Lipid and lipid nanoparticle formulations for drug delivery

Abstract

The present invention relates to lipids and compositions thereof.SOLUTION: In various embodiments of the invention, the compositions are lipid nanoparticle compositions that are used to deliver a variety of nucleic acid molecules and / or therapeutics to a selected target, such as cells for gene delivery, and / or to prevent or treat a disease or disorder in a subject in need thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 923,258, filed October 18, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under DP2 TR002776 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] Background of the Invention Naked mRNA rapidly degrades and cannot easily cross cell membranes, necessitating a delivery method for functional uptake by T cells. Currently, electroporation (EP) is clinically used to effectively deliver mRNA to various cells, including T cells (Smits E et al., 2004, Leukemia, 18:1898-1902; Barrett DM et al., 2011, Hum Gene Ther, 22:1575-1586; DiTommaso T et al., 2018, PNAS, 115). However, it has several drawbacks. Membrane disruption during EP carries the risk of loss of cellular contents and cytotoxicity, while consistent membrane penetration throughout the cell for uniform delivery cannot be guaranteed. This can result in decreased viability and altered behavior of the surviving cell population (DiTommaso T et al., 2018, PNAS, 115; Dullaers M et al., 2004, Mol Ther, 10:768-779; Singh N et al., 2014, Cancer Immunol Res, 2:1059-1070). Therefore, further studies of the long-term expression and intracellular behavior of transgenes after electroporation are needed to understand the potential risks of this nucleic acid delivery method (Lambricht L et al., 2016, Expert Opin Drug Deliv, 13: 295-310; Nickoloff JA et al., 1995, Animal Cell Electroporation and Electrofusion Protocols Methods in Molecular Biology, 273-280).

[0004] In summary, T cells are obviously difficult to transfect. Therefore, the most commonly utilized method for delivering mRNA to T cells is EP. EP uses an electric pulse to open pores in the cell membrane, allowing anything in solution (in this case, mRNA) along with the cell to enter the cytosol. While EP is effective at transporting mRNA into cells, it is prone to toxicity to T cells, can cause changes in genome expression, and does not allow for in vivo translation. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there is a need in the art for improved compositions and methods for delivering sequences and / or drugs to cells or subjects in need thereof. The present invention fulfills this unmet need. [Means for solving the problem]

[0006] (Brief summary of the invention) In one embodiment, this invention pertains, in part, to a compound having the structure of Formula (I) or a salt thereof: [ka]

[0007] In some embodiments, A1 and A2 are independently C, C(H), N, S, or P. In some embodiments, each L1, L2, L3, L4, L5, and L6 is independently C, C(H), C(H)(R 19 ), O, N(H), or N(R 19 In some embodiments, each R, R, R 3a , R 3b , R 4a , R 4b , R 5a , R 5b , R 6a , R 6b , R 7a , R 7b , R8a , R 8b , R 9a , R 9b , R 10a , R 10b , R 11a , R 11b , R 12a , R 12b , R 13a , R 13b , R 14a , R 14b , R 15a , R 15b , R 16 , R 17 , R 18 , and R 19 are independently H, halogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, -Y(R 20 ) z' (R 21 ) Z" -cycloalkyl, substituted -Y(R 20 ) z' (R 21 ) Z" -cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, -Y(R 20 ) z' (R 21 ) Z" -heterocycloalkyl, substituted -(R 20 ) z' (R 21 ) Z" -heterocycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, -Y(R 20 ) z' (R 21 ) Z" -cycloalkenyl, substituted -Y(R 20 ) z' (R 21 ) Z" -cycloalkenyl, alkynyl, substituted alkynyl, cycloalkynyl, substituted cycloalkynyl, -Y(R 20 ) z' (R 21 ) Z" -cycloalkynyl, substituted -Y(R 20 ) z' (R 21 ) Z"-cycloalkynyl, aryl, substituted aryl, -Y(R 20 ) z' (R 21 ) Z" -aryl, substituted -Y(R 20 ) z' (R 21 ) Z" -aryl, heteroaryl, substituted heteroaryl, -Y(R 20 ) z' (R 21 ) Z" -heteroaryl, substituted -Y(R 20 ) z' (R 21 ) Z" -heteroaryl, alkoxycarbonyl, linear alkoxycarbonyl, branched alkoxycarbonyl, amido, amino, aminoalkyl, aminoalkenyl, aminoalkynyl, aminoaryl, aminoacetate, acyl, hydroxyl, hydroxyalkyl, hydroxyalkenyl, hydroxyalkynyl, hydroxyaryl, alkoxy, carboxyl, carboxylate, ester, -Y(R 20 ) z' (R 21 ) Z" -ester, -Y(R 20 ) z' (R 21 ) Z" , =O, -NO2, -CN, or sulfoxy.

[0008] In some embodiments, Y is C, N, O, S, or P. In some embodiments, each R 20 and R 21is independently H, halogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, cycloalkynyl, substituted cycloalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxycarbonyl, linear alkoxycarbonyl, branched alkoxycarbonyl, amido, amino, aminoalkyl, aminoalkenyl, aminoalkynyl, aminoaryl, aminoacetate, acyl, hydroxyl, hydroxyalkyl, hydroxyalkenyl, hydroxyalkynyl, hydroxyaryl, alkoxy, carboxyl, carboxylate, ester, =0, -NO, -CN, or sulfoxy.

[0009] In some embodiments, each Z' and Z" is independently an integer represented by 0, 1, or 2. In some embodiments, each m, n, o, p, q, r, s, t, u, v, w, and x is independently an integer represented by 0, 1, 2, 3, 4, or 5.

[0010] In some embodiments, the compound having the structure of Formula (I) is a compound having the following structure: [ka] [ka] [ka] [ka] [ka] [ka]

[0011] In some embodiments, each R, R, R, R, and R is independently H, halogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, cycloalkynyl, substituted cycloalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxycarbonyl, linear alkoxycarbonyl, branched alkoxycarbonyl, amido, amino, aminoalkyl, aminoalkenyl, aminoalkynyl, aminoaryl, aminoacetate, acyl, hydroxyl, hydroxyalkyl, hydroxyalkenyl, hydroxyalkynyl, hydroxyaryl, alkoxy, carboxyl, carboxylate, or ester.

[0012] In some embodiments, each m, n, o, p, and q is independently an integer from 0 to 25. In some embodiments, each r, s, t, u, v, w, and x is independently an integer represented by 0, 1, 2; 3, 4, or 5.

[0013] In some embodiments, the compound having the structure of Formula (I) is a compound having the following structure: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0014] In various embodiments, the compound having the structure of Formula (I) is an ionizable lipid.

[0015] In another aspect, the present invention relates, in part, to lipid nanoparticles (LNPs) comprising one or more compounds of the invention. In various embodiments, the LNPs comprise one or more compounds of the invention in a concentration range of about 1 mol% to about 100 mol%. In some embodiments, the LNPs comprise one or more compounds of the invention in a concentration range of about 10 mol% to about 50 mol%.

[0016] In some embodiments, the LNPs further comprise at least one helper lipid. In some embodiments, the LNPs comprise at least one helper lipid in a concentration range of about 0.01 mol% to about 99.9 mol%. In some embodiments, the LNPs comprise at least one helper lipid in a concentration range of about 0.5 mol% to about 50 mol%.

[0017] In some embodiments, the helper lipid is a phospholipid, a cholesterol lipid, a polymer, or any combination thereof.

[0018] In some embodiments, the phospholipid is dioleoylphosphatidylethanolamine (DOPE) or a derivative thereof, distearoylphosphatidylcholine (DSPC) or a derivative thereof, distearoylphosphatidylethanolamine (DSPE) or a derivative thereof, stearoyloleoylphosphatidylcholine (SOPC) or a derivative thereof, 1-stearioyl-2-oleoyl-phosphatidylethanolamine (SOPE) or a derivative thereof, N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) or a derivative thereof, or any combination thereof. In some embodiments, the LNP comprises phospholipids in a concentration range of about 15 mol% to about 50 mol%.

[0019] In some embodiments, the cholesterol lipid is cholesterol or a derivative thereof, hi some embodiments, the LNP comprises cholesterol lipid in a concentration range of about 20 mol % to about 50 mol %.

[0020] In some embodiments, the polymer is polyethylene glycol (PEG) or a derivative thereof. In some embodiments, the LNP comprises the polymer in a concentration range of about 0.5 mol % to about 10 mol %.

[0021] In some embodiments, the LNP comprises at least one nucleic acid molecule, a therapeutic agent, or any combination thereof. In one embodiment, the nucleic acid molecule is a therapeutic agent.

[0022] In some embodiments, the nucleic acid molecule is a DNA molecule or an RNA molecule. In some embodiments, the nucleic acid molecule is a cDNA, mRNA, miRNA, siRNA, modified RNA, antagomir, antisense molecule, peptide, therapeutic peptide, targeting nucleic acid, or any combination thereof.

[0023] In one embodiment, the mRNA encodes luciferase.

[0024] In another embodiment, the mRNA encodes one or more antigens. In some embodiments, the antigen comprises at least one viral antigen, bacterial antigen, fungal antigen, parasitic antigen, influenza antigen, tumor-associated antigen, tumor-specific antigen, or any combination thereof.

[0025] In some embodiments, the nucleic acid molecule comprises a promoter or regulatory sequence.

[0026] In one embodiment, the LNP further comprises an adjuvant.

[0027] In some embodiments, the nucleic acid molecule, therapeutic agent, or combination thereof is encapsulated within a compound of the invention.

[0028] In one embodiment, this invention pertains, in part, to a composition comprising at least one compound having the structure of Formula (I), at least one LNP of the invention, or any combination thereof. In one embodiment, the composition is a vaccine.

[0029] In another aspect, the present invention relates, in part, to a method for delivering a nucleic acid molecule, a therapeutic agent, or a combination thereof to a subject in need thereof. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of one or more LNPs or compositions of the present invention. In some embodiments, the LNPs or compositions deliver the nucleic acid molecule, the therapeutic agent, or a combination thereof to a target.

[0030] In some embodiments, the target is an immune cell, a T cell, a resident T cell, a B cell, a natural killer (NK) cell, a cancerous cell, a cell associated with a disease or disorder, a tissue associated with a disease or disorder, brain tissue, central nervous system tissue, lung tissue, apical surface tissue, an epithelial cell, an endothelial cell, liver tissue, intestinal tissue, colon tissue, small intestine tissue, large intestine tissue, feces, bone marrow, a macrophage, spleen tissue, muscle tissue, joint tissue, a tumor cell, diseased tissue, lymph node tissue, lymphatic circulation, or any combination thereof.

[0031] In some embodiments, the LNP or composition is administered by an intradermal delivery route, a subcutaneous delivery route, an intramuscular delivery route, an intraventricular delivery route, an intrathecal delivery route, an oral delivery route, an intravenous delivery route, an intratracheal delivery route, an intraperitoneal delivery route, an intrauterine delivery route, or any combination thereof.

[0032] In one embodiment, the method comprises a single administration of the LNPs or composition. In some embodiments, the method comprises multiple administrations of the LNPs or composition.

[0033] In various embodiments, the method treats or prevents at least one viral infection, bacterial infection, fungal infection, parasitic infection, influenza infection, cancer, arthritis, heart disease, cardiovascular disease, neurological disorder or disease, genetic disease, autoimmune disease, fetal disease, genetic disease affecting fetal development, or any combination thereof.

[0034] In another aspect, the invention relates, in part, to a method of preventing or treating a disease or disorder in a subject in need thereof, hi one embodiment, the method comprises administering to the subject a therapeutically effective amount of one or more LNPs or compositions of the invention.

[0035] In some embodiments, the LNP or composition delivers a nucleic acid molecule, a therapeutic agent, or a combination thereof to a cell.

[0036] In yet another aspect, the invention relates, in part, to a method for delivering a nucleic acid molecule to a cell, hi one embodiment, the method comprises administering to the cell a therapeutically effective amount of one or more LNPs or compositions of the invention.

[0037] In one embodiment, the method is a gene delivery method. The following detailed description of the preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments presently preferred of the invention. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief explanation of the drawings]

[0038] [Figure 1]Figure 1, including Figure 1A and Figure 1B, shows a schematic of the LNP formulation. Figure 1A shows a schematic of the components used to generate LNPs by microfluidic mixing and the expected structure of the resulting LNPs. Figure 1B shows the size (z-average) distribution of a representative sample of C14-4 (also known as C14-494) LNPs, shown to be approximately 70 nm in diameter using dynamic light scattering. Error bars represent the standard deviation of three samples. [Figure 2] Figure 2, including Figures 2A-2C, shows representative epoxide-terminated alkyl chains and representative polyamine cores used to generate the library of lipids screened in this study. The lipids were created by Michael addition chemistry. The invention described herein is named C14-4 (also referred to as C14-494) in this figure. Figure 2A shows representative structures of lipid tails used to generate the ionizable lipid library. Figure 2B shows representative structures of amine cores used to generate the ionizable lipid library. Figure 2C shows a schematic of the Michael addition chemistry used to react excess lipid tails with the amine core to synthesize ionizable lipids. [Figure 3]Figure 3, including Figures 3A-3E, shows representative luciferase expression under various conditions. Results are normalized to untreated cells with background subtracted. For Figures 3B, 3D, and 3E, n=3. Figure 3A shows representative luciferase expression in Jurkat cells after 48 hours of treatment with the LNP library and lipofectamine at a dose of 30 ng / 60,000 cells, demonstrating that LNP was the best performing. Results were normalized to untreated cells with background subtracted. *=p<0.05 by paired Student's T-test vs. lipofectamine, n=4. Figure 3B shows representative luciferase expression in Jurkat cells treated with the five top-performing LNP formulations to determine the best LNP formulation. Results were normalized to untreated cells with background subtracted. *=p<0.05 in Tukey's multiple comparison test between C14-4 (also known as C14-494) and each of the other formulations. Figure 3C shows a table listing the representative diameter (z-average), polydispersity index, and mRNA concentration (±standard deviation) of the five top-performing LNP formulations. Figure 3D shows luciferase expression over time in Jurkat cells treated with 30 ng / 60,000 cells of C14-4 (also known as C14-494) for 24 hours, confirming transient expression of the protein. Results were normalized to expression at 24 hours and background subtracted. Figure 3E shows representative viability of Jurkat cells treated with 30 ng mRNA / 60,000 cells for 48 hours using Lipofectamine or C14-4 (also called C14-494), demonstrating minimal toxicity associated with C14-4 (also called C14-494) LNPs. [Figure 4]Figure 4, including Figures 4A-4C, shows representative luciferase expression under various conditions. For Figures 4A and 4C, luciferase expression was normalized to the lowest treatment (75 ng / 60,000 cells), and viability was normalized to no treatment with background subtraction. n=3. Figure 4A shows representative luciferase expression and viability of primary T cells treated with crude C14-4 (also known as C14-494) LNPs for 24 hours. Figure 4B shows representative results of a TNS assay to determine the LNP pKa for crude and purified C14-4 (also known as C14-494) LNPs encapsulating luciferase mRNA. The pKa was calculated as the pH corresponding to half of the maximum TNS fluorescence value. Figure 4C shows representative luciferase expression and viability of primary T cells treated with crude or purified C14-4 (also known as C14-494), demonstrating increased luciferase expression without increased toxicity. *=p<0.05 by paired Student's T-test. [Figure 5] Figure 5 shows representative named structures of the amine cores used to generate the ionizable lipid library. [Figure 6] Figure 6 shows the representative diameter (z-average), PDI, and mRNA concentration for each LNP formulation, demonstrating the narrow range of LNP size, monodispersity, and similar mRNA loading across LNP formulations. [Figure 7] Figure 7 shows a representative comparison of the properties of crude and purified C14-4 (also called C14-494) LNPs encapsulating luciferase mRNA. Mean n=3 and ± standard deviation. [Figure 8]Figure 8, comprising Figures 8A and 8B, shows representative Library A formulations with different molar ratios that were screened, along with data demonstrating that ionizable lipids (e.g., C14-494) remain fully ionizable when incorporated into LNPs. In this study, the "S2" formulation was set as the standard C14-494 formulation of the excipient. Figure 8A shows representative manufacturing parameters for a representative Library A formulation, expressed in terms of molar ratios. Figure 8B shows representative pKa ratios for Library A formulations. The pKa values ​​for Library A using the TNS assay indicated that all were still fully ionizable. [Figure 9] Figure 9 shows representative manufacturing parameters for representative Library A formulations with different molar ratios. The selection of these representative formulations was chosen based on the results of Library A screening. [Figure 10] Figure 10 shows representative normalized delivery efficiencies for both libraries, with values ​​greater than 1 (dashed line) indicating improved delivery efficiencies over the positive control. [Figure 11] Figure 11 shows representative normalized cell viability for Library A formulations. The dashed line indicates 100% viability. [Figure 12] Figure 12 shows representative normalized cell viability for Library B formulations. The dashed line indicates 100% viability. [Figure 13] Figure 13, comprising Figures 13A and 13B, shows representative mRNA delivery and viability excipient compositions: in vitro library. Jurkats were treated with 30 ng / 60,0000 cells for 24 hours. Library A is shown in orange for comparison, and Library B is shown in blue. Figure 13A shows a representative mRNA delivery excipient composition: in vitro library. Figure 13B shows a representative viability delivery excipient composition: in vitro library. [Figure 14] Figure 14 shows representative results showing the relative luciferase activity for B10 and Lipofectamine. [Figure 15]Figure 15, including Figures 15A and 15B, shows representative luminescence and viability results for various representative formulations at various concentrations / doses. Jurkats were treated with luciferase-encoding mRNA for 24 hours (the "S2" formulation was set as the standard C14-494 formulation of the vehicle). *Normalized to 0 ng for luminescence and toxicity—values ​​graphed in Figures 15A and 15B for all treatment groups are normalized to the untreated group. More specifically, the luminescence and toxicity readings for each treatment group were a measure of luminescence. The raw value (luminescence) for each treatment was divided by the raw value (luminescence) measured in the untreated cell group. Thus, the graphed values ​​represent delivery or toxicity relative to untreated cells. This allowed for the elimination of background luminescence, which varied between experiments. Furthermore, this experiment was completed at three separate times with three separate Jurkat cell populations / passages (three biological replicates), and for each experiment, cells were plated in three wells (three technical replicates). This ensures that the results are reproducible (biological replicates) and reliable (technical replicates). Figure 15A shows representative luminescence results for various representative formulations at various concentrations. Figure 15B shows representative viability results for various representative formulations at various concentrations. [Figure 16] Figure 16, comprising Figures 16A-16C, shows representative excipient compositions: ex vivo for Patient A, Patient B, and Patient C. Figure 16A shows representative excipient compositions: ex vivo for Patient A. Figure 16B shows representative excipient compositions: ex vivo for Patient B. Figure 16C shows representative excipient compositions: ex vivo for Patient C. DETAILED DESCRIPTION OF THE INVENTION

[0039] Detailed Description The present invention relates to lipids and lipid nanoparticles (LNPs) and compositions thereof. In some embodiments, the composition comprises at least one lipid of the present invention and at least one helper lipid. In certain embodiments, the present invention provides compositions comprising at least one lipid or LNP for delivering various nucleic acid molecules and / or therapeutic agents to cells. Thus, in various embodiments, the present invention relates to methods of gene delivery using compositions comprising at least one lipid or LNP. In certain embodiments, the present invention provides compositions comprising at least one lipid or LNP for preventing or treating various diseases or disorders in subjects in need thereof.

[0040] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0041] As used herein, each of the following terms has the meaning associated with it in this section.

[0042] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0043] As used herein, "about" when referring to a measurable value such as an amount, duration, etc. is meant to encompass a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are appropriate for carrying out the disclosed methods.

[0044] "Alkyl" refers to a straight or branched hydrocarbon chain radical, consisting solely of carbon and hydrogen atoms, which may be saturated or unsaturated (i.e., containing one or more double and / or triple bonds) and which has from 1 to 24 carbon atoms (C1-C 24 alkyl), 1 to 12 carbon atoms (C1-C 12 alkyl), 1 to 8 carbon atoms (C1-C8 alkyl), or 1 to 6 carbon atoms (C1-C6 alkyl), which is attached to the remainder of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless otherwise stated, alkyl groups are optionally substituted. The term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain hydrocarbon having the specified number of carbon atoms (i.e., C 1-6 means 1 to 6 carbon atoms), including straight chain, branched chain, or cyclic substituents.

[0045] The term "substituted alkyl," as used herein, refers to alkyl substituted with one, two, or three substituents selected from the group consisting of halogen, -OH, alkoxy, -NH, -N(CH), -C(=O)OH, trifluoromethyl, -C≡N, -C(=O)O(C-C)alkyl, -C(=O)NH, -SONH, -C(=NH)NH, and -NO, as defined above; preferably, one or two substituents selected from halogen, -OH, alkoxy, -NH, trifluoromethyl, -N(CH), and -C(=O)OH; more preferably, one or two substituents selected from halogen, alkoxy, and -OH. Examples of substituted alkyl include, but are not limited to, 2,2-difluoropropyl, 2-carboxycyclopentyl, and 3-chloropropyl.

[0046] "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain connecting the rest of the molecule to a radical group consisting only of carbon and hydrogen, which radical group may be saturated or unsaturated (i.e., contain one or more double bonds (alkenylene) and / or triple bonds (alkynylene)), and may contain, for example, 1 to 24 carbon atoms (C1-C 24 Alkylene, 1 to 15 carbon atoms (C1-C 15 Alkylene, 1 to 12 carbon atoms (C1-C 12 The alkylene chain may have 1 to 8 carbon atoms (C-C alkylene), 1 to 6 carbon atoms (C-C alkylene), 2 to 4 carbon atoms (C-C alkylene), or 1 to 2 carbon atoms (C-C alkylene), for example, methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. The alkylene chain is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double 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 stated otherwise in the specification, an alkylene chain can be optionally substituted.

[0047] "Cycloalkyl" or "carbocyclic ring" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical, consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, and which is saturated or unsaturated and attached to the remainder of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethylbicyclo[2.2.1]heptanyl, and the like. Unless otherwise specified, cycloalkyl groups are optionally substituted.

[0048] A "cycloalkylene" is a divalent cycloalkyl group. Unless stated otherwise in the specification, a cycloalkylene group can be optionally substituted.

[0049] The term "heteroalkyl," as used herein, by itself or in combination with another term, means, unless otherwise stated, a stable linear or branched alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, Si, P, and S, where the nitrogen and sulfur atoms can be optionally oxidized and the nitrogen heteroatom can be optionally quaternized. The heteroatom can be located at any position of the heteroalkyl group, including between the remainder of the heteroalkyl group and the fragment to which it is attached, and at the most distal carbon atom of the heteroalkyl group. Examples include -O-CH-CH-CH, -CH-CH-CH-OH, -CH-CH-NH-CH, -CH-S-CH-CH, and -CHCH-S(=O)-CH. Up to two heteroatoms can be consecutive, for example, -CH-NH-OCH or -CH-CH-SS-CH.

[0050] "Heterocyclyl" or "heterocycle" refers to a stable 3- to 18-membered non-aromatic ring radical, which consists of 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless stated otherwise in the specification, the heterocyclyl radical can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon, or sulfur atoms in the heterocyclyl radical can be optionally oxidized; and the nitrogen atom can be optionally quaternized; and the heterocyclyl radical can be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless otherwise specified, heterocyclyl groups may be optionally substituted.

[0051] The term "aromatic" as used herein refers to a carbocyclic or heterocyclic ring having one or more polyunsaturated rings and having aromatic character, i.e., having (4n+2) delocalized π electrons, where n is an integer.

[0052] As used herein, the term "aryl," used alone or in combination with other terms, means, unless otherwise specified, a carbocyclic aromatic system containing one or more rings (typically one, two, or three rings), where such rings may be pendant (e.g., biphenyl) or fused (e.g., naphthalene). Examples include phenyl, anthracyl, and naphthyl. Preferred are phenyl and naphthyl, and most preferred is phenyl.

[0053] As used herein, the term "heteroaryl" or "heteroaromatic" refers to an aryl group containing at least one heteroatom selected from N, O, Si, P, and S; wherein the nitrogen and sulfur atoms can be optionally oxidized and the nitrogen atom can be optionally quaternized. Heteroaryl groups can be substituted or unsubstituted. Heteroaryl groups can be attached to the remainder of the molecule through a heteroatom. Polycyclic heteroaryls can contain one or more rings that are partially saturated. Examples include tetrahydroquinoline, 2,3-dihydrobenzofuryl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4 ... Examples of quinolyl include thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.

[0054] Examples of non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazoline, pyrazolidine, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane, 4,7-dihydro-1,3-dioxepine, and hexamethylene oxide.

[0055] Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl (especially 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (especially 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (especially 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl.

[0056] Examples of polycyclic heterocycles include indolyl (especially 3-, 4-, 5-, 6-, and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (especially 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (especially 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (especially 3-, 4- , 5-, 6-, and 7-benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (especially 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (especially 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl (especially 2-benzimidazolyl), benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolidinyl, and quinolizidinyl.

[0057] The foregoing lists of heterocyclyl and heteroaryl moieties are intended to be representative and not limiting.

[0058] As used herein, the term "aminoaryl" refers to an aryl moiety that contains an amino moiety. Such amino moieties may include, but are not limited to, primary amines, secondary amines, tertiary amines, masked amines, or protected amines. Such tertiary amines, masked amines, or protected amines may be converted to primary amine or secondary amine moieties. Furthermore, the amine moiety may include amine-like moieties that have similar chemical properties to the amine moiety, including, but not limited to, chemical reactivity.

[0059] As used herein, the terms "alkoxy," "alkylamino," and "alkylthio" are used in their ordinary sense to refer to alkyl groups linked to the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.

[0060] As used herein, the term "alkoxy," used alone or in combination with other terms, unless otherwise specified, refers to an alkyl group having the specified number of carbon atoms as defined above, connected to the remainder of the molecule via an oxygen atom, such as methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy), and higher homologs and isomers. (C1-C3)alkoxy, particularly ethoxy and methoxy, are preferred.

[0061] The terms “halo” or “halogen,” as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably fluorine, chlorine, or bromine, and more preferably fluorine or chlorine.

[0062] As used herein, the term "substituted" means that at least one hydrogen atom has been replaced with an atom that is not hydrogen, such as, but not limited to: a halogen atom, e.g., F, Cl, Br, I; an oxo group (=O); a hydroxyl group (-OH); an alkoxy group (-OR a , where R a is C1-C 12 alkyl or cycloalkyl); carboxyl group (-OC(=O)Ra or -C(=O)OR a , where R a is H, C1-C 12 alkyl, or cycloalkyl); an amine group (—NR a R b , where R a and R b are independently H, C1-C 12 alkyl, or cycloalkyl); C1-C 12 and any of the above groups (e.g., alkyl, cycloalkyl, or heterocyclyl) substituted by a bond to a cycloalkyl group. In some embodiments, the substituent is C-C 12 In 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. In another embodiment, the substituent is a carboxyl group. In another embodiment, the substituent is an amine group.

[0063] The term "nanoparticle" as used herein refers to particles having a particle size on the nanometer scale, less than 1 micrometer. For example, nanoparticles may have a particle size of up to about 50 nm. In another example, nanoparticles may have a particle size of up to about 10 nm. In another example, nanoparticles may have a particle size of up to about 6 nm. As used herein, "nanoparticle" refers to many nanoparticles, including, but not limited to, nanoclusters, nanovesicles, micelles, lamellar-shaped particles, polymersomes, dendrimers, and other nano-sized particles of various other small products known to those of skill in the art. The shape and composition of nanoparticles can be induced during atomic condensation by selectively promoting the growth of specific crystal planes to produce spheres, rods, wires, disks, cages, core-shell structures, and many other shapes. The definition and understanding of entities within the scope of nanocapsules are known to those of skill in the art, and such definitions are incorporated herein by reference and for purposes of understanding the general nature of the subject matter of this application.

[0064] As used herein, "nucleic acid" refers to any nucleic acid, regardless of whether it is composed of deoxyribonucleosides or ribonucleosides, and whether it is composed of phosphodiester bonds or modified bonds such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate, or sulfone bonds, and combinations of such bonds. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil). The term "nucleic acid" generally refers to large polynucleotides.

[0065] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or it can exist in a non-native environment, such as a host cell.

[0066] An "isolated nucleic acid" refers to a nucleic acid segment or fragment separated from sequences that flank it in its naturally occurring state, i.e., a DNA fragment removed from the sequences that normally flank the fragment, i.e., the sequences that flank the fragment in the genome in which it naturally occurs. The term also applies to nucleic acids that have been substantially purified from other components that naturally accompany the nucleic acid, i.e., RNA or DNA, or protein, in a cell. Thus, the term includes recombinant DNA or RNA that is incorporated into, for example, a vector, an autonomously replicating plasmid or virus, or into genomic DNA or RNA of a prokaryote or eukaryote, or exists as a separate molecule independent of other sequences (i.e., as a cDNA or genomic or cDNA fragment generated by PCR or restriction enzyme digestion). It also includes recombinant DNA or RNA that is part of a hybrid gene encoding additional polypeptide sequences.

[0067] The "coding region" of an mRNA molecule also consists of nucleotide residues of the mRNA molecule that coincide with the anticodon region of a transfer RNA molecule during translation of the mRNA molecule, or that encode a stop codon. Thus, a coding region can include nucleotide residues that include codons for amino acid residues that are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).

[0068] The term "DNA" as used herein is defined as deoxyribonucleic acid.

[0069] The term "RNA" as used herein is defined as ribonucleic acid.

[0070] "Encoding" refers to the inherent property of a particular sequence of nucleotides within a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties that result therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually set forth in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0071] An "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or liposomally contained) RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating a recombinant polynucleotide.

[0072] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If a position in both of two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percentage of homology between two sequences is a function of [(number of matching or homologous positions shared by the two sequences) / (number of compared positions)] × 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, comparisons are performed when the two sequences are aligned to maximize homology.

[0073] "Immunogen" refers to any substance introduced into the body to generate an immune response. The substance may be a physical molecule such as a protein, or encoded by a vector such as DNA, mRNA, or a virus.

[0074] In the context of the present invention, the following abbreviations are used for commonly occurring nucleosides (nucleobases linked to a ribose or deoxyribose sugar via an N-glycosidic bond): "A" refers to adenosine, "C" refers to cytidine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0075] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The term nucleotide sequence encoding a protein or RNA may also include introns, to the extent that the nucleotide sequence encoding the protein may, in some versions, contain introns.

[0076] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may contain introns. Furthermore, the nucleotide sequence may contain modified nucleosides that can be translated by the translational machinery in a cell.

[0077] The term "polynucleotide," as used herein, is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Therefore, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes, conventional cloning techniques, and PCR™, and synthetic means.

[0078] In certain instances, the polynucleotide or nucleic acid of the present invention is a "nucleic acid," which refers to a nucleic acid containing at least one modified nucleoside. A "modified nucleoside" refers to a nucleoside having a modification. For example, in RNA, more than 100 different nucleoside modifications have been identified (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197).

[0079] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, of which there are many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. A polypeptide can be a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0080] The term "recombinant polypeptide," as used herein, is defined as a polypeptide produced using recombinant DNA or RNA methodologies.

[0081] The term "recombinant DNA" as used herein is defined as DNA that is produced by joining fragments of DNA from different sources.

[0082] The term "recombinant RNA" as used herein is defined as RNA that is produced by joining fragments of RNA from different sources.

[0083] As used herein, the term "identical" refers to two or more sequences or subsequences that are identical.

[0084] Furthermore, as used herein, the term "substantially identical" refers to two or more sequences that have a certain percentage of contiguous units that are the same when compared and aligned for maximum correspondence over a comparison window, or region designated by a comparison algorithm or manual alignment and visual inspection. By way of example only, two or more sequences may be "substantially identical" if the contiguous units over the designated region are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical. Such percentages describe the "percent identity" of two or more sequences. Sequence identity may exist over a region of at least about 75-100 contiguous units in length, a region of about 50 contiguous units in length, or, if not specified, over the entire sequence. This definition also refers to the complement of a test sequence.

[0085] As used herein, a "variant" refers to a nucleic acid or peptide sequence that differs in sequence from a reference nucleic acid or peptide sequence, respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions, and truncations. Sequence changes in peptide variants are usually limited or conservative, such that the sequences of the reference peptide and variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, or deletions in any combination. A nucleic acid or peptide variant may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring nucleic acid and peptide variants can be made by mutagenesis techniques or by direct synthesis. In various embodiments, the variant sequence is at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85% identical to the reference sequence.

[0086] As used herein, a "fragment" is defined as at least a portion of the variable region of an immunoglobulin molecule that binds to its target, i.e., the antigen-binding region. Some of the constant region of the immunoglobulin may be included.

[0087] As used herein, the term "linkage" refers to a bond or chemical moiety formed from a chemical reaction between a functional group of a linker and another molecule. Such bonds can include, but are not limited to, covalent and non-covalent bonds, while such chemical moieties can include, but are not limited to, esters, carbonates, imines, phosphate esters, hydrazones, acetals, orthoesters, peptide bonds, and oligonucleotide linkages. A hydrolytically stable linkage means that the linkage is substantially stable in water and does not react with water at useful pH values, including, but not limited to, physiological conditions, for extended periods of time, perhaps even indefinitely. A hydrolytically unstable or degradable linkage means that the linkage is degradable in water or aqueous solutions, including, for example, blood. An enzymatically unstable or degradable linkage means that the linkage can be degraded by one or more enzymes. By way of example only, PEG and related polymers can contain degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more terminal functional groups of the polymer molecule. Such degradable linkages include, but are not limited to, ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on the biologically active agent, which are hydrolyzed under physiological conditions to release the biologically active agent. Other hydrolyzable linkages include, but are not limited to, carbonate linkages; imine linkages resulting from the reaction of an amine with an aldehyde; phosphate linkages formed by the reaction of an alcohol with a phosphate group; hydrazone linkages, which are the reaction product of a hydrazide with an aldehyde; acetal linkages, which are the reaction product of an aldehyde with an alcohol; orthoester linkages, which are the reaction product of a formate with an alcohol; peptide linkages, which are formed by an amine group at the terminus of a polymer such as PEG and a carboxyl group on a peptide; and oligonucleotide linkages, which are formed by, but are not limited to, a phosphoramidite group at the terminus of a polymer and a 5' hydroxyl group on an oligonucleotide.

[0088] As used herein, the term "gene" refers to a nucleic acid molecule that encodes a protein or functional RNA (e.g., tRNA). A gene can include regions that do not code for the ultimate protein or RNA product, such as 5' or 3' untranslated regions, introns, ribosome binding sites, promoter or enhancer regions, or other related and / or regulatory sequence regions.

[0089] The terms "gene expression" and "expression" are used interchangeably herein and refer to the process by which heritable information from a gene, such as a DNA sequence, is made into a functional gene product, such as a protein or RNA.

[0090] As used herein, the term "promoter" or "regulatory sequence" refers to a nucleic acid sequence that is required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence, and in other instances, this sequence may also include an enhancer sequence and other regulatory elements that are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0091] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when it is placed into a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA or RNA sequences are contiguous and, where necessary, join two protein-coding regions in the same reading frame.

[0092] The term "specifically binds" as used herein with respect to antibodies refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. However, such cross-species reactivity does not in itself change the specific classification of the antibody. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not in itself change the specific classification of the antibody. In some cases, the terms "specific binding" or "specifically binds" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than proteins in general. If an antibody is specific for epitope "A," then in a reaction involving labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0093] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an adaptive immune response. This immune response may include antibody production, activation of specific immunogenic competent cells, or both. A skilled artisan will understand that virtually any macromolecule, including any protein or peptide, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA or RNA. A skilled artisan will understand that any DNA or RNA containing a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an adaptive immune response will therefore encode an "antigen," as that term is used herein. Furthermore, a skilled artisan will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences can be arranged in various combinations to induce a desired immune response. Furthermore, a skilled artisan will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be synthetically produced or derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0094] The term "adjuvant" as used herein is defined as any molecule that enhances the antigen-specific adaptive immune response.

[0095] "Disease" refers to a state of health in an animal where the animal is unable to maintain homeostasis and where the animal's health continues to deteriorate if the disease is not ameliorated.

[0096] In contrast, an animal "disorder" is a state of health in which the animal is able to maintain homeostasis, but the animal's state of health is less favorable than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily result in a further decline in the animal's state of health.

[0097] As used herein, "cancer" refers to the abnormal growth or division of cells. Generally, the growth and / or lifespan of cancer cells exceeds and is uncoordinated with the growth and / or lifespan of surrounding normal cells and tissues. Cancers can be benign, pre-malignant, or malignant. Cancers can arise in a variety of cells and tissues, including the oral cavity (e.g., mouth, tongue, pharynx, etc.), digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, liver, bile duct, bladder, pancreas, etc.), respiratory system (e.g., larynx, lung, bronchi, etc.), bones, joints, skin (e.g., basal cell, squamous cell, meningioma, etc.), breast, reproductive system (e.g., uterus, ovaries, prostate, testes, etc.), urinary system (e.g., bladder, kidney, ureter, etc.), eyes, nervous system (e.g., brain), endocrine system (e.g., thyroid), and hematopoietic system (e.g., lymphoma, myeloma, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, etc.).

[0098] As used herein, "effective amount" means an amount that provides a therapeutic or prophylactic benefit.

[0099] As used herein, the term "therapeutic" means treatment and / or prophylaxis. A therapeutic benefit is obtained by suppressing, reducing, ameliorating, or eradicating at least one sign or symptom of a disease or disorder state.

[0100] The term "therapeutically effective amount" refers to an amount of a compound of interest that elicits the biological or medical response in a tissue, system, or subject that is being sought by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes an amount of a compound that, when administered, is sufficient to prevent the onset of, or alleviate to some extent, one or more signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity of the subject being treated, and the age, weight, etc.

[0101] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cell thereof or any multicellular organism or cell thereof susceptible to the methods described herein, whether in vitro or in situ. In certain non-limiting embodiments, the patient, subject, or individual is a human. In certain non-limiting embodiments, the patient, subject, or individual is a fetus. In certain non-limiting embodiments, the patient, subject, or individual is an embryo.

[0102] As used herein, the term "modulate" means to mediate a detectable increase or decrease in the level of a response in a subject compared to the level of the response in an otherwise identical, untreated subject. The term encompasses perturbing and / or influencing a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0103] "Treating" a disease, as the term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0104] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0105] As used herein, the terms "under transcriptional control" or "operably linked" mean that the promoter is in the correct location and orientation relative to the polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0106] A "vector" is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0107] "Optional" or "optionally" (e.g., optionally substituted) means that the subsequently described neutrophil may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur. For example, "optionally substituted alkyl" means that the alkyl radical may or may not be substituted, and the description includes both substituted alkyl radicals and alkyl radicals without substitution.

[0108] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0109] explanation The present invention relates, in part, to novel lipids and lipid nanoparticles (LNPs), and compositions thereof. In some embodiments, the compositions comprise at least one lipid of the present invention and at least one helper lipid. The present invention also relates, in part, to the discovery that the novel lipids, LNPs, and / or compositions thereof deliver mRNA molecules to T cells with enhanced efficiency and reduced toxicity. Thus, in some embodiments, the present invention also relates to methods of delivering nucleic acid molecules and / or therapeutic agents to targets (e.g., cells) using compositions comprising at least one lipid or LNP. In various embodiments, the present invention relates to methods of gene delivery using compositions comprising at least one lipid or LNP. In certain embodiments, the present invention provides methods of preventing or treating a disease or disorder in a subject in need thereof using compositions comprising at least one lipid or LNP.

[0110] Lipids and Lipid Nanoparticles (LNPs) The present invention relates in part to novel lipid compounds. In one embodiment, the novel lipid compounds are ionizable lipid compounds. In various embodiments, the novel lipid compounds are compounds having the structure of Formula (I) or salts thereof: [ka]

[0111] In some embodiments, A1 is C, C(H), N, S, or P. In some embodiments, A2 is C, C(H), N, S, or P.

[0112] In some embodiments, L is C, C(H), C(H)(R 19 ), O, N(H), or N(R 19 In some embodiments, L2 is C, C(H), C(H)(R 19 ), O, N(H), or N(R 19 In some embodiments, L3 is C, C(H), C(H)(R 19 ), O, N(H), or N(R19 In some embodiments, L4 is C, C(H), C(H)(R 19 ), O, N(H), or N(R 19 In some embodiments, L5 is C, C(H), C(H)(R 19 ), O, N(H), or N(R 19 In some embodiments, L6 is C, C(H), C(H)(R 19 ), O, N(H), or N(R 19 )

[0113] In some embodiments, R, R, R 3a , R 3b , R 4a , R 4b , R 5a , R 5b , R 6a , R 6b , R 7a , R 7b , R 8a , R 8b , R 9a , R 9b , R 10a , R 10b , R 11a , R 11b , R 12a , R 12b , R 13a , R 13b , R 14a , R 14b , R 15a , R 15b , R 16 , R 17 , R 18 , or R 19 is H, halogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, -Y(R 20 ) z' (R 21 ) Z" -cycloalkyl, substituted -Y(R 20 ) z' (R 21 ) Z" -cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, -Y(R 20 ) z' (R21 ) Z" -heterocycloalkyl, substituted -(R 20 ) z' (R 21 ) Z" -heterocycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, -Y(R 20 ) z' (R 21 ) Z" -cycloalkenyl, substituted -Y(R 20 ) z' (R 21 ) Z" -cycloalkenyl, alkynyl, substituted alkynyl, cycloalkynyl, substituted cycloalkynyl, -Y(R 20 ) z' (R 21 ) Z" -cycloalkynyl, substituted -Y(R 20 ) z' (R 21 ) Z" -cycloalkynyl, aryl, substituted aryl, -Y(R 20 ) z' (R 21 ) Z" -aryl, substituted -Y(R 20 ) z' (R 21 ) Z" -aryl, heteroaryl, substituted heteroaryl, -Y(R 20 ) z' (R 21 ) Z" -heteroaryl, substituted -Y(R 20 ) z' (R 21 ) Z" -heteroaryl, alkoxycarbonyl, linear alkoxycarbonyl, branched alkoxycarbonyl, amido, amino, aminoalkyl, aminoalkenyl, aminoalkynyl, aminoaryl, aminoacetate, acyl, hydroxyl, hydroxyalkyl, hydroxyalkenyl, hydroxyalkynyl, hydroxyaryl, alkoxy, carboxyl, carboxylate, ester, -Y(R 20 ) z' (R 21 ) Z"-ester, -Y(R 20 ) z' (R 21 ) Z" , =O, -NO2, -CN, or sulfoxy.

[0114] In various embodiments, Y is C, N, O, S, or P.

[0115] In some embodiments, R 20 or R 21 is H, halogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, cycloalkynyl, substituted cycloalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxycarbonyl, linear alkoxycarbonyl, branched alkoxycarbonyl, amido, amino, aminoalkyl, aminoalkenyl, aminoalkynyl, aminoaryl, aminoacetate, acyl, hydroxyl, hydroxyalkyl, hydroxyalkenyl, hydroxyalkynyl, hydroxyaryl, alkoxy, carboxyl, carboxylate, ester, =0, -NO2, -CN, or sulfoxy.

[0116] In some embodiments, z' is an integer represented by 0, 1, 2, or 3. In some embodiments, z" is an integer represented by 0, 1, 2, or 3.

[0117] In some embodiments, m, n, o, p, q, r, s, t, u, v, w, or x is an integer between 0 and 25. In various embodiments, m, n, o, p, q, r, s, t, u, v, w, or x is an integer between 0, 1, 2, 3, 4, or 5.

[0118] In some embodiments, the lipid compound is a compound having the following structure: [ka] [ka] [ka] [ka] [ka] [ka]

[0119] Thus, in various embodiments, R1, R2, R3, R4, or R5 is H, halogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, cycloalkynyl, substituted cycloalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxycarbonyl, linear alkoxycarbonyl, branched alkoxycarbonyl, amido, amino, aminoalkyl, aminoalkenyl, aminoalkynyl, aminoaryl, aminoacetate, acyl, hydroxyl, hydroxyalkyl, hydroxyalkenyl, hydroxyalkynyl, hydroxyaryl, alkoxy, carboxyl, carboxylate, or ester.

[0120] In some embodiments, m, n, o, p, or q is an integer from 0 to 25. In some embodiments, r, s, t, u, v, w, or x is an integer represented by 0, 1, 2, 3, 4, and 5.

[0121] In some embodiments, the lipid compound is a compound having the following structure: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0122] In various embodiments, the present invention also includes lipid nanoparticles (LNPs). In some embodiments, the LNPs comprise one or more lipids described herein.

[0123] In various embodiments, the LNPs comprise one or more lipids of the present invention in a concentration range of about 0.1 mol% to about 100 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention in a concentration range of about 1 mol% to about 100 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention in a concentration range of about 10 mol% to about 70 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention in a concentration range of about 10 mol% to about 50 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention in a concentration range of about 15 mol% to about 45 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention in a concentration range of about 35 mol% to about 40 mol%.

[0124] For example, in some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 1 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 2 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 5 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 5.5 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 10 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 12 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 15 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 20 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 25 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 30 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 35 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 37 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 40 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 45 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 50 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 60 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 70 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 80 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 90 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 95 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 95.5 mol%. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 99 mol%.In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 99.9 mol %. In some embodiments, the LNPs comprise one or more lipids of the present invention at a concentration of about 100 mol %.

[0125] In various embodiments, the LNP further comprises at least one helper compound. In some embodiments, the helper compound is a helper lipid, a helper polymer, or any combination thereof. In some embodiments, the helper lipid is a phospholipid, a cholesterol lipid, a polymer, a cationic lipid, a neutral lipid, a charged lipid, a steroid, a steroid analog, a polymer-bound lipid, a stabilizing lipid, or any combination thereof.

[0126] In various embodiments, the LNPs comprise one or more helper compounds in a concentration range of about 0 mol% to about 100 mol%. In some embodiments, the LNPs comprise one or more helper compounds in a concentration range of about 0.01 mol% to about 99.9 mol%. In some embodiments, the LNPs comprise one or more helper compounds in a concentration range of about 0.1 mol% to about 90 mol%. In some embodiments, the LNPs comprise one or more helper compounds in a concentration range of about 0.1 mol% to about 70 mol%. In some embodiments, the LNPs comprise one or more helper compounds in a concentration range of about 5 mol% to about 95 mol%. In some embodiments, the LNPs comprise one or more helper compounds in a concentration range of about 0.5 mol% to about 50 mol%. In some embodiments, the LNPs comprise one or more helper compounds in a concentration range of about 0.5 mol% to about 47 mol%. In some embodiments, the LNPs comprise one or more helper compounds in a concentration range of about 2.5 mol % to about 47 mol %.

[0127] For example, in some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 0.01 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 0.1 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 0.5 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 1 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 1.5 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 2 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 2.5 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 5 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 10 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 12 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 15 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 16 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 20 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 25 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 30 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 35 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 37 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 40 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 45 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 46.5 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 47 mol %.In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 50 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 60 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 63 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 70 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 80 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 90 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 95 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 95.5 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 99 mol%. In some embodiments, the LNPs comprise one or more helper compounds at a concentration of about 100 mol%.

[0128] In some embodiments, the phospholipid is dioleoylphosphatidylethanolamine (DOPE) or a derivative thereof, distearoylphosphatidylcholine (DSPC) or a derivative thereof, distearoylphosphatidylethanolamine (DSPE) or a derivative thereof, stearoyloleoylphosphatidylcholine (SOPC) or a derivative thereof, 1-stearioyl-2-oleoyl-phosphatidylethanolamine (SOPE) or a derivative thereof, N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) or a derivative thereof, or any combination thereof.

[0129] For example, in some embodiments, the LNPs comprise phospholipids in a concentration range of about 0 mol% to about 100 mol%. In some embodiments, the LNPs comprise phospholipids in a concentration range of about 15 mol% to about 50 mol%. In some embodiments, the LNPs comprise phospholipids in a concentration range of about 10 mol% to about 40 mol%. In some embodiments, the LNPs comprise phospholipids in a concentration range of about 16 mol% to about 40 mol%.

[0130] In some embodiments, the cholesterol lipid is cholesterol or a derivative thereof. For example, in some embodiments, the LNPs comprise cholesterol lipid in a concentration range of about 0 mol% to about 100 mol%. In some embodiments, the LNPs comprise cholesterol lipid in a concentration range of about 20 mol% to about 50 mol%. In some embodiments, the LNPs comprise cholesterol lipid in a concentration range of about 20 mol% to about 47 mol%. In some embodiments, the LNPs comprise cholesterol lipid at a concentration of about 47 mol% and DOPE at a concentration of about 16 mol%.

[0131] In some embodiments, the polymer is polyethylene glycol (PEG) or a derivative thereof. For example, in some embodiments, the LNP comprises a polymer in a concentration range of about 0 mol% to about 100 mol%. In some embodiments, the LNP comprises a polymer in a concentration range of about 0.5 mol% to about 10 mol%. In some embodiments, the LNP comprises a polymer in a concentration range of about 0.5 mol% to about 2.5 mol%.

[0132] The term " cationic lipid " used herein refers to a lipid that is cationic or becomes cationic (protonated) when the pH is lower than the pK of the ionizable group of lipid, but gradually becomes more neutral at higher pH values.At pH values ​​lower than pK, lipid can bind with negatively charged nucleic acid.In certain embodiments, cationic lipid comprises zwitterionic lipid, which becomes positively charged with decreasing pH.

[0133] In some embodiments, the cationic lipid comprises any of several lipid species that carry a net positive charge at a selected pH, such as physiological pH. Such lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1 Cationic lipids that can be used in the present invention include N-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). Additionally, many commercially available preparations of cationic lipids are available that can be used in the present invention. These include, for example, LIPOFECTIN® (a commercially available cationic liposome containing DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE) from GIBCO / BRL, Grand Island, NY); LIPOFECTAMINE® (a commercially available cationic liposome containing N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE) from GIBCO / BRL); and TRANSFECTAM® (a commercially available cationic lipid containing dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.).The following lipids are cationic and positively charged at sub-physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0134] In one embodiment, cationic lipid is amino lipid.Suitable amino lipid useful in the present invention includes those described in WO2012 / 016184, the entirety of which is incorporated herein by reference.Representative amino lipid includes but is not limited to 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl). , 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanediol (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

[0135] The term "neutral lipid" refers to any of a number of lipid species that exist in either uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0136] Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl Neutral lipids include ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), distearoyl-phosphatidylethanolamine (DSPE)-maleimide-PEG, distearoyl-phosphatidylethanolamine (DSPE)-maleimide-PEG2000, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearyl-2-oleoyl-phosphatidylethanolamine (SOPE), stearoyloleoylphosphatidylcholine (SOPC) and 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (transDOPE).In one embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0137] In some embodiments, the composition comprises a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0138] A "steroid" is a compound containing the following carbon skeleton: [ka]

[0139] In certain embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of the cationic lipid.

[0140] The term "anionic lipid" refers to lipids that are negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0141] The term "polymer-conjugated lipid" refers to a molecule that contains both a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that contains both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-s-DMG), etc.

[0142] In certain embodiments, the LNP comprises an additional stabilizing lipid that is a polyethylene glycol-lipid (PEGylated lipid). Suitable polyethylene glycol-lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxypoly(ethylene glycol) 2000 )carbamyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG. In another embodiment, the LNP comprises a PEGylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), a PEGylated phosphatidylethanolamine (PEG-PE), a PEG diacylglycerol succinate (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.

[0143] In certain embodiments, the additional lipid is present in the LNP in an amount of about 1 mol% to about 10 mol%. In one embodiment, the additional lipid is present in the LNP in an amount of about 1 mol% to about 5 mol%. In one embodiment, the additional lipid is present in the LNP at about 1 mol% or about 2.5 mol%.

[0144] The term "lipid nanoparticle" refers to a particle having at least one dimension on the order of nanometers (e.g., 1 to 1,000 nm) that comprises one or more lipids, such as lipids of formulae (I) to (XV).

[0145] In various embodiments, the lipid nanoparticles have an average diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm.

[0146] In various embodiments, the lipids or LNPs of the present invention are substantially non-toxic.

[0147] In various embodiments, the lipids or LNPs described herein are easily transported to target tissues.For example, in various embodiments, the lipids or LNPs described herein are easily transported through cell membranes into cells.In various embodiments, the lipids or LNPs described herein are efficiently transported through cell membranes into cells.In some embodiments, the lipids or LNPs described herein are transported through cell membranes into cells with enhanced efficiency.

[0148] Lipid and LNP Composition In various embodiments, the present invention also provides compositions comprising one or more lipids or LNPs described herein. In various embodiments, the compositions comprise one or more nucleic acid molecules, one or more therapeutic agents, or any combination thereof. In some embodiments, the nucleic acid molecules, therapeutic agents, or any combination thereof are encapsulated in lipids. In some embodiments, the nucleic acid molecules, therapeutic agents, or any combination thereof are encapsulated in LNPs.

[0149] In one embodiment, the nucleic acid molecule is a DNA molecule. In one embodiment, the nucleic acid molecule is an RNA molecule. In some embodiments, the nucleic acid molecule is a DNA molecule or an RNA molecule. Examples of such nucleic acids include, but are not limited to, cDNA, mRNA, miRNA, siRNA, modified RNA, antagomir, antisense molecules, peptides, therapeutic peptides, targeting nucleic acids, and any combination thereof.

[0150] In one embodiment, the mRNA encodes luciferase.

[0151] In various embodiments, the nucleic acid molecule is a therapeutic agent. In some embodiments, the therapeutic agent is an isolated nucleic acid. In various embodiments, the isolated nucleic acid molecule is a DNA molecule or an RNA molecule. In various embodiments, the isolated nucleic acid molecule is a cDNA, mRNA, miRNA, siRNA, antagomir, or antisense molecule. In one embodiment, the isolated nucleic acid molecule encodes a therapeutic peptide. In some embodiments, the therapeutic agent is an siRNA, miRNA, or antisense molecule that inhibits a targeting nucleic acid.

[0152] In one embodiment, the composition comprises a promoter or regulatory sequence. In one embodiment, the nucleic acid comprises a promoter or regulatory sequence such that the nucleic acid can direct expression of the nucleic acid. Thus, in one embodiment, a composition comprising a metabolite-based polymer or polymer particle of the present invention comprises an expression vector, and the present invention comprises a method for introducing exogenous DNA into a cell or tissue of interest with co-expression of the exogenous DNA in the cell or tissue of interest.

[0153] In one embodiment, the nucleic acid molecule is mRNA.In one embodiment, the composition comprises mRNA.In one embodiment, the composition comprises mRNA encapsulated in LNP.In various embodiments, the composition comprising mRNA encapsulated in LNP has certain advantages over isolated mRNA, including, for example, increased stability, reduced or no natural immunogenicity, and enhanced translation.

[0154] In one embodiment, the RNA is modified RNA. In another embodiment, 0.1% to 100% of the residues in the modified RNA of the present invention are modified. In another embodiment, 0.1% of the residues are modified. In another embodiment, the percentage of modified residues is 0.2%. In another embodiment, the percentage is 0.3%. In another embodiment, the percentage is 0.4%. In another embodiment, the percentage is 0.5%. In another embodiment, the percentage is 0.6%. In another embodiment, the percentage is 0.8%. In another embodiment, the percentage is 1%. In another embodiment, the percentage is 1.5%. In another embodiment, the percentage is 2%. In another embodiment, the percentage is 2.5%. In another embodiment, the percentage is 3%. In another embodiment, the percentage is 4%. In another embodiment, the percentage is 5%. In another embodiment, the percentage is 6%. In another embodiment, the percentage is 8%. In another embodiment, the percentage is 10%. In another embodiment, the percentage is 12%. In another embodiment, the percentage is 14%. In another embodiment, the percentage is 16%. In another embodiment, the percentage is 18%. In another embodiment, the percentage is 20%. In another embodiment, the percentage is 25%. In another embodiment, the percentage is 30%. In another embodiment, the percentage is 35%. In another embodiment, the percentage is 40%. In another embodiment, the percentage is 45%. In another embodiment, the percentage is 50%. In another embodiment, the percentage is 60%. In another embodiment, the percentage is 70%. In another embodiment, the percentage is 80%. In another embodiment, the percentage is 90%. In another embodiment, the percentage is 100%.

[0155] In another embodiment, the percentage is less than 5%. In another embodiment, the percentage is less than 3%. In another embodiment, the percentage is less than 1%. In another embodiment, the percentage is less than 2%. In another embodiment, the percentage is less than 4%. In another embodiment, the percentage is less than 6%. In another embodiment, the percentage is less than 8%. In another embodiment, the percentage is less than 10%. In another embodiment, the percentage is less than 12%. In another embodiment, the percentage is less than 15%. In another embodiment, the percentage is less than 20%. In another embodiment, the percentage is less than 30%. In another embodiment, the percentage is less than 40%. In another embodiment, the percentage is less than 50%. In another embodiment, the percentage is less than 60%. In another embodiment, the percentage is less than 70%.

[0156] In another embodiment, 0.1% of the residues of a given nucleoside (i.e., uridine, cytidine, guanosine, or adenosine) are modified. In another embodiment, the percentage of a given nucleotide that is modified is 0.2%. In another embodiment, the percentage is 0.3%. In another embodiment, the percentage is 0.4%. In another embodiment, the percentage is 0.5%. In another embodiment, the percentage is 0.6%. In another embodiment, the percentage is 0.8%. In another embodiment, the percentage is 1%. In another embodiment, the percentage is 1.5%. In another embodiment, the percentage is 2%. In another embodiment, the percentage is 2.5%. In another embodiment, the percentage is 3%. In another embodiment, the percentage is 4%. In another embodiment, the percentage is 5%. In another embodiment, the percentage is 6%. In another embodiment, the percentage is 8%. In another embodiment, the percentage is 10%. In another embodiment, the percentage is 12%. In another embodiment, the percentage is 14%. In another embodiment, the percentage is 16%. In another embodiment, the percentage is 18%. In another embodiment, the percentage is 20%. In another embodiment, the percentage is 25%. In another embodiment, the percentage is 30%. In another embodiment, the percentage is 35%. In another embodiment, the percentage is 40%. In another embodiment, the percentage is 45%. In another embodiment, the percentage is 50%. In another embodiment, the percentage is 60%. In another embodiment, the percentage is 70%. In another embodiment, the percentage is 80%. In another embodiment, the percentage is 90%. In another embodiment, the percentage is 100%.

[0157] In another embodiment, the percentage of a given nucleotide that is modified is less than 8%. In another embodiment, the percentage is less than 10%. In another embodiment, the percentage is less than 5%. In another embodiment, the percentage is less than 3%. In another embodiment, the percentage is less than 1%. In another embodiment, the percentage is less than 2%. In another embodiment, the percentage is less than 4%. In another embodiment, the percentage is less than 6%. In another embodiment, the percentage is less than 12%. In another embodiment, the percentage is less than 15%. In another embodiment, the percentage is less than 20%. In another embodiment, the percentage is less than 30%. In another embodiment, the percentage is less than 40%. In another embodiment, the percentage is less than 50%. In another embodiment, the percentage is less than 60%. In another embodiment, the percentage is less than 70%.

[0158] In another embodiment, RNA encapsulated in the LNPs of the present invention is translated intracellularly more efficiently than isolated RNA molecules having the same sequence. In another embodiment, RNA encapsulated in the LNPs exhibits an enhanced ability to be translated by target cells. In another embodiment, translation is enhanced 2-fold compared to its unmodified counterpart. In another embodiment, translation is enhanced 3-fold. In another embodiment, translation is enhanced 5-fold. In another embodiment, translation is enhanced 7-fold. In another embodiment, translation is enhanced 10-fold. In another embodiment, translation is enhanced 15-fold. In another embodiment, translation is enhanced 20-fold. In another embodiment, translation is enhanced 50-fold. In another embodiment, translation is enhanced 100-fold. In another embodiment, translation is enhanced 200-fold. In another embodiment, translation is enhanced 500-fold. In another embodiment, translation is enhanced 1000-fold. In another embodiment, translation is enhanced 2000-fold. In another embodiment, the factor is 10-1000-fold. In another embodiment, the factor is 10-100-fold. In another embodiment, the factor is 10-200-fold. In another embodiment, the factor is 10-300 fold. In another embodiment, the factor is 10-500 fold. In another embodiment, the factor is 20-1000 fold. In another embodiment, the factor is 30-1000 fold. In another embodiment, the factor is 50-1000 fold. In another embodiment, the factor is 100-1000 fold. In another embodiment, the factor is 200-1000 fold. In another embodiment, translation is enhanced by any other significant amount or range of amounts.

[0159] In certain embodiments, mRNA does not activate pathophysiological pathways, is highly efficient in translation almost immediately after delivery, and serves as a template for continuous in vivo protein production that continues for several days (Kariko et al., 2008, Mol Ther 16:1833-1840; Kariko et al., 2012, Mol Ther 20:948-953). In certain instances, the antigen encoded by the mRNA encapsulated in LNP induces greater production of antigen-specific antibodies compared to the antigen encoded by isolated mRNA.

[0160] In one embodiment, the nucleic acid molecule encodes an antigen. In one embodiment, the nucleic acid molecule encodes multiple antigens. In some embodiments, the mRNA encodes one or more antigens. In one embodiment, the therapeutic agent is an antigen.

[0161] In various embodiments, the antigen comprises a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, an influenza antigen, a tumor-associated antigen, a tumor-specific antigen, or any combination thereof. In one embodiment, the invention includes a nucleic acid molecule encoding an adjuvant.

[0162] In one embodiment, the antigen is encoded by the nucleic acid sequence of a nucleic acid molecule. In certain embodiments, the nucleic acid sequence comprises DNA, RNA, cDNA, variants thereof, fragments thereof, or combinations thereof. In one embodiment, the nucleic acid sequence comprises a modified nucleic acid sequence. For example, in one embodiment, the nucleic acid sequence encoding the antigen comprises RNA, as described in detail elsewhere herein. In certain instances, the nucleic acid sequence comprises an additional sequence encoding a linker or tag sequence linked to the antigen by a peptide bond.

[0163] In certain embodiments, the antigen encoded by the nucleic acid molecule comprises a protein, peptide, fragment thereof, or variant thereof, or a combination thereof, from any number of organisms, such as viruses, parasites, bacteria, fungi, or mammals. For example, in certain embodiments, the antigen is associated with autoimmune disease, allergy, or asthma. In another embodiment, the antigen is associated with cancer, herpes, influenza, hepatitis B, hepatitis C, human papillomavirus (HPV), Ebola, pneumococcus, Haemophilus influenzae, meningococcus, dengue fever, tuberculosis, malaria, norovirus, or human immunodeficiency virus (HIV). In certain embodiments, the antigen comprises a consensus sequence based on the amino acid sequences of two or more different organisms. In certain embodiments, the nucleic acid sequence encoding the antigen is optimized for effective translation in the organism to which the composition is delivered.

[0164] In one embodiment, the antigen comprises a tumor-specific antigen or a tumor-associated antigen, such that the antigen induces an adaptive immune response against the tumor. In one embodiment, the antigen comprises a tumor-specific antigen or a fragment of a tumor-associated antigen, such that the antigen induces an adaptive immune response against the tumor. In a particular embodiment, the tumor-specific antigen or tumor-associated antigen is a mutant variant of a host protein.

[0165] Thus, in one embodiment, the composition comprises an antigen. In one embodiment, the composition comprises a nucleic acid sequence encoding the antigen. For example, in a specific embodiment, the composition comprises RNA encoding the antigen. The antigen can be any molecule or compound, including, but not limited to, a polypeptide, peptide, or protein that induces an adaptive immune response in a subject.

[0166] In one embodiment, the antigen comprises a polypeptide or peptide associated with a pathogen, such that the antigen induces an adaptive immune response against the antigen and therefore the pathogen. In one embodiment, the antigen comprises a fragment of a polypeptide or peptide associated with a pathogen, such that the antigen induces an adaptive immune response against the pathogen.

[0167] In certain embodiments, the antigen comprises an amino acid sequence that is substantially homologous to the amino acid sequence of an antigen described herein and that retains the immunogenic function of the original amino acid sequence. For example, in certain embodiments, the amino acid sequence of the antigen has a degree of identity to the original amino acid sequence of at least 60%, advantageously at least 70%, preferably at least 85%, and more preferably at least 95%.

[0168] Viral antigens In one embodiment, the antigen comprises a viral antigen, or a fragment thereof, or a variant thereof. In a specific embodiment, the viral antigen is derived from a virus from one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. In certain embodiments, the viral antigen is a papillomavirus, such as human papillomavirus (HPV), human immunodeficiency virus (HIV), poliovirus, hepatitis B virus, hepatitis C virus, smallpox virus (variola major and variola minor), vaccinia virus, influenza virus, rhinovirus, dengue virus, equine encephalitis virus, rubella virus, yellow fever virus, Norwalk virus, hepatitis A virus, human T-cell leukemia virus (HTLV-I), hairy cell leukemia virus (HTLV-II), California encephalitis virus, or the like. The virus may be derived from viruses such as rabies, hantavirus (hemorrhagic fever), rabies, Ebola, Marburg, measles, mumps, respiratory syncytial virus (RSV), herpes simplex 1 (oral herpes), herpes simplex 2 (genital herpes), varicella zoster (varicella-zoster, also known as chickenpox), cytomegalovirus (CMV), including human CMV, Epstein-Barr virus (EBV), flaviviruses, foot and mouth disease, chikungunya, Lassa, arenaviruses, or viruses that cause cancer.

[0169] hepatitis antigen In one embodiment, the antigen comprises a hepatitis virus antigen (i.e., a hepatitis antigen), or a fragment thereof, or a variant thereof. In a specific embodiment, the hepatitis antigen comprises an antigen or immunogen from hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and / or hepatitis E virus (HEV). In a specific embodiment, the hepatitis antigen is a full-length protein or an immunogenic fragment of a full-length protein.

[0170] In one embodiment, the hepatitis antigen comprises an antigen derived from HAV. For example, in certain embodiments, the hepatitis antigen comprises an HAV capsid protein, an HAV nonstructural protein, a fragment thereof, a variant thereof, or a combination thereof.

[0171] In one embodiment, the hepatitis antigen comprises an antigen derived from HCV. For example, in certain embodiments, the hepatitis antigen comprises an HCV nucleocapsid protein (i.e., core protein), an HCV envelope protein (e.g., E1 and E2), an HCV nonstructural protein (e.g., NS1, NS2, NS3, NS4a, NS4b, NS5a, and NS5b), a fragment thereof, a variant thereof, or a combination thereof.

[0172] In one embodiment, the hepatitis antigen comprises an antigen derived from HDV. For example, in certain embodiments, the hepatitis antigen comprises an HDV delta antigen, a fragment thereof, or a variant thereof.

[0173] In one embodiment, the hepatitis antigen comprises an antigen derived from HEV. For example, in certain embodiments, the hepatitis antigen comprises an HEV capsid protein, a fragment thereof, or a variant thereof.

[0174] In one embodiment, the hepatitis antigen comprises an antigen derived from HBV. For example, in certain embodiments, the hepatitis antigen comprises an HBV core protein, an HBV surface protein, an HBV DNA polymerase, an HBV protein encoded by gene X, a fragment thereof, a variant thereof, or a combination thereof. In certain embodiments, the hepatitis antigen comprises an HBV genotype A core protein, an HBV genotype B core protein, an HBV genotype C core protein, an HBV genotype D core protein, an HBV genotype E core protein, an HBV genotype F core protein, an HBV genotype G core protein, an HBV genotype H core protein, an HBV genotype A surface protein, an HBV genotype B surface protein, an HBV genotype C surface protein, an HBV genotype D surface protein, an HBV genotype E surface protein, an HBV genotype F surface protein, an HBV genotype G surface protein, an HBV genotype H surface protein, a fragment thereof, a variant thereof, or a combination thereof.

[0175] Human papillomavirus (HPV) antigens In one embodiment, the antigen comprises a human papillomavirus (HPV) antigen, or a fragment thereof, or a variant thereof. For example, in certain embodiments, the antigen comprises an antigen from HPV types 16, 18, 31, 33, 35, 45, 52, and 58, which cause cervical cancer, rectal cancer, and / or other cancers. In one embodiment, the antigen comprises an antigen from HPV types 6 and 11, which cause genital warts and are known to be responsible for head and neck cancer. For example, in certain embodiments, the HPV antigen comprises the HPV E6 or E7 domain from any HPV type, or a fragment or variant thereof.

[0176] RSV antigen In one embodiment, the antigen comprises a RSV antigen, or a fragment thereof, or a variant thereof. For example, in a specific embodiment, the RSV antigen comprises a human RSV fusion protein (also referred to herein as "RSV F," "RSV F protein," and "F protein"), or a fragment or variant thereof. In one embodiment, the human RSV fusion protein is conserved between RSV subtypes A and B. In a specific embodiment, the RSV antigen comprises a RSV F protein from the RSV Long strain (GenBank AAX23994.1), or a fragment or variant thereof. In one embodiment, the RSV antigen comprises a RSV F protein from the RSV A2 strain (GenBank AAB59858.1), or a fragment or variant thereof. In a specific embodiment, the RSV antigen is a RSV F protein monomer, dimer, or trimer, or a fragment or variant thereof. According to the present invention, in a specific embodiment, the RSV F protein is a pre-fusion form or a post-fusion form.

[0177] In one embodiment, the RSV antigen comprises a human RSV attachment glycoprotein (also referred to herein as "RSV G," "RSV G protein," and "G protein"), or a fragment or variant thereof. Human RSV G proteins differ between RSV subtypes A and B. In one embodiment, the antigen comprises an RSV G protein from the RSV Long strain (GenBank AAX23993), or a fragment or variant thereof. In one embodiment, the RSV antigen comprises an RSV G protein from RSV subtype B isolate H5601, RSV subtype B isolate H1068, RSV subtype B isolate H5598, RSV subtype B isolate H1123, or a fragment or variant thereof.

[0178] In another embodiment, the RSV antigen comprises a human RSV nonstructural protein 1 ("NS1 protein"), or a fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises a RSV NS1 protein from the RSV Long strain (GenBank AAX23987.1), or a fragment or variant thereof. In one embodiment, the RSV antigen comprises a RSV nonstructural protein 2 ("NS2 protein"), or a fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises a RSV NS2 protein from the RSV Long strain (GenBank AAX23988.1), or a fragment or variant thereof. In one embodiment, the RSV antigen comprises a human RSV nucleocapsid ("N") protein, or a fragment or variant thereof. For example, in one embodiment, the RSV antigen is the RSV N protein from the RSV Long strain (GenBank AAX23989.1), or a fragment or variant thereof. In one embodiment, the RSV antigen comprises a human RSV phosphoprotein ("P") protein, or a fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises a RSV P protein from the RSV Long strain (GenBank AAX23990.1), or a fragment or variant thereof. In one embodiment, the RSV antigen comprises a human RSV matrix protein ("M") protein, or a fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises a RSV M protein from the RSV Long strain (GenBank AAX23991.1), or a fragment or variant thereof.

[0179] In yet another embodiment, the RSV antigen comprises a human RSV small hydrophobic ("SH") protein, or a fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises an RSV SH protein from the RSV Long strain (GenBank AAX23992.1), or a fragment or variant thereof. In one embodiment, the RSV antigen comprises a human RSV matrix protein 2-1 ("M2-1") protein, or a fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises an RSV M2-1 protein from the RSV Long strain (GenBank AAX23995.1), or a fragment or variant thereof. In one embodiment, the RSV antigen comprises an RSV matrix protein 2-2 ("M2-2") protein, or a fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises an RSV M2-2 protein from the RSV Long strain (GenBank AAX23997.1), or a fragment or variant thereof. In one embodiment, the RSV antigen comprises an RSV polymerase L ("L") protein, or a fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises an RSV L protein from the RSV Long strain (GenBank AAX23996.1), or a fragment or variant thereof.

[0180] Influenza antigen In one embodiment, the antigen comprises an influenza antigen, or a fragment thereof, or a variant thereof. The influenza antigen is one that can induce an adaptive immune response in a mammal against one or more influenza serotypes. In certain embodiments, the antigen comprises the full-length translation product hemagglutinin (HA)0, subunit HA1, subunit HA2, variants thereof, fragments thereof, or combinations thereof. In certain embodiments, the influenza hemagglutinin antigen is derived from one or more strains of influenza A serotype H1, influenza A serotype H2, or influenza B.

[0181] In one embodiment, the influenza antigen comprises at least one antigenic epitope that may be effective against a particular influenza immunogen against which an immune response can be induced. In certain embodiments, the antigen may represent the entire repertoire of immunogenic sites and epitopes present in intact influenza virus.

[0182] In some embodiments, the influenza antigen comprises an H1HA, H2HA, H3HA, H5HA, or BHA antigen. In certain embodiments, the influenza antigen comprises neuraminidase (NA), matrix protein, nucleoprotein, M2 ectodomain-nucleoprotein (M2e-NP), variants thereof, fragments thereof, or combinations thereof.

[0183] Human immunodeficiency virus (HIV) antigens In one embodiment, the antigen comprises an HIV antigen, or a fragment thereof, or a variant thereof.

[0184] In certain embodiments, the HIV antigen comprises an envelope (Env) protein, or a fragment or variant thereof. For example, in certain embodiments, the HIV antigen comprises an Env protein selected from gp120, gp41, or a combination thereof.

[0185] In certain embodiments, the HIV antigens comprise at least one of nef, gag, pol, vif, vpr, vpu, tat, rev, or a fragment or variant thereof.

[0186] HIV antigens can be derived from any strain of HIV. For example, in certain embodiments, HIV antigens include antigens from HIV groups M, N, O, and P, and subtype A, HIV subtype B, HIV subtype C, HIV subtype D, subtype E, subtype F, subtype G, subtype H, subtype J, or subtype K. In one embodiment, the HIV antigen includes Env from the HIV-R3A strain (R3A-Env), or a fragment or variant thereof.

[0187] Parasite antigens In certain embodiments, the antigen comprises a parasitic antigen, or a fragment or variant thereof. In certain embodiments, the parasite is a protozoan, a helminth, or an ectoparasite. In certain embodiments, the helminth (i.e., worm) is a flatworm (e.g., a fluke and a tapeworm), an thornyhead, or a roundworm (e.g., a pinworm). In certain embodiments, the ectoparasite is a louse, a flea, a tick, or a mite.

[0188] In certain embodiments, the parasite is any parasite that causes the following diseases: acanthamoeba keratitis, amebiasis, ascariasis, babesiosis, balantidiosis, raccoon ascariasis, Chagas' disease, clonorchiasis, cochliomyiasis, cryptosporidiosis, diphyllobothriasis, dracunculiasis, echinococcosis, elephantiasis, enterobiasis, fascioliasis, filariasis, giardiasis, gnathostomiasis, hymenococcosis, isosporosis, Katayama fever, leishmaniasis, Lyme disease, malaria, tumefacilitatoriasis, myiasis, onchocerciasis, pediculosis, scabies, schistosomiasis, sleeping sickness, strongyloidiasis, taeniasis, toxocariasis, toxoplasmosis, trichinellosis, and trichuriasis.

[0189] In certain embodiments, the parasite is Acanthamoeba, Anisakis, Ascaris, Brynii, Balantidium coli, Bedbug, Tapeworm (Cestworm), Chigger, Screwworm, Entamoeba histolytica, Fasciola, Giardia lamblia, Hookworm, Leishmania, Dog ringworm, Liver fluke, Bacillus loa, Paragonimus westermani, Helminth, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, Tick, Tapeworm, Toxoplasma gondii, Trypanosoma, Trichuris trichiura, or Wuchereria bancrofti.

[0190] Malaria antigens In one embodiment, the antigen comprises a malaria antigen (i.e., a PF antigen or PF immunogen), or a fragment thereof, or a variant thereof. For example, in one embodiment, the antigen comprises an antigen derived from a parasite that causes malaria. In one embodiment, the parasite that causes malaria is Plasmodium falciparum.

[0191] In some embodiments, the malaria antigen comprises one or more of the Plasmodium falciparum immunogens CS; LSA1; TRAP; CelTOS; and Ama1. The immunogen can be full-length or an immunogenic fragment of the full-length protein.

[0192] bacterial antigen In one embodiment, the antigen comprises a bacterial antigen, or a fragment or variant thereof. In certain embodiments, the bacteria is from any of the following phyla: Acidobacteria, Actinomycetes, Aquifex, Bacteroides, Caldicellicum, Chlamydia, Green Sulfur Bacteria, Green Non-Sulfur Bacteria, Chrysiogenes, Cyanobacteria, Deferibacter, Deinococcus Thermus, Dictyoglomus, Yersinia Microbia, Fibrobacter, Firmicutes, Fusobacteria, Gemmatimonas, Lentisphaera, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistes, Tenericutes, Thermodesulfobacteria, Thermotoga, and Verrucomicrobium.

[0193] In certain embodiments, the bacterium is a gram-positive bacterium or a gram-negative bacterium. In certain embodiments, the bacterium is an aerobic bacterium or an anaerobic bacterium. In certain embodiments, the bacterium is an autotrophic bacterium or a heterotrophic bacterium. In certain embodiments, the bacterium is a mesophilic bacterium, a neutral bacterium, an extremophilic bacterium, an acidophilic bacterium, an alkaliphilic bacterium, a thermophilic bacterium, a psychrophilic bacterium, a halophilic bacterium, or an osmophilic bacterium.

[0194] In certain embodiments, the bacteria is Bacillus anthracis, antibiotic-resistant bacteria, pathogenic bacteria, food poisoning bacteria, infectious bacteria, Salmonella, Staphylococcus, Streptococcus, or Clostridium difficile. In certain embodiments, the bacteria is Mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile.

[0195] Mycobacterium tuberculosis antigen In one embodiment, the antigen comprises a Mycobacterium tuberculosis antigen (i.e., a TB antigen or TB immunogen), or a fragment or variant thereof. The TB antigen can be derived from the Ag85 family of TB antigens, e.g., Ag85A and Ag85B. The TB antigen can be derived from the Esx family of TB antigens, e.g., EsxA, EsxB, EsxC, EsxD, EsxE, EsxF, EsxH, EsxO, EsxQ, EsxR, EsxS, EsxT, EsxU, EsxV, and EsxW.

[0196] fungal antigen In one embodiment, the antigen comprises a fungal antigen, or a fragment or variant thereof. In a specific embodiment, the fungus is Aspergillus species, Blastomyces dermatitidis, Candida yeast (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, dermatophytes, Fusarium species, Histoplasma capsulatum, Mucor, Pneumocystis pneumoniae, Sporothrix schenckii, Exerohirum, or Cladosporium.

[0197] tumor antigens In certain embodiments, the antigen comprises a tumor antigen, including, for example, a tumor-associated antigen or a tumor-specific antigen. In the context of the present invention, a "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to an antigen common to a particular hyperproliferative disorder. In certain aspects, the hyperproliferative disorder antigen of the present invention is derived from cancer, including, but not limited to, primary or metastatic melanoma, mesothelioma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinoma, such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, etc.

[0198] Tumor antigens are proteins produced by tumor cells that induce immune responses, particularly T cell-mediated immune responses. In one embodiment, the tumor antigens of the present invention comprise one or more antigenic cancer epitopes that are immunogenically recognized by tumor-infiltrating lymphocytes (TILs) derived from mammalian cancer tumors. The choice of antigen will depend on the specific type of cancer to be treated or prevented by the compositions of the present invention.

[0199] Tumor antigens are known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin, and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0200] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express many proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase, and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute truly tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens, such as CD19, CD20, and CD37, are other candidate target antigens for B-cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies, but with limited success.

[0201] The type of tumor antigen referred to in the present invention can also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and do not exist in other cells in the body. TAA-associated antigens are not unique to tumor cells, but are instead expressed by normal cells under conditions that cannot induce a state of immune tolerance to the antigen. Antigen expression in tumors can occur under conditions that allow the immune system to respond to the antigen. TAA can be an antigen expressed in normal cells during fetal development, when the immune system is immature and unable to respond, or it can be an antigen that is usually present at very low levels in normal cells but is expressed at much higher levels in tumor cells.

[0202] Non-limiting examples of TSA or TAA antigens include: differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA, and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, and BT. These include AA, CA125, CA15-3, CA27.29, BCAA, CA195, CA242, CA-50, CAM43, CD68, P1, CO-029, FGF-5, G250, Ga733, EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, Mac-2 binding protein, cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0203] In preferred embodiments, antigens include, but are not limited to, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, MAGE A3 TCR, and the like.

[0204] In certain embodiments, the nucleic acid molecule encodes an antigen that induces an adaptive immune response against the antigen. In certain embodiments, the therapeutic agent is an antigen that induces an adaptive immune response against the antigen.

[0205] Nucleotide sequences encoding the antigens or adjuvants described herein may alternatively include sequence changes relative to the original nucleotide sequence, such as substitutions, insertions, and / or deletions of one or more nucleotides, provided that the resulting polynucleotide encodes a polynucleotide according to the invention. Thus, the scope of the present invention includes nucleotide sequences that are substantially homologous to the nucleotide sequences described herein and that encode an antigen or adjuvant of interest.

[0206] As used herein, a nucleotide sequence is "substantially homologous" to any nucleotide sequence described herein if the nucleotide sequence has a degree of identity of at least 60%, advantageously at least 70%, preferably at least 85%, and more preferably at least 95% to any nucleotide sequence described herein. Nucleotide sequences that are substantially homologous to a nucleotide sequence encoding an antigen can typically be isolated from an antigen-producing organism based on the information contained in the nucleotide sequence, for example, by introducing conservative or non-conservative substitutions. Other examples of possible modifications include the insertion of one or more nucleotides in the sequence, the addition of one or more nucleotides to either end of the sequence, or the deletion of one or more nucleotides at any end or within the sequence. The degree of identity between two polynucleotides can be determined using computer algorithms and methods well known to those skilled in the art.

[0207] In one embodiment, the present invention relates to a construct comprising a nucleotide sequence encoding an antigen. In one embodiment, the construct comprises multiple nucleotide sequences encoding multiple antigens. For example, in certain embodiments, the construct encodes one or more, two or more, five or more, ten or more, fifteen or more, or twenty or more antigens. In one embodiment, the present invention relates to a construct comprising a nucleotide sequence encoding an adjuvant. In one embodiment, the construct comprises a first nucleotide sequence encoding an antigen and a second nucleotide sequence encoding an adjuvant.

[0208] In one embodiment, the composition comprises multiple constructs, each encoding one or more antigens. In certain embodiments, the composition comprises one or more, two or more, five or more, ten or more, fifteen or more, or twenty or more constructs. In one embodiment, the composition comprises a first construct comprising a nucleotide sequence encoding an antigen and a second construct comprising a nucleotide sequence encoding an adjuvant.

[0209] In another specific embodiment, the construct is operably linked to a translational control element. The construct can incorporate operably linked regulatory sequences for expression of the nucleotide sequence of the present invention, thus forming an expression cassette.

[0210] In one embodiment, the composition of the present invention comprises in vitro transcribed (IVT) RNA. For example, in a specific embodiment, the composition of the present invention comprises an IVT RNA encoding an antigen, wherein the antigen induces an adaptive immune response. In a specific embodiment, the antigen is at least one of a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a tumor-specific antigen, or a tumor-associated antigen. However, the present invention is not limited to a specific antigen or combination of antigens.

[0211] For example, in one embodiment, the composition comprises a nucleic acid molecule encoding an antigen encapsulated within an LNP. In certain instances, the LNP enhances cellular uptake of the nucleic acid molecule.

[0212] In one embodiment, the nucleic acid molecule is an IVT RNA encoding an antigen. Thus, in one embodiment, a composition of the invention comprises an IVT RNA encoding an antigen. In one embodiment, a composition of the invention comprises an IVT RNA encoding multiple antigens. In one embodiment, a composition of the invention comprises an IVT RNA encoding an adjuvant. In one embodiment, a composition of the invention comprises an IVT RNA encoding one or more antigens and one or more adjuvants.

[0213] In one embodiment, the composition comprises a nucleic acid molecule encoding an adjuvant. Thus, in one embodiment, the composition comprises an adjuvant. In one embodiment, the nucleic acid molecule encoding the adjuvant is IVT RNA. In one embodiment, the nucleic acid molecule encoding the adjuvant is RNA.

[0214] Exemplary adjuvants include, but are not limited to, alpha interferon, gamma interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosal-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 (including IL-15 with the signal sequence deleted, optionally containing a signal peptide from IgE). Other genes that may be useful adjuvants include those encoding MCP-I, MIP-Ia, MIP-Ip, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-I, VLA-I, Mac-1, pl50.95, PECAM, ICAM-I, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant IL-18, CD40, C D40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-I, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase-ICE, Fos, c-jun, Sp-I, Ap-I, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-I, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, anti-CTLA4-sc, anti-LAG3-Ig, anti-TIM3-Ig, and functional fragments thereof.

[0215] In some embodiments, the composition further comprises cationic lipid and one or more excipients selected from neutral lipid, charged lipid, steroid, and polymer-bound lipid.In some embodiments, nucleic acid molecule is encapsulated in the lipid portion of lipid nanoparticle, or in the aqueous space enclosed by some or all of the lipid portion of lipid nanoparticle, thus protecting it from the enzyme degradation or other undesirable effects induced by the host organism or cell mechanism, such as harmful immune response.

[0216] In one embodiment, the composition comprises one or more cationic lipids and one or more stabilizing lipids, including neutral lipids and pegylated lipids.

[0217] In one embodiment, the composition comprises cationic lipid.The term " cationic lipid " used herein refers to the lipid that is cationic or becomes cationic (protonated) when pH is lower than the pK of the ionizable group of lipid, but gradually becomes more neutral at higher pH values.At pH values ​​lower than pK, lipid can bind with negatively charged nucleic acid.In certain embodiments, cationic lipid comprises zwitterionic lipid, which becomes positively charged with decreasing pH.

[0218] In certain embodiments, cationic lipid comprises any of several lipid species that are net positively charged at selected pH, such as physiological pH.Such lipids include but are not limited to N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1 Cationic lipids that can be used in the present invention include N-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). Additionally, many commercially available preparations of cationic lipids are available that can be used in the present invention. These include, for example, LIPOFECTIN® (a commercially available cationic liposome containing DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE) from GIBCO / BRL, Grand Island, NY); LIPOFECTAMINE® (a commercially available cationic liposome containing N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE) from GIBCO / BRL); and TRANSFECTAM® (a commercially available cationic lipid containing dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.).The following lipids are cationic and positively charged at sub-physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0219] In one embodiment, cationic lipid is amino lipid.Suitable amino lipid useful in the present invention includes those described in WO2012 / 016184, the entirety of which is incorporated herein by reference.Representative amino lipid includes but is not limited to 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl). , 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanediol (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

[0220] In certain embodiments, the cationic lipid is present in the composition in an amount of about 30 to about 95 mol%. In one embodiment, the cationic lipid is present in the composition in an amount of about 30 to about 70 mol%. In one embodiment, the cationic lipid is present in the composition in an amount of about 40 to about 60 mol%. In one embodiment, the cationic lipid is present in the composition in an amount of about 50 mol%. In one embodiment, the composition contains only cationic lipid.

[0221] In certain embodiments, the composition comprises one or more additional lipids that stabilize the particle formation during particle formation. Suitable stabilizing lipids include neutral lipids and anionic lipids.

[0222] The term "neutral lipid" refers to any of a number of lipid species that exist in either uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0223] Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl Neutral lipids include ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), distearoyl-phosphatidylethanolamine (DSPE)-maleimide-PEG, distearoyl-phosphatidylethanolamine (DSPE)-maleimide-PEG2000, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearyl-2-oleoyl-phosphatidylethanolamine (SOPE), stearoyloleoylphosphatidylcholine (SOPC) and 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (transDOPE).In one embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0224] In some embodiments, the composition comprises a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0225] In various embodiments, the composition further comprises a steroid or steroid analog. A "steroid" is a compound containing the following carbon skeleton: [ka]

[0226] In certain embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of the cationic lipid.

[0227] The term "anionic lipid" refers to lipids that are negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0228] In certain embodiments, the composition comprises a glycolipid (e.g., monosialoganglioside GM1). In certain embodiments, the composition comprises a sterol, such as cholesterol.

[0229] In some embodiments, the composition comprises a polymer-conjugated lipid. The term "polymer-conjugated lipid" refers to a molecule that comprises both a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that comprises both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-s-DMG) and the like.

[0230] In certain embodiments, the LNP comprises an additional stabilizing lipid that is a polyethylene glycol-lipid (PEGylated lipid). Suitable polyethylene glycol-lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxypoly(ethylene glycol) 2000 )carbamyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG. In another embodiment, the LNP is a PEGylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinate (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate. PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxyl)propyl)carbamate or 2,3-di(tetradecanoxyl)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of cationic lipid to PEGylated lipid ranges from about 100:1 to about 25:1.

[0231] In certain embodiments, the additional lipid is present in the LNP in an amount of about 1 mol% to about 10 mol%. In one embodiment, the additional lipid is present in the LNP in an amount of about 1 mol% to about 5 mol%. In one embodiment, the additional lipid is present in the LNP at about 1 mol% or about 1.5 mol%.

[0232] In certain embodiments, the nucleic acid molecule, when present in the lipid nanoparticle, is resistant to degradation by nucleases in aqueous solution.

[0233] In various embodiments, the composition comprises one or more transfection reagents. In another embodiment, the transfection reagent is a lipid-based transfection reagent. In another embodiment, the transfection reagent is a protein-based transfection reagent. In another embodiment, the transfection reagent is a polyethylenimine-based transfection reagent. In another embodiment, the transfection reagent is calcium phosphate. In another embodiment, the transfection reagent is Lipofectin®, Lipofectamine®, or TransIT®. In another embodiment, the transfection reagent is any other transfection reagent known in the art.

[0234] In another embodiment, the transfection reagent forms liposomes. In another embodiment, liposomes increase intracellular stability, increase uptake efficiency, and improve biological activity. In another embodiment, liposomes are hollow spherical vesicles composed of lipids arranged in a manner similar to that of the lipids that make up cell membranes. These, in another embodiment, have an internal aqueous space for entrapment of water-soluble compounds and range in size from 0.05 to several microns in diameter. In another embodiment, liposomes can deliver RNA to cells in a biologically active form.

[0235] In various embodiments, the compositions of the present invention can include any lipid capable of forming particles to which one or more nucleic acid molecules are attached or in which one or more nucleic acid molecules are encapsulated. The term "lipid" refers to a group of organic compounds that are derivatives (e.g., esters) of fatty acids and are generally insoluble in water but soluble in many organic solvents. Lipids are generally classified into at least three classes: (1) "simple lipids," which include oils, fats, and waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0236] In certain embodiments, the composition comprises one or more targeting moieties that can target the LNP to a cell, cell population, tissue, or any combination thereof of interest. For example, in one embodiment, the targeting moiety is a ligand that directs the LNP to a receptor found on the cell surface.

[0237] In certain embodiments, the composition comprises one or more internalization domains. For example, in one embodiment, the composition comprises one or more domains that bind to cells and induce internalization of the LNP. For example, in one embodiment, the one or more internalization domains bind to receptors found on the cell surface and induce receptor-mediated uptake of the LNP. In certain embodiments, the LNP can bind to a biomolecule in vivo, where the biomolecule bound to the LNP can be recognized by a cell surface receptor and induce internalization. For example, in one embodiment, the LNP binds to systemic ApoE, which results in the uptake of the LNP and associated cargo (e.g., one or more nucleic acid molecules, one or more therapeutic agents, or any combination thereof).

[0238] RNA is produced by in vitro transcription using synthetically produced plasmid DNA templates. DNA of interest from any source can be directly converted into a template for in vitro mRNA synthesis by PCR using appropriate primers and RNA polymerase. The source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences, or any other suitable source of DNA. In one embodiment, the desired template for in vitro transcription is an antigen capable of inducing an adaptive immune response, including, for example, an antigen associated with a pathogen or tumor, as described elsewhere herein. In one embodiment, the desired template for in vitro transcription is an adjuvant capable of enhancing the adaptive immune response.

[0239] In one embodiment, the DNA used for PCR contains an open reading frame. The DNA may be derived from a natural DNA sequence from the genome of an organism. In one embodiment, the DNA is a full-length gene of interest, a portion of the gene. The gene may include some or all of the 5' and / or 3' untranslated regions (UTRs). The gene may include exons and introns. In one embodiment, the DNA used for PCR is a human gene. In another embodiment, the DNA used for PCR is a human gene including 5' and 3' UTRs. In another embodiment, the DNA used for PCR is a gene from a pathogenic or symbiotic organism, including bacteria, viruses, parasites, and fungi. In another embodiment, the DNA used for PCR is derived from a pathogenic or symbiotic organism, including bacteria, viruses, parasites, and fungi, including 5' and 3' UTRs. Alternatively, the DNA may be an artificial DNA sequence that is not normally expressed in naturally occurring organisms. An exemplary artificial DNA sequence is one that contains portions of a gene that are linked together to form an open reading frame encoding a fusion protein. The portions of DNA that are linked together can be from a single organism or multiple organisms.

[0240] Genes that can be used as DNA sources for PCR include genes that encode polypeptides that induce or enhance an organism's adaptive immune response. Preferred genes are those useful for short-term treatments or when there are safety concerns regarding dosage or the expressed gene.

[0241] In various embodiments, the plasmid is used to generate a template for in vitro transcription of mRNA used in transfection.

[0242] Chemical structures capable of promoting stability and / or translation efficiency can also be used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is between 0 and 3,000 nucleotides in length. The length of the 5' and 3' UTR sequences added to the coding region can be varied by various methods, including, but not limited to, designing PCR primers that anneal to different regions of the UTR. Using this approach, those skilled in the art can modify the length of the 5' and 3' UTRs required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0243] The 5' and 3' UTRs can be the naturally occurring endogenous 5' and 3' UTRs of the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating UTR sequences into the forward and reverse primers or by any other modification of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying RNA stability and / or translation efficiency. For example, it is known that AU-rich elements in the 3' UTR sequence can reduce mRNA stability. Therefore, the 3' UTR can be selected or designed to increase the stability of the transcribed RNA based on the properties of UTRs well known in the art.

[0244] In one embodiment, the 5'UTR can contain the Kozak sequence of the endogenous gene. Alternatively, if a non-endogenous 5'UTR is added to the gene of interest by PCR as described above, the consensus Kozak sequence can be redesigned by adding a 5'UTR sequence. Although the Kozak sequence can increase the translation efficiency of some RNA transcripts, it does not appear to be necessary for all RNAs to enable efficient translation. The requirement for a Kozak sequence for many mRNAs is known in the art. In another embodiment, the 5'UTR can be derived from an RNA virus whose RNA genome is stable in cells. In another embodiment, various nucleotide analogs can be used in the 3' or 5'UTR to prevent exonuclease degradation of mRNA.

[0245] To enable RNA synthesis from a DNA template without the need for gene cloning, a transcription promoter must be added to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame to be transcribed. In one preferred embodiment, the promoter is a T7 RNA polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3, and SP6 promoters are known in the art.

[0246] In a preferred embodiment, the mRNA has both a cap at the 5' end and a 3' poly(A) tail, which determine ribosome binding, translation initiation, and mRNA stability within the cell. On circular DNA templates, such as plasmid DNA, RNA polymerase generates long concatemeric products that are not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the end of the 3' UTR results in normal-sized mRNA, which, when polyadenylated after transcription, is effective for eukaryotic transfection.

[0247] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003)).

[0248] The traditional method for incorporating polyA / T stretches into DNA templates is molecular cloning. However, polyA / T sequences incorporated into plasmid DNA can lead to plasmid instability, which can be ameliorated by using recombination-incompetent bacterial cells for plasmid propagation.

[0249] The poly(A) tail of RNA can be further extended after in vitro transcription using poly(A) polymerases such as Escherichia coli poly(A) polymerase (E-PAP) or yeast poly(A) polymerase. In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides increases the RNA translation efficiency by approximately two-fold. Furthermore, attachment of different chemical groups to the 3' end can enhance mRNA stability. Such attachments can include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further enhance RNA stability.

[0250] The 5' cap also provides stability to the mRNA molecule. In a preferred embodiment, the RNA produced by this method contains a 5' cap1 structure. Such a cap1 structure can be generated using vaccinia capping enzyme and 2'-O-methyltransferase enzyme (CellScript, Madison, WI). Alternatively, the 5' cap can be provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0251] Nucleic acid sequences encoding antigens or adjuvants can be obtained using standard techniques, such as recombinant methods known in the art, by screening libraries from cells which express the gene, by deriving the gene from a vector known to contain the same, or by direct isolation from cells or tissues containing the same. Alternatively, the gene of interest can be produced synthetically.

[0252] Nucleic acids can be cloned into many types of vectors, including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0253] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle).

[0254] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo) is contemplated. In another embodiment, the nucleic acid can be associated with a lipid. Lipid-bound nucleic acids can be encapsulated within the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, bound to the liposome via a linking molecule attached to both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a lipid-containing liquid, mixed with the lipid, combined with the lipid, contained in a suspension in the lipid, comprised in or complexed with a micelle, or associated with the lipid. Lipid, lipid / RNA, or lipid / expression vector-associated compositions are not limited to a particular structure in solution. For example, they can exist as micelles in a bilayer structure or in a "collapsed" structure. They can also simply be dispersed in the solution or form aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic lipids. For example, lipids include the lipid droplets naturally occurring in the cytoplasm and a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0255] Suitable lipids for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO. Dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY). Cholesterol ("Chol") can be obtained from Calbiochem-Behring. Dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components self-rearrange to form a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions with structures in solution that differ from the typical vesicle structure are also included. For example, lipids may adopt a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0256] LNP vaccine In one embodiment of the present invention, the compositions described herein are vaccines. For a composition to be useful as a vaccine, it must induce an adaptive immune response to an antigen in a cell, tissue, or mammal (e.g., a human). In certain instances, the vaccine induces a protective immune response in a mammal. As used herein, an "immunogenic composition" can include an antigen (e.g., a peptide or polypeptide), a nucleic acid encoding the antigen, a cell expressing or presenting the antigen or a cellular component, or a combination thereof. In certain embodiments, the composition comprises or encodes all or a portion of any peptide antigen described herein, or an immunogenically functional equivalent thereof. In another embodiment, the composition is a mixture containing an additional immunostimulatory agent or a nucleic acid encoding such an agent. Immunostimulatory agents include, but are not limited to, additional antigens, immunomodulatory agents, antigen-presenting cells, or adjuvants. In another embodiment, one or more additional agents are covalently linked to the antigen or immunostimulatory agent, in any combination. In certain embodiments, the antigenic composition is bound to or comprises an HLA anchor motif amino acid.

[0257] In the context of the present invention, the term "vaccine" refers to a substance that induces immunity when inoculated into an animal.

[0258] Vaccines of the present invention may vary in the composition of nucleic acids and / or cellular components. In a non-limiting example, the nucleic acid encoding the antigen may also be formulated with an adjuvant. Of course, it will be understood that the various compositions described herein may further comprise additional components. For example, one or more vaccine components may be contained in lipids, liposomes, or lipid nanoparticles. In another non-limiting example, the vaccine may include one or more adjuvants. Vaccines of the present invention and their various components may be prepared and / or administered by any method disclosed herein or that would be known to one of skill in the art in light of the present disclosure.

[0259] The induction of immunity by expression of an antigen can be detected by observing the response of all or part of the host's immune system to the antigen in vivo or in vitro.

[0260] For example, methods for detecting the induction of cytotoxic T lymphocytes are well known. Foreign substances that enter the body are presented to T cells and B cells by the action of APCs. T cells that respond specifically to the antigens presented by APCs differentiate into cytotoxic T cells (also called cytotoxic T lymphocytes or CTLs) upon stimulation with the antigen. These antigen-stimulated cells then proliferate. This process is referred to herein as "activation" of T cells. Therefore, CTL induction by epitopes of polypeptides, peptides, or combinations thereof can be evaluated by presenting epitopes of polypeptides, peptides, or combinations thereof to T cells by APCs and detecting the induction of CTLs. Furthermore, APCs have the ability to activate B cells, CD4+ T cells, CD8+ T cells, macrophages, eosinophils, and NK cells.

[0261] Methods for evaluating the CTL induction effect using dendritic cells (DCs) as APCs are well known in the art. DCs are a representative APC with strong CTL induction activity. In the method of the present invention, epitopes of a polypeptide, peptide, or combination thereof are first expressed by DCs, and then the DCs are contacted with T cells. Detection of T cells with cytotoxic activity against the target cells after contact with DCs indicates that the epitopes of the polypeptide, peptide, or combination thereof have the activity of inducing cytotoxic T cells. Furthermore, the induced immune response can also be tested by measuring IFN-gamma produced and released by CTLs in the presence of antigen-presenting cells carrying immobilized peptides or peptide combinations, visualized using an anti-IFN-gamma antibody, such as in an ELISPOT assay.

[0262] Besides DCs, peripheral blood mononuclear cells (PBMCs) can also be used as APCs. It has been reported that the induction of CTLs is enhanced by culturing PBMCs in the presence of GM-CSF and IL-4. Similarly, CTLs have been shown to be induced by culturing PBMCs in the presence of keyhole limpet hemocyanin (KLH) and IL-7.

[0263] Antigens confirmed to have CTL-inducing activity by these methods are antigens that activate DCs and subsequently induce CTLs. Furthermore, CTLs that acquire cytotoxicity upon antigen presentation by APCs can be used as vaccines against antigen-related disorders.

[0264] The induction of immunity by expression of an antigen can be further confirmed by observing the induction of antibody production against the antigen. For example, if antibodies against the antigen are induced in an experimental animal immunized with a composition encoding the antigen, and if antigen-related pathologies are suppressed by these antibodies, the composition is determined to induce immunity.

[0265] The induction of immunity by antigen expression can be further confirmed by observing the induction of CD4+ T cells. Although CD4+ T cells can also lyse target cells, they primarily serve to induce other types of immune responses, such as CTL and antibody production. The types of CD4+ T cell help are Th1, Th2, Th9, Th17, T regulatory, or T follicular helper (T fh Each subtype of CD4+ T cell supply contributes to a particular type of immune response. Of particular interest to the present invention are T fh Subtyping is useful for generating high affinity antibodies.

[0266] Pharmaceutical LNP compositions The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing into association the active ingredient with the carrier or one or more other accessory ingredients, and then, if necessary or desired, shaping or packaging the product into the desired single or multi-dosage unit.

[0267] Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for medical administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to all types of animals. Modifications to pharmaceutical compositions suitable for administration to humans to make the compositions suitable for administration to various animals are well understood, and an ordinarily skilled veterinary pharmacologist can design and implement such modifications, if any, with routine experimentation. Subjects to which the pharmaceutical compositions of the present invention are intended for administration include, but are not limited to, humans and other primates, non-human primates, and commercially relevant mammals, such as cows, pigs, horses, sheep, cats, and dogs.

[0268] Pharmaceutical compositions useful in the methods of the invention may be prepared, packaged, or sold in a formulation suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intraventricular, intradermal, intramuscular, subcutaneous, intracerebroventricular, intrathecal, intratracheal, intraperitoneal, intrauterine delivery, or another route of administration, or any combination thereof. Other contemplated formulations include engineered nanoparticles, liposomal preparations, resealed red blood cells containing the active ingredient, and immunogenic-based formulations.

[0269] The pharmaceutical compositions of the present invention can be prepared, packaged, or sold in bulk as a unit dose or as a plurality of unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient that would be administered to a subject, or a convenient fraction of such a dose, for example, one-half or one-third of such a dose.

[0270] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary depending on the identity, size, and condition of the subject being treated, as well as the route by which the composition is administered. By way of example, the composition may contain from 0.1% to 100% (w / w) of the active ingredient.

[0271] In addition to the active ingredient, the pharmaceutical compositions of the present invention may further comprise one or more additional pharmaceutically active substances.

[0272] Controlled- or sustained-release formulations of the pharmaceutical compositions of the invention can be prepared using conventional techniques.

[0273] As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical disruption of a subject's tissue, and any route of administration of a pharmaceutical composition via tissue disruption. Thus, parenteral administration includes, but is not limited to, administering a pharmaceutical composition by injection of the composition, by applying the composition through a surgical incision, by applying the composition through a tissue-penetrating non-surgical wound, and the like. In some embodiments, parenteral administration includes, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intrauterine delivery, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intraventricular administration, and kidney dialysis infusion techniques.

[0274] Pharmaceutical compositions suitable for parenteral administration include the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients, including, but not limited to, suspending agents, stabilizers, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water), after which the reconstituted composition is administered parenterally.

[0275] Pharmaceutical compositions can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. These suspensions or solutions can be prepared according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations can be prepared using a non-toxic, parenterally-acceptable diluent or solvent, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils, such as synthetic mono- or diglycerides. Other useful parenterally-administrable formulations include those comprising the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may include pharmaceutically acceptable polymers or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts.

[0276] Pharmaceutical compositions of the present invention can be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such formulations can comprise dry particles, including the active ingredient, having diameters ranging from about 0.5 to about 7 nanometers, preferably from about 1 to about 6 nanometers. Such compositions are conveniently in the form of a dry powder for administration using a device comprising a dry powder reservoir capable of directing a stream of propellant to disperse the powder, or using a self-propelling solvent / powder dispensing container, such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. Preferably, such powders comprise particles in which at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions preferably include a solid fine powder diluent, such as sugar, and are conveniently provided in unit dosage form.

[0277] Low-boiling propellants generally include liquid propellants having a boiling point below 65°F at atmospheric pressure. Generally, the propellant may comprise 50-99.9% (w / w) of the composition, and the active ingredient may comprise 0.1-20% (w / w) of the composition. The propellant may further comprise additional components such as a liquid nonionic or solid anionic surfactant, or a solid diluent (preferably having a particle size on the same order as the particles containing the active ingredient).

[0278] Pharmaceutical compositions suitable for parenteral administration include the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients, including, but not limited to, suspending agents, stabilizers, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water), after which the reconstituted composition is administered parenterally.

[0279] Pharmaceutical compositions can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. These suspensions or solutions can be prepared according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations can be prepared using a non-toxic, parenterally-acceptable diluent or solvent, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils, such as synthetic mono- or diglycerides. Other useful parenterally-administrable formulations include those comprising the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may include pharmaceutically acceptable polymers or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts.

[0280] As used herein, "additional ingredients" includes, but is not limited to, one or more of the following: excipients; surfactants; dispersing agents; inert diluents; granulating and disintegrating agents; binders; lubricants; sweeteners; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0281] Therapeutic compounds or compositions of the invention can be administered prophylactically (i.e., to prevent a disease or disorder) or therapeutically (i.e., to treat a disease or disorder) to subjects suffering from or at risk of (or susceptible to) developing a disease or disorder. Such subjects can be identified using standard clinical methods. In the context of the present invention, prophylactic administration occurs before overt clinical symptoms of the disease appear, such that the disease or disorder is prevented or its progression is delayed. In the medical context, the term "preventing" encompasses any activity that reduces the burden of mortality or morbidity due to a disease. Prevention can occur at primary, secondary, or tertiary levels of prevention. Primary prevention avoids the onset of disease, while secondary and tertiary levels include activities aimed at reducing the adverse effects of an already established disease and reducing disease-related complications by preventing the progression of the disease and the appearance of symptoms, as well as restoring function.

[0282] Delivery method In one aspect, the present invention provides a method for delivering a nucleic acid molecule, a therapeutic agent, or any combination thereof, to a desired target. Examples of such targets include, but are not limited to, immune cells, T cells, resident T cells, B cells, natural killer (NK) cells, cancerous cells, cells associated with a disease or disorder, disease or disease-associated tissue, brain tissue, central nervous system tissue, lung tissue, apical surface tissue, epithelial cells, endothelial cells, liver tissue, intestinal tissue, colon tissue, small intestine tissue, large intestine tissue, feces, bone marrow, macrophages, spleen tissue, muscle tissue, joint tissue, tumor cells, diseased tissue, lymph node tissue, lymphatic circulation, or any combination thereof. In various embodiments, the method comprises administering a therapeutically effective amount of one or more compositions of the present invention.

[0283] For example, in some embodiments, the present invention provides methods for delivering a nucleic acid molecule, a therapeutic agent, or any combination thereof, to a cell, including, but not limited to, a T cell, a B cell, a natural killer (NK) cell, a cancerous cell, a cell associated with a disease or disorder, and any combination thereof.

[0284] In one embodiment, the method is a gene delivery method.

[0285] In one embodiment, this method involves the IVT RNA described herein, which can be introduced into a target of interest (e.g., a cell, tissue, etc.) as a form of transient transfection using an LNP composition of the invention.

[0286] In one embodiment, the method comprises a single administration of the composition. In one embodiment, the method comprises multiple administrations of the composition.

[0287] In some embodiments, the composition is administered by an intradermal delivery route, a subcutaneous delivery route, an intramuscular delivery route, an intraventricular delivery route, an intrathecal delivery route, an oral delivery route, an intravenous delivery route, an intratracheal delivery route, an intraperitoneal delivery route, an intrauterine delivery route, or any combination thereof.

[0288] In some embodiments, methods for delivery of nucleic acid molecules, therapeutic agents, or any combination thereof, to a target of interest (e.g., a cell, a tissue, etc.), comprising administering a therapeutically effective amount of a composition of the invention, are carried out simultaneously with any of a number of different methods, e.g., commercially available methods, including, but not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), ECM830(BTX) (Harvard Instruments, Boston, Mass.), or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), TransIT®-mRNA transfection kit (Mirus, Madison WI), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or a "gene gun" (e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70). (2001)).

[0289] In certain instances, expressing proteins by delivering encoding mRNA offers numerous advantages over methods using proteins, plasmid DNA, or viral vectors. During mRNA transfection, the coding sequence for the desired protein is the only substance delivered to the cell, avoiding all side effects associated with plasmid backbones, viral genes, and viral proteins. More importantly, unlike DNA- and viral-based vectors, mRNA does not risk integration into the genome, and protein production begins immediately after mRNA delivery. For example, high levels of circulating protein have been measured within 15–30 minutes of in vivo injection of encoding mRNA. In certain embodiments, using mRNA rather than protein also offers numerous advantages. Proteins often have short half-lives in the circulation, necessitating frequent administration, whereas mRNA provides a template for continuous protein production over several days. Protein purification can be problematic and may contain aggregates and other impurities that can cause adverse effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050:257-265).

[0290] A variety of assays can be performed to confirm the presence of an mRNA sequence in a host cell, including, for example, "molecular biological" assays known to those skilled in the art, such as Northern blotting and RT-PCR, and "biochemical" assays, such as detecting the presence or absence of specific peptides, to identify agents within the scope of the invention by immunogenic means (ELISA and Western blot) or by the assays described herein.

[0291] In one aspect, the present invention also discloses a method for delivering a nucleic acid molecule, a therapeutic agent, or any combination thereof to a subject in need thereof. In various embodiments, the method comprises administering to the subject a therapeutically effective amount of one or more compositions of the present invention. In various embodiments, the method comprises a composition of the present invention delivering the nucleic acid molecule, the therapeutic agent, or any combination thereof to cells, tissues, or both of the subject.

[0292] treatment method The present invention provides a method for inducing an adaptive immune response in a subject, comprising administering an effective amount of a composition of the present invention. For example, in some embodiments, the composition comprises one or more lipids or LNPs of the present invention. In some embodiments, the composition comprises one or more antigens, one or more nucleic acids encoding one or more antigens, or any combination thereof, and one or more lipids or LNPs of the present invention.

[0293] In one embodiment, the method provides a subject with immunity to an infection, cancer, or disease or disorder associated with the antigen. Thus, the present invention provides methods for treating or preventing an infection, cancer, or disease or disorder associated with an antigen. Exemplary antigens and associated infections, diseases, and tumors are described elsewhere herein.

[0294] For example, the method may be used to treat or prevent viral infections, bacterial infections, fungal infections, parasitic infections, arthritis, heart disease, cardiovascular disease, neurological disorders or diseases, genetic diseases, autoimmune diseases, fetal diseases, genetic diseases affecting fetal development, or cancer, depending on the type of antigen in the administered composition.

[0295] The following are non-limiting examples of cancers that can be treated by the disclosed methods and compositions: acute lymphoblastic; acute myeloid leukemia; adrenocortical carcinoma; adrenocortical carcinoma, childhood; appendix cancer; basal cell carcinoma; cholangiocarcinoma, extrahepatic; bladder cancer; bone cancer; osteosarcoma and malignant fibrous histiocytoma; brain stem glioma, childhood; brain tumor, adult; brain tumor, brain stem glioma, childhood; brain tumor, atypical teratoma / rhabdoid tumor of the central nervous system, childhood; central nervous system embryonal tumor; cerebellar astrocytoma; cerebral astrocytoma / malignant glioma; craniopharyngioma; epidermoma; ependymoma; medulloblastoma; medulloepithelioma; moderately differentiated pineal parenchymal tumor; supratentorial tumor Primitive neuroectodermal tumors and pineoblastic tumors; visual pathway and hypothalamic gliomas; brain and spinal cord tumors; breast cancer; bronchial tumors; Burkitt's lymphoma; carcinoid tumors; carcinoid tumors, gastrointestinal tract; atypical malformations / rhabdoid tumors of the central nervous system; central nervous system embryonal tumors; central nervous system lymphomas; cerebellar astrocytoma; cerebral astrocytoma / malignant glioma, childhood; cervical cancer; chordoma, childhood; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; esophageal cancer; Ewing family tumors; extragonadal germ cell tumors; extrahepatic bile duct cancer; eye Cancer of the eye, intraocular melanoma;eye cancer, retinoblastoma;gallbladder cancer;gastric (stomach) cancer;gastrointestinal carcinoid tumor;gastrointestinal stromal tumor (gist);germ cell tumor, extracranial;germ cell tumor, extragonadal;germ cell tumor, ovarian;gestational trophoblastic tumor;glioma;glioma, pediatric brain stem;glioma, pediatric brain astrocytoma;glioma, pediatric visual pathway and hypothalamus;hairy cell leukemia;head and neck cancer;hepatocellular (liver) cancer;histiocytosis, Langerhans cell;Hodgkin's lymphoma;hypopharyngeal cancer;hypothalamic and visual pathway glioma;intraocular melanoma;pancreatic islet cell tumor;kidney (renal cell) cancer;Langerhans cell histiocytosis;laryngeal cancer;leukemia , acute lymphoblastic; leukemia, acute myeloid; leukemia, chronic lymphocytic; leukemia, chronic myeloid; leukemia, hairy cell; lip and oral cancer; liver cancer; lung cancer, non-small cell; lung cancer, small cell; lymphoma, AIDS-related; lymphoma, Burkitt's; lymphoma, cutaneous T-cell; lymphoma, non-Hodgkin's lymphoma; lymphoma, primary central nervous system; macroglobulinemia, Waldenstrom; malignant fibrous histiocytoma and osteosarcoma of bone; medulloblastoma; melanoma; melanoma, intraocular (eye); Merkel cell carcinoma; mesothelioma; metastatic squamous cell carcinoma of the neck with occult primary; oral cancer; multiple endocrine neoplasia syndrome, (childhood);Multiple myeloma / plasma cell neoplasms; mycoses; mycosis fungoides; myelodysplastic syndromes; myelodysplastic / myeloproliferative disorders; myeloid leukemia, chronic; myeloid leukemia, adult acute; myeloid leukemia, childhood acute; myeloma, multiple; myeloproliferative disorders, chronic; nasal cavity and paranasal sinus cancer; nasopharyngeal carcinoma; neuroblastoma; non-small cell lung cancer; oral cancer; oral cancer; oropharyngeal cancer; osteosarcoma and malignant fibrous histiocytoma of bone; ovarian cancer; ovarian epithelial carcinoma; ovarian germ cell tumors; low-grade tumors of the ovary; pancreatic cancer; pancreatic cancer, islet cell tumors; papillomatosis; parathyroid carcinoma; penile cancer; pharyngeal cancer; pheochromocytoma; moderately differentiated pineal parenchymal tumors; pineoblastoma and supratentorial primitive neuroectodermal tumors; pituitary tumors; plasma cell neoplasms / multiple myeloma; pleuropulmonary blastoma; primary central nervous system lymphoma; prostate cancer; orthotopic Intestinal cancer; renal cell (kidney) cancer; renal pelvis and ureter, transitional cell carcinoma; respiratory tract cancer involving the nut gene on chromosome 15; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; sarcoma, Ewing's sarcoma; sarcoma, Kaposi's sarcoma; sarcoma, soft tissue; sarcoma, uterine; Sézary syndrome; skin cancer (non-melanoma); skin cancer (melanoma); skin cancer, Merkel cell; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma, occult primary, squamous cell carcinoma of the cervix with metastasis; gastric (stomach) cancer; supratentorial primitive neuroectodermal tumor; T-cell lymphoma, skin; testicular cancer; throat cancer; thymoma and thymic carcinoma; thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; trophoblastic tumor, pregnancy; urethral cancer; uterine cancer, endometrium; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenstrom's macroglobulinemia; and Wilms' tumor.

[0296] In one embodiment, the composition is administered to a subject with an infectious disease, disease, heart disease, cardiovascular disease, neurological disorder or disease, genetic disease, autoimmune disease, or antigen-associated cancer. In one embodiment, the composition is administered to a subject at risk of developing an infectious disease, disease, heart disease, cardiovascular disease, neurological disorder or disease, genetic disease, autoimmune disease, or antigen-associated cancer. For example, the composition can be administered to a subject at risk of contact with a virus, bacteria, fungus, parasite, or the like. In one embodiment, the composition is administered to a subject who is likely to develop cancer due to genetic factors, environmental factors, or the like.

[0297] In some embodiments, the composition is administered by an intradermal delivery route, a subcutaneous delivery route, an intramuscular delivery route, an intracerebroventricular delivery route, an intrathecal delivery route, an oral delivery route, an intravenous delivery route, an intratracheal delivery route, an intraperitoneal delivery route, an intrauterine delivery route, or any combination thereof.

[0298] In another embodiment, the compositions of the present invention comprising RNA encoding an antigen induce a significantly more adaptive immune response than unmodified in vitro synthesized RNA molecules having the same sequence. In another embodiment, the compositions exhibit a two-fold greater adaptive immune response than their unmodified counterparts. In another embodiment, the adaptive immune response is increased three-fold. In another embodiment, the adaptive immune response is increased five-fold. In another embodiment, the adaptive immune response is increased seven-fold. In another embodiment, the adaptive immune response is increased ten-fold. In another embodiment, the adaptive immune response is increased fifteen-fold. In another embodiment, the adaptive immune response is increased twenty-fold. In another embodiment, the adaptive immune response is increased fifty-fold. In another embodiment, the adaptive immune response is increased one hundred-fold. In another embodiment, the adaptive immune response is increased two hundred-fold. In another embodiment, the adaptive immune response is increased five hundred-fold. In another embodiment, the adaptive immune response is increased one thousand-fold. In another embodiment, the adaptive immune response is increased two thousand-fold. In another embodiment, the adaptive immune response is increased by another fold difference.

[0299] In another embodiment, "inducing a significantly greater adaptive immune response" refers to a detectable increase in the adaptive immune response. In another embodiment, this term refers to a fold increase in the adaptive immune response (e.g., one of the fold increases listed above). In another embodiment, this term refers to the ability of the composition of the present invention comprising RNA to induce an effective adaptive immune response even when administered at a lower dose or frequency than an isolated RNA molecule of the same species. In another embodiment, the increase is such that the composition of the present invention comprising RNA can be administered using a single dose to induce an effective adaptive immune response.

[0300] In another embodiment, the composition of the present invention comprising RNA exhibits significantly less natural immunogenicity than an isolated in vitro synthesized RNA molecule having the same sequence. In another embodiment, the composition of the present invention comprising RNA exhibits a 2-fold less natural immune response than its isolated counterpart. In another embodiment, the natural immunogenicity is reduced by 3-fold. In another embodiment, the natural immunogenicity is reduced by 5-fold. In another embodiment, the natural immunogenicity is reduced by 7-fold. In another embodiment, the natural immunogenicity is reduced by 10-fold. In another embodiment, the natural immunogenicity is reduced by 15-fold. In another embodiment, the natural immunogenicity is reduced by 20-fold. In another embodiment, the natural immunogenicity is reduced by 50-fold. In another embodiment, the natural immunogenicity is reduced by 100-fold. In another embodiment, the natural immunogenicity is reduced by 200-fold. In another embodiment, the natural immunogenicity is reduced by 500-fold. In another embodiment, the natural immunogenicity is reduced by 1000-fold. In another embodiment, the natural immunogenicity is reduced by 2000-fold. In another embodiment, the natural immunogenicity is reduced by another fold difference.

[0301] In another embodiment, "exhibiting significantly less natural immunogenicity" refers to a detectable reduction in natural immunogenicity. In another embodiment, this term refers to a fold reduction in natural immunogenicity (e.g., one of the fold reductions listed above). In another embodiment, this term refers to a reduction such that an effective amount of the composition of the present invention comprising RNA can be administered without inducing a detectable natural immune response. In another embodiment, this term refers to a reduction such that the composition of the present invention comprising RNA can be repeatedly administered without inducing a natural immune response sufficient to detectably reduce the production of the recombinant protein. In another embodiment, the reduction is such that the composition of the present invention comprising RNA can be repeatedly administered without inducing a natural immune response sufficient to eliminate the detectable production of the recombinant protein.

[0302] In one embodiment, the present invention relates, in part, to a method of preventing or treating a disease or disorder in a subject in need thereof. In various embodiments, the method comprises administering to the subject a therapeutically effective amount of a composition of the present invention. In some embodiments, the composition delivers a nucleic acid molecule, a therapeutic agent, or a combination thereof to a desired target (e.g., a cell, a tissue, etc.).

[0303] In one embodiment, the method comprises administering a composition comprising one or more nucleic acid molecules encoding one or more antigens and one or more adjuvants. In one embodiment, the method comprises administering a composition comprising a first nucleic acid molecule encoding one or more antigens and a second nucleic acid molecule encoding one or more adjuvants. In one embodiment, the method comprises administering a first composition comprising one or more nucleic acid molecules encoding one or more antigens and a second composition comprising one or more nucleic acid molecules encoding one or more adjuvants.

[0304] In certain embodiments, the method comprises administering to the subject multiple nucleic acid molecules encoding multiple antigens, adjuvants, or combinations thereof.

[0305] In certain embodiments, the methods of the invention allow for sustained expression of the antigens or adjuvants described herein for at least several days after administration, however, in certain embodiments, the methods also provide for transient expression, since the nucleic acid is not integrated into the subject's genome.

[0306] In certain embodiments, the method comprises administering RNA that provides stable expression of an antigen or adjuvant described herein. In some embodiments, administration of the RNA induces an effective adaptive immune response while eliciting little or no innate immune response.

[0307] The administration of the compositions of the present invention in the therapeutic method can be achieved in several different ways using methods known in the art. In one embodiment, the method of the present invention comprises systemic administration to the subject, including, for example, enteral or parenteral administration. In certain embodiments, the method comprises intradermal delivery of the composition. In another embodiment, the method comprises intravenous delivery of the composition. In some embodiments, the method comprises intramuscular delivery of the composition. In one embodiment, the method comprises subcutaneous delivery of the composition. In one embodiment, the method comprises inhalation of the composition. In one embodiment, the method comprises intranasal delivery of the composition.

[0308] It will be appreciated that the compositions of the present invention can be administered to a subject alone or in combination with another agent.

[0309] Accordingly, the therapeutic and prophylactic methods of the present invention encompass the use of pharmaceutical compositions encoding the antigens, adjuvants, or combinations thereof described herein to practice the methods of the present invention. Pharmaceutical compositions useful for practicing the present invention can be administered to deliver doses ranging from ng / kg / day to 100 mg / kg / day. In one embodiment, the present invention contemplates the administration of a dose that results in a concentration of the compound of the present invention in a mammal ranging from 10 nM to 10 μM.

[0310] Typically, the dosage that can be administered to a mammal, preferably a human, in the methods of the present invention ranges from 0.01 μg to about 50 mg per kilogram of mammalian body weight, although the exact dosage administered will vary depending on many factors, including, but not limited to, the type of mammal and the type of condition being treated, the mammal's age, and the route of administration. Preferably, the dosage of the compound will range from about 0.1 μg to about 10 mg per kilogram of mammalian body weight. More preferably, the dosage will range from about 1 μg to about 1 mg per kilogram of mammalian body weight.

[0311] The composition may be administered to the mammal several times daily, or less frequently, such as once daily, once weekly, once every two weeks, once a month, or even less frequently, such as once every few months or once a year or less. The frequency of administration will be readily apparent to one of skill in the art and will depend on any number of factors, including, but not limited to, the type and severity of the disease being treated, the species and age of the mammal, etc.

[0312] In certain embodiments, administration of the compositions or vaccines of the present invention can be performed by a single dose or can be boosted by multiple doses.

[0313] In one embodiment, the invention includes a method comprising administering one or more compositions encoding one or more antigens or adjuvants described herein. In certain embodiments, the method has an additive effect, where the overall effect of administering the combination is approximately equal to the sum of the effects of administering each antigen or adjuvant. In another embodiment, the method has a synergistic effect, where the overall effect of administering the combination is greater than the sum of the effects of administering each antigen or adjuvant.

[0314] In one embodiment, this method comprises systemic administration of the composition to the subject, including, for example, intradermal administration. In certain embodiments, this method comprises administering multiple doses to the subject. In another embodiment, this method comprises administering a single dose of the composition, wherein the single dose is effective to induce an adaptive immune response.

[0315] Experimental Example The present invention will be described in further detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and, unless otherwise specified, are not intended to be limiting. Therefore, the present invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass all modifications that become evident as a result of the teachings provided herein.

[0316] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following examples therefore specifically point out preferred embodiments of the present invention, and are not to be construed as limiting the remainder of the disclosure. [Example]

[0317] Example 1: Ionizable lipid nanoparticles for mRNA-based T cell engineering Nanoparticle (NP)-based delivery systems, composed of lipid- and polymer-based materials, offer a promising avenue to overcome the challenges faced using mechanical and viral cell engineering approaches (DiTommaso T et al., 2018, PNAS, 115; Hajj KA et al., 2017, Nat Rev Mater, 2; McKinlay CJ et al., 2018, PNAS, 115:E5859-E5866, Mukalel AJ et al., 2019, Cancer Lett, 458:102-112). NPs have many potential advantages, including the ability to stabilize nucleic acid cargo, aid intracellular delivery, and reduce toxicity (Pardi N et al., 2018, Nat Rev Drug Discov, 17:261-279; Fornaguera C et al., 2018, Adv Healthc Mater, 7:1-11; Zhang R et al., 2018, J Control Release, 292:256-276; Islam MA et al., 2015, Biomater Sci, 1519-1533). Several studies have been conducted on polymer-based NPs for mRNA delivery to cells, with promising results, including reduced toxicity compared to EPs (CJ et al., 2018, PNAS, 115:E5859-E5866; Olden BR et al., 2018, J Control Release, 282:140-147; Moffett HF et al., 2017, Nat Commun, 8:389; Demoulins T et al., 2016, Biol Med, 12:711-722; Anderson DG et al., 2003, Angew Chem Int Ed Engl, 42:3153-3158).

[0318] However, ionizable lipid nanoparticle (LNP) delivery systems, with approval of Alnylam's Onpattro (Pardi N et al., 2018, Nat Rev Drug Discov, 17:261-279; Garber K et al., 2018, Nat Biotechnol, 36:777), have made clinical advances over polymers in the context of RNA delivery. Furthermore, LNPs possess an ionizable lipid core, which remains neutral at physiologically relevant pH but accumulates charge in acidic environments such as endosomes, ultimately aiding in endosomal escape and resulting in potent intracellular nucleic acid delivery (Hajj KA et al., 2017, Nat Rev Mater, 2; Kauffman KJ et al., 2016, J Control Release, 240:227-234; Oberli MA et al., 2017, Nano Lett, 17:1326-1335; Fan YN et al., 2018, Biomater Sci Royal Society of Chemistry, 6:3009-3018). It has been validated in a variety of cell types, including immune cells, with minimal toxicity, and previous studies in lymphocyte delivery have shown that LNPs deliver mRNA more effectively than commercially available lipofectamine (Hajj KA et al., 2017, Nat Rev Mater, 2; McKinlay CJ et al., 2018, PNAS, 115:E5859-E5866; Zhang R et al., 2018, J Control Release, 292:256-276; Kauffman KJ et al., 2016, J Control Release, 240:227-234; Oberli MA et al., 2017, Nano Lett, 17:1326-1335; Love KT et al., 2010, Proc Natl Acad Sci, 107:9915-9915).

[0319] Furthermore, the easily tunable composition of LNPs allows for tuning of their physicochemical properties to maximize uptake into specific cell types, and their ionizable nature allows for electrostatic complexation with negatively charged nucleic acid cargoes (Hajj KA et al., 2017, Nat Rev Mater, 2; McKinlay CJ et al., 2018, PNAS, 115:E5859-E5866; Zhang R et al., 2018, J Control Release, 292:256-276; Kauffman KJ et al., 2016, J Control Release, 240:227-234; Love KT et al., 2010, Proc Natl Acad Sci, 107:9915-9915; Kauffman KJ et al., 2015, Nano Lett, 15:7300-7306).

[0320] More specifically, a diverse library of 24 LNPs was generated (Figure 2A), characterized (Figure 2B), and screened for luciferase mRNA delivery to Jurkat cells, an immortalized human T cell line. Ionizable lipids were first synthesized by Michael addition chemistry, in which a polyamine core was reacted with excess epoxide-terminated alkyl chains of various lengths (Figure 2C). The lipids were then evaluated for mRNA delivery to T cells.

[0321] To prepare LNPs, ionizable lipids were combined in ethanol with three other excipients: (i) cholesterol for LNP stability and membrane fusion, (ii) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DOPE) to strengthen the LNP bilayer structure and promote endosomal escape, and (iii) C14-PEG to reduce aggregation and nonspecific endocytosis (Granot Y et al., 2017, Semin Immunol, 34:68-77; Varkouhi AK et al., 2011, J Control Release, 151:220-228; Mui BL et al., 2013, Mol Ther Acids, 2:1-8). This ethanol phase was then mixed with the aqueous mRNA phase in a microfluidic device (Figure 1). These excipients and their molar ratios were selected based on previously optimized LNP formulations for mRNA delivery, which generally utilized (i) DOPE as the phospholipid component, (ii) reduced molar percentages of ionizable lipids, and (iii) increased concentrations of cholesterol and lipid-PEG (Kauffman KJ et al., 2015, Nano Lett, 15: 300-7306; Ball RL et al., 2018, Nano Lett, 18:3814-3822). The ratio of components was kept constant throughout these experiments because changes in the molar ratio of excipients affect the physicochemical properties and ultimately the potent delivery of LNPs (Kauffman KJ et al., 2015, Nano Lett, 15: 300-7306; Ball RL e al., 2018, Nano Lett, 18:3814-3822; Cheng Q et al., 2018, Adv Mater, 30:1805308).

[0322] To evaluate the ability of LNPs to deliver functional mRNA, luciferase was selected as the encoded reporter protein. This screening revealed seven LNP formulations that enhanced mRNA delivery compared to the commonly used transfection reagent, Lipofectamine (Cardarelli F et al., 2016, Sci Reports, 6:25879). Furthermore, when 24 LNPs were screened for mRNA delivery to Jurkat cells (immortalized human T cells), the best LNP formulation, C14-4 LNP, was selected for further development due to its potent delivery and low toxicity. Next, optimizing C14-4 LNPs for transfection of primary T cells showed that purified saturated ionized lipids yielded improved mRNA delivery compared to crude products.

[0323] Characterization of the LNP library In this study, ionizable lipid nanoparticles (LNPs) were investigated for mRNA delivery to T cells. LNPs were chosen because they have been shown to deliver mRNA intracellularly with high efficacy and low toxicity to a wide range of cell and tissue targets in vivo and ex vivo. Recently, LNPs have been utilized for nucleic acid delivery to a wide range of immune cell types (Berdeja JG et al., 2017, J Clin Oncol, 35; Oberli MA et al., 2017, Nano Lett, 17:1326-1335; Love KT et al., 2010, Proc Natl Acad Sci, 107:9915-9915; Kauffman KJ et al., 2015, Nano Lett, 15:7300-7306; Midoux P et al., 2014, Expert Rev Vaccines, 14:221-234; Lokugamage MP et al., 2019, Adv Mater, 1902251:1-8).

[0324] To specifically examine mRNA delivery to T cells, we first synthesized ionizable lipid materials via Michael addition chemistry to generate a library of 24 different LNP formulations, in which a polyamine core was reacted with an excess of epoxy-terminated alkyl chains of various lengths (Figures 2 and 5). The specific ionizable lipids synthesized in this library are structural analogs of ionizable lipids previously formulated into LNPs and shown to deliver siRNA and mRNA to immune cells (Oberli MA et al., 2017, Nano Lett, 17:1326-1335; Love KT et al., 2010, Proc Natl Acad Sci, 107:9915-9915; Leuschner F et al., 2012, Nat Biotechnol, 29:1005-1010).

[0325] Lipids were evaluated for mRNA delivery specifically to T cells, rather than across a broad range of cell types. To prepare LNPs, ionizable lipids were combined in ethanol with three other excipients: (i) cholesterol for LNP stability and membrane fusion, (ii) DOPE to strengthen the LNP bilayer structure and promote endosomal escape, and (iii) C14-PEG to reduce aggregation and nonspecific endocytosis (Granot Y et al., 2017, Semin Immunol, 34:68-77; Varkouhi AK et al., 2011, J Control Release, 151:220-228; Mui BL et al., 2013, Mol Ther Acids, 2:1-8). This ethanol phase was then mixed with the aqueous mRNA phase in a microfluidic device (Figure 1A). These excipients and their molar ratios were selected based on previously optimized LNP formulations for mRNA delivery, which generally utilized (i) DOPE as the phospholipid component, (ii) reduced molar percentages of ionizable lipids, and (iii) increased concentrations of cholesterol and lipid-PEG (Kauffman KJ et al., 2015, Nano Lett, 15: 300-7306; Ball RL et al., 2018, Nano Lett, 18:3814-3822). The ratio of components was kept constant throughout these experiments because changes in the molar ratio of excipients affect the physicochemical properties and ultimately the potent delivery of LNPs (Kauffman KJ et al., 2015, Nano Lett, 15: 300-7306; Ball RL e al., 2018, Nano Lett, 18:3814-3822; Cheng Q et al., 2018, Adv Mater, 30:1805308).

[0326] The resulting LNPs were then characterized for size and mRNA concentration using dynamic light scattering (DLS) and A260 absorbance measurements. LNP diameters, reported as z-average measurements, ranged from 51.05 to 97.01 nm, with PDIs less than 0.3 (Figure 6). mRNA concentrations, measured as A260 absorbance, showed consistency across LNP formulations, ranging from 33.3 to 48.3 ng / µL. Taken together, these results confirmed the preparation of 24 different LNP formulations encapsulating mRNA used in this study for T cell delivery.

[0327] Screening of LNPs for mRNA delivery to Jurkat cells To assess the ability of LNPs to deliver functional mRNA, we selected luciferase as the encoded reporter protein. After the addition of luciferin, only luciferase protein translated from mRNA reacts to generate a luminescent signal, producing a readily detectable output that correlates with functional mRNA delivery (Hajj KA et al., 2019, Small, 15:1-7). The luciferase mRNA used in these experiments utilizes N1-methyl-pseudo-U and 5-methyl-C modifications, which have been shown to enhance mRNA translation and successful encapsulation within LNPs (Pardi N et al., 2015, J Control Release, 217:345-351; Svitkin YV et al., 2017, Nucleic Acids Res, 45:6023-6036; Trixl L et al., 2018, WIREs RNA, 10:1-17). These modifications may alter mRNA encapsulation within the LNP, mRNA delivery, and overall immunogenicity, so further testing of optimized modifications for these specific LNP delivery vehicles can be considered in future studies (Pardi N et al., 2015, J Control Release, 217:345-351; Zhang R et al., 2018, J Control Release, 292:256-276; Kariko K et al., 2005, Immunity, 23:165-175; Li J et al., 2017, ACS Nano, 11:2531-2544; Shen X et al., 2018, Nucleic Acids Res, 46:1584-1600; Sahin U et al., 2014, Nat Rev Drug Discovery,13:759-780).

[0328] Functional delivery of luciferase mRNA was observed using Jurkat cells, an immortalized human T cell line commonly used to study T cell behavior (Olden BR et al., 2018, J Control Release, 282:140-147; Abraham RT et al., 2004, Nat Rev Immunol, 4:1-8; Cancer P et al., 2018, Nucleic Acid Ther, 28:285-296). Jurkat cells were treated with luciferase mRNA-encapsulated LNPs at a concentration of 30 ng / 60,000 cells. After 48 hours, luciferase expression was assessed by luminescence measurement. Luminescence measurements from LNP formulations were normalized to untreated cell populations and compared with commercially available Lipofectamine, a commonly used transfection reagent widely considered the gold standard in vitro (Cardarelli F et al., 2016, Sci Reports, pp. 1–8; Wang T et al., 2018, Molecules, pp. 23). Library screening revealed seven LNP formulations that resulted in significantly higher luciferase expression than Lipofectamine, demonstrating improved ability to deliver luciferase mRNA to Jurkat cells (Figure 3A). Of these seven, three formulations had ionizable lipids with C12 tails, three with C14 tails, and one with C16 tails. Polyamine cores 3, 6, and 7 did not enhance transfection compared to Lipofectamine, regardless of lipid tail length. However, polyamine cores 2, 4, and 5 all have a similar structure with only one ring and an additional oxygen and were responsible for producing the five formulations with the highest luciferase expression, namely, C14-4, C14-2, C14-5, C16-2, and C12-4 LNPs.

[0329] Next, these top five LNP formulations were compared across a wide range of mRNA concentrations to determine both the top LNP formulation and the optimal LNP dose for Jurkat cell transfection. Results confirmed that C14-4 LNP, the best-performing LNP formulation from the original library screening, induced the highest luciferase expression among the top five formulations (Figure 3B). This increase in luciferase expression was significant compared to all other LNP formulations at doses above 20 ng, indicating that the optimal dose of C14-4 LNP in Jurkat cells was 30 ng. Because the C14-4 LNP formulation has a diameter of 70.17 nm and a concentration of 35.6 ng / µL, the improved performance of C14-4 LNP does not reflect differences in size or mRNA concentration (Figure 3C). C14-4 LNP toxicity to Jurkat cells was minimal, and cell viability was comparable to that of lipofectamine-treated and untreated cells, with viability measured at over 95% after treatment with C14-4 LNP.

[0330] To further validate the transient expression of mRNA delivered via C14-4, luciferase expression in Jurkat cells treated with LNPs was monitored over a 96-hour period. Results showed a 23% decrease in expression at 48 hours compared to 24 hours, an 84% decrease by 72 hours, and no detectable expression by 96 hours (Figure 3D). This confirmed transient luciferase expression and supported the use of the 24-hour time point for subsequent experiments. Collectively, these results confirmed the selection of C14-4 LNPs as the top formulation for mRNA delivery and provided an optimized in vitro transfection method for C14-4 LNPs.

[0331] Lipid nanoparticle-mediated mRNA delivery to primary human T cells The best-performing C14-4 LNPs were utilized for mRNA delivery to primary human T cells, demonstrating translational potential beyond that of the Jurkat cell line. Limitations of the Jurkat cell line include its exclusive CD4+ T cell derivation, whereas primary T cells also contain the CD8+ phenotype (Abraham RT et al., 2004, Nat Rev Immunol, 4:1-8). However, primary T cells require activation to achieve transfection (Barrett DM et al., 2011, Hum Gene Ther, 22:1575-1586; Harrer DC et al., 2017, BMC Cancer, 17:551). Dynabeads, widely used magnetic beads coated with CD3 and CD28 antibodies, were used to activate T cells using a method similar to that used in clinical trials (Hajj KA et al., 2019, Small, 15:1-7; Wang X et al., 2016, Mol Ther Oncolytics, 3:1-7; Lee DW et al., 2015, Lancet, 385:517-528). Isolated T cells were suspended at a 1:1 ratio of CD4+:CD8+ and treated with C14-4 LNPs containing luciferase mRNA at a wide range of concentrations. After 24 hours, luciferase expression and cell viability were quantified (Figure 4A). LNPs induced luciferase expression in T cells in a dose-dependent manner, indicating successful delivery of luciferase mRNA to T cells. Furthermore, slight toxicity was observed only at the highest dose, indicating the biocompatibility of C14-4 LNPs with primary cells.

[0332] To further test the potential of C14-4 for mRNA delivery to T cells, we purified the fully saturated ionizable lipid by flash chromatography and utilized the purified product to create C14-4 LNPs. These purified C14-4 LNPs were compared with C14-4 LNPs made from crude C14-4 ionizable lipids to verify which structure is responsible for potent mRNA delivery. DLS and A260 absorbance characterization of the purified C14-4 LNPs revealed a diameter of 65.19 nm and an mRNA concentration of 29.8 ng / µL, which was not significantly different from LNPs made with the crude C14-4 product (Figure 7). Using the Ribogreen assay to evaluate each formulation's ability to encapsulate mRNA, we found that the crude and purified formulations had similar encapsulation efficiencies, 92.5% and 86.3%, respectively. Finally, the surface ionization (pKa) of the two LNP formulations was assessed using a TNS assay. pKa, defined as the pH at which LNPs are 50% protonated, indicates how pH affects the surface charge and stability of LNPs (Hajj KA et al., 2019, Small, 15:1-7). Because the pKa of ionizable lipids is less than 7, they become charged in acidic endosomal compartments, releasing the encapsulated mRNA (Hajj KA et al., 2019, Small, 15:1-7; Zhang J et al., 2011, Langmuir, 27:9473-9483). Both crude and purified C14-4 LNPs proved to be ionizable, with the purified formulation having a slightly higher pKa value (Figure 4B).

[0333] Next, crude C14-4 LNPs and purified C14-4 LNPs were compared for their ability to deliver mRNA to primary T cells. T cells were suspended in a 1:1 ratio of CD4+ and CD8+ T cells, activated with Dynabeads, and then treated with LNPs. Crude and purified C14-4 LNPs encapsulating luciferase mRNA were tested for luciferase expression and viability at two concentrations (Figure 4C). At both concentrations, purified C14-4 LNPs significantly increased luciferase expression compared to crude LNP formulations, and both formulations had minimal impact on cell viability. Overall, the increased luciferase expression without increased toxicity indicates that purified C14-4 LNPs are the best-performing formulation for primary T cell mRNA delivery.

[0334] In summary, the data described herein disclose novel ionizable lipids and novel LNP formulations effective for mRNA delivery to T cells. The present invention addresses, in part, the problem of targeted delivery of mRNA to T cells using a novel LNP system. The present invention discloses, in part, an ionizable lipid called C14-4 and its LNP formulation (comprising cholesterol, phospholipid, and PEG components) that have been utilized for potent delivery of mRNA to T cells. Both crude lipids and purified fully saturated lipids were used. The studies described herein also demonstrate the ability of C14-4 and C14-4 LNP formulations to deliver mRNA to T cells with low toxicity, and the enhanced efficacy of the current gold standard reagent, Lipofectamine, gives C14-4 the potential to change the way T cells are engineered. This has application in the clinical field, where the present invention has future commercial potential, but it is also applicable in laboratory-based / research settings, as T cells are particularly difficult to transfect.

[0335] Additional research is underway to use these lipid nanoparticles for in vivo mRNA delivery to T cells, possibly with the addition of antibody-based targeting agents or other targeting ligands. Further optimization of the LNP formulation will be tested in terms of excipient ratios (varying the molar ratios of phospholipids, cholesterol, PEG, and C14-4) and the ratio of ionizable lipid to mRNA. This could involve the introduction of new excipients or modifications to the C14-4 lipid itself, such as using branched alkyl chains instead of linear ones. Furthermore, other mRNA cargoes beyond luciferase will be explored.

[0336] We now describe the materials and methods used in these experiments.

[0337] lipid synthesis Ionizable lipids were synthesized by reacting epoxide-terminated alkyl chains (Avanti Polar Lipids) with a polyamine core (Enamine, Monmouth Junction, NJ) using Michael addition chemistry. The components were combined with a 7-fold excess of alkyl chains and mixed with a magnetic stir bar at 80°C for 48 hours. The crude product was then transferred to a Rotavapor R-300 (BUCHI, Newark, DE) to evaporate the solvent, and the lipids were suspended in ethanol. Finally, to purify the highest-performing lipids (C14-4), the lipid fractions were separated using a CombiFlash® Nextgen 300+ chromatography system (Teledyne ISCO, Lincoln, NE), and the saturated lipid fraction was identified by molecular weight using liquid chromatography-mass spectrometry.

[0338] Preparation and characterization of LNPs To synthesize LNPs, an aqueous phase containing mRNA and an ethanol phase containing lipid and cholesterol components were mixed using a microfluidic device as previously described (Chen D et al., 2012, J Am Chem Soc, 134:6948-6951). Briefly, the aqueous phase was prepared using 10 mM citrate buffer and 1 mg / mL luciferase mRNA with N1-methyl-pseudo-U and 5-methyl-C substitutions (Trilink Biotechnologies, San Diego, CA). To prepare the ethanol phase, the ionizable lipid, 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine (DOPE) (Avanti Polar Lipids, Alabaster, AL), cholesterol (Sigma, St. Louis, MO), and lipid-anchored PEG (Avanti Polar Lipids) components were combined at set molar ratios of 35%, 16%, 46.5%, and 2.5%, respectively. The ethanol and aqueous phases were mixed in a 3:1 ratio within the microfluidic device using a Pump33DS syringe pump (Harvard Apparatus, Holliston, MA) (Chen D et al., 2012, J Am Chem Soc, 134:6948-6951). After mixing, the LNPs were dialyzed against 1x PBS for 2 hours and then sterilized with a 0.22 μm filter. The diameter (z-average) and polydispersity index (PDI) of LNPs suspended in 1x PBS were then measured in triplicate using dynamic light scattering (DLS) performed on a Zetasizer Nano (Malvern Instruments, Malvern, UK). The mRNA concentration of each LNP formulation was obtained using a NanoDrop ND-1000 spectrophotometer (ThermoFisher, Waltham, MA).

[0339] Further analysis of the best-performing LNP formulations included Quant-iT Ribogreen (ThermoFisher) and 6-(p-toluidinyl)naphthalene-2-sulfonic acid (TNS) assays to determine the LNP encapsulation efficiency and pKa, respectively. Quant-iT Ribogreen was performed as previously described (Heyes J et al., 2005, J Control Release, 107:276-287). Briefly, equal concentrations of LNP were treated with Triton X-100 (Sigma) to dissolve the LNP or left untreated. After 10 minutes, the groups were plated in triplicate in a 96-well plate along with RNA standards. Fluorescent Ribogreen reagent was added according to the manufacturer's instructions, and the resulting fluorescence was measured using a plate reader. RNA content was quantified by comparing the readings to a standard curve, and encapsulation efficiency was calculated. To determine LNP pKa, the TNS assay was used to measure surface ionization as previously described (Hajj KA et al., 2019, Small, 15:1-7). A buffer solution of 150 mM sodium chloride, 20 mM sodium phosphate, 25 mM ammonium citrate, and 20 mM ammonium acetate was adjusted to reach pH values ​​ranging from 2 to 12 in 0.5 increments. LNP was added in triplicate to each pH-adjusted solution in a 96-well plate. TNS was then added to each well to reach a final TNS concentration of 6 μM, and the resulting fluorescence was read on a plate reader. The pKa was then calculated as the pH at which the fluorescence intensity was 50% of its maximum (reflecting 50% protonation).

[0340] mRNA transfection of Jurkat cells Jurkat cells (ATCC TIB-152), an immortalized human T cell line (Abraham RT et al., 2004, Nat Rev Immunol, 4:1-8), were cultured in RPMI-1640 medium supplemented with L-glutamine (ThermoFisher), 10% fetal bovine serum, and 1% penicillin-streptomycin. Cells were plated at 60,000 cells per well of a 96-well plate in 60 μL of medium and immediately treated with 60 μL of LNP diluted to various concentrations in PBS. Lipofectamine MessengerMAX transfection reagent (ThermoFisher), used as a positive control, was combined with mRNA for 10 minutes according to the manufacturer's protocol, and the same mRNA concentration as in the LNP group was used to treat the wells. After 48 hours of incubation, cells were centrifuged at 300 × g for 4 minutes and resuspended in 50 μL of 1× lysis buffer (Promega, Madison, WI) and 100 μL of luciferase assay substrate (Promega). Luminescence was then quantified using an Infinite M Plex plate reader (Tecan, Morrisville, NC). Luminescence signals from each group were normalized to either untreated cells or the lowest concentration treatment group, and background measurements were subtracted from wells containing reagent but no cells. To assess cytotoxicity, Jurkat cells were plated under the same conditions and treated with either C14-4 or Lipofectamine at 30 ng of mRNA per 60,000 cells. After 48 hours, 60 μL of CellTiter-Glo (Promega) was added to each well, and luminescence, corresponding to ATP production, was quantified using a plate reader. Luminescence signals from each group were normalized to untreated cells and background subtracted.

[0341] mRNA transfection of primary T cells Primary T cells (CD3+) were obtained from the University of Pennsylvania Human Immunology Core and pooled at a 1:1 CD4:CD8 ratio. Cells to be treated with LNP were then activated overnight with Human Tactivator CD3 / CD28 Dynabeads (ThermoFisher) at a 3:1 bead-to-cell ratio. After activation, cells were plated in 96-well plates at 60,000 cells per well in 60 μL of medium and treated with LNP at various mRNA concentrations. For electroporation, T cells were washed three times with medium, resuspended at 108 cells / mL, and mixed with transcribed mRNA at a concentration of 100 μg of mRNA per mL of T cells. Cells were then electroporated in 2 mm cuvettes using an ECM830 Electro Square Wave Porator (Harvard Apparatus BTX). In experiments using luciferase mRNA treatment, luminescence was assessed after 48 hours and toxicity after 24 hours using the same protocol as above.

[0342] Example 2: Engineering lipid nanoparticles for T cell delivery Further optimization of C14-4 formulation parameters regarding excipient ratios has already begun. Two libraries of formulations have been generated (Library A and then Library B, based on the results of Library A; a representative formulation from Library A is designated A#, and a representative formulation from Library B is designated B#) are attached. Both were created using C14-4 lipids, but some of the new formulations showed enhanced mRNA delivery in T cell lines compared to the original C14-4 formulation without increased toxicity.

[0343] Ionizable LNPs have shown great promise as vehicles for the intracellular delivery of therapeutic macromolecules, including nucleic acids (Mukalel AJ, 2019, Cancer Lett, 458:102-112). While numerous LNP formulations exist, they utilize common excipients: cholesterol for membrane stability, phospholipids to aid endosomal escape, and polyethylene glycol (PEG) to reduce immunogenicity (Reichmuth AM, 2016, Ther Deliv, 7:319-334). Combinations of various excipients can significantly alter the physicochemical properties of LNPs, thereby potentially affecting their delivery capabilities (Kauffman K., 2015, Nano Lett, 15:7300-7306). During this study, two libraries of LNPs were engineered for T cell targeting (Figures 8 and 9). Because formulations were selected using an orthogonal DOE design, a wide range of component variation was observed across only 16 representative formulations.

[0344] Each formulation contained various molar ratios of ionizable lipids, cholesterol, helper lipids, and lipid-conjugated PEG. The z-average diameter and pKa of each formulation were determined using dynamic light scattering, 2-(p-toluidinyl)naphthalene-6-sulfonic acid (TNS) assay, and absorbance measurement at 260 nm, respectively. Jurkat cells, immortalized human T cell lines, were treated with each formulation for 48 hours to assess intracellular delivery in vitro. The cytotoxicity of each formulation was also assessed using a commercially available Cell-Titer Glo assay.

[0345] Data for the optimized formulations are shown in Figures 8 and 9. The in vitro delivery efficiency of each formulation was evaluated using a standard luciferase expression assay. Briefly, LNPs containing mRNA encoding firefly luciferase were delivered to Jurkat cells, immortalized human T cell lines, at an mRNA concentration of 30 ng per 60,000 cells. After 48 hours of incubation, the cells were lysed and treated with firefly luciferin. The extent of LNP-mediated transfection was then measured as luminescence intensity using a plate reader. The cytotoxicity of each formulation was similarly evaluated using a commercially available Cell-Titer Glo assay.

[0346] Characterization of Library A revealed several delivery trends related to excipient composition. The optimal excipient conditions for Library A led to the development of next-generation Library B. Library B formulations demonstrated significantly better encapsulation efficiency and larger z-average diameters than Library A formulations, and several Library B formulations outperformed the best-performing Library A formulations, supporting the above trends. Furthermore, all Library B formulations were observed to exhibit viability rates greater than 80% over 48 hours. Thus, the development of multiple highly potent LNP formulations for intracellular delivery to T cells has been reported. These LNPs may be useful for future T cell engineering applications, including cancer immunotherapy.

[0347] Example 3: Modifying the excipient composition of LNPs to improve their ability to deliver mRNA to T cells (with minimal toxicity) This example shows in vitro and ex vitro data obtained with representative Library A and Library B formulations. In in vitro studies, Library A, containing 16 representative formulations of C14-494 with various excipient concentrations (e.g., Figure 8A), was screened for its ability to deliver luciferase mRNA to the Jurkat cell line (immortalized human T cells). Library B, generated based on the results of Library A (e.g., Figure 9), was also screened in in vitro studies in Jurkat cells. Further ex vivo studies focused on the delivery of luciferase mRNA to primary T cells using representative, top-performing formulations from Libraries A and B.

[0348] More specifically, Jurkat cells were treated with 0 ng / 60,0000 cells for 24 hours. Adjustments made to Library B based on data from Library A resulted in more "hit" formulations (i.e., formulations that achieved higher delivery than the standard formulation S2) and reduced the overall toxicity of the LNP formulations. After 24 hours of incubation with LNPs (containing luciferase mRNA), luciferase activity was measured using a luciferase assay (Figure 13A). Percent viability was measured at the same time points using the Cell Titer Glo assay (Figure 13B). Each bar contains three biological replicates (three technical replicates each) and was normalized to the 0 ng treatment.

[0349] A comparison with Lipofectamine (Figure 14) showed that formulation B10 outperformed this commercial standard. Furthermore, toxicity testing results showed that both were non-toxic to Jurkat.

[0350] Additionally, Jurkats were treated with luciferase-encoding mRNA for 24 hours to assess luminescence and viability of various representative formulations at various concentrations / doses (Figures 15A and 15B). Additionally, three different primary patient T cell samples were activated overnight and treated with standard, A16, or B10 formulations (Figures 16A-16C). Luciferase-encoding mRNA delivery values ​​were normalized to 0 ng treatment. Donor variability resulted in different overall luciferase readings.

[0351] The disclosures of all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. While the present invention has been disclosed with reference to specific embodiments, it will be apparent that other embodiments and modifications of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention. It is intended that the appended claims be construed to include all such embodiments and equivalent variations. Some aspects of the invention are described below. 1. A compound having the structure of formula (I) or a salt thereof: [ka] wherein A1 and A2 are independently selected from the group consisting of C, C(H), N, S, and P; where L1, L2, L3, L4, L5, and L6 are C, C(H)2, C(H)(R 19 ), O, N(H), and N(R 19 ) independently selected from the group consisting of: Here, each R1, R2, R 3a , R 3b , R 4a , R 4b , R 5a , R 5b , R 6a , R 6b , R 7a , R 7b , R 8a , R 8b , R 9a , R 9b , R 10a , R10b , R 11a , R 11b , R 12a , R 12b , R 13a , R 13b , R 14a , R 14b , R 15a , R 15b , R 16 , R 17 , R 18 , and R 19 is H, halogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, -Y(R 20 ) z' (R 21 ) Z" -cycloalkyl, substituted -Y(R 20 ) z' (R 21 ) Z" -cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, -Y(R 20 ) z' (R 21 ) Z" -heterocycloalkyl, substituted -(R 20 ) z' (R 21 ) Z" -heterocycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, -Y(R 20 ) z' (R 21 ) Z" -cycloalkenyl, substituted -Y(R 20 ) z' (R 21 ) Z" -cycloalkenyl, alkynyl, substituted alkynyl, cycloalkynyl, substituted cycloalkynyl, -Y(R 20 ) z' (R 21 ) Z" -cycloalkynyl, substituted -Y(R 20 ) z' (R 21 ) Z" -cycloalkynyl, aryl, substituted aryl, -Y(R 20 ) z' (R 21 ) Z"-aryl, substituted -Y(R 20 ) z' (R 21 ) Z" -aryl, heteroaryl, substituted heteroaryl, -Y(R 20 ) z' (R 21 ) Z" -heteroaryl, substituted -Y(R 20 ) z' (R 21 ) Z" -heteroaryl, alkoxycarbonyl, linear alkoxycarbonyl, branched alkoxycarbonyl, amido, amino, aminoalkyl, aminoalkenyl, aminoalkynyl, aminoaryl, aminoacetate, acyl, hydroxyl, hydroxyalkyl, hydroxyalkenyl, hydroxyalkynyl, hydroxyaryl, alkoxy, carboxyl, carboxylate, ester, -Y(R 20 ) z' (R 21 ) Z" -ester, -Y(R 20 ) z' (R 21 ) Z" independently selected from the group consisting of ═O, —NO2, —CN, and sulfoxy; wherein Y is selected from the group consisting of C, N, O, S, or P; Here, each R 20 and R 21is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, cycloalkynyl, substituted cycloalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxycarbonyl, linear alkoxycarbonyl, branched alkoxycarbonyl, amido, amino, aminoalkyl, aminoalkenyl, aminoalkynyl, aminoaryl, aminoacetate, acyl, hydroxyl, hydroxyalkyl, hydroxyalkenyl, hydroxyalkynyl, hydroxyaryl, alkoxy, carboxyl, carboxylate, ester, ═O, —NO, —CN, and sulfoxy; wherein each Z′ and Z″ is independently an integer of 0, 1, or 2; and wherein each m, n, o, p, q, r, s, t, u, v, w, and x is independently an integer of 0, 1, 2, 3, 4, or 5. 2. The compound according to item 1, wherein the compound having the structure of formula (I) is a compound having a structure selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] [ka] wherein each R, R, R, R, and R is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, cycloalkynyl, substituted cycloalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxycarbonyl, linear alkoxycarbonyl, branched alkoxycarbonyl, amido, amino, aminoalkyl, aminoalkenyl, aminoalkynyl, aminoaryl, aminoacetate, acyl, hydroxyl, hydroxyalkyl, hydroxyalkenyl, hydroxyalkynyl, hydroxyaryl, alkoxy, carboxyl, carboxylate, and ester; wherein m, n, o, p, and q are each independently an integer from 0 to 25; and where r, s, t, u, v, w, and x are each independently integers represented by 0, 1, 2, 3, 4, and 5. 3. The compound according to item 1, wherein the compound having the structure of formula (I) is a compound having a structure selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] wherein each R, R, R, R, and R is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, cycloalkynyl, substituted cycloalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxycarbonyl, linear alkoxycarbonyl, branched alkoxycarbonyl, amido, amino, aminoalkyl, aminoalkenyl, aminoalkynyl, aminoaryl, aminoacetate, acyl, hydroxyl, hydroxyalkyl, hydroxyalkenyl, hydroxyalkynyl, hydroxyaryl, alkoxy, carboxyl, carboxylate, and ester; and Here, m, n, o, p, and q are each independently an integer of 0 to 25. 4. The compound according to item 1, wherein the compound having the structure of formula (I) is an ionizable lipid. 5. Lipid nanoparticles (LNPs) comprising one or more compounds according to item 1. 6. The LNP according to item 5, wherein the LNP comprises one or more compounds having the structure of formula (I) or salts thereof in a concentration range of about 1 mol % to about 100 mol %. 7. The LNP according to item 6, wherein the LNP comprises one or more compounds having the structure of formula (I) or salts thereof in a concentration range of about 10 mol % to about 50 mol %. 8. The LNP of item 5, wherein the LNP further comprises at least one helper lipid. 9. The LNP according to item 8, wherein the LNP comprises at least one helper lipid in a concentration range of about 0.01 mol% to about 99.9 mol%. 10. The LNP according to item 9, wherein the LNP comprises at least one helper lipid in a concentration range of about 0.5 mol% to about 50 mol%. 11. The LNP of item 8, wherein the helper lipid is selected from the group consisting of a phospholipid, a cholesterol lipid, a polymer, and any combination thereof. 12. The LNP of item 11, wherein the phospholipid is selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE) or a derivative thereof, distearoylphosphatidylcholine (DSPC) or a derivative thereof, distearoylphosphatidylethanolamine (DSPE) or a derivative thereof, stearoyloleoylphosphatidylcholine (SOPC) or a derivative thereof, 1-stearioyl-2-oleoyl-phosphatidylethanolamine (SOPE) or a derivative thereof, N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) or a derivative thereof, and any combination thereof. 13. The LNP according to item 11, wherein the LNP comprises phospholipids in a concentration range of about 15 mol% to about 50 mol%. 14. The LNP of item 11, wherein the cholesterol lipid is cholesterol or a derivative thereof. 15. The LNP according to item 11, wherein the LNP comprises cholesterol lipids in a concentration range of about 20 mol% to about 50 mol%. 16. The LNP of item 11, wherein the polymer is polyethylene glycol (PEG) or a derivative thereof. 17. The LNP of item 11, wherein the LNP comprises the polymer in a concentration range of about 0.5 mol% to about 10 mol%. 18. The LNP of item 11, wherein the LNP comprises at least one selected from the group consisting of a nucleic acid molecule, a therapeutic agent, and any combination thereof. 19. The LNP of item 18, wherein the nucleic acid molecule is a therapeutic agent. 20. The LNP of item 18, wherein the nucleic acid molecule is a DNA molecule or an RNA molecule. 21. The LNP of item 18, wherein the nucleic acid molecule is selected from the group consisting of cDNA, mRNA, miRNA, siRNA, modified RNA, antagomir, antisense molecule, peptide, therapeutic peptide, targeting nucleic acid, and any combination thereof. 22. The LNP of item 21, wherein the mRNA encodes luciferase. 23. The LNP according to item 21, wherein the mRNA encodes one or more antigens. 24. The LNP according to item 23, wherein the antigen comprises at least one selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, an influenza antigen, a tumor-associated antigen, and a tumor-specific antigen. 25. The LNP of item 18, wherein the nucleic acid molecule comprises a promoter or regulatory sequence. 26. The LNP according to item 18, wherein the LNP further comprises an adjuvant. 27. The LNP of item 18, wherein the nucleic acid molecule, therapeutic agent, or combination thereof is encapsulated within a compound having the structure of formula (I) or a salt thereof. 28. A composition comprising at least one compound according to item 1, at least one LNP according to item 5, or any combination thereof. 29. The composition according to item 28, wherein the composition is a vaccine. 30. A method for delivering a nucleic acid molecule, a therapeutic agent, or a combination thereof to a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one LNP or composition thereof according to item 5, wherein the LNP or composition thereof delivers the nucleic acid molecule, therapeutic agent, or combination thereof to a target. 31. The method of item 30, wherein the nucleic acid molecule is a therapeutic agent. 32. The method according to item 30, wherein the nucleic acid molecule is a DNA molecule or an RNA molecule. 33. The method according to item 30, wherein the nucleic acid molecule is selected from the group consisting of cDNA, mRNA, miRNA, siRNA, antagomir, antisense molecule, peptide, therapeutic peptide, targeting nucleic acid, and any combination thereof. 34. The method of item 33, wherein the mRNA encodes luciferase. 35. The method of item 30, wherein the target is selected from the group consisting of immune cells, T cells, resident T cells, B cells, natural killer (NK) cells, cancerous cells, cells associated with a disease or disorder, tissue associated with a disease or disorder, brain tissue, central nervous system tissue, lung tissue, apical surface tissue, epithelial cells, endothelial cells, liver tissue, intestinal tissue, colon tissue, small intestine tissue, large intestine tissue, feces, bone marrow, macrophages, spleen tissue, muscle tissue, joint tissue, tumor cells, diseased tissue, lymph node tissue, lymphatic circulation, and any combination thereof. 36. The method of item 33, wherein the mRNA encodes one or more antigens. 37. The method according to item 36, wherein the antigen comprises at least one selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, an influenza antigen, a tumor-associated antigen, and a tumor-specific antigen. 38. The method according to item 30, wherein the nucleic acid molecule comprises a promoter or regulatory sequence. 39. The method according to item 30, wherein the LNP or composition thereof further comprises an adjuvant. 40. The method of claim 30, wherein the nucleic acid molecule, therapeutic agent, or combination thereof is encapsulated within the compound of claim 1. 41. The method of item 30, wherein the LNP composition is a vaccine. 42. The method of item 30, wherein the LNP or composition thereof is administered by a delivery route selected from the group consisting of an intradermal delivery route, a subcutaneous delivery route, an intramuscular delivery route, an intracerebroventricular delivery route, an intrathecal delivery route, an oral delivery route, an intravenous delivery route, an intratracheal delivery route, an intraperitoneal delivery route, an intrauterine delivery route, and any combination thereof. 43. The method of item 30, wherein the method comprises a single administration of the LNP composition. 44. The method of item 30, wherein the method comprises multiple administrations of the LNP composition. 45. The method according to item 30, wherein the method treats or prevents at least one selected from the group consisting of a viral infection, a bacterial infection, a fungal infection, a parasitic infection, an influenza infection, cancer, arthritis, heart disease, cardiovascular disease, a neurological disorder or disease, a genetic disease, an autoimmune disease, a fetal disease, a genetic disease affecting fetal development, and any combination thereof. 46. ​​A method for preventing or treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one LNP or composition thereof according to item 5. 47. The method of item 46, wherein the LNP or composition thereof delivers a nucleic acid molecule, a therapeutic agent, or a combination thereof to a cell. 48. A method for delivering a nucleic acid molecule to a cell, comprising administering to the cell a therapeutically effective amount of at least one LNP or composition thereof according to item 5. 49. The method according to item 48, wherein the method is a gene delivery method.

Claims

1. A compound selected from the group consisting of: 【Chemistry 1】

2. A lipid nanoparticle (LNP) comprising at least one compound according to claim 1.

3. The LNP of claim 2, wherein at least one of the following is true: (a) the LNPs comprise the at least one compound of claim 1 or a salt thereof in a concentration range of about 1 mol % to about 100 mol %; and (b) the LNPs comprise the at least one compound of claim 1 or a salt thereof in a concentration range of about 10 mol % to about 50 mol %.

4. The LNP of claim 2 , wherein the LNP further comprises at least one helper lipid.

5. The LNP of claim 4, wherein at least one of the following is true: (a) the LNPs comprise the at least one helper lipid in a concentration range of about 0.01 mol% to about 99.9 mol%; (b) the LNPs comprise the at least one helper lipid in a concentration range of about 0.5 mol% to about 50 mol%; and (c) the at least one helper lipid is selected from the group consisting of phospholipids, cholesterol lipids, polymers, and any combination thereof.

6. The LNP of claim 5, wherein at least one of the following is true: (a) the at least one helper lipid is a phospholipid, and the phospholipid is selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE) or a derivative thereof, distearoylphosphatidylcholine (DSPC) or a derivative thereof, distearoylphosphatidylethanolamine (DSPE) or a derivative thereof, stearoyloleoylphosphatidylcholine (SOPC) or a derivative thereof, 1-stearioyl-2-oleoyl-phosphatidylethanolamine (SOPE) or a derivative thereof, N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) or a derivative thereof, and any combination thereof; (b) the at least one helper lipid is a phospholipid, and the LNPs comprise the phospholipid in a concentration range of about 15 mol% to about 50 mol%; (c) the cholesterol lipid is cholesterol or a derivative thereof; (d) the at least one helper lipid is a cholesterol lipid, and the LNPs comprise the cholesterol lipid in a concentration range of about 20 mol% to about 50 mol%; (e) the polymer is polyethylene glycol (PEG) or a derivative thereof; (f) the at least one helper lipid is a polymer, and the LNP comprises the polymer in a concentration range of about 0.5 mol% to about 10 mol%.

7. The LNP of claim 2 , wherein the LNP comprises at least one selected from the group consisting of a nucleic acid molecule, a therapeutic agent, and any combination thereof.

8. 8. The LNP of claim 7, wherein at least one of the following is true: (a) the nucleic acid molecule is a therapeutic agent; (b) the nucleic acid molecule is a DNA molecule or an RNA molecule; (c) the nucleic acid molecule is selected from the group consisting of cDNA, mRNA, miRNA, siRNA, modified RNA, antagomir, antisense molecule, peptide, therapeutic peptide, targeting nucleic acid, and any combination thereof, optionally wherein the mRNA encodes luciferase, optionally wherein the mRNA encodes one or more antigens, and optionally wherein the antigen comprises at least one selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, an influenza antigen, a tumor-associated antigen, and a tumor-specific antigen; and (d) the nucleic acid molecule comprises a promoter or regulatory sequence.

9. The LNP of claim 2 , wherein the LNP further comprises an adjuvant.

10. A composition comprising the LNP of claim 2.

11. 3. The LNP or composition thereof of claim 2, for use in a method for delivering a nucleic acid molecule, a therapeutic agent, or a combination thereof to a subject in need thereof, wherein the LNP or composition thereof delivers the nucleic acid molecule, the therapeutic agent, or a combination thereof to a target.

12. 12. The LNP or composition thereof according to claim 11, wherein at least one of the following is true: (a) the nucleic acid molecule is a therapeutic agent; (b) the nucleic acid molecule is a DNA molecule or an RNA molecule; (c) the nucleic acid molecule is selected from the group consisting of cDNA, mRNA, miRNA, siRNA, antagomir, antisense molecule, peptide, therapeutic peptide, targeting nucleic acid, and any combination thereof, optionally wherein the mRNA encodes luciferase, optionally wherein the mRNA encodes one or more antigens, optionally wherein the antigens comprise at least one selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, an influenza antigen, a tumor-associated antigen, and a tumor-specific antigen; (d) the target is selected from the group consisting of an immune cell, a T cell, a resident T cell, a B cell, a natural killer (NK) cell, a cancerous cell, a cell associated with a disease or disorder, a tissue associated with a disease or disorder, brain tissue, central nervous system tissue, lung tissue, apical surface tissue, an epithelial cell, an endothelial cell, liver tissue, intestinal tissue, colon tissue, small intestine tissue, large intestine tissue, feces, bone marrow, a macrophage, spleen tissue, muscle tissue, joint tissue, a tumor cell, a diseased tissue, lymph node tissue, lymphatic circulation, and any combination thereof; and (e) the nucleic acid molecule comprises a promoter or regulatory sequence.

13. The LNP or composition thereof of claim 10, wherein the LNP or composition thereof further comprises an adjuvant.

14. The LNP or composition thereof according to claim 10 or 13, wherein the LNP composition is a vaccine.

15. The LNP or composition thereof according to claim 10, wherein at least one of the following is true: (a) the LNPs or compositions thereof are administered by a delivery route selected from the group consisting of an intradermal delivery route, a subcutaneous delivery route, an intramuscular delivery route, an intraventricular delivery route, an intrathecal delivery route, an oral delivery route, an intravenous delivery route, an intratracheal delivery route, an intraperitoneal delivery route, an intrauterine delivery route, and any combination thereof; (b) the method comprises a single administration of the LNP composition; (c) the method comprises multiple administrations of the LNP composition; (d) the method treats or prevents at least one selected from the group consisting of a viral infection, a bacterial infection, a fungal infection, a parasitic infection, an influenza infection, cancer, arthritis, heart disease, cardiovascular disease, a neurological disorder or disease, a genetic disease, an autoimmune disease, a fetal disease, a genetic disease affecting fetal development, and any combination thereof.

16. 3. The LNP of claim 2 for use in a method for preventing or treating a disease or disorder in a subject in need thereof.

17. The LNP of claim 16, wherein the LNP or composition thereof delivers a nucleic acid molecule, a therapeutic agent, or a combination thereof to a cell.

18. 3. The LNP of claim 2 for use in a method for delivering a nucleic acid molecule to a cell.

19. 19. The LNP or composition thereof of claim 18, wherein the method is a gene delivery method.

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