a linear C 11 In one embodiment, R 2 is a linear C 12 In one embodiment, R 2 is a linear C 13 In one embodiment, R 2 is a linear C 14 In one embodiment, R 2 is a linear C 15 In one embodiment, R 2 is a linear C 16 It is alkyl.

[0188] In one embodiment, R 2 is branch C 14 ~C 24 In one embodiment, R 2 is branch C 14 ~C 19 In one embodiment, R 2 -R 7 -CH(R 8 )(R 9 ) where R 7 is a C0-C5 alkylene, and R 8 and R 9 are independently C2 to C 10 In one embodiment, R 2 -R 7 -CH(R 8 )(R 9 ) where R 7 is a C0-C1 alkylene, and R 8 and R 9 are independently C4 to C8 alkyl.

[0189] In one embodiment, R 2 is a branched C alkyl. In one embodiment, R 2 is a branched C alkyl. In one embodiment, R 2 is branch C 10 In one embodiment, R 2 is branch C 12 In one embodiment, R 2 is branch C 14 In one embodiment, R 2 is branch C 15 In one embodiment, R 2 is branch C 16 In one embodiment, R 2 is branch C 17 In one embodiment, R 2 is branch C 18 In one embodiment, R 2 is branch C 19 In one embodiment, R 2 is branch C 20 In one embodiment, R 2 is branch C 22 In one embodiment, R 2 is branch C 24 In one embodiment, R 2 is branch C 26 In one embodiment, R 2 is branch C 28 In one embodiment, R 2 is branch C 30 In one embodiment, R 2 is branch C 32 In one embodiment, R 2 is branch C 34 In one embodiment, R 2 is branch C 36 In one embodiment, R 2 is branch C 38 In one embodiment, R 2 is branch C40 In one embodiment, R 2 is branch C 42 It is alkyl.

[0190] In one embodiment, R 1 , R 2 are each independently of the following structure: [ka] It is one of them.

[0191] In one embodiment, R x is C1-C6 alkyl. In one embodiment, R x is methyl. In one embodiment, R x is ethyl. In one embodiment, R x is C alkyl. In one embodiment, R x is C4 alkyl. In one embodiment, R x is C alkyl. In one embodiment, R x is a C6 alkyl.

[0192] In one embodiment, R x -G 3 -R 3 is.

[0193] In one embodiment, G 3 is C2~C 12 In one embodiment, G is an alkylene. 3 is a C2-C4 alkylene. 3 is a C2-C5 alkylene. 3 is a C6-C9 alkylene. 3 is C 10 ~C 12 In one embodiment, G is an alkylene. 3 is a C alkylene. In one embodiment, G 3 is a C alkylene. In one embodiment, G 3 is a C4 alkylene. In one embodiment, G 3is a C5 alkylene. In one embodiment, G 3 is a C alkylene. In one embodiment, G 3 is a C7 alkylene. In one embodiment, G 3 is a C alkylene. In one embodiment, G 3 is a C alkylene. In one embodiment, G 3 is C 10 In one embodiment, G is an alkylene. 3 is C 11 In one embodiment, G is an alkylene. 3 is C 12 It is alkylene.

[0194] In one embodiment, R 3 is -N(R 4 )R 5 is.

[0195] In one embodiment, R 4 is C1~C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C 10 It is aryl.

[0196] In one embodiment, R 4 is C1~C 12 In one embodiment, R 4 is C1-C8 alkyl. In one embodiment, R 4 is C1-C6 alkyl. In one embodiment, R 4 is C1-C4 alkyl. In one embodiment, R 4 is methyl. In one embodiment, R 4 is ethyl. In one embodiment, R 4 is n-propyl. In one embodiment, R 4 is iso-propyl. In one embodiment, R 4 is n-butyl. In one embodiment, R 4 is n-pentyl. In one embodiment, R 4 is n-hexyl. In one embodiment, R 4is n-octyl. In one embodiment, R 4 is n-nonyl.

[0197] In one embodiment, R 4 is C3-C8 cycloalkyl. In one embodiment, R 4 is cyclopropyl. In one embodiment, R 4 is cyclobutyl. In one embodiment, R 4 is cyclopentyl. In one embodiment, R 4 is cyclohexyl. In one embodiment, R 4 is cycloheptyl. In one embodiment, R 4 is cyclooctyl.

[0198] In one embodiment, R 4 is C-C cycloalkenyl. In one embodiment, R 4 is cyclopropenyl. In one embodiment, R 4 is cyclobutenyl. In one embodiment, R 4 is cyclopentenyl. In one embodiment, R 4 is cyclohexenyl. In one embodiment, R 4 is cycloheptenyl. In one embodiment, R 4 is cyclooctenyl.

[0199] In one embodiment, R 4 is C6~C 10 In one embodiment, R 4 is phenyl.

[0200] In one embodiment, R 4 is non-substituted.

[0201] In one embodiment, R 4 is 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, wherein R g is independently in each occurrence H or C1-C6 alkyl; R h is independently in each occurrence C1-C6 alkyl; R i is independently in each occurrence C1-C6 alkylene.

[0202] In one embodiment, R 4 is substituted with one or more hydroxyls. In one embodiment, R 4 is substituted with one hydroxyl. In one embodiment, R 4 is substituted with one or more hydroxyl and one or more oxo. In one embodiment, R 4 is substituted with one hydroxyl and one oxo.

[0203] In one embodiment, R 5 is C1~C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C 10 It is aryl.

[0204] In one embodiment, R 5 is C1~C 12 In one embodiment, R 5 is C1-C8 alkyl. In one embodiment, R 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 is methyl. In one embodiment, R 5 is ethyl. In one embodiment, R 5 is n-propyl. In one embodiment, R 5is iso-propyl. In one embodiment, R 5 is n-butyl. In one embodiment, R 5 is n-pentyl. In one embodiment, R 5 is n-hexyl. In one embodiment, R 5 is n-octyl. In one embodiment, R 5 is n-nonyl.

[0205] In one embodiment, R 5 is C3-C8 cycloalkyl. In one embodiment, R 5 is cyclopropyl. In one embodiment, R 5 is cyclobutyl. In one embodiment, R 5 is cyclopentyl. In one embodiment, R 5 is cyclohexyl. In one embodiment, R 5 is cycloheptyl. In one embodiment, R 5 is cyclooctyl.

[0206] In one embodiment, R 5 is C-C cycloalkenyl. In one embodiment, R 5 is cyclopropenyl. In one embodiment, R 5 is cyclobutenyl. In one embodiment, R 5 is cyclopentenyl. In one embodiment, R 5 is cyclohexenyl. In one embodiment, R 5 is cycloheptenyl. In one embodiment, R 4 is cyclooctenyl.

[0207] In one embodiment, R 5 is C6~C 10 In one embodiment, R 5 is phenyl.

[0208] In one embodiment, R 4 and R 5 together with the nitrogen to which they are attached form a cyclic moiety.

[0209] In one embodiment, the cyclic moiety (R 4 and R 5

[0049] In 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.

[0210] In one embodiment, the cyclic moiety (R 4 and R 5 (formed together with the nitrogen attached thereto by) is azetidin-1-yl. In one embodiment, the cyclic moiety is pyrrolysin-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 azocan-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 G 3 It is for.

[0211] As used herein and unless otherwise specified, R 5 The substitution pattern for R 4 and R 5 The term also applies to cyclic moieties formed by, together with the nitrogen attached thereto,

[0212] In one embodiment, R 5 is non-substituted.

[0213] In one embodiment, R 5 is 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, wherein R g is independently in each occurrence H or C1-C6 alkyl; R h is independently in each occurrence C1-C6 alkyl; R i is independently in each occurrence C1-C6 alkylene.

[0214] In one embodiment, R 5 is substituted with one or more hydroxyls. In one embodiment, R 5 is substituted with one hydroxyl.

[0215] In one embodiment, R 5 is substituted with one or more hydroxyl and one or more oxo. In one embodiment, R 5 is substituted with one hydroxyl and one oxo. In one embodiment, R 5 is a C2-C4 alkyl substituted with hydroxy. 5 is -CH2CH2OH.

[0216] In one embodiment, R 3 -OR 6 is.

[0217] In one embodiment, R 6 is hydrogen (i.e., R 3 is —OH). In one embodiment, R 6 is C1~C 12 In one embodiment, R 6 is C1-C8 alkyl. In one embodiment, R 6 is C1-C6 alkyl. In one embodiment, R 6is C1-C4 alkyl. In one embodiment, R 6 is methyl. In one embodiment, R 6 is ethyl. In one embodiment, R 6 is C3-C8 cycloalkyl. In one embodiment, R 6 is C-C cycloalkenyl. In one embodiment, R 6 is C6~C 10 In one embodiment, R 6 is phenyl. In one embodiment, R 6 is unsubstituted. In one embodiment, R 6 has been substituted.

[0218] In one embodiment, the cationic lipid is [ka] is.

[0219] It is understood that any embodiment of the compounds provided herein defined above, and any particular substituents and / or variables of the compounds provided herein defined above, can be independently combined with the substituents and / or variables of other embodiments and / or compounds to form embodiments not specifically defined above. Additionally, when a list of substituents and / or variables is recited for any particular group or variable, it is understood that individual substituents and / or variables may be deleted from a particular embodiment and / or claim, and the remaining list of substituents and / or variables is deemed to be within the scope of the embodiments provided herein.

[0220] In describing the present invention, it is understood that combinations of substituents and / or variables of the depicted formulae are permissible only if such combinations result in stable compounds.

[0221] 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).

[0222] In one embodiment, the lipid nanoparticles comprise 0.5 mole percent or less of phospholipids. In one embodiment, the lipid nanoparticles comprise 0.01 mole percent or less of phospholipids, 0.05 mole percent or less of phospholipids, 0.1 mole percent or less of phospholipids, 0.15 mole percent or less of phospholipids, 0.2 mole percent or less of phospholipids, 0.25 mole percent or less of phospholipids, 0.3 mole percent or less of phospholipids, 0.35 mole percent or less of phospholipids, 0.4 mole percent or less of phospholipids, 0.45 mole percent or less of phospholipids, or 0.5 mole percent or less of phospholipids. In one embodiment, the lipid nanoparticles are free of phospholipids.

[0223] In one embodiment, the lipid nanoparticles have a lipid:nucleic acid mass ratio of about 9:1 to about 20:1. In one embodiment, the lipid nanoparticles have a lipid:nucleic acid mass ratio of about 9:1 to about 15:1. In one embodiment, the lipid nanoparticles have 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.

[0224] In one embodiment, the size and encapsulation efficiency of the LNPs provided herein are comparable to that of conventional LNP formulations. In one embodiment, the LNPs provided herein have improved in vitro and / or in vivo mRNA delivery efficiency as assessed by protein expression levels compared to conventional LNP formulations.

[0225] In one embodiment, LNPs are vesicles comprising one or more lipid bilayers. In one embodiment, LNPs comprise two or more concentric bilayers separated by aqueous compartments. The lipid bilayers may be functionalized and / or cross-linked to one another. The lipid bilayers may comprise one or more ligands, proteins, or channels.

[0226] In one embodiment, the nanoparticles contain one or more nucleic acid molecules as a therapeutic payload.

[0227] 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.

[0228] 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.

[0229] In one embodiment, the mRNA is a monocistronic mRNA. In one embodiment, the mRNA is a multicistronic mRNA. In one embodiment, the mRNA is a multicistronic mRNA containing two or more open reading frames (ORFs). In one embodiment, the multicistronic mRNA encodes two identical peptides or proteins. In one embodiment, the multicistronic mRNA encodes two different peptides or proteins.

[0230] In one embodiment, each peptide or protein encoded by the multicistronic mRNA comprises at least one epitope of a selected antigen, hi one embodiment, different peptides or proteins encoded by the multicistronic mRNA each comprise at least one epitope of a different antigen.

[0231] In one embodiment, the at least one epitope can be at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten epitopes of the antigen.

[0232] In one embodiment, the nucleic acid is a small interfering RNA (siRNA).

[0233] In one embodiment, the siRNA selectively silences a gene associated with a particular disease, disorder, or condition when a nanoparticle composition comprising the siRNA is administered to a subject in need of silencing the gene associated with the particular disease, disorder, or condition.

[0234] In one embodiment, the nucleic acid is an immunomodulatory siRNA.

[0235] In one embodiment, the siRNA comprises a sequence that is complementary to an mRNA sequence that encodes a protein product of interest.

[0236] In one embodiment, the nucleic acid molecule contains only standard 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-standard nucleobase.

[0237] In one embodiment, the nucleic acid is chemically modified. In one embodiment, the nucleic acid comprises at least one chemical modification to the nucleobase. Exemplary modifications to the nucleobase include, but are not limited to, one or more substitutions or modifications, including but not limited to, alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitutions, one or more ring condensation or ring opening, oxidation, and / or reduction. In one embodiment, the nucleic acid comprises at least one chemical modification to the sugar group. In one embodiment, the nucleic acid comprises at least one chemical modification to the internucleoside linkage.

[0238] In one aspect, the nucleic acid molecules described herein are formulated for in vitro and in vivo gene delivery. In particular, in one embodiment, the nucleic acid molecules are formulated in lipid nanoparticles. In one embodiment, the lipid nanoparticles encapsulate the nucleic acid molecules within a lipid shell. In one embodiment, the lipid shell protects the nucleic acid molecules from degradation. In one embodiment, the lipid nanoparticles also facilitate the transport of the encapsulated nucleic acid molecules to intracellular compartments and / or mechanisms where they exert their intended therapeutic or preventive function. In certain embodiments, the nucleic acid, when present in the lipid nanoparticles, is resistant to nuclease degradation in aqueous solution. Lipid nanoparticles containing nucleic acids and methods for their preparation are known in the art, such as those disclosed in U.S. Patent Publication No. 2004 / 0142025, U.S. Patent Publication No. 2007 / 0042031, PCT Publication No. WO2017 / 004143, PCT Publication No. WO2015 / 199952, PCT Publication No. WO2013 / 016058, and PCT Publication No. WO2013 / 086373, the entire disclosures of each of which are incorporated herein by reference in their entirety for all purposes.

[0239] In one embodiment, the largest dimension of the LNP compositions provided herein is 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.

[0240] In one embodiment, provided herein are lipid nanoparticles having a nanoparticle size of about 40 nm to about 150 nm. In one embodiment, the nanoparticle size is about 50 nm to about 100 nm. In one embodiment, the nanoparticle size is about 40 nm to about 120 nm. In one embodiment, the nanoparticle size is about 40 nm to about 100 nm. In one embodiment, the nanoparticle size is about 50 nm to about 150 nm. In one embodiment, the nanoparticle size is about 60 nm to about 150 nm. In one embodiment, the nanoparticle size is about 60 nm to about 120 nm. In one embodiment, the nanoparticle size is about 70 nm to about 120 nm. In one embodiment, the nanoparticle size is about 80 nm to about 120 nm.

[0241] As used herein, and unless otherwise specified, the size of a nanoparticle refers to the average size of a group of nanoparticles or a plurality of such nanoparticles. In one embodiment, the size of the three-lipid component lipid nanoparticles provided herein (containing no phospholipid component or a reduced / insignificant amount of phospholipid component) is increased compared to a reference formulation, where the reference formulation differs from the lipid nanoparticles in that (i) the reference formulation contains phospholipids, and (ii) the molar ratio of cationic lipid:steroid:phospholipid:polymer-conjugated lipid is about 50:38.5:10:1.5. In one embodiment, the three-lipid component nanoparticles provided herein have a size increase 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 size of the nanoparticles is increased by at least about 50% compared to the reference formulation. In one embodiment, the three-lipid component nanoparticles provided herein have a size increase of not more than about 90%, not more than about 100%, or not more than about 110%. In one embodiment, the size (of the three lipid component nanoparticles provided herein) is about 90 nm to about 150 nm. In one embodiment, the size is about 100 nm to about 140 nm. In one embodiment, the size is about 110 nm to about 130 nm. In one embodiment, the size is about 100 nm to about 110 nm. In one embodiment, the size is about 110 nm to about 120 nm. In one embodiment, the size is about 120 nm to about 130 nm.

[0242] In one embodiment, the nanoparticles are about 40 nm in size. In one embodiment, the nanoparticles are about 50 nm in size. In one embodiment, the nanoparticles are about 55 nm in size. In one embodiment, the nanoparticles are about 60 nm in size. In one embodiment, the nanoparticles are about 65 nm in size. In one embodiment, the nanoparticles are about 70 nm in size. In one embodiment, the nanoparticles are about 75 nm in size. In one embodiment, the nanoparticles are about 80 nm in size. In one embodiment, the nanoparticles are about 85 nm in size. In one embodiment, the nanoparticles are about 90 nm in size. In one embodiment, the nanoparticles are about 95 nm in size. In one embodiment, the nanoparticles are about 100 nm in size. In one embodiment, the nanoparticles are about 105 nm in size. In one embodiment, the nanoparticles are about 110 nm in size. In one embodiment, the nanoparticles are about 115 nm in size. In one embodiment, the nanoparticles are about 120 nm in size. In one embodiment, the nanoparticles are about 125 nm in size. In one embodiment, the nanoparticles are about 125 nm in size. In one embodiment, the size of the nanoparticles is about 130 nm. In one embodiment, the size of the nanoparticles is about 135 nm. In one embodiment, the size of the nanoparticles is about 140 nm. In one embodiment, the size of the nanoparticles is about 145 nm. In one embodiment, the size of the nanoparticles is about 150 nm. In one embodiment, the size of the nanoparticles is about 155 nm.

[0243] In one embodiment, the lipid nanoparticles provided herein have an encapsulation efficiency of at least about 80% for nucleic acids. In one embodiment, the lipid nanoparticles provided herein have an encapsulation efficiency of at least about 80% for mRNA. In one embodiment, the lipid nanoparticles provided herein have an encapsulation efficiency of at least about 80% for DNA. In one embodiment, the lipid nanoparticles provided herein have an encapsulation efficiency of at least about 80% for siRNA.

[0244] In one embodiment, the lipid nanoparticles provided herein have an encapsulation efficiency of nucleic acids 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%.

[0245] 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%.

[0246] In one embodiment, the nanoparticles have an encapsulation efficiency of at least about 90% for nucleic acids. In one embodiment, the nanoparticles have an encapsulation efficiency of at least about 90% for mRNA. In one embodiment, the nanoparticles have an encapsulation efficiency of at least about 90% for DNA. In one embodiment, the nanoparticles have an encapsulation efficiency of at least about 90% for siRNA.

[0247] In one embodiment, the nucleic acid encodes RNA or protein, and the amount of RNA or protein expressed from the nucleic acid in the nanoparticle in a mammalian cell or mammalian tissue is greater than the amount of RNA or protein expressed from the nucleic acid in a reference formulation, and the reference formulation differs from the lipid nanoparticle in that (i) the reference formulation comprises a phospholipid, and (ii) the molar ratio of cationic lipid:steroid:phospholipid:polymer-conjugated lipid is approximately 50:38.5:10:1.5.

[0248] In one embodiment, the amount of phospholipids in the reference formulation is greater than 5 mol% of the total lipids present in the reference formulation. In one embodiment, the amount of phospholipids in the reference formulation is greater than 10 mol% of the total lipids present in the reference formulation. In one embodiment, the amount of phospholipids in the reference formulation is greater than 15 mol% of the total lipids present in the reference formulation. In one embodiment, the amount of phospholipids in the reference formulation is greater than 20 mol% of the total lipids present in the reference formulation. In one embodiment, the amount of phospholipids in the reference formulation is greater than 25 mol% of the total lipids present in the reference formulation. In one embodiment, the amount of phospholipids in the reference formulation is greater than 30 mol% of the total lipids present in the reference formulation. In one embodiment, the amount of phospholipids in the reference formulation is greater than 35 mol% of the total lipids present in the reference formulation. In one embodiment, the amount of phospholipids in the reference formulation is greater than 40 mol% of the total lipids present in the reference formulation. In one embodiment, the amount of phospholipids in the reference formulation is greater than 45 mol% of the total lipids present in the reference formulation. In one embodiment, the amount of phospholipid in the reference formulation is greater than 50 mol % of the total lipid present in the reference formulation.

[0249] In one embodiment, the amount of phospholipid in the reference formulation is about 10 mol % of the total lipid present in the reference formulation.

[0250] In one embodiment, the reference formulation comprises DSPC. In one embodiment, the reference formulation comprises DOPE.

[0251] In one embodiment, the amount of RNA or protein expressed from the nucleic acid in the nanoparticles provided herein is at least about 50% greater than the amount of RNA or protein expressed from the nucleic acid in a reference formulation.

[0252] In one embodiment, the amount of RNA or protein expressed from the nucleic acid 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 from the nucleic acid in a reference formulation.

[0253] In one embodiment, the amount of RNA or protein expressed from a nucleic acid in a nanoparticle provided herein is at least about 100% greater than the amount of RNA or protein expressed from a nucleic acid in a reference formulation. In one embodiment, the amount of RNA or protein expressed from a nucleic acid in a nanoparticle provided herein is at least about 150% greater than the amount of RNA or protein expressed from a nucleic acid in a reference formulation. In one embodiment, the amount of RNA or protein expressed from a nucleic acid in a nanoparticle provided herein is at least about 200% greater than the amount of RNA or protein expressed from a nucleic acid in a reference formulation. In one embodiment, the amount of RNA or protein expressed from a nucleic acid in a nanoparticle provided herein is at least about 250% greater than the amount of RNA or protein expressed from a nucleic acid in a reference formulation. In one embodiment, the amount of RNA or protein expressed from a nucleic acid in a nanoparticle provided herein is at least about 300% greater than the amount of RNA or protein expressed from a nucleic acid in a reference formulation.

[0254] In one embodiment, the amount of RNA or protein expressed from the nucleic acid in a nanoparticle 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% greater than the amount of RNA or protein expressed from the nucleic acid in a reference formulation.

[0255] In one embodiment, provided herein is a pharmaceutical composition comprising a lipid nanoparticle provided herein and a pharmaceutically acceptable excipient, in one embodiment, the excipient is approved for human and veterinary use.

[0256] In one embodiment, one or more excipients can comprise more than 50% of the total mass or volume of the pharmaceutical composition, including the nanoparticle composition, For example, one or more excipients can comprise about 50%, 60%, 70%, 80%, 90%, or more of the pharmaceutical composition.

[0257] In one embodiment, the pharmaceutical composition may contain from 0.1% to 100% (w / w) of one or more nanoparticle compositions.

[0258] 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.

[0259] In one embodiment, provided herein is a method for expressing mRNA in a mammalian cell or mammalian tissue, the method comprising delivering a lipid nanoparticle provided herein or a pharmaceutical composition provided herein to a mammalian cell or mammalian tissue, wherein the nucleic acid is mRNA, and the delivered mRNA is expressed in the mammalian cell or mammalian tissue.

[0260] 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.

[0261] 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.

[0262] In one embodiment, the cells are in the liver, in one embodiment, the cells are in the lung, in one embodiment, the cells are in the spleen, in one embodiment, the cells are in the colon, in one embodiment, the cells are in the kidney.

[0263] 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.

[0264] In one embodiment, the cell is a genetically engineered cell.

[0265] In one embodiment, the cells are stem cells, including, but not limited to, pluripotent stem cells, mesenchymal stem cells, neural stem cells, and hematopoietic stem cells.

[0266] In one embodiment, the cell is a cancer cell.

[0267] 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.

[0268] In one embodiment, provided herein is a method for treating diseases or disorders caused by impaired protein expression in humans, the method comprising administering to humans a therapeutically effective amount of lipid nanoparticles provided herein or pharmaceutical compositions provided herein, wherein the nucleic acid is mRNA encoding the protein.In one embodiment, the expression level of the protein is increased 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%.

[0269] In one embodiment, provided herein is a method for treating a disease or disorder caused by overexpression of a protein in a human, the method comprising administering to a human a therapeutically effective amount of lipid nanoparticles provided herein or pharmaceutical compositions 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%.

[0270] In one embodiment, the disease or disorder is a viral infection. In one embodiment, the disease or disorder is a liver disease. In one embodiment, the disease or disorder is cancer.

[0271] In one embodiment, administration is intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, subcutaneous, or intradermal administration. In one embodiment, administration is intravenous administration. In one embodiment, administration is intramuscular administration. In one embodiment, administration is oral administration.

[0272] In one embodiment, provided herein is a method for preventing disease in a subject by administering to the subject a vaccine comprising a therapeutically effective amount of lipid nanoparticles provided herein or a pharmaceutical composition provided herein, wherein the nucleic acid encodes an antigen associated with the disease, thereby inducing an immune response in the subject against the target disease. In one embodiment, the immune response is an adaptive immune response or an innate immune response, or both.

[0273] In one embodiment, the vaccine is administered before the onset of clinical symptoms of the target disease, thereby immunizing or reducing the susceptibility of the vaccinated person to developing the disease, hi one embodiment, the vaccine is administered after the onset of clinical symptoms of the target disease, thereby alleviating symptoms and / or preventing recurrence.

[0274] 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.

[0275] In one embodiment, the pathogen is a coronavirus (e.g., SARS, SARS-Cov-2, MERS), influenza, measles, human papillomavirus (HPV), rabies, meningitis, whooping cough, tetanus, plague, hepatitis, or tuberculosis.

[0276] In one embodiment, the therapeutic payload comprises a nucleic acid sequence (e.g., mRNA) encoding a pathogenic protein, or antigenic fragment or epitope thereof, unique to a pathogen. When administered to a vaccinated subject, the vaccine allows for expression of the encoded pathogenic protein (or antigenic fragment or epitope thereof), thereby inducing immunity in the subject against the pathogen.

[0277] 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 eliciting an immune response in humans against the target disease.

[0278] In one embodiment, the infectious disease is a viral infection. In one embodiment, the infectious disease is a bacterial infection.

[0279] In one embodiment, the nucleic acid is an mRNA that encodes a polypeptide present in the antigen.

[0280] 6.3.1 Therapeutic payloads The nanoparticle compositions described herein can include one or more therapeutic and / or prophylactic agents. These therapeutic and / or prophylactic agents are sometimes referred to in this disclosure as "therapeutic payloads" or "payloads." In some embodiments, therapeutic payloads can be administered in vivo or in vitro using nanoparticles as delivery vehicles.

[0281] In one embodiment, the therapeutic payload is a nucleic acid provided herein. In one embodiment, the nanoparticle composition comprises a therapeutic payload in addition to a nucleic acid provided herein. In one embodiment, the nanoparticle composition comprises a therapeutic payload in place of a nucleic acid provided herein.

[0282] In some embodiments, the nanoparticle compositions contain, as a therapeutic payload, small molecule compounds (e.g., small molecule drugs), such as antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antitumor agents (e.g., cyclophosphamide, methotrexate, and streptozotocin), anticancer ... agents) (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs such as methotrexate and purine and pyrimidine analogs), anti-infectives, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blocking agents (e.g., propranolol, timolol, and labetalol), antihypertensives (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), ), anticonvulsants (e.g., phenytoin), antihistamines (diphenhydramine, chlorpheniramine, promethazine), antibiotics / antibacterials (e.g., gentamicin, 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 contrast media.

[0283] 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, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, 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 (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium.

[0284] In other embodiments, the therapeutic payload of the nanoparticle composition is an antimetabolite (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), an alkylating agent (e.g., mechlorethamine, thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine, vinblastine, taxol, and maytansinoids).

[0285] In some embodiments, the nanoparticle compositions include biomolecules, such as peptides and polypeptides, as therapeutic payloads. The biomolecules forming part of the nanoparticle compositions can be either naturally occurring or synthetic. For example, in some embodiments, the therapeutic payloads of the nanoparticle compositions can include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), VIR factors, luteinizing hormone-releasing hormone (LHRH) analogs, interferons, heparin, hepatitis B surface antigen, typhoid vaccine, cholera vaccine, and peptides and polypeptides.

[0286] 6.3.1.1 Additional Nucleic Acid Description In some embodiments, the nanoparticle compositions comprise 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 compositions as a therapeutic payload include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), including messenger RNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, vectors, etc. In certain embodiments, the therapeutic payload comprises RNA. RNA molecules that can be included in the nanoparticle compositions as therapeutic payloads include, but are not limited to, shortomers, agomils, antagomirs, antisense, ribozymes, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and other forms of RNA molecules known in the art. In certain embodiments, the RNA is mRNA.

[0287] In one embodiment, the nanoparticle composition 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 as a therapeutic payload include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), including messenger RNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, vectors, etc. In certain embodiments, the therapeutic payload comprises RNA. RNA molecules that can be included in the nanoparticle compositions as therapeutic payloads include, but are not limited to, shortomers, agomils, antagomirs, antisense, ribozymes, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and other forms of RNA molecules known in the art. In certain embodiments, the RNA is mRNA.

[0288] In another embodiment, the nanoparticle composition comprises an siRNA molecule as a therapeutic payload. In particular, in one embodiment, the siRNA molecule can selectively interfere with and downregulate the expression of a gene of interest. For example, in one embodiment, the siRNA payload selectively silences a gene associated with a particular disease, disorder, or condition when the nanoparticle composition comprising the siRNA is administered to a subject in need of silencing the gene associated with the particular disease, disorder, or condition. In one embodiment, the siRNA molecule comprises a sequence complementary to an mRNA sequence encoding a protein product of interest. In one embodiment, the siRNA molecule is an immunomodulatory siRNA.

[0289] In one embodiment, the nanoparticle composition comprises an shRNA molecule or a vector encoding an shRNA molecule as a therapeutic payload. In particular, in one embodiment, the therapeutic payload produces shRNA in target cells when administered to the target cells. The structure and mechanism of shRNA are well known in the relevant technical field.

[0290] In one embodiment, the nanoparticle composition comprises an mRNA molecule as a therapeutic payload. In particular, in one embodiment, the mRNA molecule encodes a polypeptide of interest, including any naturally occurring or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA can be of any size and have any secondary structure or activity. In one embodiment, the polypeptide encoded by the mRNA payload can have a therapeutic effect when expressed in cells.

[0291] In some embodiments, a nucleic acid molecule of the present disclosure comprises an mRNA molecule. In certain embodiments, a nucleic acid molecule comprises at least one coding region (e.g., an open reading frame (ORF)) encoding a peptide or polypeptide of interest. In one embodiment, a nucleic acid molecule further comprises at least one untranslated region (UTR). In certain embodiments, the untranslated region (UTR) is located upstream (towards the 5' end) of the coding region and is referred to herein as a 5'-UTR. In certain embodiments, the untranslated region (UTR) is located downstream (towards the 3' end) of the coding region and is referred to herein as a 3'-UTR. In certain embodiments, a nucleic acid molecule comprises both a 5'-UTR and a 3'-UTR. In one embodiment, the 5'-UTR comprises a 5' cap structure. In one embodiment, a nucleic acid molecule comprises a Kozak sequence (e.g., in the 5'-UTR). In one embodiment, a nucleic acid molecule comprises a polyA region (e.g., in the 3'-UTR). In one embodiment, a nucleic acid molecule comprises a polyadenylation signal (e.g., in the 3'-UTR). In one embodiment, the nucleic acid molecule comprises a stabilizing region (e.g., in the 3'-UTR). In one embodiment, the nucleic acid molecule comprises 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 the 5'-UTR and / or 3'-UTR). In one embodiment, the nucleic acid molecule comprises one or more intron 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.

[0292] Code Region In one embodiment, a nucleic acid molecule of the present disclosure comprises 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 comprises at least two ORFs, each encoding a peptide or protein. In embodiments where the coding region comprises two or more ORFs, the encoded peptides and / or proteins can be the same as or different from one another. In one embodiment, the multiple ORFs in the coding region are separated by non-coding sequences. In a specific embodiment, the non-coding sequence separating the two ORFs comprises an internal ribosome entry site (IRES).

[0293] Without being bound by theory, it is contemplated that an internal ribosome entry site (IRES) can act as the only ribosome binding site or can function as one of multiple ribosome binding sites of an mRNA. An mRNA molecule containing two or more functional ribosome binding sites can encode several peptides or polypeptides that are independently translated by the ribosome (e.g., a multicistronic mRNA). Thus, in one embodiment, a nucleic acid molecule (e.g., an mRNA) of the present disclosure comprises one or more internal ribosome entry sites (IRES). Examples of IRES sequences that can be used in connection with the present disclosure include, but are not limited to, those derived from picomaviruses (e.g., FMDV), plague viruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia viruses (MLV), simian immunodeficiency viruses (SIV), or cricket paralysis viruses (CrPV).

[0294] In various embodiments, a nucleic acid molecule of the present disclosure encodes 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 can be the same or different. In one embodiment, a nucleic acid molecule of the present disclosure encodes a dipeptide (e.g., chamocine and anserine). In one embodiment, a nucleic acid molecule encodes a tripeptide. In one embodiment, a nucleic acid molecule encodes a tetrapeptide. In one embodiment, a nucleic acid molecule encodes a pentapeptide. In one embodiment, a nucleic acid molecule encodes a hexapeptide. In one embodiment, a nucleic acid molecule encodes a heptapeptide. In one embodiment, a nucleic acid molecule encodes an octapeptide. In one embodiment, a nucleic acid molecule encodes a nonapeptide. In one embodiment, a nucleic acid molecule encodes a decapeptide. In one embodiment, a nucleic acid molecule encodes a peptide or polypeptide having at least about 15 amino acids. In one embodiment, a nucleic acid molecule encodes a peptide or polypeptide having at least about 50 amino acids. In one embodiment, a nucleic acid molecule encodes a peptide or polypeptide having at least about 100 amino acids. In one embodiment, the nucleic acid molecule encodes a peptide or polypeptide having at least about 150 amino acids. In one embodiment, the nucleic acid molecule encodes a peptide or polypeptide 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.

[0295] In one embodiment, a nucleic acid molecule of the present disclosure is at least about 30 nucleotides (nt) in length. In one embodiment, a nucleic acid molecule is at least about 35 nt in length. In one embodiment, a nucleic acid molecule is at least about 40 nt in length. In one embodiment, a nucleic acid molecule is at least about 45 nt in length. In one embodiment, a nucleic acid molecule is at least about 50 nt in length. In one embodiment, a nucleic acid molecule is at least about 55 nt in length. In one embodiment, a nucleic acid molecule is at least about 60 nt in length. In one embodiment, a nucleic acid molecule is at least about 65 nt in length. In one embodiment, a nucleic acid molecule is at least about 70 nt in length. In one embodiment, a nucleic acid molecule is at least about 75 nt in length. In one embodiment, a nucleic acid molecule is at least about 80 nt in length. In one embodiment, a nucleic acid molecule is at least about 85 nt in length. In one embodiment, a nucleic acid molecule is at least about 90 nt in length. In one embodiment, a nucleic acid molecule is at least about 95 nt in length. In one embodiment, a nucleic acid molecule is at least about 100 nt in length. In one embodiment, the nucleic acid molecule is at least about 120 nt in length. In one embodiment, the nucleic acid molecule is at least about 140 nt in length. In one embodiment, the nucleic acid molecule is at least about 160 nt in length. In one embodiment, the nucleic acid molecule is at least about 180 nt in length. In one embodiment, the nucleic acid molecule is at least about 200 nt in length. In one embodiment, the nucleic acid molecule is at least about 250 nt in length. In one embodiment, the nucleic acid molecule is at least about 300 nt in length. In one embodiment, the nucleic acid molecule is at least about 400 nt in length. In one embodiment, the nucleic acid molecule is at least about 500 nt in length. In one embodiment, the nucleic acid molecule is at least about 600 nt in length. In one embodiment, the nucleic acid molecule is at least about 700 nt in length. In one embodiment, the nucleic acid molecule is at least about 800 nt in length. In one embodiment, the nucleic acid molecule is at least about 900 nt in length. In one embodiment, the nucleic acid molecule is at least about 1000 nt in length. In one embodiment, the nucleic acid molecule is at least about 1100 nt in length. In one embodiment, the nucleic acid molecule is at least about 1200 nt in length. In one embodiment, the nucleic acid molecule is at least about 1300 nt in length. In one embodiment, the nucleic acid molecule is at least about 1400 nt in length.In one embodiment, the nucleic acid molecule is at least about 1500 nt in length. In one embodiment, the nucleic acid molecule is at least about 1600 nt in length. In one embodiment, the nucleic acid molecule is at least about 1700 nt in length. In one embodiment, the nucleic acid molecule is at least about 1800 nt in length. In one embodiment, the nucleic acid molecule is at least about 1900 nt in length. In one embodiment, the nucleic acid molecule is at least about 2000 nt in length. In one embodiment, the nucleic acid molecule is at least about 2500 nt in length. In one embodiment, the nucleic acid molecule is at least about 3000 nt in length. In one embodiment, the nucleic acid molecule is at least about 3500 nt in length. In one embodiment, the nucleic acid molecule is at least about 4000 nt in length. In one embodiment, the nucleic acid molecule is at least about 4500 nt in length. In one embodiment, the nucleic acid molecule is at least about 5000 nt in length.

[0296] In certain embodiments, the therapeutic payload comprises a vaccine composition (e.g., a genetic vaccine) described herein. In one embodiment, the therapeutic payload comprises a compound capable of inducing immunity against one or more target conditions or diseases. In one embodiment, the target condition is associated with or caused by infection by a pathogen, such as coronavirus (e.g., 2019-nCoV), influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis, and tuberculosis. 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, unique to the pathogen. When administered to a vaccinated subject, the vaccine allows for expression of the encoded pathogenic protein (or antigenic fragment or epitope thereof), thereby inducing immunity in the subject against the pathogen.

[0297] In one embodiment, the target pathology is associated with or caused by neoplastic proliferation 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) specific to the cancer, or an antigenic fragment or epitope thereof. When administered to a vaccinated subject, the vaccine allows expression of the encoded TAA (or antigenic fragment or epitope thereof), thereby eliciting immunity in the subject against tumor cells expressing the TAA.

[0298] 5' cap structure Without being bound by theory, it is believed that the 5' cap structure of a polynucleotide is involved in nuclear export and increased polynucleotide stability, and binds to mRNA cap-binding protein (CBP), which associates with polyA-binding protein to form mature circular mRNA species, thereby contributing to intracellular polynucleotide stability and translation competence. The 5' cap structure further assists in the removal of 5'-proximal introns during mRNA splicing. Thus, in one embodiment, a nucleic acid molecule of the present disclosure comprises a 5' cap structure.

[0299] Nucleic acid molecules can be 5'-end capped by a cell's endogenous transcription machinery to generate a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue and the transcribed sense nucleotide at the 5' end of the polynucleotide. This 5'-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue. The ribose sugar of the terminal and / or ante-terminal transcribed nucleotide at the 5' end of the polynucleotide can also optionally be 2'-O-methylated. 5'-decapping and cleavage of the guanylate cap structure by hydrolysis can target nucleic acid molecules, such as mRNA molecules, for degradation.

[0300] In one embodiment, a nucleic acid molecule of the present disclosure contains one or more modifications to the native 5' cap structure produced by endogenous processes. 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 potentially increase the translation efficiency of the polynucleotide.

[0301] Exemplary modifications to the native 5' cap structure include the creation of a non-hydrolyzable cap structure, which prevents decapping and thus increases the half-life of the polynucleotide. In one embodiment, modified nucleotides may be used during the capping reaction because hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphorodiester bond. For example, in one embodiment, vaccinia capping enzyme from New England Biolabs (Ipswich, Mass.) may be used with α-thio-guanosine nucleotides according to the manufacturer's instructions to create phosphorothioate linkages in the 5'-ppp-5' cap. Additional modified guanosine nucleotides, such as α-methyl-phosphonate nucleotides and selenophosphate nucleotides, may also be used.

[0302] Additional exemplary modifications to the native 5' cap structure also include modifications at the 2' and / or 3' positions of the capped guanosine triphosphate (GTP), replacement of the sugar ring oxygen (which created a carbocyclic ring) with a methylene moiety (CH), modifications in the triphosphate bridge portion of the cap structure, or modifications in the nucleobase (G) portion.

[0303] Additional exemplary modifications to the native 5' cap structure include, but are not limited to, 2'-O-methylation of the ribose sugar of the 5'-terminal and / or pre-5'-terminal nucleotide of a polynucleotide (as described above) on the 2'-hydroxy group of the sugar. Multiple distinct 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 may be used in connection with the present disclosure further include those described in International Patent Publications WO2008127688, WO2008016473, and WO2011015347, the contents of which are incorporated herein by reference in their entireties.

[0304] In various embodiments, the 5'-end cap can comprise a cap analog. Cap analogs, also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ in their chemical structure from the natural (i.e., endogenous, wild-type, or physiological) 5' cap while retaining cap function. Cap analogs can be synthesized chemically (i.e., non-enzymatically) or enzymatically and / or linked to a polynucleotide.

[0305] For example, the anti-reverse cap analog (ARCA) cap contains two guanosines linked by a 5'-5'-triphosphate group, with one guanosine containing an N7-methyl group as well as a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m 7The N7-methylated and 3'-O-methylated guanosine forms the 5'-terminal nucleotide 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). The 3'-O atom of the other, unmodified guanosine becomes linked to the 5'-terminal nucleotide of the capped polynucleotide (e.g., mRNA). The N7-methylated and 3'-O-methylated guanosine provide 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).

[0306] In one embodiment, the cap analog can be a dinucleotide cap analog. As a non-limiting example, the dinucleotide cap analog can be modified with boranophosphate or phosphoroselenoate groups at different phosphate positions, such as the dinucleotide cap analogs described in U.S. Patent No. 8,519,110, the contents of which are incorporated herein by reference in their entirety.

[0307] In one embodiment, the cap analog can be an 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 which are incorporated herein by reference). In another embodiment, a cap analog useful in connection with the nucleic acid molecules of the present disclosure is a 4-chloro / bromophenoxyethyl analog.

[0308] In various embodiments, the cap analog can comprise 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.

[0309] Without being bound by theory, it is contemplated that cap analogs allow for the simultaneous capping of polynucleotides, whereas in in vitro transcription reactions, up to 20% of transcripts remain uncapped. This, along with the structural differences of cap analogs from the native 5' cap structure of polynucleotides produced by the endogenous cellular transcription machinery, may lead to reduced translational competence and reduced cellular stability.

[0310] Thus, in one embodiment, nucleic acid molecules of the present disclosure can also be post-transcriptionally capped using enzymes to generate more authentic 5' cap structures. As used herein, the phrase "more authentic" refers to characteristics that closely reflect or mimic endogenous or wild-type characteristics, either structurally or functionally. That is, "more authentic" characteristics better represent endogenous, wild-type, natural, or physiological cellular functions and / or structures compared to prior art synthetic characteristics or analogs, or are superior in one or more respects to corresponding endogenous, wild-type, natural, or physiological characteristics. Non-limiting examples of more authentic 5' cap structures useful in connection with nucleic acid molecules of the present disclosure are those that exhibit, among other things, enhanced cap-binding protein binding, increased half-life, reduced susceptibility to 5' endonucleases, and / or reduced 5' decapping compared to synthetic 5' cap structures (or wild-type, natural, or physiological 5' cap structures) known in the art. For example, in one embodiment, a recombinant vaccinia virus capping enzyme and a recombinant 2'-O-methyltransferase enzyme can create a classical 5'-5'-triphosphate linkage between the 5'-terminal nucleotide of a polynucleotide and a guanosine cap nucleotide, where the cap guanosine contains an N7 methylation and the 5'-terminal nucleotide of the polynucleotide contains a 2'-O-methyl. Such a structure is referred to as a Cap 1 structure. This cap results in higher translational competence, cellular stability, and reduced activation of cellular pro-inflammatory cytokines, for example, compared to other 5'-cap analog structures known in the art. 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).

[0311] Without being bound by theory, it is contemplated that the nucleic acid molecules of the present disclosure may be post-transcriptionally capped, and that this process is more efficient such that nearly 100% of the nucleic acid molecules may be capped.

[0312] Untranslated Regions (UTRs) In one embodiment, a nucleic acid molecule of the present disclosure comprises one or more untranslated regions (UTRs). In one embodiment, a UTR is located upstream of a coding region in a nucleic acid molecule and is referred to as a 5'-UTR. In one embodiment, a UTR is located downstream of a coding region in a nucleic acid molecule and is referred to as a 3'-UTR. The sequence of a UTR can be homologous or heterologous to the sequence of the coding region found in the nucleic acid molecule. Multiple UTRs can be included in a nucleic acid molecule and can be of the same or different sequence and / or genetic origin. According to the present disclosure, any portion (including none) of a UTR in a nucleic acid molecule can be codon-optimized, any of which can independently contain one or more different structural or chemical modifications before and / or after codon optimization.

[0313] In one embodiment, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises a UTR and a coding region that are homologous to each other. In other embodiments, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises a UTR and a coding region that are heterologous to each other. In one embodiment, to monitor the activity of a UTR sequence, a nucleic acid molecule comprising a coding sequence for a UTR and a detectable probe can be administered in vitro (e.g., to a cell or tissue culture) or in vivo (e.g., to a subject), and the effect of the UTR sequence (e.g., modulation of expression levels, cellular localization of the encoded product, or half-life of the encoded product) can be measured using methods known in the art.

[0314] In one embodiment, the UTR of a nucleic acid molecule (e.g., mRNA) of the present disclosure contains at least one translational enhancer element (TEE), which functions to increase the amount of polypeptide or protein produced from the nucleic acid molecule. In one embodiment, the TEE is located in the 5'-UTR of the nucleic acid molecule. In another embodiment, the TEE is located in the 3'-UTR of the nucleic acid molecule. In yet another embodiment, at least two TEEs are located in each of the 5'-UTR and 3'-UTR of the nucleic acid molecule. In one embodiment, a nucleic acid molecule (e.g., mRNA) of the present disclosure may contain one or more copies of a TEE sequence, or may contain two or more different TEE sequences. In one embodiment, the different TEE sequences present in a nucleic acid molecule of the present disclosure can be homologous or heterologous to each other.

[0315] Various TEE sequences known in the art can be used in connection with the present disclosure. For example, in one embodiment, the TEE can be an internal ribosome entry site (IRES), an 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 may be used in connection with the present disclosure include, but are not limited to, those described in U.S. Pat. No. 7,468,275, U.S. Patent Publication Nos. 2007 / 0048776 and 2011 / 0124100, and International Patent Publication Nos. WO2007 / 025008 and WO2001 / 055369, the contents of each of which are incorporated by reference in their entirety. In one embodiment, the TEEs can be those 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 by reference in their entirety.

[0316] Additional exemplary TEEs that may be used in connection with the present disclosure include those disclosed in 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. 2009 / 0093049, U.S. Patent Publication No. 2011 / 012410 ... and TEE sequences disclosed in International Patent Publication Nos. WO2009 / 075886, WO2012 / 009644, and International Patent Publication Nos. WO1999 / 024595, WO2007 / 025008, WO2001 / 055371, EP2610341, and EP2610340, the contents of each of which are incorporated herein by reference in their entirety.

[0317] In various embodiments, the nucleic acid molecule (e.g., mRNA) of the present disclosure comprises at least one UTR comprising 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 sequences in the UTR of the nucleic acid molecule are copies of the same TEE sequence. In other embodiments, at least two TEE sequences in the UTR of the nucleic acid molecule are different TEE sequences. In one embodiment, multiple different TEE sequences are arranged in one or more repeating patterns in the UTR region of a nucleic acid molecule. For illustrative purposes only, the repeating pattern can be, for example, ABABAB, AABBAABBAABB, ABCABCABC, etc., where each capital letter (A, B, or C) represents a different TEE sequence. In one embodiment, at least two TEE sequences are contiguous with each other in the UTR of a nucleic acid molecule (i.e., there is no spacer sequence between them). In other embodiments, at least two TEE sequences are separated by a spacer sequence. In one embodiment, a UTR can include a TEE sequence-spacer sequence module that is repeated at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or more than nine times in the UTR. In any of the embodiments described in this paragraph, the UTR can be the 5'-UTR, the 3'-UTR, or both the 5'-UTR and the 3'-UTR of a nucleic acid molecule.

[0318] In one embodiment, the UTR of a nucleic acid molecule (e.g., mRNA) of the present disclosure contains at least one translational repression element that functions to reduce the amount of polypeptide or protein produced from the nucleic acid molecule. In one embodiment, the UTR of the nucleic acid molecule contains one or more miR sequences or fragments thereof (e.g., miR seed sequences) recognized by one or more microRNAs. In one embodiment, the UTR of the nucleic acid molecule contains one or more stem-loop structures that downregulate the translational activity of the nucleic acid molecule. Other mechanisms for repressing translational activity associated with a nucleic acid molecule are known in the art. In any of the embodiments described in this paragraph, the UTR can be the 5'-UTR, 3'-UTR, or both the 5'-UTR and 3'-UTR of the nucleic acid molecule.

[0319] Polyadenylation (polyA) region During natural RNA processing, long chains of adenosine nucleotides (polyA tracts) are typically added to messenger RNA (mRNA) molecules to increase the molecule's stability. Immediately after transcription, the 3' end of the transcript is cleaved, liberating a 3'-hydroxyl group. PolyA polymerase then adds chains of adenosine nucleotides to the RNA. This process, called polyadenylation, adds polyA tracts that are 100 to 250 residues long. Without being bound by theory, it is contemplated that polyA tracts may confer various advantages to the nucleic acid molecules of the present disclosure.

[0320] Thus, in one embodiment, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises a polyadenylation signal. In one embodiment, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises one or more polyadenylation (polyA) regions. In one embodiment, the polyA region consists entirely of adenine nucleotides or functional analogs thereof. In one embodiment, a nucleic acid molecule comprises at least one polyA region at its 3' end. In one embodiment, a nucleic acid molecule comprises at least one polyA region at its 5' end. In one embodiment, a nucleic acid molecule comprises at least one polyA region at its 5' end and at least one polyA region at its 3' end.

[0321] According to the present disclosure, the poly A region can have various lengths in different embodiments. In particular, in one embodiment, the poly A region of the nucleic acid molecule of this disclosure is at least 30 nucleotides in length. In one embodiment, the poly A region of the nucleic acid molecule of this disclosure is at least 35 nucleotides in length. In one embodiment, the poly A region of the nucleic acid molecule of this disclosure is at least 40 nucleotides in length. In one embodiment, the poly A region of the nucleic acid molecule of this disclosure is at least 45 nucleotides in length. In one embodiment, the poly A region of the nucleic acid molecule of this disclosure is at least 50 nucleotides in length. In one embodiment, the poly A region of the nucleic acid molecule of this disclosure is at least 55 nucleotides in length. In one embodiment, the poly A region of the nucleic acid molecule of this disclosure is at least 60 nucleotides in length. In one embodiment, the poly A region of the nucleic acid molecule of this disclosure is at least 65 nucleotides in length. In one embodiment, the poly A region of the nucleic acid molecule of this disclosure is at least 70 nucleotides in length. In one embodiment, the poly A region of the nucleic acid molecule of this disclosure is at least 75 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 80 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 85 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 90 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 95 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 100 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 110 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 120 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 130 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 140 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 150 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 160 nucleotides in length. In one embodiment, the polyA region of a nucleic acid molecule of the present disclosure is at least 170 nucleotides in length.In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 180 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 190 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 200 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 225 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 250 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 275 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 300 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 350 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 400 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 450 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 500 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 600 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 700 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 800 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 900 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 1000 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 1100 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 1200 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 1300 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 1400 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 1500 nucleotides in length. In one embodiment, the polyA region of a nucleic acid molecule of the present disclosure is at least 1600 nucleotides in length. In one embodiment, the polyA region of a nucleic acid molecule of the present disclosure is at least 1700 nucleotides in length.In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 1800 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 1900 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 2000 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 2250 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 2500 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 2750 nucleotides in length. In one embodiment, the poly A region of a nucleic acid molecule of this disclosure is at least 3000 nucleotides in length.

[0322] In one embodiment, the length of the polyA region in a nucleic acid molecule can be selected based on the total length of the nucleic acid molecule or a portion thereof (e.g., the length of the coding region or the length of the open reading frame of the nucleic acid molecule, etc.) For example, in one embodiment, the polyA 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 including the polyA region.

[0323] Without being bound by theory, it is contemplated that certain RNA-binding proteins may bind to the polyA region located at the 3' end of an mRNA molecule. These polyA-binding proteins (PABPs) can regulate mRNA expression, such as by interacting with the translation initiation machinery in cells and / or protecting the 3'-polyA tail from degradation. Thus, in one embodiment, a nucleic acid molecule (e.g., mRNA) of the present disclosure contains at least one binding site for a polyA-binding protein (PABP). In other embodiments, the nucleic acid molecule is conjugated or complexed with a PABP before being loaded into a delivery vehicle (e.g., lipid nanoparticle).

[0324] In one embodiment, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises a poly-AG quartet. A G-quartet is a cyclic hydrogen-bonded sequence of four guanosine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of a poly-A tract. The resulting polynucleotide (e.g., mRNA) can be assayed for stability, protein production, and other parameters, including half-life, at various time points. It has been discovered that a poly-AG quartet structure results in protein production equivalent to at least 75% of that seen using only a 120-nucleotide poly-A tract.

[0325] In one embodiment, the nucleic acid molecules (e.g., mRNA) of the present disclosure may contain a polyA region and may be stabilized by the addition of a 3' stabilization region. In one embodiment, a 3' stabilization region that can be used to stabilize a nucleic acid molecule (e.g., mRNA) comprises a polyA or polyAG quartet structure as described in International Patent Publication No. WO2013 / 103659, the contents of which are incorporated herein by reference in their entirety.

[0326] In other embodiments, 3' stabilizing regions that may be used in connection with nucleic acid molecules of the present disclosure include 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymine, etc. Chain-terminating nucleosides include, but are not limited to, 2',3'-dideoxynucleosides, 2'-deoxynucleosides, or O-methylnucleosides, 3'-deoxynucleosides, 2',3'-dideoxynucleosides, 3'-O-methylnucleosides, 3'-O-ethylnucleosides, 3'-arabinosides, and other alternative nucleosides known in the art and / or described herein.

[0327] secondary structure Without being bound by theory, it is contemplated that the stem-loop structure may guide RNA folding, protect the structural stability of nucleic acid molecules (e.g., mRNA), provide a recognition site for RNA-binding proteins, and serve as a substrate for enzymatic reactions. For example, the incorporation of miR and / or TEE sequences may alter 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).

[0328] Thus, in one embodiment, a nucleic acid molecule (e.g., mRNA) described herein, or a portion thereof, can adopt a stem-loop structure, such as, but not limited to, a histone stem-loop. In one embodiment, the stem-loop structure is formed from a stem-loop sequence that is about 25 or about 26 nucleotides in length, such as, but not limited to, those 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 those described in International Patent Publication Nos. WO2012 / 019780 and WO201502667, the contents of which are incorporated herein by reference. In one embodiment, the stem-loop sequence comprises a TEE described herein. In one embodiment, the stem-loop sequence comprises a miR sequence described herein. In a specific embodiment, the stem-loop sequence can comprise a miR-122 seed sequence. In a specific embodiment, the nucleic acid molecule comprises two stem-loop sequences described in International Patent Publication No. WO2021204175, the entirety of which is incorporated herein by reference.

[0329] In one embodiment, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises a stem-loop sequence located upstream (towards the 5' end) of a coding region in the nucleic acid molecule. In one embodiment, the stem-loop sequence is located within the 5'-UTR of the nucleic acid molecule. In one embodiment, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises a stem-loop sequence located downstream (towards the 3' end) of a coding region in the nucleic acid molecule. In one embodiment, the stem-loop sequence is located within the 3'-UTR of the nucleic acid molecule. In some cases, a nucleic acid molecule may contain more than one stem-loop sequence. In some embodiments, a nucleic acid molecule comprises at least one stem-loop sequence in the 5'-UTR and at least one stem-loop sequence in the 3'-UTR.

[0330] In one embodiment, the nucleic acid molecule comprising a stem-loop structure further comprises a stabilizing region. In some embodiments, the stabilizing region comprises at least one chain-terminating nucleoside, which functions to slow degradation and thus increase the half-life of the nucleic acid molecule. Exemplary chain-terminating nucleosides that may be used in connection with the present disclosure include 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymine. Nucleosides include, but are not limited to, 2',3'-dideoxynucleosides, 2'-deoxynucleosides, or O-methylnucleosides such as 3'-deoxynucleosides, 2',3'-dideoxynucleosides, 3'-O-methylnucleosides, 3'-O-ethylnucleosides, 3'-arabinosides, and other alternative nucleosides known in the art and / or described herein. In other embodiments, the stem-loop structure can be stabilized by modifications to the 3' region of the polynucleotide that can prevent and / or inhibit the addition of oligo(U) (International Patent Publication No. WO 2013 / 103659, incorporated herein by reference in its entirety).

[0331] In one embodiment, a nucleic acid molecule of the present disclosure comprises at least one stem-loop sequence and a polyA tract or polyadenylation signal. Non-limiting examples of polynucleotide sequences comprising at least one stem-loop sequence and a polyA tract or polyadenylation signal include those described in International Patent Publication Nos. WO2013 / 120497, WO2013 / 120629, WO2013 / 120500, WO2013 / 120627, WO2013 / 120498, WO2013 / 120626, WO2013 / 120499, and WO2013 / 120628, the contents of each of which are incorporated herein by reference in their entirety.

[0332] In one embodiment, the nucleic acid molecule comprising a stem-loop sequence and a polyA tract or polyadenylation signal can encode a pathogenic antigen or a fragment thereof, such as the polynucleotide sequences described in International Patent Publication Nos. WO2013 / 120499 and WO2013 / 120628 (the contents of each of which are incorporated by reference in their entirety).

[0333] In one embodiment, a nucleic acid molecule comprising a stem-loop sequence and a polyA tract or polyadenylation signal can encode a therapeutic protein, such as the polynucleotide sequences described in International Patent Publication Nos. WO2013 / 120497 and WO2013 / 120629, the contents of each of which are incorporated herein by reference in their entirety.

[0334] In one embodiment, the nucleic acid molecule comprising the stem-loop sequence and polyA tract or polyadenylation signal can encode a tumor antigen or a fragment thereof, such as the polynucleotide sequences described in International Patent Publication Nos. WO2013 / 120500 and WO2013 / 120627, the contents of each of which are incorporated herein by reference in their entirety.

[0335] In one embodiment, the nucleic acid molecule comprising the stem-loop sequence and polyA tract or polyadenylation signal can encode an allergic antigen or an autoimmune autoantigen, such as the polynucleotide sequences described in International Patent Publication Nos. WO2013 / 120498 and WO2013 / 120626, the contents of each of which are incorporated herein by reference in their entirety.

[0336] Functional Nucleotide Analogues In one embodiment, the payload nucleic acid molecules described herein contain only standard nucleotides selected from A (adenosine), G (guanosine), C (cytosine), U (urine), and T (thymidine). Without being bound by theory, it is contemplated that certain functional nucleotide analogs may confer useful properties to nucleic acid molecules. Examples of such useful properties in the context of the present disclosure include, but are not limited to, increased stability of the nucleic acid molecule, reduced immunogenicity of the nucleic acid molecule in inducing an innate immune response, enhanced production of the protein encoded by the nucleic acid molecule, increased intracellular delivery and / or retention of the nucleic acid molecule, and / or reduced cytotoxicity of the nucleic acid molecule.

[0337] Thus, in one embodiment, a payload nucleic acid molecule comprises at least one functional nucleotide analogue described herein. In one embodiment, the functional nucleotide analogue contains at least one chemical modification to the nucleobase, sugar group, and / or phosphate group. Thus, a payload nucleic acid molecule comprising at least one functional nucleotide analogue contains at least one chemical modification to the nucleobase, sugar group, and / or internucleoside linkage. Exemplary chemical modifications to the nucleobase, sugar group, or internucleoside linkage of a nucleic acid molecule are provided herein.

[0338] As described herein, a range of 0% to 100% of all nucleotides in a payload nucleic acid molecule can be a functional nucleotide analog 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%, or about 20% to about 70% of all nucleotides in a nucleic acid molecule. , about 20% to about 80%, about 20% to about 90%, about 20% to about 95%, about 20% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 90%, about 80% to about 95%, about 80% to about 100%, about 90% to about 95%, about 90% to about 100%, or about 95% to about 100% are functional nucleotide analogs as described herein. In any of these embodiments, the functional nucleotide analogue may be present at any position(s) of the nucleic acid molecule, including at the 5' end, the 3' end, and / or at one or more internal positions. In one embodiment, a single nucleic acid molecule may contain different sugar modifications, different nucleobase modifications, and / or different types of internucleoside linkages (e.g., backbone structures).

[0339] As described herein, a range of 0% to 100% of all nucleotides of the same type (e.g., all purine-containing nucleotides of one type, or all pyrimidine-containing nucleotides of one type, or all A, G, C, T, or U of one 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 20% to about 80%, about 20% to about 90%, about 20% to about 95%, about 20% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 90%, about 80% to about 95%, about 80% to about 100%, about 90% to about 95%, about 90% to about 100%, or about 95% to about 100% are functional nucleotide analogs as described herein. In any of these embodiments, the functional nucleotide analogue may be present at any position(s) of the nucleic acid molecule, including at the 5' end, the 3' end, and / or at one or more internal positions. In one embodiment, a single nucleic acid molecule may contain different sugar modifications, different nucleobase modifications, and / or different types of internucleoside linkages (e.g., backbone structures).

[0340] Modifications to nucleobases In one embodiment, functional nucleotide analogs contain non-standard nucleobases. In one embodiment, standard nucleobases in nucleotides (e.g., adenine, guanine, uracil, thymine, and cytosine) can be modified or substituted to provide one or more functional analogs of the nucleotide. Exemplary modifications to nucleobases include one or more substitutions or modifications, including, but not limited to, alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitutions, one or more condensation or ring opening, oxidation, and / or reduction.

[0341] In one embodiment, the non-standard nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouricine (ψ), pyridin-4-one ribonucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s 2 U), 4-thio-uracil (s 4 U), 4-thio-pseudouricine, 2-thio-pseudouricine, 5-hydroxy-uracil (ho 5 U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil), 3-methyl-uracil (m 3 U), 5-methoxy-uracil (mo 5 U), uracil 5-hydroxyacetic acid (cmo 5 U), uracil 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uracil (cm 5 U), 1-carboxymethyl-pseudouricine, 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-thio-uracil (nm 5 s 2U), 5-methylaminomethyl-uracil (mnm 5 U), 5-methylaminomethyl-2-thiouracil (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uracil (mnm 5 se 2 U), 5-carbamoylmethyl-uracil (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-taurinomethyl-uracil (τm 5 U), 1-taurinomethyl-pseudouricine, 5-taurinomethyl-2-thio-uracil (τm 5 5s 2 U), 1-taurinomethyl-4-thio-pseudouricine, 5-methyl-uracil (m 5 U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouricine (m 1 ψ), 1-ethyl-pseudouricine (Et 1 ψ), 5-methyl-2-thiouracil (m 5 s 2 U), 1-methyl-4-thio-pseudouricine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouricine, 3-methyl-pseudouricine (m 3 ψ), 2-thio-1-methyl-pseudouricin, 1-methyl-1-deaza-pseudouricin, 2-thio-1-methyl-1-deaza-pseudouricin, dihydrouracil (D), dihydropseudouricin, 5,6-dihydrouracil, 5-methyl-dihydrouracil (m 5 D), 2-thio-dihydrouracil, 2-thio-dihydropseudouricine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy-pseudouricine, 4-methoxy-2-thio-pseudouricine, N1-methyl-pseudouricine, 3-(3-amino-3-carboxypropyl)uracil (acp3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouricine (acp 3 ψ), 5-(isopentenylaminomethyl)uracil (m 5 U), 5-(isopentenylaminomethyl)-2-thiouracil (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-carbomethoxyvinyl)-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.

[0342] In one embodiment, the non-standard nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosines include 5-aza-cytosine, 6-aza-cytosine, pseudoisocytidine, 3-methyl-cytosine (m3C), N4-acetyl-cytosine (ac4C), 5-formyl-cytosine (f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C), 5-halo-cytosine (e.g., 5-iodo-cytosine ... -hydroxymethyl-cytosine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytosine, pyrrolo-pseudoisocytidine, 2-thio-cytosine (s2C), 2-thio-5-methyl-cytosine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine Idoisocytidine, 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- These include acetyl-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.

[0343] In one embodiment, the non-standard 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-adenine, 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-glycinyl N6-threonylcarbamoyl-adenine (g6A), N6-threonylcarbamoyl-adenine (t6A), 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 These include 1-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-pentaoxanonadecyl)-adenine, 2,8-dimethyl-adenine, N6-formyl-adenine, and N6-hydroxymethyl-adenine.

[0344] In one embodiment, the non-standard nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyobutosine (yW), peroxywyobutosine (o2yW), hydroxywyobutosine (OHyW), intermediate hydroxywyobutosine (OHyW*), 7-deaza-guanine, queosine (Q), Epoxyqueosin (oQ), galactosyl-queosin (galQ), mannosyl-queosin (manQ), 7-cyano-7-deaza-guanine (preQO), 7-aminomethyl-7-deaza-guanine (preQ1), archaeosin (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 These include 1-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.

[0345] In one embodiment, the non-standard nucleobase of a functional nucleotide analog can independently be a purine, a pyrimidine, a purine or pyrimidine analog. For example, in one embodiment, the non-standard nucleobase can be a modified adenine, cytosine, guanine, uracil, or hypoxanthine. In other embodiments, non-standard nucleobases can include, for example, naturally occurring and synthetic base derivatives, 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.

[0346] Sugar modifications In one embodiment, the functional nucleotide analog contains a non-standard sugar group. In various embodiments, the non-standard sugar group can be a five- or six-carbon sugar (e.g., pentose, ribose, arabinose, xylose, glucose, galactose, or deoxy derivatives thereof) with one or more substitutions such as halo, hydroxy, thiol, alkyl, alkoxy, alkenyloxy, alkynyloxy, cycloalkyl, aminoalkoxy, alkoxyalkoxy, hydroxyalkoxy, amino, azido, aryl, aminoalkyl, aminoalkenyl, or aminoalkynyl groups.

[0347] Generally, RNA molecules contain a ribose sugar group, which is a five-membered ring with oxygen. Exemplary, non-limiting alternative nucleotides include replacing oxygen in ribose (e.g., with S, Se, or an alkylene such as methylene or ethylene); adding a double bond (e.g., replacing ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., forming a four-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., adding an additional carbon atom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino (which also has a phosphoramidate backbone)). forming six- or seven-membered rings with carbon or heteroatoms; polycyclic forms (e.g., tricyclic and "unlocked" forms such as glycol nucleic acids (GNAs) (e.g., R-GNAs or S-GNAs, in which the ribose is replaced with a glycol unit linked to a phosphodiester bond), threose nucleic acids (TNAs, in which the ribose is replaced with α-L-threofuranosyl-(3'→2')), and peptide nucleic acids (PNAs, in which a 2-amino-ethyl-glycine linkage replaces the ribose and phosphodiester backbone).

[0348] In one embodiment, the sugar group contains one or more carbons that have the opposite stereochemical configuration of the corresponding carbon in ribose.Thus, nucleic acid molecules can contain nucleotides that contain, for example, arabinose or L-ribose as sugar.In one embodiment, nucleic acid molecules contain at least one nucleoside, where the sugar is L-ribose, 2'-O-methylribose, 2'-fluororibose, arabinose, hexitol, LNA, or PNA.

[0349] Modifications to internucleoside linkages In one embodiment, the payload nucleic acid molecules of the present disclosure may contain one or more modified internucleoside linkages (e.g., phosphate backbones). The backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with different substituents.

[0350] In one embodiment, functional nucleotide analogs can include replacing the unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of alternative phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. In phosphorodithioates, both non-linked oxygens are replaced with sulfur. The phosphate linker can also be modified by replacing the linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

[0351] Alternative nucleosides and nucleotides may include replacing one or more of the non-bridging oxygens with 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., the alpha (α), beta (β), or gamma (γ) positions) can be replaced with sulfur (thio) and methoxy. Replacement of one or more oxygen atoms at the phosphate moiety position (e.g., α-thiophosphate) provides stability (e.g., against exonucleases and endonucleases) to RNA and DNA via unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance, resulting in longer half-lives in the cellular environment.

[0352] 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.

[0353] Additional examples of nucleic acid molecules (e.g., mRNA), compositions, formulations, and / or related methods that may be used in connection with the present disclosure include WO2002 / 098443, WO2003 / 051401, WO2008 / 052770, WO2009127230, WO2006122828, WO2008 / 083949, WO2010088927, WO2010 / 037539, WO2004 / 004743, WO2 005 / 016376, WO2006 / 024518, WO2007 / 095976, WO2008 / 014979, WO2008 / 077592, WO2009 / 030481, WO2009 / 09522 6, WO2011069586, WO2011026641, WO2011 / 144358, WO2012019780, WO2012013326, WO2012089338, WO2012113513 , WO2012116811, WO2012116810, WO2013113502, WO2013113501, WO2013113736, WO2013143698, WO2013143699, W O2013143700, WO2013 / 120626, WO2013120627, WO2013120628, WO2013120629, WO2013174409, WO2014127917, WO 2015 / 024669, WO2015 / 024668, WO2015 / 024667, WO2015 / 024665, WO2015 / 024666, WO2015 / 024664, WO2015101415, WO2015101414, WO2015024667, WO2015062738, and WO2015101416, the contents of each of which are incorporated herein by reference in their entirety.

[0354] 6.3.2 Formulation The lipid nanoparticle compositions described herein can 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 described herein). Nanoparticle compositions can be designed for one or more specific applications or targets. The components of the nanoparticle composition can be selected based on the 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, a particular formulation of a nanoparticle composition can be selected for a particular application or target, for example, according to the efficacy and toxicity of a particular combination of components.

[0355] In one embodiment, the therapeutic and / or prophylactic agent encapsulated in the nanoparticles can be delivered to host cells in vitro, e.g., by contacting the host cells with the nanoparticle composition, or in vivo, e.g., by administering the nanoparticle composition to a subject containing the host cells. In one embodiment, the therapeutic nucleic acid molecule encapsulated in the nanoparticles, once delivered, can be expressed via the endogenous transcription and translation machinery of the host cells.

[0356] In one embodiment, the nanoparticle composition comprises a non-lipid component that comprises a therapeutic agent. In one embodiment, the therapeutic agent is a nucleic acid molecule. In one embodiment, the therapeutic agent is an mRNA molecule.

[0357] In one embodiment, upon delivery of the nucleic acid-containing nanoparticle composition to a host cell, the nucleic acid is expressed via the host cell's endogenous transcription and / or translation machinery to form RNA and / or protein, hi one embodiment, the expression level of the nucleic acid formulated in the LNP is increased compared to the nucleic acid formulated in a reference LNP composition.

[0358] Thus, the nanoparticle compositions described herein can be used in a method for expressing mRNA in a host cell or tissue of a host subject, the method comprising formulating mRNA in a nanoparticle composition and delivering the nanoparticle composition to a host cell or host subject, wherein the delivered mRNA is expressed in the host cell or host subject. In one embodiment, the host cell is a mammalian cell (e.g., a cell originating from a human or non-human vertebrate). In one embodiment, the host subject is a mammal (e.g., a human or non-human vertebrate). In one embodiment, delivery of the nanoparticle composition can be achieved by contacting the nanoparticle composition with a host cell in vitro. In one embodiment, delivery of the nanoparticle composition can be achieved by administering the nanoparticle composition to a host subject in vivo.

[0359] In some embodiments, the ratio of therapeutic agent to lipid in the lipid nanoparticle composition (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) ranges from 9:1 to 20:1, e.g., 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.

[0360] In one embodiment, provided herein are lipid nanoparticles for use in targeted delivery of therapeutic payloads. Nanoparticle compositions can be designed for one or more specific applications or targets. For example, nanoparticle compositions can be designed to deliver therapeutic and / or prophylactic agents, such as RNA, to specific cells, tissues, organs, or systems, or groups thereof, within a mammalian body. The physiochemical properties of the nanoparticle composition can be modified to increase selectivity for specific bodily targets. For example, particle size can be adjusted based on the fenestration size of different organs. The therapeutic and / or prophylactic agents included in the nanoparticle composition can also be selected based on the desired delivery target(s). For example, therapeutic and / or prophylactic agents can be selected for a particular indication, condition, disease, or disorder and / or for delivery (e.g., localized or specific delivery) to specific cells, tissues, organs, or systems, or groups thereof. In certain embodiments, the nanoparticle composition can contain mRNA encoding a polypeptide of interest, which can be translated intracellularly to produce the polypeptide of interest. Such compositions can be designed for specific delivery to a specific organ. In one embodiment, the composition can be designed for specific delivery to the mammalian liver. In one embodiment, the composition can be designed for specific delivery to the lungs of a mammal, hi one embodiment, the composition can be designed for specific delivery to a site of interest, such as a site of inflammation, a site of cancer, or a site of infection.

[0361] The amount of therapeutic and / or prophylactic agent in a nanoparticle composition can depend on the size, composition, desired target and / or use, or other properties of the nanoparticle composition, as well as the nature of the therapeutic and / or prophylactic agent. For example, the amount of RNA useful in a nanoparticle composition can depend on the size, sequence, and other characteristics of the RNA. The relative amounts of therapeutic and / or prophylactic agent and other components (e.g., lipids) in the nanoparticle composition can also vary. In one embodiment, the weight / weight ratio of lipid component to therapeutic and / or prophylactic agent in the nanoparticle composition can be from about 5:1 to about 60:1, e.g., 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 weight / weight ratio of lipid component to therapeutic and / or prophylactic agent can be from about 10:1 to about 40:1. In certain embodiments, the weight / weight ratio is about 20:1. The amount of therapeutic and / or prophylactic agent in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., UV-visible spectroscopy).

[0362] 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 particular N:P ratio.

[0363] Nanoparticle compositions can be characterized by various 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. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to determine particle size. Additionally, instruments such as the Zetasizer Nano ZS (Malvem Instruments Ltd, Malvem, Worcestershire, UK) can be used to measure several properties of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

[0364] In various embodiments, the average size of the nanoparticle composition can be tens of nanometers to hundreds of nanometers. For example, the average size can be about 40 nm to about 150 nm, e.g., about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In one embodiment, the lipid nanoparticle composition comprises a plurality of nanoparticles, and the average size of the plurality of nanoparticles is about 40 nm to about 150 nm. In one embodiment, the average size of the plurality of particles is about 50 nm to about 100 nm. In one embodiment, the average size of the plurality of particles is about 95 nm.

[0365] In one embodiment, the average size of a three-lipid component lipid nanoparticle composition provided herein (containing no phospholipid component or a reduced / insignificant amount of phospholipid component) is increased compared to the average size of a conventional four-lipid component lipid nanoparticle composition, e.g., a four-lipid component lipid nanoparticle having a molar ratio of cationic lipid:steroid:phospholipid:polymer-conjugated lipid of about 50:38.5:10:1.5. In one embodiment, the average size of a three-lipid component nanoparticle composition provided herein is increased by 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 a three-lipid component nanoparticle composition provided herein is increased by no more than about 90%, no more than about 100%, or no more than about 110%. In one embodiment, the average size of a three-lipid component nanoparticle composition provided herein is between about 90 nm and about 150 nm. In one embodiment, the average size (of a three-lipid component nanoparticle composition provided herein) is between about 100 nm and about 140 nm. In one embodiment, the average size is about 110 nm to about 130 nm. In one embodiment, the average size is about 100 nm to about 110 nm. In one embodiment, the average size is about 110 nm to about 120 nm. In one embodiment, the average size is about 120 nm to about 130 nm. In one embodiment, the average size is about 90 nm. In one embodiment, the average size is about 95 nm. In one embodiment, the average size is about 100 nm. In one embodiment, the average size is about 105 nm. In one embodiment, the average size is about 110 nm. In one embodiment, the average size is about 115 nm. In one embodiment, the average size is about 120 nm. In one embodiment, the average size is about 125 nm. In one embodiment, the average size is about 130 nm. In one embodiment, the average size is about 135 nm. In one embodiment, the average size is about 140 nm. In one embodiment, the average size is about 145 nm. In one embodiment, the average size is about 150 nm, hi one embodiment, the average size is about 155 nm.

[0366] The nanoparticle composition can be relatively homogeneous. The polydispersity index can be used to indicate the homogeneity of the nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25, e.g., 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 from about 0 to about 0.25. In one embodiment, the PDI of the nanoparticle composition is less than 0.1.

[0367] The zeta potential of a nanoparticle composition can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can describe the surface charge of the nanoparticle composition. Nanoparticle compositions with a relatively low positive or negative charge are generally desirable because more highly charged species can undesirably interact with cells, tissues, and other elements 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.

[0368] The encapsulation efficiency of a therapeutic and / or prophylactic agent describes the amount of therapeutic and / or prophylactic agent encapsulated by or otherwise associated with a nanoparticle composition after preparation, compared to the initial amount provided. A high encapsulation efficiency is desirable (e.g., approaching 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or prophylactic agent in a solution containing the nanoparticle composition before and after dissolving the nanoparticle composition with one or more organic solvents or surfactants. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) in solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a therapeutic and / or prophylactic agent 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%.

[0369] Nanoparticle composition can optionally comprise one or more coatings.For example, nanoparticle composition can be formulated into capsules, films or tablets with coatings.The capsules, films or tablets comprising the compositions described herein can have any useful size, tensile strength, hardness or density.

[0370] 6.3.3 Pharmaceutical Compositions According to the present disclosure, the nanoparticle composition can be formulated in whole or in part as a pharmaceutical composition. A pharmaceutical composition can include one or more nanoparticle compositions. For example, a pharmaceutical composition can include one or more nanoparticle compositions containing one or more different therapeutic and / or prophylactic agents. A pharmaceutical composition can further include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and medicaments can be found, for example, in Remington's The Science and Practice of Pharmacy, 21 stEdition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and auxiliary ingredients can be used in any pharmaceutical composition, except insofar as any of the conventional excipients or auxiliary ingredients may be incompatible with one or more of the components of the nanoparticle composition. An excipient or auxiliary ingredient may be incompatible with a component of the nanoparticle composition if its combination with the component of the nanoparticle composition could result in any undesirable biological or other adverse effect.

[0371] In one embodiment, one or more excipients or auxiliary ingredients can comprise more than 50% of the total mass or volume of a pharmaceutical composition comprising a nanoparticle composition. For example, one or more excipients or auxiliary ingredients can comprise 50%, 60%, 70%, 80%, 90%, or more of pharmaceutical practice. In one embodiment, a 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 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 Pharmacopoeias.

[0372] 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 invention will vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered. By way of example, a pharmaceutical composition may contain from 0.1% to 100% (w / w) of one or more nanoparticle compositions.

[0373] In certain embodiments, the nanoparticle compositions and / or pharmaceutical compositions of the present disclosure are refrigerated or frozen for storage and / or shipping (e.g., stored at a temperature of 4°C or below, e.g., about -150°C to about 0°C or about -80°C to 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). In certain embodiments, The present disclosure also relates to methods of maintaining or increasing the stability of a lipid nanoparticle composition and / or pharmaceutical composition by storing the nanoparticle composition and / or pharmaceutical composition at a temperature of 4°C or below, e.g., from about -150°C to about 0°C or from about -80°C to about -20°C, e.g., from 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, e.g., at temperatures below 4°C (e.g., about 4°C to -20°C), for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 1 month, at least about 2 months, at least about 4 months, at least about 6 months, at least about 8 months, at least about 10 months, at least about 12 months, at least about 14 months, at least about 16 months, at least about 18 months, at least about 20 months, at least about 22 months, or at least about 24 months. In one embodiment, the formulation is stabilized at about 4°C for at least 4 weeks. In certain embodiments, the pharmaceutical compositions of the present disclosure comprise a nanoparticle composition disclosed herein and one or more pharmaceutically acceptable carriers selected from Tris, acetate (e.g., sodium acetate), citrate (e.g., sodium acetate), saline, PBS, and sucrose. In certain embodiments, the pharmaceutical compositions of the present disclosure have a pH value of about 7 to 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 7.5 to 8 or 7 to 7.8).For example, a pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein, Tris, saline, and sucrose, and has a pH of about 7.5 to 8, suitable for storage and / or shipping at, for example, about −20° C. For example, a pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein and PBS, and has a pH of about 7 to 7.8, suitable for storage and / or shipping at, for example, about 4° C. or below. “Stability,” “stabilized,” and “stable,” in the context of the present disclosure, refer to the resistance of a nanoparticle composition and / or pharmaceutical composition disclosed herein to chemical or physical changes (e.g., degradation, particle size change, aggregation, change in encapsulation, etc.) under given conditions of manufacture, preparation, transportation, storage, and / or use, for example, when subjected to stresses such as shear forces, freeze / thaw stress, etc.

[0374] Nanoparticle compositions and / or pharmaceutical compositions comprising one or more nanoparticle compositions can be administered to any patient or subject, including those who can benefit from a therapeutic effect provided by delivery of a therapeutic and / or prophylactic agent to one or more specific cells, tissues, organs, or systems or groups thereof, such as the renal system. While the description provided herein of nanoparticle compositions and pharmaceutical compositions comprising nanoparticle compositions is primarily directed to compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to any other mammal. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to various animals are well understood, and veterinary pharmacologists of ordinary skill can design and / or perform such modifications with no more than routine experimentation, if any. Subjects to which the compositions are intended for administration include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cows, pigs, horses, sheep, cats, dogs, mice, and / or rats.

[0375] Pharmaceutical compositions containing one or more nanoparticle compositions can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparative methods involve bringing into association the active ingredient with an excipient and / or one or more other accessory ingredients, and then, as desired or necessary, dividing, shaping, and / or packaging the product into desired single- or multi-dosage units.

[0376] Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient (e.g., a nanoparticle composition). The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject and / or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

[0377] Pharmaceutical compositions can be prepared in 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.

[0378] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, 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, tetrahydrofuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters, and mixtures thereof. In addition to inert diluents, oral compositions may contain additional agents, such as additional therapeutic and / or prophylactic agents, wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, and / or fragrances. In certain embodiments for parenteral administration, the composition is mixed with a solubilizing agent, such as Cremophor™, alcohol, oil, modified oil, glycol, polysorbate, cyclodextrin, polymer, and / or combinations thereof.

[0379] Injectable preparations, for example, aqueous or oily sterile injectable suspensions, can be formulated 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 non-toxic parenterally acceptable diluents or solvents, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution (USP), and isotonic sodium chloride solution. Sterile fixed oils are commonly used as solvents or suspending media. For this purpose, any non-irritating fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid are used in the preparation of injectables.

[0380] Injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0381] The present disclosure features methods for delivering therapeutic and / or prophylactic agents to mammalian cells or organs, methods for producing a polypeptide of interest in mammalian cells, and methods for treating a disease or disorder in a mammal in need thereof, the methods comprising administering to the mammal a nanoparticle composition comprising a therapeutic and / or prophylactic agent and / or contacting mammalian cells with a nanoparticle composition comprising a therapeutic and / or prophylactic agent. [Example]

[0382] 7. Working Example The examples in this section are offered by way of illustration and not by way of limitation.

[0383] Example 1: Preparation of lipid nanoparticle formulations LNP formulations were prepared by mixing lipid and mRNA stock solutions at a 1:3 flow rate through a T-connector with a total flow rate of 12 mL / min. Lipid stock solutions were prepared in ethanol at a total lipid concentration of 12.5 mM. mRNA stock solutions were prepared in 20 mM pH 4.0 citrate buffer at a final concentration of 0.125 mg / mL. The size and distribution of all formulations were determined by dynamic light scattering (DLS). The encapsulation efficiency (EE%) of each formulation was determined by Ribogreen assay according to the kit supplier's instructions. The concentration of the formulations was determined by measuring UV absorbance at 260 nm using HPLC and comparing it to a linear standard concentration curve.

[0384] Example 2: Physical properties of lipid nanoparticle formulations Lipid nanoparticle compositions containing mRNA and variable lipid compositions are listed below in Tables 1-4 and 9. Physical characterization of LNP compositions is listed below in Tables 5-8 and 10.

[0385] The structures and names of the cationic lipids tested are listed below. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10-1] [Table 10-2] [Table 11-1] [Table 11-2]

[0386] Cryo-EM images of LNPs composed of C1 / cholesterol / DMG-PEG (60 / 38.5 / 1.5%) and C1 / cholesterol / DMG-PEG (65 / 33 / 2.0%) are provided in Figures 9A and 9B, respectively. Three-component LNP samples were concentrated 20-fold (volume change) by centrifugation using an Amicon® Ultra-4. Samples (3 μl) were deposited onto glow-discharged (Quantifoil R1.2 / 1.3) holey carbon grids and separately vitrified using a Vitrobot Mark IV (Thermo Fisher Scientific). Cryo-EM imaging was performed on a Talos F200C equipped with a Ceta 4k × 4k camera operated at an accelerating voltage of 200 kV. The results show that C1-based three-component LNPs exhibit a uniform morphology of solid spheres lacking an aqueous core, distinct from multilayered solid nanoparticles or conventional liposomes.

[0387] Example 3: In vivo protein expression. Lipid nanoparticles with different compositions (listed in Tables 1–4) encapsulating human erythropoietin (hEPO) mRNA were administered systemically to 6–8-week-old female ICR mice via tail vein injection at a dose of 0.5 mg / kg weekly, and blood samples were collected at specific time points (e.g., 6 h) after administration. Three to five mice per formulation were included in each administration. After the final sampling time, mice were euthanized by CO2 overdose. Serum was separated from whole blood by centrifugation at 5,000 g for 10 min at 4°C, flash-frozen, and stored at -80°C for analysis. To assess hEPO expression, an ELISA assay was performed using a commercially available kit (DEP00, R&D Systems) according to the manufacturer's instructions.

[0388] GFP-encoding mRNA was encapsulated in lipid-5-containing LNPs with different lipid compositions. LNPs were administered systemically to 8-10 week-old female Balb / c mice via intramuscular injection at a dose of 0.1 mg / kg. Muscles surrounding the injection site were collected 6 hours after intramuscular administration, followed by cell lysis with cell lysis buffer. Total protein from the samples was detected using the Pierce BCA Protein Assay Kit. Intramuscular GFP and IL-6 were determined by ELISA assay using the GFP SimpleStem ELISA Kit and Mouse IL-6 ELISA Kit, respectively.

[0389] Figure 1 shows normalized hEPO expression levels upon in vivo administration of three-lipid LNPs containing lipid-5. The lipid-5 formulation group also exhibits superior in vivo hEPO expression. Conventional four-lipid LNPs (L-0) were used as the reference group to normalize hEPO expression levels. In the lipid-5 formulation group, 25 different LNP formulations were found to have higher in vivo hEPO expression levels than the conventional formulation (L-0). Among these formulations, the amount of cationic lipid (lipid-5) ranged from about 40 mol% to about 70 mol%, and the amount of PEG lipid ranged from about 0.5 mol% to about 3.0 mol%. Furthermore, seven of these formulations were observed to increase in vivo gene expression levels by more than two-fold compared to the L-0 control. The data also show that three-lipid LNP formulations containing less than 40 mol% or more than 70 mol% lipid-5 and more than 3 mol% PEG lipid have relatively low in vivo activity.

[0390] Figure 2 shows the normalized hEPO expression levels upon in vivo administration of three lipid LNPs containing SM-102. The SM-102 formulation group also demonstrates a clear advantage in terms of in vivo hEPO expression levels compared to the reference formulation (S-0). In the SM-102 formulation group, 20 of the tested LNP formulations were found to have higher in vivo hEPO expression levels than the conventional formulation (S-0). Among these formulations, the amount of cationic lipid (SM-102) ranged from approximately 40 mol% to approximately 70 mol%, and the amount of PEG lipid ranged from approximately 0.5 mol% to approximately 3 mol%. Ten of these formulations were also observed to increase in vivo gene expression levels by more than twofold compared to the S-0 control. Furthermore, one of the formulations demonstrated a more than three-fold increase in in vivo hEPO expression levels compared to the reference formulation (S-0). The data also show that three-lipid LNP formulations containing amounts of SM-102 below 40 mol% or above 70 mol% and PEG lipids above 3 mol% have relatively low in vivo activity.

[0391] Based on these results, LNPs containing cationic lipids in amounts between about 40 mol% and about 70 mol% and PEG-lipids in amounts less than 3 mol% appear to be the general range in which the in vivo activity of three-lipid LNP formulations is superior to that of conventional four-lipid LNP formulations.

[0392] Figure 3 shows the normalized hEPO expression levels upon in vivo administration of three-lipid LNPs containing the cationic lipids ALC-0315 or C1. In the ALC-0315 group, 10 different LNP compositions were prepared and characterized (Tables 3 and 7). All 10 LNP formulations containing ALC-0315 were found to have higher in vivo hEPO expression levels than the conventional formulation (A-0). Five of these formulations were also observed to increase in vivo gene expression levels by more than two-fold compared to the A-0 control. Additionally, six LNP formulations containing C1 were prepared and characterized (Tables 4 and 8). These LNP formulations exhibited higher in vivo activity than the conventional formulation (C1-0).

[0393] In U.S. Patent No. 11,191,849, it was mentioned that increasing the PEG-lipid ratio to 3.3% in PL-free (phospholipid-free) formulations slightly affected LNP efficiency in terms of ApoB silencing activity. However, the data presented herein show a very different picture in terms of mRNA delivery when using three-lipid LNPs containing more than 3 mol% PEG lipids. Figure 4 shows the normalized hEPO expression levels when three-lipid LNPs containing more than 3 mol% PEG lipids are administered in vivo. All formulations containing more than 3 mol% PEG lipids showed significantly lower in vivo hEPO expression levels 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 those of conventional formulations.

[0394] Table 11 and Figure 8 show the normalized hEPO expression levels when three-lipid LNPs containing C2 were administered in vivo. The C2-containing three-lipid LNPs demonstrated favorable in vivo hEPO expression activity. Seventeen of the tested LNP formulations were found to have higher hEPO expression levels than the conventional formulation (C2-0). The cationic lipid content in the formulations ranged from about 40% to about 55%, while the PEG lipid content ranged from about 1.0% to about 2.5%. [Table 12]

[0395] Example 4: Fragment analysis Fragment analysis (FA) was performed to test the long-term stability of the LNP formulations at different temperatures, as shown in Figures 5A, 5B, and 5C. mRNA was isolated from the LNPs using the Rneasy MiNi kit (50). Fragment analysis was performed on an Agilant 5280 analyzer.

[0396] LNPs were able to protect RNA from degradation by ubiquitous RNases. Three LNPs containing lipid-5 encapsulating GFP-encoding mRNA were used for fragment analysis. These formulations were stored at room temperature (RT), 2-8°C, or -20°C for 4 months. At room temperature, mRNA purity decreased over time in all three formulations. No clear difference was observed between the conventional LNP formulation and the three-lipid LNP formulation. When these LNPs were stored at 2-8°C or -20°C, there was no significant decrease in RNA purity in all three formulations tested, even after 4 months of storage. These results indicate that the three-lipid LNP formulations have similar or equivalent properties in protecting the encapsulated RNA from degradation.

[0397] Figures 5D and 5E show the updated long-term stability of lipid-5-containing LNPs stored for up to 400 days at 2-8 °C or -20 °C, respectively. Over a period of one year under both storage conditions, no clear decline in mRNA purity was observed in lipid-5-containing LNPs.

[0398] Example 5: Intramuscular Administration We investigated intramuscular administration of two lipid 5-containing LNPs (L-50 / 1.5 and L-60 / 1.5) by comparing GFP expression levels with those of the conventional formulation (L-0). As shown in Figures 6A and 6B, there was a roughly two-fold increase in GFP expression levels in the three-lipid LNP (L-50 / 1.5 and L-60 / 1.5) intramuscularly administered group compared with the conventional formulation (L-0). The data indicate that the three-lipid LNP formulation has a significant advantage in gene expression, whether administered intramuscularly or intravenously. The cytokine interleukin-6 (IL-6) was detected to assess the effect of LNP administration on immune stimulation. When IL-6 production was normalized to GFP expression, it was found that the three-lipid LNP formulations induced relatively less IL-6 production in vivo at the same GFP expression level, indicating that the three-lipid LNP formulations (L-50 / 1.5 and L-60 / 1.5) have relatively fewer side effects than the conventional four-lipid LNP formulation (L-0).

[0399] Example 6: Weight Change After administration, some side effects, such as weight change, may be induced in mice due to immune stimulation by LNPs. Different LNP compositions containing lipid-5 were used to study the level and duration of side effects after intravenous or intramuscular administration. All LNP formulations encapsulated mRNA encoding luciferase. Following LNP administration, the weight changes of mice were tracked over a 7-day course (Figures 7A and 7B). After intravenous administration, the three-lipid LNP formulation group showed lower maximum weight loss and more rapid weight recovery. Mice intravenously injected with the three-lipid LNP formulations (L-50 / 1.5 and L-60 / 1.5) regained 100% of their body weight within two days, whereas the group intravenously injected with the conventional four-lipid LNP formulation (L-0) took more than four days to recover. The L-50 / 1.5 group showed equally rapid weight gain, indicating minimal side effects after intravenous administration of LNPs.

[0400] Intramuscular administration resulted in a rapid loss of more than 5% of body weight in all three groups within one day of administration, indicating that intramuscular administration has more severe side effects than intravenous administration in this system. Similarly, mice treated with the three-lipid LNP formulations (L-50 / 1.5 and L-60 / 1.5) took less time to regain weight after intramuscular administration than mice treated with the conventional four-lipid LNP formulation (L-0). The L-50 / 1.5 and L-60 / 1.5 groups took 7 days to fully regain their original weight, while the weight of the L-0 group only changed slightly. Mice in the L-50 / 1.5 group showed faster weight gain than the other two groups, indicating that this formulation induced relatively few side effects.

Claims

1. Lipid nanoparticles, (a) Cationic lipids in an amount of about 40 mol% to about 75 mol% of the total lipids present in the nanoparticles, (i) at least one tertiary amine or quaternary ammonium moiety, and (ii) at least one C １４ ~C ４２ The cationic lipid containing the alkyl ester portion, (b) A steroid in an amount of about 22 mol% to about 59.5 mol% of the total lipids present in the nanoparticles, (c) A polymer conjugate lipid in an amount of about 0.5 mol% to about 2.5 mol% of the total lipids present in the nanoparticles, (d) Nucleic acids and, Lipid nanoparticles, provided that the lipid nanoparticles contain 0.5 mole percent or less of phospholipids.

2. The amount of the cationic lipid is approximately 55 mol% to approximately 65 mol% of the total lipids present in the nanoparticles, or approximately 60 mol% of the total lipids present in the nanoparticles; or The amount of the polymer conjugate lipid is about 1 mol% to about 2 mol% of the total lipids present in the nanoparticles, or about 1.5 mol% of the total lipids present in the nanoparticles; or The amount of the steroid is approximately 33 mol% to approximately 44 mol% of the total lipids present in the nanoparticles; or The lipid nanoparticles according to claim 1, wherein the lipid nanoparticles do not contain phospholipids.

3. (1) The amount of cationic lipid is about 45 mol% to about 70 mol% of the total lipids present in the nanoparticles, and the amount of polymer conjugate lipid is about 1 mol% to about 2.5 mol% of the total lipids present in the nanoparticles; (2) The amount of cationic lipid is about 45 mol% to about 65 mol% of the total lipids present in the nanoparticles, and the amount of polymer conjugate lipid is about 1 mol% to about 2.5 mol% of the total lipids present in the nanoparticles; (3) The amount of cationic lipid is about 45 mol% to about 65 mol% of the total lipids present in the nanoparticles, and the amount of polymer conjugate lipid is about 1 mol% to about 2 mol% of the total lipids present in the nanoparticles; (4) The amount of cationic lipid is about 50 mol% to about 65 mol% of the total lipids present in the nanoparticles, and the amount of polymer conjugate lipid is about 0.5 mol% to about 2.5 mol% of the total lipids present in the nanoparticles; (5) The amount of cationic lipid is about 50 mol% to about 60 mol% of the total lipids present in the nanoparticles, and the amount of polymer conjugate lipid is about 0.8 mol% to about 2 mol% of the total lipids present in the nanoparticles; (6) The amount of cationic lipid is about 50 mol% to about 60 mol% of the total lipids present in the nanoparticles, and the amount of polymer conjugate lipid is about 1 mol% to about 2 mol% of the total lipids present in the nanoparticles; (7) The amount of cationic lipid is about 55 mol% to about 65 mol% of the total lipids present in the nanoparticles, and the amount of polymer conjugate lipid is about 1 mol% to about 2.5 mol% of the total lipids present in the nanoparticles; (8) The amount of cationic lipid is about 60 mol% to about 65 mol% of the total lipids present in the nanoparticles, and the amount of polymer conjugate lipid is about 1 mol% to about 2 mol% of the total lipids present in the nanoparticles; (9) The amount of cationic lipid is about 60 mol% to about 65 mol% of the total lipids present in the nanoparticles, and the amount of polymer conjugate lipid is about 1.5 mol% to about 2.5 mol% of the total lipids present in the nanoparticles; or (10) The lipid nanoparticle according to claim 1, wherein the amount of cationic lipid is about 40 mol% to about 55 mol% of the total lipids present in the nanoparticle, and the amount of polymer conjugate lipid is about 1 mol% to about 2.5 mol% of the total lipids present in the nanoparticle.

4. The polymer conjugate lipid is a polyethylene glycol (PEG) conjugate lipid, or The polymer conjugate lipid is PEG-diacylglycerol (PEG-DAG) conjugate, PEG-dialkyloxypropyl (PEG-DAA) conjugate, PEG-dimyristyloxypropyl (PEG-DMA) conjugate, PEG-distearyloxypropyl (PEG-DSA) conjugate, or a mixture thereof. The polymer conjugate lipid is DMG-PEG2000 or DMPE-PEG2000; or The steroid is a glucocorticoid, mineralocorticoid, clobetasol, cholesterol, or a cholesterol derivative; or The cationic lipid is 【Chemistry 1】 The lipid nanoparticle according to claim 1, comprising the portion wherein G 1 is a C2-C12 alkylene, X 1 is -C(=O)O- or -OC(=O)-, and R 1 is a branched C14-C42 alkyl.

5. The cationic lipid is defined by formula (I): 【Chemistry 2】 It is a substance, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, in which, G １ However, C ２ ~C １２ It is alkylene, X １ However, it is -C(=O)O- or -OC(=O)-, R １ is a branched C １４ to C ４２ alkyl, and R ｙ However, C １ ~C ６ Alkyl or -G ２ -X ２ -R ２ And, G ２ However, C ２ ~C １２ It is alkylene, X ２ However, it is -C(=O)O- or -OC(=O)-, R ２ However, linear or branched C ６ ~C ４２ It is alkyl, R ｘ However, C １ ~C ６ Alkyl or -G ３ -R ３ And, G ３ However, C ２ ~C １２ It is alkylene, R ３ However, -N(R ４ ) R ５ OR ６ And, R ４ However, C １ ~C １２ Alkyl, C ３ ~C ８ Cycloalkyl, C ３ ~C ８ Cycloalkenyl, or C ６ ~C １０ It is Ariel, R ５ However, C １ ~C １２ Alkyl, C ３ ~C ８ Cycloalkyl, C ３ ~C ８ Cycloalkenyl, or C ６ ~C １０ Is it an allele? Or R ４ and R ５ However, together with the nitrogen atoms bonded to them, they form a ring portion. R ６ However, hydrogen, C １ ~C １２ Alkyl, C ３ ~C ８ Cycloalkyl, C ３ ~C ８ Cycloalkenyl, or C ６ ~C １０ It is Ariel, Here, each alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, alkylene, and cyclic moiety is independently substituted by any choice; or The cationic lipid is defined by formula (II): 【Transformation 3】 Lipid nanoparticles according to claim 1, which are a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

6. The cationic lipid is of formula (II-A): 【Chemistry 4】 It is a substance, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, in which, G １ However, C ５ ~C ７ It is alkylene, R １ However, branch C １４ ~C １９ It is alkyl, G ２ However, C ５ ~C ７ It is alkylene, R ２ However, linear C ７ ~C １３ It is alkyl, G ３ However, C ２ ~C ４ It is alkylene; or The cationic lipid is defined by formula (II-A-1): 【Transformation 5】 It is a substance, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof; or The aforementioned cationic lipid is given by formula (II-B): 【Transformation 6】 It is a substance, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, in which, G1 is C5-C7 alkylene, R1 is a branched C14-C19 alkyl group. G2 is C5-C7 alkylene, R2 is a branched C14-C19 alkyl group. G3 is C2-C4 alkylene; or The cationic lipid is defined by formula (II-B-1): 【Transformation 7】 Lipid nanoparticles according to claim 5, which are a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

7. The cationic lipid is defined by formula (II-C): 【Transformation 8】 It is a substance, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, in which, G １ However, C ５ ~C ７ It is alkylene, R １ However, branch C １４ ~C １９ It is alkyl, G ２ However, C ５ ~C ７ It is alkylene, R ２ is a branched C １４ to C １９ alkyl, and G ３ However, C ２ ~C ４ It is alkylene, R ４ However, C ３ ~C ８ It is a cycloalkyl, R ５ is C substituted with hydroxy ２ ~C ４ alkyl; or, The cationic lipid is of formula (II-C-1): 【Chemistry 9】 The lipid nanoparticles described in claim 5, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof; or the lipid nanoparticles described in claim 5.

8. The cationic lipid is 【Chemistry 10】 The lipid nanoparticles according to claim 5.

9. The cationic lipid is of formula (III): 【Chemistry 11】 It is a substance, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, in which, G １ However, C ２ ~C １２ It is alkylene, X １ However, it is -C(=O)O- or -OC(=O)-, R １ However, branch C １４ ~C ４２ It is alkyl, G ２ However, C ２ ~C １２ It is alkylene, Each L ２ -X ２ -R ２ And, Each X ２ These are independently -C(=O)O- or -OC(=O)-, Each R ２ These can be independent, linear or branched C ６ ~C ４２ It is alkyl, m is 1 or 2, G ３ However, C ２ ~C １２ It is alkylene, R ４ However, C １ ~C １２ Alkyl, C ３ ~C ８ Cycloalkyl, C ３ ~C ８ Cycloalkenyl, or C ６ ~C １０ It is Ariel, R ５ However, C １ ~C １２ Alkyl, C ３ ~C ８ Cycloalkyl, C ３ ~C ８ Cycloalkenyl, or C ６ ~C １０ It is Ariel, Here, each alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, alkylene, and cyclic moiety is independently substituted by any choice; or The cationic lipid is of formula (IV): 【Chemistry 12】 It is a substance, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, in which, y1 is an integer between 0 and 9. Lipid nanoparticles according to claim 1.

10. The cationic lipid is of formula (IV-A): 【Chemistry 13】 It is a substance, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, in which, G １ However, C ５ ~C ７ It is alkylene, R １ However, branch C １４ ~C １９ It is alkyl, Each R ２ Independently, linear C ７ ~C １３ It is alkyl, G ３ However, C ２ ~C ４ It is alkylene, R ４ However, C ３ ~C ８ It is a cycloalkyl, R ５ However, C substituted with hydroxyl ２ ~C ４ It is alkyl, y1 is an integer between 2 and 6; or The cationic lipid is of formula (IV-A-1): 【Chemistry 14】 Lipid nanoparticles according to claim 9, which are a pharmaceutically acceptable salt thereof, a prodrug, or a stereoisomer thereof.

11. The cationic lipid is 【Chemistry 15】 The lipid nanoparticles according to claim 9.

12. The lipid nanoparticles according to claim 1, wherein the lipid nanoparticles have a lipid:nucleic acid mass ratio of about 9:1 to about 20:

1.

13. The lipid nanoparticle according to claim 1, wherein the nucleic acid is mRNA or small interfering RNA (siRNA).

14. The mRNA is mRNA that encodes an antigen, a fragment thereof, or an epitope; The mRNA is monocistronic mRNA or multicistronic mRNA; or The lipid nanoparticle according to claim 13, wherein the mRNA is an mRNA encoding an antigen, a fragment thereof, or an epitope, and the antigen is a pathogenic antigen or a tumor-associated antigen.

15. The lipid nanoparticles according to claim 1, wherein the nucleic acid is chemically modified.

16. The size of the nanoparticles is about 100 nm to about 140 nm; The lipid nanoparticles have an encapsulation efficiency of at least about 80% of the nucleic acids; The size of the nanoparticles increases compared to the reference formulation; or The nucleic acid encodes RNA or protein, and the amount of RNA or protein expressed from the nucleic acid in the nanoparticles in mammalian cells or mammalian tissue is greater than the amount of RNA or protein expressed from the nucleic acid in the reference formulation. The lipid nanoparticles according to claim 1, wherein the reference formulation differs from the lipid nanoparticles in that (i) the reference formulation contains a phospholipid, and (ii) the molar ratio of the cationic lipid:steroid:phospholipid:polymer conjugate lipid is about 50:38.5:10:1.

5.

17. The size of the aforementioned nanoparticles is approximately 110 nm to approximately 130 nm; The lipid nanoparticles have an encapsulation efficiency of at least about 90% of the nucleic acids; The size of the nanoparticles is increased by at least about 50% compared to the reference formulation; The amount of RNA or protein expressed from the nucleic acid in the nanoparticles is at least about 50% greater than the amount of RNA or protein expressed from the nucleic acid in the reference formulation; or The lipid nanoparticle according to claim 16, wherein the amount of RNA or protein expressed from the nucleic acid in the nanoparticle is at least about 100% greater than the amount of RNA or protein expressed from the nucleic acid in the reference formulation.

18. A pharmaceutical composition comprising lipid nanoparticles as described in claim 1 and a pharmaceutically acceptable excipient.

Images