Dry Nanoparticle Composition

Abstract

Provided herein is a dry composition and a method for preparing such a composition for use in delivering nucleic acid to a subject.The dry composition can include a lipid carrier, where the lipid carrier is a nanoemulsion that includes a hydrophobic core, optionally one or more inorganic nanoparticles, and one or more lipids, one or more nucleic acids, and at least one cryoprotectant.Methods of using the dry composition for these treatments are also provided.

Description

[Technical field]

[0001] cross reference This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 247,172, filed September 22, 2021, and U.S. Provisional Patent Application No. 63 / 297,449, filed January 7, 2022, the contents of each of which are incorporated by reference herein in their entirety. Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 18, 2022, is named 201953-712601_SL.txt and is 3,396 bytes in size. [Background technology]

[0002] background A variety of therapeutic and preventative products, including vaccines, are available for diseases. A challenge in the industry for such products is stability. Many of such products must be kept at low temperatures to maintain stability, requiring expensive refrigeration units or liquid nitrogen. Thus, there is a need for solutions, compositions, and uses thereof, where the therapeutic and preventative products are stable at room temperature or with minimal refrigeration requirements. Summary of the Invention [Means for solving the problem]

[0003] A quick overview Provided herein is a composition that is a dry composition.Provided herein is a composition that includes: lipid carrier, wherein lipid carrier is a nanoemulsion that includes a hydrophobic core, one or more inorganic nanoparticles, and one or more lipids; one or more nucleic acids; and at least one cryoprotectant.

[0004] Further provided herein is a pharmaceutical composition comprising the dry composition reconstituted in a suitable diluent and a pharma- ceutically acceptable carrier.

[0005] Further provided herein is a kit comprising a pharmaceutical composition comprising a dry composition provided herein; and a delivery system for administration to a subject.

[0006] Further provided herein is a vaccine delivery system comprising the pharmaceutical composition and optionally one or more vaccine adjuvants.

[0007] Further provided herein is a method for generating an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition provided herein.

[0008] Further provided herein is a method of treating or preventing a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition.

[0009] Further provided herein is a method of imaging and / or tracking delivery of one or more nucleic acids in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition.

[0010] Further provided herein is a method for preparing a lyophilized composition, the method comprising: (a) obtaining a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, optionally one or more inorganic nanoparticles, and one or more lipids; (b) incorporating one or more nucleic acids into the lipid carrier to form a lipid carrier-nucleic acid complex; (c) adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; and (d) lyophilizing the formulation to form a lyophilized composition.

[0011] Further provided herein is a method for preparing a spray-dried composition, the method comprising: (a) obtaining a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, optionally one or more inorganic nanoparticles, and one or more lipids; (b) incorporating one or more nucleic acids into the lipid carrier to form a lipid carrier-nucleic acid complex; (c) adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; and (d) spray drying the formulation to form the spray-dried composition.

[0012] Further provided herein is a method for reconstituting a lyophilized composition, the method comprising: (a) obtaining a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, optionally one or more inorganic nanoparticles, and one or more lipids; (b) incorporating one or more nucleic acids into the lipid carrier to form a lipid carrier-nucleic acid complex; (c) adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; lyophilizing the formulation to form a lyophilized composition; and (d) reconstituting the lyophilized composition in a suitable diluent.

[0013] Further provided herein is a method for reconstituting a spray-dried composition, the method comprising: (a) obtaining a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, optionally one or more inorganic nanoparticles, and one or more lipids; (b) incorporating one or more nucleic acids into the lipid carrier to form a lipid carrier-nucleic acid complex; (c) adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; (d) spray drying the formulation to form the spray-dried composition; and (e) reconstituting the spray-dried composition in a suitable diluent.

[0014] Further provided herein is a dry composition comprising a sorbitan fatty acid ester, an ethoxylated sorbitan ester, a cationic lipid, an immunostimulant, and RNA.

[0015] Further provided herein is a dry composition comprising sorbitan monostearate, polyoxyethylene (20) sorbitan monooleate, DOTAP, an immunostimulant, and RNA.

[0016] Further provided herein is a dry composition comprising: (a) a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core and one or more lipids; (b) optionally one or more nucleic acids; and (c) at least one sugar present in an amount of (i) at least about 50% by weight of the dry composition, or (ii) at least about 50 mg.

[0017] Further provided herein is a pharmaceutical composition comprising a dry composition provided herein reconstituted in a suitable diluent; and a pharma- ceutically acceptable carrier.

[0018] Further provided herein is a kit comprising a pharmaceutical composition provided herein and a delivery system for administration to a subject.

[0019] Further provided herein is a method for generating an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition provided herein.

[0020] Further provided herein is a method of treating or preventing a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition provided herein.

[0021] Further provided herein is a method for imaging and / or tracking delivery of one or more nucleic acids in a subject, the method comprising administering a therapeutically effective amount of a pharmaceutical composition described herein. [Brief description of the drawings]

[0022] [Figure 1] Figures 1A-1C show schematic diagrams of nanoparticle (NP) carriers. Figure 1A shows an oil-in-water emulsion. Figure 1B shows a nanostructured lipid carrier (NLC). Figure 1C shows a nanoparticle with inorganic nanoparticles in liquid oil.

[0023] [Diagram 2] FIG. 2 illustrates cake appearance for nanostructured lipid carrier (NLC) and lipid carrier formulations at the indicated sugar compositions at time=0 hours after lyophilization cycle #1.

[0024] [Diagram 3] FIG. 3 illustrates cake appearance for nanostructured lipid carrier (NLC) and lipid carrier formulations at the indicated sugar compositions after 24 hours at 25 degrees Celsius and 42 degrees Celsius following lyophilization cycle #1.

[0025] [Figure 4] FIG. 4 illustrates the appearance of nanostructured lipid carrier (NLC) and lipid carrier formulations at the indicated sugar compositions at 25 degrees Celsius and 42 degrees Celsius for 24 hours after lyophilization cycle #1.

[0026] [Figure 5A] 5A-5B illustrate particle size of reconstituted cakes after 1 day of storage at 25° C. and 42° C. for the indicated sugar compositions after lyophilization cycle #1. FIG. 5A shows the fold change compared to the liquid formulation at time=0 hours for the indicated sugar compositions. FIG. 5B shows the point change in PDI compared to the liquid formulation at time=0 hours for the indicated sugar compositions. [Figure 5B] Same as above.

[0027] [Figure 6]6A-6B illustrate RNA integrity by agarose gel electrophoresis at the indicated sugar compositions for lipid carrier+RNA and NLC+RNA formulations after lyophilization cycle #1. FIG. 6A shows RNA integrity by agarose gel electrophoresis at the indicated sugar compositions for lipid carrier+RNA formulations after lyophilization cycle #1. FIG. 6B shows RNA integrity by agarose gel electrophoresis at the indicated sugar compositions for lipid carrier+RNA and NLC+RNA formulations after lyophilization cycle #1.

[0028] [Figure 7-1] Figures 7A-7G illustrate SEAP expression in BHK21 cells at the indicated sugar compositions after lyophilization cycle #1 for lipid carrier + repRNA and NLC + repRNA-SEAP formulations. Figure 7A shows the relative light units (RLU) for lipid carrier + repRNA-SEAP in 10% trehalose. Figure 7B shows the relative light units (RLU) for lipid carrier + repRNA-SEAP in 5% mannitol. Figure 7C shows the relative light units (RLU) for lipid carrier + repRNA-SEAP in 10% sucrose. Figure 7D shows the relative light units (RLU) for lipid carrier + repRNA-SEAP in 20% sucrose. Figure 7E shows the relative light units (RLU) for lipid carrier + repRNA-SEAP in 5% glucose. Figure 7F shows the relative light units (RLU) for lipid carrier + repRNA-SEAP in 10% maltose. FIG. 7G shows relative light units (RLU) for NLC+repRNA-SEAP in 10% sucrose. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 7-4] Same as above.

[0029] [Figure 8]FIG. 8 illustrates the appearance of lipid carrier+RNA-SEAP cake at various time points (time=0 hours, after 6 days at 25 degrees Celsius, and after 1 month at 25 degrees Celsius) at the indicated sugar compositions after lyophilization cycle #1.

[0030] [Figure 9] FIG. 9 illustrates the appearance of lipid carrier+RNA-SEAP cake at various time points (time=0 hours, 24 hours at 42 degrees Celsius, 3 days at 42 degrees Celsius, and 1 month at 42 degrees Celsius) at the indicated sugar compositions after lyophilization cycle #1.

[0031] [Figure 10] Figures 10A-10B illustrate particle size of reconstituted cakes after 1 day of storage at the indicated sugar compositions after lyophilization cycle #3. Figure 10A shows the point change in PDI of reconstituted formulations at the indicated sugar compositions. Figure 10B shows the log10 fold change in particle size compared to fresh complexes of lipid carrier + RNA.

[0032] [Figure 11] FIG. 11 illustrates RNA integrity by agarose gel electrophoresis at the indicated sugar compositions after lyophilization cycle #3.

[0033] [Figure 12] FIG. 12 illustrates SEAP expression in BHK21 cells at the indicated sugar compositions after lyophilization cycle #3.

[0034] [Figure 13A] Figures 13A-13B show that miglyol-lipid carrier formulations induce enhanced repRNA protein production in macrophages. Figure 13A shows the absorption spectra of various formulations with Nano Luciferase-encoded replicon RNA in the first experiment. Figure 13B shows the absorption spectra of various formulations with Nano Luciferase-encoded replicon RNA in the second experiment. [Figure 13B] Same as above.

[0035] [Figure 14A] Figures 14A-14B show the reduction of macrophage TNF-alpha (α) production by miglyol formulations. Figure 14A shows the production of TNF-alpha for various formulations in a first experiment. Figure 14B shows the production of TNF-alpha for various formulations in a second experiment. [Figure 14B] Same as above.

[0036] [Figure 15] Figures 15A-15B show the correlation between enhanced macrophage protein production and low TNF-alpha production by miglyol formulations. Figure 15A shows the correlation between sec-NanoLuc and TNF-alpha for various formulations in a first experiment. Figure 15B shows the correlation between sec-NanoLuc and TNF-alpha for various formulations in a second experiment.

[0037] [Figure 16-1] 16A-16F illustrate SEAP levels in BALB / c mice injected intramuscularly with various replicates of lipid carrier-formulated DNA SEAP. FIG. 16A shows the relative light units (RLU) on day 4. FIG. 16B shows the relative light units (RLU) on day 6. FIG. 16C shows the relative light units (RLU) on day 8. FIG. 16D is a copy of the relative light units (RLU) on day 4. FIG. 16E is a copy of the relative light units (RLU) on day 6. FIG. 16F is a copy of the relative light units (RLU) on day 8. [Figure 16-2] Same as above. [Figure 16-3] Same as above.

[0038] [Figure 17] FIG. 17 is a bar graph with Z-average and polydispersity index (PDI) measurements on the Y-axis and group number on the X-axis for conditions 1-14.

[0039] [Figure 18]18A-18B show dot charts showing anti-D614G IgG levels for conditions 1-14. FIG. 18A shows a dot chart with IgG (μg / ml) on the Y-axis and group number on the X-axis for conditions 1-14. Measurements were recorded on day 14 for the IgG response of anti-D614G (1:40 dilution). FIG. 18B shows a dot chart with IgG (μg / ml) on the Y-axis and group number on the X-axis for conditions 1-14. Measurements were recorded on day 28 for the IgG response of anti-D614G (1:200 dilution).

[0040] [Figure 19] FIG. 19 shows a dot chart at day 28 with IgG (μg / ml) measurements of anti-D614G (1:200 dilution) on the Y-axis and indications of storage conditions on the X-axis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Detailed Description Various aspects are now described more fully hereinafter. Such aspects may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey its scope to those skilled in the art.

[0042] Provided herein is a method for the preparation of a therapeutically relevant composition with enhanced safety profile.In some embodiments, the composition comprises a lipid nanoparticle carrier, optionally complexed with nucleic acid.In further embodiments, the composition is lyophilized. definition

[0043] All definitions, as defined and used herein, should be understood to control for any dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meaning of the defined terms.

[0044] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter cited, and in some cases may include the entirety of the document.All references disclosed herein, including patent and non-patent literature, are hereby incorporated by reference in their entirety, as if each were individually incorporated.However, when patents, patent applications or publications containing express definitions are incorporated by reference, it should be understood that these express definitions apply to the incorporated patents, patent applications or publications in which they are found, and do not necessarily apply to the text of this application, particularly the claims of this application, and in that case, the definitions provided herein shall mean priority.

[0045] The indefinite articles "a" and "an," as used herein in the specification and the claims, unless clearly dictated to the contrary, should be understood to mean "at least one."

[0046] The term "and / or" as used herein in the specification and claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjointly present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. Other elements, whether related or unrelated to the specifically identified elements, may optionally be present other than the elements specifically identified by the "and / or" clause. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with an open-ended term such as "comprising", may refer in one embodiment to only A (including elements other than B, if desired), in another embodiment to only B (including elements other than A, if desired), in yet another embodiment to both A and B (including other elements, if desired), etc.

[0047] When used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as being inclusive, i.e., including at least one of a number or list of elements, but also including two or more, and including additional unlisted items, if necessary. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or," when used herein, shall be interpreted merely as indicating exclusive alternatives (i.e., "either one, but not both") when preceded by an exclusive term, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0048] The compositions of the present disclosure may comprise, consist essentially of, or consist of the disclosed components.

[0049] As used herein, "as needed" or "as needed" means that the situation described thereafter may or may not occur, such that the description includes instances where the situation occurs and instances where the situation does not occur.

[0050] As used herein, the term "about" or "approximately" means within a range of up to ±20% of a given value. When a particular value is described in this application and claims, unless otherwise stated, the term "about" is implicit and, in this context, means within an acceptable range of error for the particular value.

[0051] As used herein, the term "N / P ratio (or N:P)" refers to the ratio of positively charged polymeric amine (N=nitrogen) groups to negatively charged nucleic acid phosphate (P) groups. The N / P characteristics of a polymer / nucleic acid complex can affect many other properties, such as its net surface charge, size, and stability.

[0052] As used herein, "modified nucleotide" refers to a nucleotide that contains one or more chemical modifications (e.g., substitutions) in or on the nitrogenous base of the nucleoside (e.g., cytosine (C), thymine (T) or uracil (U), adenine (A) or guanine (G)).

[0053] Where a range of values ​​is provided herein, it is understood that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges, which may be independently included in the smaller ranges, subject to any specifically excluded limitations in the stated range, are also encompassed within the disclosure. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. For example, any concentration range, percentage range, ratio range, or integer range provided herein should be understood to include any integer value within the recited range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers), unless otherwise indicated. Also, any numerical range recited herein relating to any physical feature, such as polymer subunits, size, or thickness, should be understood to include any integer within the recited range, unless otherwise indicated. Nanoparticle Carrier System

[0054] Provided herein are various compositions that include single nanoparticles and multiple nanoparticles.Nanoparticles are also referred to herein as carriers or abbreviated as NP.Provided herein are nanoparticles that can be organic materials, inorganic materials, or combinations of inorganic and organic materials with diameters of less than about 1 micrometer (μm).In some embodiments, provided herein are nanoparticles that are used as delivery systems for provided herein bioactive agents.

[0055] In some embodiments, a dry composition is provided, comprising: a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles, and one or more lipids; one or more nucleic acids; and at least one cryoprotectant. The composition can be spray-dried or freeze-dried using techniques known in the art. The composition is thermally stable. For example, the composition is thermally stable at about 25 degrees Celsius, about 45 degrees Celsius, about -20 degrees Celsius, and about 2 degrees Celsius to about 8 degrees Celsius. The composition is thermally stable for at least one week, at least two weeks, and / or at least one month.

[0056] Provided herein is a method for freeze-drying the compositions described herein.In some embodiments, sublimation, or primary drying, is carried out at this point to remove unbound water; then secondary drying is carried out to sublimate bound water and reduce the material to a desired residual moisture level.Considering that the water at this stage is bound to the target rather than unbound, typically more energy is required to drive this process.Spray drying may also be used, which is often a faster process and involves the conversion of liquid formulations to dry powder in a single step.The solution is atomized into fine droplets, which are rapidly dried in a large chamber using warm gas without pause.The resulting dried particles can then be collected in a cyclone.Additional drying techniques include, for example, spray freeze drying and supercritical fluid drying.

[0057] The present disclosure provides the use of lipid carriers as carriers of one or more nucleic acids, such as RNA. In particular, a solid inorganic core in a lipid matrix with a charged coating in a buffer solution is disclosed. The use of these nanoparticles has many advantages: RNA can be complexed independently of the particle, and the particle can be designed to have a magnetic signal, for example, usable for MRI or other imaging techniques. The RNA is protected by the particle, and when given to a cell or organism, they drive the expression from the protected RNA of many types of proteins, including antigens off of.

[0058] A variety of nanoparticles and nanoparticle formulations (i.e., nanoemulsions) are used. Exemplary nanoparticles are illustrated in Figures 1A-1C. Nanoparticles provided herein may include, but are not limited to, oil-in-water emulsions, nanostructured lipid carriers (NLCs), cationic nanoemulsions (CNEs), vesicular phospholipid gels (VPGs), polymeric nanoparticles, cationic lipid nanoparticles, liposomes, gold nanoparticles, solid lipid nanoparticles (LNPs or SLNs), mixed-phase core NLCs, ionized lipid carriers, magnetic carriers, polyethylene glycol (PEG)-functionalized carriers, cholesterol-functionalized carriers, polylactic acid (PLA)-functionalized carriers, and polylactic-co-glycolic acid (PLGA)-functionalized lipid carriers.

[0059] An oil-in-water emulsion, as illustrated in FIG. 1A (not to scale), is a stable immiscible fluid containing oil droplets dispersed in water or an aqueous phase. FIG. 1B (not to scale) illustrates a nanostructured lipid carrier (NLC), which may include a blend of solid organic lipids (e.g., trimyristin) and liquid oil (e.g., squalene). In the NLC, the solid lipid is dispersed in the liquid oil. The entire nanodroplet is dispersed in the aqueous (water) phase. In some embodiments, the nanoparticles include inorganic nanoparticles as solid inorganic nanoparticles (e.g., iron oxide nanoparticles) dispersed in the liquid oil, as illustrated in FIG. 1C (not to scale). The entire nanodroplet is then dispersed as a colloid in the aqueous (water) phase. In some embodiments, the nanoparticles provided herein are dispersed in an aqueous solution. Non-limiting examples of aqueous solutions include water (e.g., sterile, distilled, deionized, ultrapure, RNAse-free, etc.), saline (e.g., Krebs, Ascaris, Dent's, Tet's saline), or 1% (w / v) dimethyl sulfoxide (DMSO) in water.

[0060] Various compositions and methods comprising lipid carriers are provided herein.The lipid carrier is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids.The hydrophobic core of lipid carrier comprises oil.In some embodiments, the oil is in liquid phase.

[0061] In some embodiments, the nanoparticles provided herein comprise a hydrophilic surface. In some embodiments, the hydrophilic surface comprises a cationic lipid. In some embodiments, the hydrophilic surface comprises an ionized lipid. In some embodiments, the nanoparticles comprise a membrane. In some embodiments, the membrane comprises a cationic lipid. In some embodiments, the nanoparticles provided herein comprise a cationic lipid. Exemplary cationic lipids for inclusion into hydrophilic surfaces include, but are not limited to, 1,2-dioleoyloxy-3(trimethylammonium)propane (DOTAP), 3β-[N-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol (DC cholesterol), dimethyldioctadecylammonium (DDA); 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), dipalmitoyl (C16:0) trimethylammonium propane (DPTAP), distearoyltrimethylammonium propane (DSTAP), N-[1-(2,3-dioleyloxy)propyl]N,N,N trimethylammonium, chloride (DOTMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 1,2-dioleoyl-3-di methylammonium-propane (DODAP), and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 1,1'-((2-(4-(2-((2-(bis(2-hydroxy-dodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), 306Oi10, tetrakis(8-methylnonyl)3 ,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate; 9A1P9, decyl(2-(dioctylammonio)ethyl)phosphate; A2-Iso5-2DC18, ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate;ALC-0315, ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); ALC-0159, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; β-sitosterol, (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17 -tetradecahydro-1H-cyclopenta[a]phenanthren-3-ol; BAME-O16B, bis(2-(dodecyldisulfanyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl) dipropionate; BHEM-cholesterol, 2-((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10, 11,12,13,14,15,16,17-Tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethane-1-aminium bromide; cKK-E12, 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione; DC-cholesterol, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol; DLin-MC3-DMA, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOSPA, 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; ePC, ethylphosphatidylcholine;FTT5, hexa(octan-3-yl) 9,9',9'',9''',9'''',9'''''-(((benzene-1,3,5-tricarbonyl)iris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanoate; Lipid H (SM-102), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate; OF-Deg-Lin, (((3,6-dioxopiperazine-2,5-diyl)bis(butane- 4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl)(9Z,9'Z,9''Z,9''Z,12Z,12'Z,12''Z,12''Z)-tetrakis(octadeca-9,12-dienoate); PEG2000-DMG, (R)-2,3-bis(myristoyloxy)propyl-1-(methoxypoly(ethylene glycol)2000)carbamate; TT3, or N1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide. Other examples of suitable classes of lipids include, but are not limited to, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG); and PEGylated lipids (e.g., DSPE-PEG), including PEGylated versions of any of the above lipids. In some embodiments, the nanoparticles provided herein comprise DOTAP;

[0062] In some embodiments, the nanoparticles provided herein comprise oil. In some embodiments, the oil is in a liquid phase. Non-limiting examples of oils that can be used include alpha-tocopherol, coconut oil, grape seed oil, lauroyl polyoxyl glyceride, mineral oil, monoacylglycerol, palm kernel oil, olive oil, paraffin oil, peanut oil, propolis, squalene, squalane, solanesol, soy lecithin, soybean oil, sunflower oil, triglyceride, or vitamin E. In some embodiments, the nanoparticles provided herein comprise triglyceride. Exemplary triglycerides include, but are not limited to, capric triglyceride, caprylic triglyceride, caprylic and capric triglycerides, triglyceride esters, and myristate triglyceride.

[0063] In some embodiments, the nanoparticles provided herein comprise a liquid organic material and a solid inorganic material. In some embodiments, the nanoparticles provided herein comprise inorganic particles. In some embodiments, the inorganic particles are solid inorganic particles. In some embodiments, the nanoparticles provided herein comprise inorganic particles within a hydrophobic core. In some embodiments, the oil is in the solid phase. In some embodiments, the oil comprises solanesol.

[0064] In some embodiments, the nanoparticles provided herein comprise a metal. In some embodiments, the nanoparticles provided herein comprise a metal in a hydrophobic core. The metal can be, but is not limited to, a metal salt, a metal oxide, a metal hydroxide, or a metal phosphate. In some embodiments, the nanoparticles provided herein comprise a metal salt, but is not limited to, a metal salt, a metal oxide, a metal hydroxide, a metal phosphate, or a metal salt. In some embodiments, the nanoparticles provided herein comprise a metal salt, but is not limited to, a metal salt, a metal oxide, a metal hydroxide, a metal salt ... x (PO4) y ), calcium phosphate (Ca3(PO4)2), calcium hydroxyapatite (Ca 10(PO4)6(OH)2), iron gluconate, or iron sulfate. The inorganic particles may be formed from one or more of the same or different metals (any metal including transition metals). In some embodiments, the inorganic particles are transition metal oxides. In some embodiments, the transition metal is magnetite (Fe3O4), maghemite (y-Fe2O3), wustite (FeO), or hematite (alpha (α)-Fe2O3). In some embodiments, the metal is aluminum hydroxide or aluminum oxyhydroxide, and a phosphate-terminated lipid or surfactant, such as oleic acid, oleylamine, SDS, TOPO, or DSPA, is used to coat the inorganic solid nanoparticles before being mixed with the liquid oil to form the hydrophobic core.

[0065] In some embodiments, the metal can include a paramagnetic, superparamagnetic, ferrimagnetic, or ferromagnetic compound, hi some embodiments, the metal is superparamagnetic iron oxide (Fe3O4).

[0066] In some embodiments, the nanoparticles provided herein comprise cationic lipids, oils, and inorganic particles. In some embodiments, the nanoparticles provided herein comprise DOTAP; squalene and / or glyceryl trimyristate-dynasan; and iron oxide. In some embodiments, the nanoparticles provided herein further comprise a surfactant. Thus, in some embodiments, the nanoparticles provided herein comprise cationic lipids, oils, inorganic particles, and surfactants.

[0067] A surfactant is a compound that reduces the surface tension between two liquids or between a liquid and a solid component of the nanoparticles provided herein. Surfactants can be hydrophobic, hydrophilic, or amphiphilic. In some embodiments, the nanoparticles provided herein include a hydrophobic surfactant. Exemplary hydrophobic surfactants that can be used include, but are not limited to, sorbitan monolaurate (SPAN® 20), sorbitan monopalmitate (SPAN® 40), sorbitan monostearate (SPAN® 60), sorbitan tristearate (SPAN® 65), sorbitan monooleate (SPAN® 80), and sorbitan trioleate (SPAN® 85). Suitable hydrophobic surfactants include those having a hydrophilic-lipophilic balance (HLB) value of 10 or less, e.g., 5 or less, 1-5, or 4-5. For example, the hydrophobic surfactant can be a sorbitan ester having an HLB value of 1 to 5, or 4 to 5. In some embodiments, the nanoparticles provided herein comprise a hydrophilic surfactant, also referred to as an emulsifier.

[0068] In some embodiments, the nanoparticles provided herein comprise polysorbates. Polysorbates are oily liquids derived from ethoxylated sorbitan (a derivative of sorbitol) esterified with fatty acids. In some embodiments, the nanoparticles or lipid carriers provided herein comprise hydrophilic surfactants. Exemplary hydrophilic surfactants that can be used include, but are not limited to, polysorbates, such as TWEEN®, Kolliphor, Scattics, Alkest, or Canarcel; polyoxyethylene sorbitan esters (polysorbates); polysorbate 80 (polyoxyethylene sorbitan monooleate, or TWEEN® 80); polysorbate 60 (polyoxyethylene sorbitan monostearate, or TWEEN® 60); polysorbate 40 (polyoxyethylene sorbitan monopalmitate, or TWEEN® 40); and polysorbate 20 (polyoxyethylene sorbitan monolaurate, or TWEEN® 20). In one embodiment, the hydrophilic surfactant is Polysorbate 80.

[0069] The nanoparticles provided herein comprise a hydrophobic core surrounded by a lipid membrane (e.g., a cationic lipid such as DOTAP). In some embodiments, the hydrophobic core comprises one or more inorganic particles; a phosphate-terminated lipid; and a surfactant.

[0070] The inorganic solid nanoparticles described herein may be surface modified prior to mixing with liquid oil. For example, if the surface of the inorganic solid nanoparticles is hydrophilic, the inorganic solid nanoparticles may be coated with hydrophobic molecules (or surfactants) to promote the miscibility of the inorganic solid nanoparticles with the liquid oil in the "oil" phase of the nanoemulsion particle. In some embodiments, the inorganic particles are coated with capping ligands, phosphate-terminated lipids, and / or surfactants. In some embodiments, the hydrophobic core comprises a phosphate-terminated lipid. Exemplary phosphate-terminated lipids that may be used include, but are not limited to, trioctylphosphine oxide (TOPO) or distearylphosphatidic acid (DSPA). In some embodiments, the hydrophobic core comprises a surfactant that is a phosphorus-terminated surfactant, a carboxylate-terminated surfactant, a sulfate-terminated surfactant, or an amine-terminated surfactant. A typical carboxylate-terminated surfactant includes oleic acid. A typical amine-terminated surfactant includes oleylamine. In some embodiments, the surfactant is distearyl phosphatidic acid (DSPA), oleic acid, oleylamine, or sodium dodecyl sulfate (SDS). In some embodiments, the inorganic solid nanoparticles are metal oxides, such as iron oxide, and are coated with a surfactant, such as oleic acid, oleylamine, SDS, DSPA, or TOPO, before mixing with the liquid oil to form a hydrophobic core.

[0071] In some embodiments, the hydrophobic core comprises one or more inorganic particles containing at least one metal hydroxide or oxyhydroxide particle, optionally coated with a phosphate-terminated lipid, phosphorus-terminated surfactant, carboxylate-terminated surfactant, sulfate-terminated surfactant, or amine-terminated surfactant; and a naturally occurring or synthetic squalene-containing liquid oil; a cationic lipid comprising DOTAP; a hydrophobic surfactant comprising a sorbitan ester selected from the group consisting of sorbitan monostearate, sorbitan monooleate, and sorbitan trioleate; and a hydrophilic surfactant comprising a polysorbate.

[0072] In some embodiments, the hydrophobic core comprises one or more inorganic nanoparticles containing aluminum hydroxide or aluminum oxyhydroxide nanoparticles, optionally coated with TOPO, as well as a naturally occurring or synthetic squalene-containing liquid oil; the cationic lipid DOTAP; a hydrophobic surfactant including sorbitan monostearate; and a hydrophilic surfactant including polysorbate 80.

[0073] In some embodiments, the hydrophobic core is comprised of one or more inorganic particles containing at least one metal hydroxide or oxyhydroxide particle, optionally coated with a phosphate-terminated lipid, phosphorus-terminated surfactant, carboxylate-terminated surfactant, sulfate-terminated surfactant, or amine-terminated surfactant; and a liquid oil containing naturally occurring or synthetic squalene; a cationic lipid containing DOTAP; a hydrophobic surfactant containing a sorbitan ester selected from the group consisting of sorbitan monostearate, sorbitan monooleate, and sorbitan trioleate; and a hydrophilic surfactant containing polysorbate. In some embodiments, the hydrophobic core is comprised of one or more inorganic nanoparticles containing aluminum hydroxide or aluminum oxyhydroxide nanoparticles, optionally coated with TOPO, and a liquid oil containing naturally occurring or synthetic squalene; a cationic lipid DOTAP; a hydrophobic surfactant containing sorbitan monostearate; and a hydrophilic surfactant containing polysorbate 80. In some embodiments, the nanoparticles provided herein may comprise about 0.2% to about 40% w / v squalene, about 0.001% to about 10% w / v iron oxide nanoparticles, about 0.2% to about 10% w / v DOTAP, about 0.25% to about 5% w / v sorbitan monostearate, and about 0.5% to about 10% w / v polysorbate 80. In some embodiments, the nanoparticles provided herein may comprise about 2% to about 6% w / v squalene, about 0.01% to about 1% w / v iron oxide nanoparticles, about 0.2% to about 1% w / v DOTAP, about 0.25% to about 1% w / v sorbitan monostearate, and about 0.5% to about 5% w / v polysorbate 80. In some embodiments, the nanoparticles provided herein may comprise about 0.2% to about 40% w / v squalene, about 0.001% to about 10% w / v aluminum hydroxide or aluminum oxyhydroxide nanoparticles, about 0.2% to about 10% w / v DOTAP, about 0.25% to about 5% w / v sorbitan monostearate, and about 0.5% to about 10% w / v polysorbate 80.In some embodiments, the nanoparticles provided herein may comprise about 2% to about 6% w / v squalene, about 0.01% to about 1% w / v aluminum hydroxide or aluminum oxyhydroxide nanoparticles, about 0.2% to about 1% w / v DOTAP, about 0.25% to about 1% w / v sorbitan monostearate, and about 0.5% to about 5% w / v polysorbate 80.

[0074] In some embodiments, the compositions described herein comprise at least one nanoparticle formulation described in Table 1. In some embodiments, the compositions described herein comprise any one of NP-1 to NP-30. In some embodiments, the compositions described herein comprise any one of NP-1 to NP-31. In some embodiments, the nanoparticles provided herein are mixed with the nucleic acids provided herein. In some embodiments, the nanoparticles provided herein are made by homogenization and sonication techniques. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0075] In some embodiments, the nanoparticles provided herein comprise sorbitan monostearate (e.g., SPAN® 60), polysorbate 80 (e.g., TWEEN® 80), DOTAP, squalene, and are free of solid particles. In some embodiments, the nanoparticles provided herein comprise sorbitan monostearate (e.g., SPAN® 60), polysorbate 80 (e.g., TWEEN® 80), DOTAP, squalene, and iron oxide particles. In some embodiments, the nanoparticles provided herein comprise an immune stimulant. In some embodiments, the immune stimulant is squalene. In some embodiments, the immune stimulant is a medium chain triglyceride. In some embodiments, the immune stimulant is Miglyol 810 or Miglyol 812. Miglyol 810 is a triglyceride ester of saturated caprylic and capric fatty acids and glycerol. Miglyol 812 is a triglyceride ester of saturated coconut oil / palm kernel oil derived caprylic and capric fatty acids and vegetable derived glycerol. In some embodiments, the immune stimulant may reduce the total amount of protein produced but increase the immune response to the compositions provided herein (e.g., when delivered as a vaccine). In some embodiments, the immune stimulant may increase the total amount of protein produced but decrease the immune response to the compositions provided herein.

[0076] The nanoparticles provided herein can be of various average diameter sizes. In some embodiments, the nanoparticles provided herein have an average diameter (z-average hydrodynamic diameter, measured by dynamic light scattering) ranging from about 20 nanometers (nm) to about 200 nm. In some embodiments, the z-average diameter of the nanoparticles ranges from about 20 nm to about 150 nm, about 20 nm to about 100 nm, about 20 nm to about 80 nm, or about 20 nm to about 60 nm. In some embodiments, the z-average diameter of the nanoparticles ranges from about 40 nm to about 200 nm, about 40 nm to about 150 nm, about 40 nm to about 100 nm, about 40 nm to about 90 nm, about 40 nm to about 80 nm, or about 40 nm to about 60 nm. In one embodiment, the z-average diameter of the nanoparticles is about 40 nm to about 80 nm. In some embodiments, the z-average diameter of the nanoparticles is about 40 nm to about 60 nm. In some embodiments, the nanoparticles are up to 100 nm in diameter. In some embodiments, the nanoparticles are 50-70 nm in diameter. In some embodiments, the nanoparticles are 40-80 nm in diameter. In some embodiments, the inorganic particles (e.g., iron oxide) within the hydrophobic core of the nanoparticles can have an average diameter (number weighted average diameter) ranging from about 3 nm to about 50 nm. For example, the inorganic particles can have an average diameter of about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, or about 50 nm.

[0077] The nanoparticles provided herein may be characterized by their polydispersity index (PDI), which is an indication of their quality with respect to size distribution. In some embodiments, the average polydispersity index (PDI) of the nanoparticles provided herein ranges from about 0.1 to about 0.5. In some embodiments, the average PDI of the nanoparticles may range from about 0.2 to about 0.5, about 0.1 to about 0.4, about 0.2 to about 0.4, about 0.2 to about 0.3, or about 0.1 to about 0.3.

[0078] In some embodiments, the nanoparticles provided herein comprise a molar ratio of oil to surfactant ranging from about 0.1:1 to about 20:1, from about 0.5:1 to about 12:1, from about 0.5:1 to about 9:1, from about 0.5:1 to about 5:1, from about 0.5:1 to about 3:1, or from about 0.5:1 to about 1:1. In some embodiments, the nanoparticles provided herein comprise a ratio of hydrophilic surfactant to lipid ranging from about 0.1:1 to about 2:1, from about 0.2:1 to about 1.5:1, from about 0.3:1 to about 1:1, from about 0.5:1 to about 1:1, or from about 0.6:1 to about 1:1. In some embodiments, the nanoparticles provided herein comprise a ratio of hydrophobic surfactant to lipid ranging from about 0.1:1 to about 5:1, about 0.2:1 to about 3:1, about 0.3:1 to about 2:1, about 0.5:1 to about 2:1, or about 1:1 to about 2:1. In some embodiments, the nanoparticles provided herein comprise about 0.2% to about 40% w / v liquid oil, about 0.001% to about 10% w / v inorganic solid nanoparticles, about 0.2% to about 10% w / v lipid, about 0.25% to about 5% w / v hydrophobic surfactant, and about 0.5% to about 10% w / v hydrophilic surfactant. In some embodiments, the lipid comprises a cationic lipid, the oil comprises squalene, and / or the hydrophobic surfactant comprises a sorbitan ester. In some embodiments, the nanoparticles provided herein comprise a ratio of esters that results in a hydrophilic-lipophilic balance of 8 to 11. In some embodiments, the nucleic acids provided herein are incorporated into, associated with, or complexed with the lipid carriers provided herein to form lipid carrier-nucleic acid complexes. In some embodiments, the lipid carrier-nucleic acid complexes are formed via non-covalent interactions or via reversible covalent interactions. nucleic acid

[0079] In some embodiments, the composition described herein comprises one or more nucleic acids.In some embodiments, the nucleic acid is DNA or RNA.Various types of RNA, including RNA that modulates natural immune response, RNA that codes for protein or antigen, silencing RNA, microRNA, tRNA, self-replicating RNA, etc., can be associated with lipid carrier for delivery.In a specific aspect of the present disclosure, the RNA is self-replicating RNA.

[0080] Provided herein are compositions comprising nanoparticles and nucleic acids. In some embodiments, the nucleic acid is in a complex with the nanoparticle. In some embodiments, the nucleic acid is in a complex with the membrane of the nanoparticle. In some embodiments, the nucleic acid is in a complex with the hydrophilic surface of the nanoparticle. In some embodiments, the nucleic acid is within the nanoparticle. In some embodiments, the nucleic acid is within the hydrophobic core.

[0081] In some embodiments, the one or more nucleic acids encode an RNA or DNA polymerase. In some embodiments, the one or more nucleic acids encode an RNA-dependent RNA polymerase. In some embodiments, the one or more nucleic acids encode elements for self-replication, such as an RNA polymerase (e.g., VEEV polymerase).

[0082] The self-replicating nucleotide sequence generally contains at least one or more genes selected from the group consisting of viral replicase, viral protease, viral helicase and other non-structural viral proteins, and also contains 5'- and 3'-terminal cis-active replication sequences, and antigen sequences encoding cancer-related proteins. The subgenomic promoter that directs the expression of the heterologous sequence may be included in the self-replicating nucleotide sequence. If desired, the heterologous sequence may be fused in frame with other coding regions in the self-replicating RNA and / or may be under the control of an internal ribosome entry site (IRES).

[0083] In various embodiments, the self-replicating nucleotide sequence is a self-replicating RNA molecule. The self-replicating RNA molecule of the present disclosure can be designed so that it cannot induce the production of infectious virus particles. This can be achieved, for example, by removing one or more viral genes that code for structural proteins that are necessary for the production of virus particles in the self-replicating RNA. For example, when the self-replicating RNA molecule is based on an alphavirus, such as Sindbis virus (SIN), Semliki Forest virus and Venezuelan equine encephalitis virus (VEE), one or more genes that code for viral structural proteins, such as capsid and / or envelope glycoproteins, can be removed. If desired, the self-replicating RNA molecule of the present disclosure can be designed to induce the production of infectious virus particles that are attenuated or pathogenic, or to generate virus particles that are capable of a single round of subsequent infection.

[0084] When a self-replicating RNA molecule is delivered to a vertebrate cell without any protein, it can result in the generation of multiple daughter RNAs by transcription from itself (or from its own antisense copy). After delivery to a cell, the self-replicating RNA can be directly translated, which then provides an RNA-dependent RNA polymerase that generates transcripts from the delivered RNA. In this way, the delivered RNA results in the generation of multiple daughter RNAs. These transcripts can be antisense to the delivered RNA and can themselves be translated to provide in situ expression of the encoded cancer-associated protein, or can be transcribed to provide additional transcripts with the same sense as the delivered RNA that are translated to provide in situ expression of the encoded cancer-associated protein.

[0085] Optionally, the self-replicating RNA can contain chemical modifications in or on the sugar moiety of the nucleoside (e.g., ribose, deoxyribose, modified ribose, modified deoxyribose, six-membered sugar analogs, or open-ring sugar analogs) or at the phosphate.

[0086] The self-replicating RNA molecules of the present disclosure may contain one or more modified nucleotides, and thus have improved stability, resistance to in vivo degradation and clearance, and other advantages. Without wishing to be bound by any particular theory, it is believed that the self-replicating RNA molecules containing modified nucleotides avoid or reduce the stimulation of endosomes and cytoplasmic immune receptors when the self-replicating RNA is delivered to cells. This allows self-replication, amplification, and expression of proteins to occur. This also reduces safety concerns compared to self-replicating RNAs that do not contain modified nucleotides, since the self-replicating RNAs containing modified nucleotides reduce the activation of the innate immune system and the subsequent undesirable consequences (e.g., inflammation at the injection site, irritation at the injection site, pain, etc.). It is also believed that the RNA molecules generated as a result of self-replication are recognized as foreign nucleic acids by cytoplasmic immune receptors. Therefore, the self-replicating RNA molecules containing modified nucleotides provide efficient amplification of RNA in host cells, and expression of cancer-related proteins, as well as adjuvant effects.

[0087] The RNA sequence can be modified, for example, with respect to its codon usage frequency to increase the translation efficiency and half-life of the RNA. A poly-A tail (e.g., of about 30 or more adenosine residues) can be attached to the 3' end of the RNA to increase its half-life. The 5' end of the RNA can be capped with a modified ribonucleotide or its derivative having a m7G(5')ppp(5')N structure (cap 0 structure), which can be incorporated during RNA synthesis or enzymatically engineered after RNA transcription (e.g., by using vaccinia virus capping enzyme (VCE), which consists of mRNA triphosphatase, guanylyl-transferase and guanine-7-methyltransferase, which catalyzes the construction of the N7-monomethylated cap 0 structure). The cap structure can provide stability and translation efficiency to the RNA molecule. The 5' cap of the RNA molecule can be further modified by 2'-O-methyltransferase, which results in the generation of a cap 1 structure (m7Gppp[m2'-O]N), which can further increase the translation efficiency. The Cap1 structure may also increase in vivo potency.

[0088] As used herein, "modified nucleotide" refers to a nucleotide that contains one or more chemical modifications (e.g., substitutions) in or on the nitrogenous base of the nucleoside (e.g., cytosine (C), thymine (T) or uracil (U), adenine (A) or guanine (G)). Optionally, the self-replicating RNA molecule can contain chemical modifications in or on the sugar moiety of the nucleoside (e.g., ribose, deoxyribose, modified ribose, modified deoxyribose, six-membered sugar analogs, or open-ring sugar analogs) or at the phosphate.

[0089] A self-replicating RNA molecule may preferably contain at least one modified nucleotide that is not part of the 5' cap (e.g., in addition to a modification that is part of the 5'' cap). Thus, a self-replicating RNA molecule may contain a modified nucleotide at a single position, may contain a particular modified nucleotide (e.g., pseudouridine, N6-methyladenosine, 5-methylcytidine, 5-methyluridine) at two or more positions, or may contain 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified nucleotides (e.g., at each of one or more positions). Preferably, a self-replicating RNA molecule includes modified nucleotides that contain modifications on or in the nitrogenous base, but do not contain modified sugar or phosphate moieties.

[0090] In some examples, 0.001% to 99% or 100% of the nucleotides in the self-replicating RNA molecule are modified nucleotides, for example, 0.001% to 25%, 0.01% to 25%, 0.1% to 25%, or 1% to 25% of the nucleotides in the self-replicating RNA molecule are modified nucleotides.

[0091] In other examples, 0.001% to 99% or 100% of specific unmodified nucleotides in the self-replicating RNA molecule are replaced with modified nucleotides. For example, about 1% of nucleotides in the self-replicating RNA molecule containing uridine can be modified, for example, by replacing uridine with pseudouridine. In other examples, a desired amount (percentage) of 2, 3, or 4 specific nucleotides (nucleotides containing uridine, cytidine, guanosine, or adenine) in the self-replicating RNA molecule are modified nucleotides. For example, 0.001% to 25%, 0.01% to 25%, 0.1% to 25%, or 1% to 25% of specific nucleotides in the self-replicating RNA molecule are modified nucleotides. In other examples, between 0.001% and 20%, between 0.001% and 15%, between 0.001% and 10%, between 0.01% and 20%, between 0.01% and 15%, between 0.1% and 25%, between 0.01% and 10%, between 1% and 20%, between 1% and 15%, between 1% and 10%, or about 5%, about 10%, about 15%, about 20% of a particular nucleotide in a self-replicating RNA molecule are modified nucleotides.

[0092] It is preferred that less than 100% of the nucleotides in the self-replicating RNA molecule are modified nucleotides. It is also preferred that less than 100% of a particular nucleotide in the self-replicating RNA molecule is modified nucleotide. Thus, preferred self-replicating RNA molecules contain at least some unmodified nucleotides.

[0093] Modified nucleobases that can be incorporated into modified nucleosides and nucleotides and present in RNA molecules include m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine); Am (2-1-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyl adenosine); ms2i6A (2-methylthio-N6-methyladenosine); o-N6isopentenyl adenosine;io6A(N6-(cis-hydroxyisopentenyl)adenosine);ms2io6A(2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine);g6A(N6-glycinylcarbamoyl adenosine);t6A(N6-threonylcarbamoyl adenosine);ms2t6A(2-methylthio-N6-threonylcarbamoyl adenosine);m6t6A(N6-methyl-N6-threonylcarbamoyl adenosine);hn6A(N6-hydroxynorvalylcarbamoyl adenosine sine;ms2hn6A(2-methylthio-N6-hydroxynorvalylcarbamoyl adenosine);Ar(p)(2'-O-ribosyladenosine (phosphate));I(inosine);m1I(1-methylinosine);m'Im(1,2'-O-dimethylinosine);m3C(3-methylcytidine);Cm(2T-O-methylcytidine);s2C(2-thiocytidine);ac4C(N4-acetylcytidine);f5C(5-phonylcytidine);m5Cm(5,2-O-dimethylcytidine);ac4Cm(N4-acetyl2TOmethyl Cytidine;k2C(lysidine);m1G(1-methylguanosine);m2G(N2-methylguanosine);m7G(7-methylguanosine);Gm(2'-O-methylguanosine);m22G(N2,N2-dimethylguanosine);m2Gm(N2,2'-O-dimethylguanosine);m22Gm(N2,N2,2'-O-trimethylguanosine);Gr(p)(2'-O-ribosylguanosine(phosphate));yW(uybutosine);o2yW(peroxyuybutosine);OHyW(hydroxyuybutosine);OHyW *(unmodified hydroxyubutosine); imG (ubutosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G *(Archaeosin);D(Dihydrouridine);m5Um(5,2'-O-Dimethyluridine);s4U(4-Thiouridine);m5s2U(5-Methyl-2-Thiouridine);s2Um(2-Thio-2'-O-Methyluridine);acp3U(3-(3-Amino-3-Carboxypropyl)uridine);hoSU(5-Hydroxyuridine);moSU(5-Methoxyuridine);cmo5U(Uridine 5-Oxyacetic Acid);mcmo5U(Uridine 5-Oxyacetic Acid Methyl Ester);chm5U(5-(Carboxyhydroxymethyl)uridine) uridine);mchm5U(5-(carboxyhydroxymethyl)uridine methyl ester);mcm5U(5-methoxycarbonylmethyluridine);mcm5Um(S-methoxycarbonylmethyl-2-O-methyluridine);mcm5s2U(5-methoxycarbonylmethyl-2-thiouridine);nm5s2U(5-aminomethyl-2-thiouridine);mnm5U(5-methylaminomethyluridine);mnm5s2U(5-methylaminomethyl-2-thiouridine);mnm5se2U(5-methylaminomethyl-2-selenouridine) ncm5U(5-carbamoylmethyluridine); ncm5Um(5-carbamoylmethyl-2'-O-methyluridine); cmnm5U(5-carboxymethylaminomethyluridine); cnmm5Um(5-carboxymethylaminomethyl-2-LO-methyluridine); cmnm5s2U(5-carboxymethylaminomethyl-2-thiouridine); m62A(N6,N6-dimethyladenosine); Tm(2'-O-methylinosine); m4C(N4-methylcytidine); m4Cm(N4,2-O-dimethylcytidine); hm5C (5-hydroxymethylcytidine);m3U(3-methyluridine);cm5U(5-carboxymethyluridine);m6Am(N6,TO-dimethyladenosine);rn62Am(N6,N6,O-2-trimethyladenosine);m2'7G(N2,7-dimethylguanosine);m2'2'7G(N2,N2,7-trimethylguanosine);m3Um(3,2T-O-dimethyluridine);m5D(5-methyldihydrouridine);f5Cm(5-formyl-2'-O-methylcytidine);m1Gm(1,2'-O-dimethylguanosine);m'Am (1,2-O-dimethyladenosine) irinomethyluridine; tm5s2U (S-taurinomethyl-2-thiouridine); imG-14 (4-demethylguanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil , 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N; 2 -dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (abasic residue), m5C, m5U, m6A, s2U, W, or 2'-O-methyl-U. Any one or any combination of these modified nucleobases may be included in the self-replicating RNA of the present disclosure. Many of these modified nucleobases and their corresponding ribonucleosides are available from commercial suppliers.

[0094] In some embodiments, the RNA molecule, optionally the self-replicating RNA molecule, comprises phosphoramidate, phosphorothioate, and / or methylphosphonate linkages.

[0095] Self-replicating RNA molecules containing at least one modified nucleotide can be prepared using any suitable method. Several suitable methods are known in the art for generating RNA molecules containing modified nucleotides. For example, self-replicating RNA molecules containing modified nucleotides can be prepared by transcribing (e.g., in vitro transcription) DNA encoding the self-replicating RNA molecule using a suitable DNA-dependent RNA polymerase, such as T7 phage RNA polymerase, SP6 phage RNA polymerase, T3 phage RNA polymerase, etc., or mutants of these polymerases that allow efficient incorporation of modified nucleotides into RNA molecules. The transcription reaction contains nucleotides and modified nucleotides, as well as other components that support the activity of the selected polymerase, such as suitable buffers and suitable salts. The incorporation of nucleotide analogs into self-replicating RNA can be manipulated, for example, to modify the stability of such RNA molecules, increase resistance to RNase, establish replication after introduction into suitable host cells (the "infectivity" of RNA), and / or induce or reduce innate and adaptive immune responses.

[0096] The presence and / or amount of one or more modified nucleotides in a self-replicating RNA molecule can be determined using any suitable method. For example, the self-replicating RNA can be digested (e.g., using nuclease P1) and dephosphorylated (e.g., using a suitable phosphatase such as CIAP) to monophosphates, and the resulting nucleosides can be analyzed by reverse-phase HPLC (e.g., using a YMC Pack ODS-AQ column (5 microns, 4.6 x 250 mm) and a gradient of 30% B (0-5 min) to 100% B (5-13 min) and 100% B (13-40 min), flow rate (0.7 ml / min), UV detection (wavelength: 260 nm), column temperature (30°C), elution using Buffer A (20 mM acetic acid-ammonium acetate pH 3.5), Buffer B (20 mM acetic acid-ammonium acetate pH 3.5 / methanol [90 / 10]).

[0097] The self-replicating RNA may be associated with a delivery system. The self-replicating RNA may be administered with or without an adjuvant.

[0098] One or more nucleic acids may be incorporated into / associated with / complexed with the lipid carrier to form a lipid carrier-nucleic acid complex, which may be formed via non-covalent interactions or via reversible covalent interactions.

[0099] In some embodiments, the compositions provided herein comprise one or more nucleic acids. In some embodiments, the compositions provided herein comprise two or more nucleic acids. In some embodiments, the compositions provided herein comprise at least one DNA. In some embodiments, the compositions provided herein comprise at least one RNA. In some embodiments, the compositions provided herein comprise at least one DNA and at least one RNA. In some embodiments, the nucleic acids provided herein are present in an amount of more than 5 ng to about 1 mg. In some embodiments, the nucleic acids provided herein are present in an amount of up to about 25, 50, 75, 100, 150, 175 ng. In some embodiments, the nucleic acids provided herein are present in an amount of up to about 1 mg. In some embodiments, the nucleic acids provided herein are present in an amount of about 0.05 μg, 0.1 μg, 0.2 μg, 0.5 μg, 1 μg, 5 μg, 10 μg, 12.5 μg, 15 μg, 25 μg, 40 μg, 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 mg. In some embodiments, the nucleic acid provided herein is present in an amount of 0.05 μg, 0.1 μg, 0.2 μg, 0.5 μg, 1 μg, 5 μg, 10 μg, 12.5 μg, 15 μg, 25 μg, 40 μg, 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 mg. In some embodiments, the nucleic acid provided herein is present in an amount of about 5 μg, about 10 μg, about 25 μg, about 50 μg, or about 100 μg. In some embodiments, the nucleic acid provided herein is present in an amount of up to about 5 μg, about 10 μg, about 25 μg, about 50 μg, or 100 μg. In some embodiments, the nucleic acid is at least about 200, 250, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 nucleotides in length.In some embodiments, the nucleic acid is at most about 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 nucleotides in length. In some embodiments, the nucleic acid is at most about 7500, 10,000, 15,000, or 20,000 nucleotides in length. cryoprotectant

[0100] In some embodiments, the compositions and methods provided herein include at least one cryoprotectant. Exemplary cryoprotectants for inclusion are, but are not limited to, sucrose, maltose, trehalose, mannitol, or glucose, and any combination thereof. In some embodiments, additional or alternative cryoprotectants for inclusion are sorbitol, ribitol, erythritol, threitol, ethylene glycol, or fructose. In some embodiments, additional or alternative cryoprotectants for inclusion are dimethylsulfoxide (DMSO), glycerol, propylene glycol, ethylene glycol, 3-O-methyl-D-glucopyranose (3-OMG), polyethylene glycol (PEG), 1,2-propanediol, acetamide, trehalose, formamide, sugars, proteins, and carbohydrates. In some embodiments, the cryoprotectant is present at about 1% w / v to about 20% w / v, preferably about 10% w / v to about 20% w / v, more preferably about 10% w / v. In certain aspects of the present disclosure, the cryoprotectant is sucrose. In some aspects of the present disclosure, the cryoprotectant is maltose. In some aspects of the present disclosure, the cryoprotectant is trehalose. In some aspects of the present disclosure, the cryoprotectant is mannitol. In some aspects of the present disclosure, the cryoprotectant is glucose. In some embodiments, the cryoprotectant is present in an amount of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 325, 350, 375, 400, 450, 500 mg or more. In some embodiments, the cryoprotectant is present in an amount of about 50 to about 500 mg. In some embodiments, the cryoprotectant is present in an amount of about 200 to about 300 mg. In some embodiments, the cryoprotectant is present in an amount of about 250 mg. In some embodiments, the cryoprotectant is present in an amount of at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more percent by weight of the lyophilized composition.In some embodiments, the cryoprotectant is present in an amount of about 95% by weight of the lyophilized composition. In some embodiments, the cryoprotectant is present in an amount of 80-98%, 85-98%, 90-98%, or 94-96% by weight of the lyophilized composition. In some embodiments, the cryoprotectant is a sugar. In some embodiments, the sugar is sucrose, maltose, trehalose, mannitol, or glucose. In some embodiments, the sugar is sucrose. In some embodiments, sucrose is present in an amount of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 325, 350, 375, 400, 450, 500 mg or more. In some embodiments, sucrose is present in an amount of about 50 to about 500 mg. In some embodiments, sucrose is present in an amount of about 200 to about 300 mg. In some embodiments, sucrose is present in an amount of about 250 mg. In some embodiments, sucrose is present in an amount of at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more percent by weight of the lyophilized composition. In some embodiments, sucrose is present in an amount of about 95% by weight of the lyophilized composition. In some embodiments, sucrose is present in an amount of 80-98%, 85-98%, 90-98%, or 94-96% by weight of the lyophilized composition.

[0101] In some embodiments, the compositions provided herein are thermally stable. A composition is considered to be thermally stable if the composition maintains its properties, such as the ability to resist the effects of heat or cold and protect nucleic acid molecules from degradation at a given temperature. In some embodiments, the compositions and vaccines provided herein are thermally stable at about 25 degrees Celsius or standard room temperature. In some embodiments, the compositions and vaccines provided herein are thermally stable at about 45 degrees Celsius. In some embodiments, the compositions and vaccines provided herein are thermally stable at about -20 degrees Celsius. In some embodiments, the compositions and vaccines provided herein are thermally stable at about 2 degrees Celsius to about 8 degrees Celsius. In some embodiments, the compositions and vaccines provided herein are thermally stable at temperatures of at least about -80 degrees Celsius, at least about -20 degrees Celsius, at least about 0 degrees Celsius, at least about 2 degrees Celsius, at least about 4 degrees Celsius, at least about 6 degrees Celsius, at least about 8 degrees Celsius, at least about 10 degrees Celsius, at least about 20 degrees Celsius, at least about 25 degrees Celsius, at least about 30 degrees Celsius, at least about 37 degrees Celsius, and up to 45 degrees Celsius. In some embodiments, the compositions and vaccines provided herein are thermally stable for at least about 5 days, at least about 1 week, at least about 2 weeks, at least about 1 month, and up to 3 months. In some embodiments, the compositions and vaccines provided herein are stored at temperatures of at least about 4°C and up to 37 degrees Celsius for at least about 5 days, at least about 1 week, at least about 2 weeks, at least about 1 month, and up to 3 months. In some embodiments, the compositions and vaccines provided herein are stored at a temperature of at least about 20 degrees Celsius and up to 25 degrees Celsius for at least about 5 days, at least about 1 week, at least about 2 weeks, at least about 1 month, and up to 3 months. Combination Compositions

[0102] Provided herein are compositions comprising a nucleic acid as described herein and a lipid carrier as described herein. In some embodiments, the lipid carrier comprises NP-1. In some embodiments, the lipid carrier comprises NP-2. In some embodiments, the lipid carrier comprises NP-3. In some embodiments, the lipid carrier comprises NP-4. In some embodiments, the lipid carrier comprises NP-5. In some embodiments, the lipid carrier comprises NP-6. In some embodiments, the lipid carrier comprises NP-7. In some embodiments, the lipid carrier comprises NP-8. In some embodiments, the lipid carrier comprises NP-9. In some embodiments, the lipid carrier comprises NP-10. In some embodiments, the lipid carrier comprises NP-11. In some embodiments, the lipid carrier comprises NP-12. In some embodiments, the lipid carrier comprises NP-13. In some embodiments, the lipid carrier comprises NP-14. In some embodiments, the lipid carrier comprises NP-15. In some embodiments, the lipid carrier comprises NP-16. In some embodiments, the lipid carrier comprises NP-17. In some embodiments, the lipid carrier comprises NP-18. In some embodiments, the lipid carrier comprises NP-18. In some embodiments, the lipid carrier comprises NP-19. In some embodiments, the lipid carrier comprises NP-20. In some embodiments, the lipid carrier comprises NP-21. In some embodiments, the lipid carrier comprises NP-22. In some embodiments, the lipid carrier comprises NP-23. In some embodiments, the lipid carrier comprises NP-24. In some embodiments, the lipid carrier comprises NP-25. In some embodiments, the lipid carrier comprises NP-26. In some embodiments, the lipid carrier comprises NP-27. In some embodiments, the lipid carrier comprises NP-28. In some embodiments, the lipid carrier comprises NP-28. In some embodiments, the lipid carrier comprises NP-29. In some embodiments, the lipid carrier comprises NP-30. In some embodiments, the lipid carrier comprises NP-31. In some embodiments, the lipid carrier comprises any of NP-1-NP-31 and a cryoprotectant.In some embodiments, the cryoprotectant is a sugar as described herein. The compositions provided herein may be characterized by the nitrogen to phosphate (N:P) molar ratio. The N:P ratio is determined by the amount of cationic lipid in the nitrogen-containing nanoparticle and the amount of nucleic acid used in the composition containing negatively charged phosphate. The lipid carrier to nucleic acid molar ratio can be selected to increase the delivery efficiency of the nucleic acid, increase the ability of the nucleic acid-carrying nanoemulsion composition to induce an immune response against an antigen, or increase the ability of the nucleic acid-carrying nanoemulsion composition to induce the production of antibody titers against an antigen in a subject. In some embodiments, the compositions provided herein have a molar ratio of lipid carrier to nucleic acid, which may be characterized by the molar ratio of nitrogen to phosphate, which may range from about 0.01:1 to about 1000:1, e.g., from about 0.2:1 to about 500:1, from about 0.5:1 to about 150:1, from about 1:1 to about 150:1, from about 1:1 to about 125:1, from about 1:1 to about 100:1, from about 1:1 to about 50:1, from about 1:1 to about 50:1, from about 5:1 to about 50:1, from about 5:1 to about 25:1, or from about 10:1 to about 20:1. In some embodiments, the molar ratio of lipid carrier to nucleic acid, characterized by the molar ratio of nitrogen to phosphate (N:P), ranges from about 1:1 to about 150:1, from about 5:1 to about 25:1, or from about 10:1 to about 20:1. In some embodiments, the N:P molar ratio of the nanoemulsion composition is about 15: 1. In some embodiments, the nanoparticles comprise a nucleic acid provided herein covalently attached to a membrane.

[0103] The compositions provided herein may be characterized by the molar ratio of oil to surfactant. In some embodiments, the ratio of oil to surfactant is the molar ratio of squalene: cationic lipid, hydrophobic surfactant, and hydrophilic surfactant. In some embodiments, the ratio of oil to surfactant is the molar ratio of squalene: DOTAP, hydrophobic surfactant, and hydrophilic surfactant. In some embodiments, the ratio of oil to surfactant is the molar ratio of squalene: DOTAP, sorbitan monostearate, and polysorbate 80. In some embodiments, the molar ratio of oil to surfactant is in the range of about 0.1:1 to about 20:1, about 0.5:1 to about 12:1, about 0.5:1 to about 9:1, about 0.5:1 to about 5:1, about 0.5:1 to about 3:1, or about 0.5:1 to about 1:1. In some embodiments, the molar ratio of oil to surfactant is at least about 0.1:1, at least about 0.2:1, at least about 0.3:1, at least about 0.4:1, at least about 0.5:1, at least about 0.6:1, at least about 0.7:1. In some embodiments, the molar ratio of oil to surfactant is at least about 0.4:1 to at most 1:1.

[0104] The compositions provided herein may be characterized by the ratio of hydrophilic surfactant to cationic lipid. In some embodiments, the ratio of hydrophilic surfactant to cationic lipid ranges from about 0.1:1 to about 2:1, from about 0.2:1 to about 1.5:1, from about 0.3:1 to about 1:1, from about 0.5:1 to about 1:1, or from about 0.6:1 to about 1:1. The compositions provided herein may be characterized by the ratio of hydrophobic surfactant to lipid (e.g., cationic lipid). In some embodiments, the ratio of hydrophobic surfactant to lipid ranges from about 0.1:1 to about 5:1, from about 0.2:1 to about 3:1, from about 0.3:1 to about 2:1, from about 0.5:1 to about 2:1, or from about 1:1 to about 2:1. In some embodiments, the cationic lipid is DOTAP.

[0105] Further provided herein is a dry composition comprising a sorbitan fatty acid ester, an ethoxylated sorbitan ester, a cationic lipid, an immunostimulant, and an RNA. Further provided herein is a dry composition comprising a sorbitan monostearate (e.g., SPAN® 60), a polysorbate 80 (e.g., TWEEN® 80), a DOTAP, an immunostimulant, and an RNA. Pharmaceutical Compositions

[0106] In one aspect, the present disclosure provides pharmaceutical compositions comprising lipid carriers and, optionally, the nucleic acid described herein.Optionally, pharmaceutical compositions can comprise pharmaceutically acceptable carriers or excipients.As used herein, the term "pharmaceutically acceptable carriers or excipients" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, etc., that are compatible with pharmaceutical administration.

[0107] The pharmaceutical composition comprises the dry composition reconstituted in a suitable diluent and a pharma- ceutically acceptable carrier. The diluent is aqueous. In a preferred embodiment, the diluent is water.

[0108] The present disclosure also provides a kit comprising the pharmaceutical composition and a delivery system for administration to a subject. The subject may be a mammal. In a preferred embodiment, the subject is a human.

[0109] In another aspect, the present disclosure provides a vaccine delivery system comprising a pharmaceutical composition described herein and, optionally, one or more vaccine adjuvants.

[0110] By complexing the nucleic acid with the lipid carrier, the composition can be delivered to a cell. The cell can be in a subject in need. For example, if the nucleic acid is a protein antigen or encodes a protein antigen, the composition carrying the nucleic acid can induce an immune response in the subject against the antigen. The composition can contribute by inducing antibody titers against the antigen in the subject, for example, by inducing neutralizing antibody titers in the subject.

[0111] The present disclosure also relates to a method for generating an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition.

[0112] In one embodiment, a composition containing a lipid carrier, when administered to a subject in an effective amount, is capable of eliciting an immune response against an antigen that is equal to or greater than the immune response elicited when the nucleic acid is administered to a subject without the lipid carrier.

[0113] Without being bound by theory, a hydrophobic surfactant in a nanoemulsion composition may contribute to increasing the ability of the nanoemulsion composition to deliver a nucleic acid to a cell, or to increasing the ability of the nanoemulsion composition to retain a nucleic acid that elicits an immune response in a subject against an antigen (where the nucleic acid is or encodes a protein antigen). For example, a hydrophobic surfactant in a nanoemulsion composition may contribute to increasing the ability of the nanoemulsion composition to retain a nucleic acid.

[0114] Another aspect of the present disclosure relates to a method of treating or preventing an infection or disease in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition.

[0115] As discussed above, if the inorganic solid nanoparticles contain a reporter element detectable via imaging methods, the resulting nanoemulsion particles can be imaged and tracked after the nanoemulsion particles are administered to the body. For example, the inorganic solid nanoparticles can contain a reporter element detectable via magnetic resonance imaging (MRI), such as a paramagnetic, superparamagnetic, ferrimagnetic, or ferromagnetic compound.

[0116] Accordingly, one aspect of the present disclosure also relates to a method of imaging and / or tracking nucleic acid delivery in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition.

[0117] The present disclosure also provides methods for preparing and reconstituting the freeze-dried and spray-dried compositions.

[0118] In some aspects of the present disclosure, the compositions and methods are useful for treating a variety of diseases and conditions.

[0119] Non-limiting examples of infectious diseases may include viral and non-viral infectious diseases. Examples of viral infectious diseases include, but are not limited to, human papillomavirus (HPV), herpes simplex virus (HSV), varicella zoster virus (VZV), influenza virus, influenza virus types and subtypes, yellow fever virus (YFV), Zika virus, West Nile virus, chikungunya virus, dengue fever, respiratory syncytial virus (RSV), human immunodeficiency virus (HIV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and variants thereof.

[0120] Exemplary diseases include cancers associated with melanoma associated antigen A genes, such as MAGE-A1, MAGE-A3, and tyrosinase-related protein (TYRP) genes, such as TYRP-1.

[0121] In some aspects of the disclosure, the compositions and methods include one or more nucleic acids encoding an antigen, wherein the antigen is derived from a bacterial infection, a bacterial disease, a viral infection, a viral disease, a protozoal infection, a protozoal disease, a non-communicable disease, one or more cancers, or an autoimmune disease.

[0122] In some embodiments, the antigen is derived from a virus selected from the group consisting of human papillomavirus (HPV), herpes simplex virus (HSV), varicella zoster virus (VZV), influenza virus, influenza virus types and subtypes, yellow fever virus (YFV), Zika virus, West Nile virus, chikungunya virus, dengue fever, respiratory syncytial virus (RSV), human immunodeficiency virus (HIV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and variants thereof. Administration and Dosage

[0123] In certain aspects, the dry compositions of the present disclosure can be used to deliver nucleic acid (e.g., RNA, optionally self-replicating RNA) to a cell or subject, such as a mammal, including but not limited to, a human, dog, cat, livestock (e.g., cows, sheep, goats, pigs), horses, etc. Exemplary amounts of total nucleic acid for incorporation in the compositions described herein include about 1, 2, 2.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30, 35, 40, 45, 50 micrograms (μg) or more.

[0124] In some embodiments, the formulations described herein are prepared in a single container for administration.In some embodiments, the formulations described herein are prepared in two containers for administration, separating the formulation from the nanoparticle carrier.

[0125] As used herein, "container" includes vessels, vials, ampoules, tubes, cups, boxes, bottles, flasks, jars, dishes, wells of single- or multi-well instruments, reservoirs, tanks, etc., or other devices in which the compositions disclosed herein may be contained, stored, and / or transported, and accessed to remove contents. Examples of such containers include glass and / or plastic sealed or resealable tubes and ampoules, including those with rubber stoppers or other sealing means compatible with withdrawal of contents using a needle and syringe. In some implementations, the container is RNase-free.

[0126] In preferred embodiments, the dry composition is freeze-dried, hi some embodiments, the dry composition is spray-dried.

[0127] In some embodiments, the lyophilized composition is reconstituted by the methods described herein. In other embodiments, the spray-dried composition is reconstituted by the methods described herein.

[0128] In some embodiments, the pharmaceutical compositions provided herein are in a form that allows the composition to be administered to a subject. In some embodiments, the pharmaceutical composition is in the form of a solid, semi-solid, liquid or gas (aerosol).

[0129] Any suitable route of administration may be used to deliver the nucleic acid to a cell or subject. In some embodiments, the pharmaceutical compositions described herein are formulated for administration and / or for use in administration via intratumoral, subcutaneous, intradermal, intramuscular, inhalation / intranasal, intravenous, intraperitoneal, intracranial, or intrathecal routes. In some embodiments, the pharmaceutical composition is administered parenterally. In some embodiments, the pharmaceutical composition is administered transdermally. In other embodiments, the pharmaceutical composition is administered intramuscularly. In other embodiments, the pharmaceutical composition is administered intradermally. In other embodiments, the pharmaceutical composition is administered transdermally. In other embodiments, the pharmaceutical composition is administered subcutaneously. In other embodiments, the pharmaceutical composition is administered intranasally, e.g., via a nasal spray. In other embodiments, the pharmaceutical composition is administered orally, e.g., via drops.

[0130] In certain aspects, any suitable dosage form may be used for delivery of the pharmaceutical compositions described herein. In some embodiments, the pharmaceutical compositions are provided in an injectable dosage form, such as a solution or suspension.

[0131] In other embodiments, the composition is provided in a dosage form that can be delivered via an inhaler, e.g., a solution, suspension, or powder, and the dosage form is formulated for delivery via an inhaler, such as a metered dose inhaler, a soft mist inhaler, a nebulizer, or a dry powder inhaler.

[0132] In other embodiments, the composition is provided in a dosage form that can be delivered nasally, e.g., a solution, suspension, or powder, and the dosage form is formulated for nasal delivery via an atomizer or nasal pump bottle, or other suitable device for delivery of a pharmaceutical composition administered nasally.

[0133] In certain embodiments, pharma- ceutically acceptable carriers, preservatives, and / or other excipients may be used in the dosage forms described herein for delivery of the compositions of the present disclosure.

[0134] The present disclosure also provides a dry composition that does not contain one or more inorganic nanoparticles. The dry composition comprises a sorbitan fatty acid ester, an ethoxylated sorbitan ester, a cationic lipid, an immunostimulant, and an RNA. In a specific embodiment, the dry composition comprises sorbitan monostearate, polyoxyethylene (20) sorbitan monooleate, DOTAP, an immunostimulant, and an RNA.

[0135] In some embodiments, the immune stimulant may decrease the total amount of protein produced, but increase the immune response to the vaccine. In some embodiments, the immune stimulant may increase the total amount of protein produced, but decrease the immune response to the vaccine. In some embodiments, the immune stimulant is squalene. In some embodiments, the immune stimulant is caprylic / capric triglyceride (MIGLYOL® 810) or triglyceride esters of saturated coconut oil / palm kernel oil-derived caprylic and capric fatty acids and vegetable-derived glycerol (MIGLYOL® 812N). Exemplary embodiments

[0136] Provided herein are dry compositions comprising: (a) a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles, and one or more lipids; (b) one or more nucleic acids; and (c) at least one cryoprotectant. Further provided herein are dry compositions, the composition being lyophilized. Further provided herein are dry compositions, the composition being spray dried. Further provided herein are dry compositions, the composition being thermally stable. Further provided herein are dry compositions, the composition being thermally stable at about 25 degrees Celsius. Further provided herein are dry compositions, the composition being thermally stable at about 45 degrees Celsius. Further provided herein are dry compositions, the composition being thermally stable at about -20 degrees Celsius. Further provided herein are dry compositions, the composition being thermally stable at about 2 degrees Celsius to about 8 degrees Celsius. Further provided herein are dry compositions, the composition being thermally stable for at least one week, at least two weeks, and / or at least one month. Further provided herein are dry compositions, the composition's hydrophobic core comprising an oil. Further provided herein is a dry composition, wherein the oil comprises at least one of α-tocopherol, lauroyl polyoxylglyceride, monoacylglycerol, propolis, squalene, mineral oil, grape seed oil, olive oil, paraffin oil, peanut oil, soybean oil, sunflower oil, soy lecithin, triglyceride, vitamin E, caprylic / capric triglyceride, triglyceride esters of saturated coconut / palm kernel oil-derived caprylic and capric fatty acids and vegetable-derived glycerol, dihydroisosqualene (DHIS), farnesene, and squalane. Further provided herein is a dry composition, wherein the one or more inorganic nanoparticles are selected from the group consisting of metal salts, metal oxides, metal hydroxides, metal phosphates, and any combination thereof. Further provided herein is a dry composition, wherein the one or more lipids are selected from the group consisting of cationic lipids, anionic lipids, neutral lipids, and any combination thereof. Further provided herein is a dry composition, wherein the one or more lipids are cationic lipids.Cationic lipids include 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP); 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol); dimethyldioctadecylammonium (DDA); 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP); dipalmitoyl (C16:0) trimethylammonium propane (DPTAP); distearoyltrimethylammonium propane (DSTAP); N-[1-(2,3-dioleyloxy)propyl]-N,N,N trimethylammonium chloride (DOTMA); N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dioleoyl-3-dimethylammonium Further provided herein is a dry composition selected from the group consisting of N-decyl-N,N-dimethyldecane-1-aminium bromide (DDAB); 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA); ethylphosphatidylcholine (ePC); and any combination thereof. Further provided herein is a dry composition, wherein the lipid carrier optionally comprises at least one surfactant.Further provided herein is a dry composition, wherein the at least one surfactant is selected from the group consisting of hydrophobic surfactants, hydrophilic surfactants, and any combination.Further provided herein is a dry composition, wherein the hydrophobic surfactant comprises a sorbitan ester.Further provided herein is a dry composition, wherein the sorbitan ester is selected from the group consisting of sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, and sorbitan trioleate; and the hydrophilic surfactant comprises a polysorbate. Further provided herein is a dry composition, wherein the lipid carrier has a z-average hydrodynamic diameter ranging from about 40 nm to about 150 nm with an average polydispersity index ranging from about 0.1 to 0.4. Further provided herein is a dry composition, wherein the one or more nucleic acids are RNA. Further provided herein is a dry composition, wherein the RNA is a self-replicating RNA. Further provided herein is a dry composition, wherein the one or more nucleic acids are incorporated into or complexed with the lipid carrier to form a lipid carrier-nucleic acid complex. Further provided herein is a dry composition, wherein the lipid carrier-nucleic acid complex is formed via non-covalent interactions or via reversible covalent interactions. Further provided herein is a dry composition, wherein the molar ratio of lipid carrier to one or more nucleic acids, characterized by the nitrogen to phosphate (N:P) molar ratio, ranges from about 1:1 to about 150:1. Further provided herein is a dry composition, wherein the at least one cryoprotectant is selected from the group consisting of sucrose, maltose, trehalose, mannitol, glucose, and any combination thereof. Further provided herein is a dry composition, wherein the at least one cryoprotectant is sucrose. Further provided herein is a dry composition, wherein the at least one cryoprotectant is about 1% w / v to about 20% w / v. Further provided herein is a dry composition, wherein the at least one cryoprotectant is about 1% w / v to about 40% w / v. Further provided herein is a dry composition, wherein the at least one cryoprotectant is about 10% w / v to about 20% w / v. Further provided herein is a dry composition, wherein the at least one cryoprotectant is about 10% w / v. One or more inorganic nanoparticles may be aluminum oxide (Al2O3), aluminum oxyhydroxide, iron oxide, titanium dioxide, silicon dioxide (SiO2), aluminum hydroxyphosphate (Al(OH).x (PO4) y ), calcium phosphate (Ca3(PO4)2), calcium hydroxyapatite (Ca 10 Further provided herein is a composition, wherein the one or more inorganic nanoparticles comprise magnetite (Fe3O4), maghemite (y-Fe2O3), wustite (FeO), or hematite (alpha (α)-Fe2O3), or any combination thereof.

[0137] Further provided herein is a pharmaceutical composition comprising the dry composition provided herein reconstituted in a suitable diluent; and a pharma- ceutically acceptable carrier. Further provided herein is a pharmaceutical composition in which the suitable diluent is aqueous. Further provided herein is a pharmaceutical composition in which the suitable diluent is water.

[0138] Further provided herein is a kit comprising a pharmaceutical composition provided herein or a dry composition provided herein; and a delivery system for administration to a subject.

[0139] Further provided herein is a vaccine delivery system comprising a pharmaceutical composition provided herein and, optionally, one or more vaccine adjuvants.

[0140] Further provided herein is a method for generating an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition provided herein.

[0141] Further provided herein is a method of treating or preventing a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition provided herein.

[0142] Further provided herein is a method for imaging and / or tracking delivery of one or more nucleic acids in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition provided herein.

[0143] Further provided herein is a method for preparing a lyophilized composition, the method comprising: (a) obtaining a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, optionally one or more inorganic nanoparticles, and one or more lipids; (b) incorporating one or more nucleic acids into the lipid carrier to form a lipid carrier-nucleic acid complex; (c) adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; and (d) lyophilizing the formulation to form a lyophilized composition.

[0144] Further provided herein is a method for preparing a spray-dried composition, the method comprising: (a) obtaining a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, optionally one or more inorganic nanoparticles, and one or more lipids; (b) incorporating one or more nucleic acids into the lipid carrier to form a lipid carrier-nucleic acid complex; (c) adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; and (d) spray drying the formulation to form the spray-dried composition.

[0145] Further provided herein is a method for reconstituting a freeze-dried composition, the method comprising the steps of: (a) obtaining a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, optionally one or more inorganic nanoparticles, and one or more lipids; (b) incorporating one or more nucleic acids into the lipid carrier to form a lipid carrier-nucleic acid complex; (c) adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; freeze-drying the formulation to form a freeze-dried composition; and (d) reconstituting the freeze-dried composition in a suitable diluent. Further provided herein is a method in which the suitable diluent is aqueous. Further provided herein is a method in which the suitable diluent is water. Further provided herein is a method in which the freeze-dried composition is thermally stable. Further provided herein is a method in which the freeze-dried composition is thermally stable for at least one week, at least two weeks, and / or at least one month. Further provided herein is a method in which the freeze-dried composition is thermally stable at about 25 degrees Celsius. Further provided herein is a method, wherein the freeze-dried composition is thermally stable at about 45 degrees Celsius. Further provided herein is a method, wherein the freeze-dried composition is thermally stable at about -20 degrees Celsius. Further provided herein is a method, wherein the freeze-dried composition is thermally stable at about 2 degrees Celsius to about 8 degrees Celsius. The one or more inorganic nanoparticles may be aluminum oxide (Al2O3), aluminum oxyhydroxide, iron oxide, titanium dioxide, silicon dioxide (SiO2), aluminum hydroxyphosphate (Al(OH) x (PO4) y ), calcium phosphate (Ca3(PO4)2), calcium hydroxyapatite (Ca 10 Further provided herein is a composition, wherein the one or more inorganic nanoparticles comprise magnetite (Fe3O4), maghemite (y-Fe2O3), wustite (FeO), or hematite (alpha (α)-Fe2O3), or any combination thereof.

[0146] Further provided herein is a method for reconstituting a spray-dried composition, the method comprising the steps of: (a) obtaining a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core, optionally one or more inorganic nanoparticles, and one or more lipids; (b) incorporating one or more nucleic acids into said lipid carrier to form a lipid carrier-nucleic acid complex; (c) adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; (d) spray drying the formulation to form a spray-dried composition; and (e) reconstituting the spray-dried composition in a suitable diluent. Further provided herein is a method in which the diluent is aqueous. Further provided herein is a method in which the diluent is water. Further provided herein is a method in which the lyophilized composition is thermally stable. Further provided herein is a method in which the lyophilized composition is thermally stable at about 25 degrees Celsius. Further provided herein is a method in which the lyophilized composition is thermally stable at about 45 degrees Celsius. Further provided herein is a method in which the lyophilized composition is thermally stable at about -20 degrees Celsius. Further provided herein is a method, wherein the lyophilized composition is thermally stable at about 2 degrees Celsius to about 8 degrees Celsius. Further provided herein is a method, wherein the hydrophobic core comprises an oil. Further provided herein is a method, wherein the oil comprises at least one of alpha-tocopherol, lauroyl polyoxylglyceride, monoacylglycerol, propolis, squalene, mineral oil, grape seed oil, olive oil, paraffin oil, peanut oil, soybean oil, sunflower oil, soy lecithin, triglyceride, vitamin E, caprylic / capric triglyceride, triglyceride esters of saturated coconut / palm kernel oil-derived caprylic and capric fatty acids and vegetable-derived glycerol, dihydroisosqualene (DHIS), farnesene, and squalane. Further provided herein is a method, wherein the one or more lipids are selected from the group consisting of cationic lipids, anionic lipids, neutral lipids, and any combination thereof. Further provided herein is a method, wherein the one or more lipids comprises a cationic lipid.Cationic lipids include 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP); 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol); dimethyldioctadecylammonium (DDA); 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP); dipalmitoyl (C16:0) trimethylammonium propane (DPTAP); distearoyltrimethylammonium propane (DSTAP); N-[1-(2,3-dioleyloxy)propyl]-N,N,N trimethylammonium chloride (DOTMA); N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dioleoyl-3-dimethylammonium and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA); 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); N-decyl-N,N-dimethyldecane-1-aminium bromide (DDAB); 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA); ethylphosphatidylcholine (ePC); and any combination thereof. Further provided herein is a method, wherein the lipid carrier optionally comprises one or more surfactants. Further provided herein is a method, wherein the one or more surfactants are selected from the group consisting of hydrophobic surfactants, hydrophilic surfactants, and any combination thereof.Further provided herein is a method in which the hydrophobic surfactant comprises a sorbitan ester selected from the group consisting of sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, and sorbitan trioleate; and the hydrophilic surfactant comprises a polysorbate. Further provided herein is a method in which the lipid carrier has a z-average hydrodynamic diameter in the range of about 40 nm to about 150 nm with an average polydispersity index in the range of about 0.1 to about 0.4. Further provided herein is a method in which the one or more nucleic acids are RNA. Further provided herein is a method in which the RNA is a self-replicating RNA. Further provided herein is a method in which the one or more nucleic acids are incorporated into or complexed with the lipid carrier to form a lipid carrier-nucleic acid complex. Further provided herein is a method in which the lipid carrier-nucleic acid complex is formed via non-covalent interactions or via reversible covalent interactions. Further provided herein is a method, wherein the at least one cryoprotectant is selected from the group consisting of sucrose, maltose, trehalose, mannitol, glucose, and any combination thereof. Further provided herein is a method, wherein the at least one cryoprotectant is sucrose. Further provided herein is a method, wherein the at least one cryoprotectant is about 1% w / v to about 20% w / v. Further provided herein is a method, wherein the at least one cryoprotectant is about 10% w / v to about 20% w / v. Further provided herein is a method, wherein the at least one cryoprotectant is about 10% w / v. Further provided herein is a method, wherein the one or more inorganic nanoparticles are selected from the group consisting of aluminum oxide (Al2O3), aluminum oxyhydroxide, iron oxide, titanium dioxide, silicon dioxide (SiO2), aluminum hydroxyphosphate (Al(OH). x (PO4) y ), calcium phosphate (Ca3(PO4)2), calcium hydroxyapatite (Ca 10Further provided herein is a composition comprising one or more inorganic nanoparticles comprising magnetite (Fe3O4), maghemite (y-Fe2O3), wustite (FeO), or hematite (alpha (α)-Fe2O3), or a combination thereof.

[0147] Further provided herein is a dry composition comprising a sorbitan fatty acid ester, an ethoxylated sorbitan ester, a cationic lipid, an immunostimulant, and RNA. Further provided herein is a dry composition in which the sorbitan fatty acid ester is sorbitan monostearate. Further provided herein is a dry composition in which the ethoxylated sorbitan ester is polyoxyethylene (20) sorbitan monooleate. Further provided herein is a dry composition in which the cationic lipid is DOTAP. Further provided herein is a dry composition in which the immunostimulant is squalene. Further provided herein is a dry composition in which the ratio of esters results in a hydrophilic-lipophilic balance of 8-11. Further provided herein is a dry composition in which the ratio of esters and lipids results in a particle size of 30 nm-200 nm. Further provided herein is a dry composition in which the ratio of esters and lipids results in a particle size of 40 nm-70 nm. The one or more inorganic nanoparticles may be aluminum oxide (Al2O3), aluminum oxyhydroxide, iron oxide, titanium dioxide, silicon dioxide (SiO2), aluminum hydroxyphosphate (Al(OH) x (PO4) y ), calcium phosphate (Ca3(PO4)2), calcium hydroxyapatite (Ca 10 Further provided herein is a composition, wherein the one or more inorganic nanoparticles comprise magnetite (Fe3O4), maghemite (y-Fe2O3), wustite (FeO), or hematite (alpha (α)-Fe2O3), or any combination thereof.

[0148] Further provided herein is a dry composition comprising sorbitan monostearate, polyoxyethylene (20) sorbitan monooleate, DOTAP, an immunostimulant, and RNA. Further provided herein is a dry composition in which the immunostimulant reduces the total amount of protein produced, but increases the immune response to a vaccine. Further provided herein is a dry composition in which the immunostimulant reduces the total amount of protein produced, but increases the immune response to a vaccine. Further provided herein is a dry composition in which the ester ratio produces a hydrophilic-lipophilic balance of 8-11. Further provided herein is a dry composition in which the particle size is 30 nm-200 nm. Further provided herein is a dry composition in which the N:P ratio is 5-35. Further provided herein is a dry composition in which the immunostimulant increases the total amount of protein produced, but decreases the immune response to a vaccine. Further provided herein is a dry composition, wherein the immune stimulant is at least one of caprylic / capric triglyceride, or triglyceride esters of saturated coconut oil / palm kernel oil-derived caprylic and capric fatty acids and vegetable-derived glycerol. The one or more inorganic nanoparticles may be aluminum oxide (Al2O3), aluminum oxyhydroxide, iron oxide, titanium dioxide, silicon dioxide (SiO2), aluminum hydroxyphosphate (Al(OH) x (PO4) y ), calcium phosphate (Ca3(PO4)2), calcium hydroxyapatite (Ca 10 Further provided herein is a composition, wherein the one or more inorganic nanoparticles comprise magnetite (Fe3O4), maghemite (y-Fe2O3), wustite (FeO), or hematite (alpha (α)-Fe2O3), or any combination thereof.

[0149] Further provided herein is a dry composition comprising: (a) a lipid carrier, the lipid carrier being a nanoemulsion comprising a hydrophobic core and one or more lipids; (b) optionally one or more nucleic acids; and (c) at least one sugar present in an amount of (i) at least about 50% by weight of the dry composition, or (ii) at least about 50 mg. Further provided herein is a composition that is lyophilized. Further provided herein is a composition that is thermally stable at about 25 degrees Celsius. Further provided herein is a composition that is thermally stable at about 45 degrees Celsius. Further provided herein is a composition that is thermally stable at about -20 degrees Celsius. Further provided herein is a composition that is thermally stable at about 2 degrees Celsius to about 8 degrees Celsius. Further provided herein is a composition that is thermally stable for at least one week, at least two weeks, and / or at least one month. Further provided herein is a composition, the hydrophobic core comprises an oil. Further provided herein is a composition in which the oil comprises at least one of α-tocopherol, lauroyl polyoxyl glyceride, monoacyl glycerol, propolis, squalene, mineral oil, grape seed oil, olive oil, paraffin oil, peanut oil, soybean oil, sunflower oil, soy lecithin, triglyceride, vitamin E, caprylic / capric triglyceride, triglyceride esters of saturated coconut / palm kernel oil-derived caprylic and capric fatty acids and plant-derived glycerol, dihydroisosqualene (DHIS), farnesene, and squalane. Further provided herein is a composition in which the one or more lipids are selected from the group consisting of cationic lipids, anionic lipids, neutral lipids, and any combination thereof. Further provided herein is a composition in which the one or more lipids comprise cationic lipids.Cationic lipids include 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP); 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol); dimethyldioctadecylammonium (DDA); 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP); dipalmitoyl (C16:0) trimethylammonium propane (DPTAP); distearoyltrimethylammonium propane (DSTAP); N-[1-(2,3-dioleyloxy)propyl]-N,N,N trimethylammonium chloride (DOTMA); N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dioleoyl-3-dimethylammonium chloride (DSTAP); Further provided herein is a composition selected from the group consisting of N-decyl-N,N-dimethyldecane-1-aminium bromide (DDAB); 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA); ethylphosphatidylcholine (ePC); and any combination thereof. Further provided herein is a composition, wherein the lipid carrier comprises at least one surfactant. Further provided herein is a composition, wherein the at least one surfactant is selected from the group consisting of hydrophobic surfactants, hydrophilic surfactants, and any combination thereof.Further provided herein are compositions in which the hydrophobic surfactant comprises a sorbitan ester selected from the group consisting of sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, and sorbitan trioleate; and the hydrophilic surfactant comprises a polysorbate. Further provided herein are compositions in which the lipid carrier has a z-average hydrodynamic diameter in the range of about 40 nm to about 150 nm with an average polydispersity index in the range of about 0.1 to about 0.4. Further provided herein are compositions in which the one or more nucleic acids are DNA. Further provided herein are compositions in which the one or more nucleic acids are RNA. Further provided herein are compositions in which the RNA is a self-replicating RNA. Further provided herein are compositions in which the hydrophobic core comprises one or more inorganic nanoparticles. Further provided herein are compositions in which the one or more inorganic nanoparticles are selected from the group consisting of metal salts, metal oxides, metal hydroxides, metal phosphates, and any combination thereof. Further provided herein are compositions in which one or more nucleic acids are incorporated into or complexed with a lipid carrier to form a lipid carrier-nucleic acid complex. Further provided herein are compositions in which the lipid carrier-nucleic acid complex is formed via non-covalent interactions or via reversible covalent interactions. Further provided herein are compositions in which the molar ratio of lipid carrier to one or more nucleic acids, characterized by the nitrogen to phosphate (N:P) molar ratio, ranges from about 1:1 to about 150:1. Further provided herein are compositions in which at least one sugar is selected from the group consisting of sucrose, maltose, trehalose, mannitol, glucose, and any combination thereof. Further provided herein are compositions wherein at least one sugar is present in an amount of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 mg, or more.Further provided herein is a composition, wherein the at least one sugar is present in an amount of 50 mg to 250 mg. Further provided herein is a composition, wherein the at least one sugar is present in an amount of at least about 250 mg. Further provided herein is a composition, wherein the sugar is present in an amount of at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more by weight of the composition. Further provided herein is a composition, wherein the sugar is present in an amount of 80-98%, optionally 94-96% by weight of the composition. Further provided herein is a composition, wherein the sugar is present in an amount of about 95% by weight of the composition. Further provided herein is a composition, wherein the at least one sugar comprises sucrose. The one or more inorganic nanoparticles are selected from the group consisting of aluminum oxide (Al2O3), aluminum oxyhydroxide, iron oxide, titanium dioxide, silicon dioxide (SiO2), aluminum hydroxyphosphate (Al(OH). x (PO4) y ), calcium phosphate (Ca3(PO4)2), calcium hydroxyapatite (Ca 10 Further provided herein is a composition, wherein the one or more inorganic nanoparticles comprise magnetite (Fe3O4), maghemite (y-Fe2O3), wustite (FeO), or hematite (alpha (α)-Fe2O3), or any combination thereof.

[0150] Further provided herein is a composition comprising: a nucleic acid present in an amount of up to about 200 micrograms (μg); a cationic lipid present in a concentration of up to about 1.5 mg / ml; an iron oxide present in a concentration of up to about 0.01 mg / ml; squalene present in a concentration of up to about 1.88 mg / ml; sorbitan monostearate present in a concentration of up to about 1.86 mg / ml; polysorbate 80 present in a concentration of up to about 1.86 mg / ml; sucrose present in a concentration of up to about 50 mg / ml; and optionally citric acid monohydrate present in a concentration of up to about 2.1 mg / ml. Further provided herein is a composition in which the nucleic acid is RNA or DNA. Further provided herein is a composition in which the nucleic acid is RNA and present in an amount of up to about 50 μg.

[0151] Further provided herein is a kit comprising the pharmaceutical composition provided herein and a delivery system for administration to a subject.Further provided herein is a kit comprising two or more separate units, each comprising a lipid carrier and a nucleic acid.Further provided herein is a kit comprising a unit comprising a lipid carrier and a nucleic acid.Further provided herein is a kit further comprising a unit comprising a reagent for hydration of the dry composition.Further provided herein is a kit, wherein the reagent for hydration comprises water.

[0152] Further provided herein is a method for generating an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition provided herein.

[0153] Further provided herein is a method of treating or preventing a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition provided herein.

[0154] Further provided herein is a method for imaging and / or tracking delivery of one or more nucleic acids in a subject, the method comprising administering a therapeutically effective amount of a pharmaceutical composition described herein.

[0155] The following examples are presented to more clearly illustrate to those skilled in the art the principles and practice of the embodiments disclosed herein, and should not be construed as limiting the scope of any claimed embodiments. Unless otherwise stated, all parts and percentages are by weight. EXAMPLES

[0156] Example 1 General preparation techniques and materials used The following materials were used in the preparation of the lipid carrier, which is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids.

[0157] The agents include squalene, sorbitan monostearate (SPAN® 60), polyoxyethylene (20) sorbitan monooleate (TWEEN® 80), DOTAP chloride, iron oxide nanoparticles, and sodium citrate dihydrate. Typically, chloroform was added to iron oxide nanoparticles having a number-weight average diameter of 5 nm. The chloroform was evaporated in a fume hood, leaving a dry coating of iron oxide nanoparticles. SPAN® 60, squalene, and DOTAP chloride were added to the iron oxide nanoparticles to prepare the "oil" phase. The oil phase was sonicated for 30 minutes in a water bath preheated to 60°C. Separately, in a 1 liter glass bottle, the "water" phase was prepared by adding TWEEN® 80 to a sodium citrate dihydrate solution prepared with Milli-Q water. The water phase was stirred for 30 minutes to completely dissolve the TWEEN® 80. After complete dissolution of TWEEN® 80, the aqueous phase was transferred to a beaker and incubated in a water bath preheated to 60°C. The preheated aqueous phase was added to the heated oil phase. The mixture was immediately emulsified using a VWR 200 homogenizer (VWR International) until a homogenous colloid with a milky appearance was produced. The colloid was then processed by passing the fluid through the Y-shaped interaction chamber of an LM10 microfluidizer at 20,000 psi. The fluid was passed until the z-average hydrodynamic diameter was 59 nm with a polydispersity index of 0.2 as measured by dynamic light scattering (Malvern Zetasizer Nano S). The microfluidized lipid carrier sample was finally filtered through a polyethersulfone (PES) syringe filter with a pore size of 200 nm. Example 2 Preparation of lipid carrier + RNA-secreted embryonic alkaline phosphatase (SEAP) complex.

[0158] Lipid carrier + RNA-SEAP complexes were prepared and aliquoted in triplicate for lyophilization. The Fe-lipid carrier described elsewhere herein contains 37.5 mg / ml squalene (SEPPIC), 37 mg / ml SPAN® 60 (Millipore Sigma), 37 mg / ml TWEEN® 80 (Fisher Chemical), 30 mg / ml DOTAP chloride (LIPOID), 0.2 mg Fe / ml 12 nm oleic acid-coated iron oxide nanoparticles (Imagion Biosystems, San Diego, CA, USA) and 10 mM sodium citrate dihydrate (Fisher Chemical). Lipid samples were omitted for comparison. Samples were collected and selected for reconstitution. The appearance of the lyophilized cakes was then recorded. All lyophilized cakes were then reconstituted in 0.7 ml of milliQ water. The physicochemical properties of the reconstituted materials were then measured, including (i) particle size, (ii) size distribution, (iii) RNA integrity, (iv) DOTAP and squalene content, and (v) in vitro protein expression measurements. The results are shown below. Table 2 discloses the materials used in the preparation of lipid carrier + RNA-SEAP complexes. [Table 2]

[0159] The conditions for freeze-drying are shown in Tables 3 to 5 as follows. [Table 3] [Table 4] [Table 5]

[0160] Diluent Preparation: Diluents containing sugar and citrate were prepared as outlined in Table 6. Each sugar was weighed into a 50ml RNase-free conical tube. Approximately 35-40ml of nuclease-free water was added to dissolve the sugar, using slight heating and sonication if necessary. Pipette in 0.5ml of 1M Na-citrate, pH=6 solution. After all sugar was dissolved and the solution was clear, QS in the conical tube with nuclease-free water to the 50ml mark. Diluents were filtered through a 0.22μm STERIFLIP® and aseptically capped to maintain sterility. [Table 6]

[0161] Preparation of precomplex formulations: Lipid carrier "DS" is 30 mg DOTAP / ml bulk solution and refers to the Fe-lipid carrier formulation, the preparation of which is described in Example 8.

[0162] The 10x lipid carrier "DS" (30mg DOTAP / ml) was diluted in the respective diluent to make 3mg DOTAP / ml lipid carrier "DP" except for the 5x 50% sucrose composition lipid carrier which was diluted to make 2x 6mg DOTAP / ml lipid carrier "DP". The target RNA concentration in the liquid formulation was 50ng / μl and complexed with the lipid carrier with an N:P of 15. This simulates an RNA dose of 25μg per vial. Table 7 discloses the preparation of the pre-complexed lipid carrier complex. Unused lipid carriers were stored at 2-8 degrees Celsius. [Table 7]

[0163] Table 8 discloses the preparation of pre-complexed nanostructured lipid carrier (NLC) complexes. NLC was used as a control. Unused NLC was stored at 2-8 degrees Celsius for fresh complex control. [Table 8]

[0164] The preparation of RNA pre-complex is disclosed in Table 9. RNA stocks were prepared, divided into approximately 7.5 ml or 0.63 ml for 50% sucrose per aliquot and stored at -80 degrees Celsius. [Table 9]

[0165] The preparation of lipid carrier-RNA complexes is disclosed in Table 9. RNA stocks were prepared. The volume of RNA diluted per complexation per lyo cycle was (+5%) and the lipid carrier diluted was (+5%).

[0166] Lipid carrier+RNA or NLC+RNA complexes were prepared by mixing 1:1 volume of each dilution formulation listed in Tables 8 and 9 above with the corresponding "Composition ID" diluted RNA disclosed in Table 10. The complexes were equilibrated at room temperature for 30 minutes before being subjected to a lyophilization cycle or long-term storage conditions. [Table 10] Example 3 Particle size measurement.

[0167] The formulation was diluted 100-fold by adding 900 μl of milliQ water to 10 μl of GLB51-F04-20-02 in a disposable sizing cuvette DTS0012. Vortex and remove bubbles by gently tapping the cuvette. Particle size was measured by the MALDS method on a zetasizer ULTRA. A minimum of five measurements were collected. The average z-average diameter and average PDI were recorded from the backscatter (173°) measurements. Example 4 RNA integrity by agarose gel electrophoresis.

[0168] Samples were prepared by diluting 50ng / μl complex 5-fold with 10ng / μl nuclease-free water:12μl complex + 48μl water. RNA was extracted by adding 60μl phenol:chloroform and inverting 10-15 times. RNA was denatured by centrifuging for 15 minutes, removing 10μl of supernatant, and mixing with 10μl glyoxal loading dye followed by heating to 50 degrees Celsius for 30 minutes. 10μl was loaded per lane. Example 5 SEAP expression in BHK21 cells.

[0169] Cell transfection: 1.5 × 10 cells were transfected into a 96-well plate one day before transfection. 5 The RNA:lipid carrier mixture and 50 μL of Opti-MEM™ (Thermo Fisher Scientific, Waltham, MA USA) were added to the BHK21 (baby hamster kidney fibroblast) cells. The cells were incubated for 4 hours. The RNA:lipid carrier and Opti-MEM™ medium were removed by pipetting and replaced with DMEM growth medium. The cells were incubated overnight.

[0170] Assessment of SEAP expression in supernatants: SEAP levels in supernatants were measured using the SEAP Reporter Assay Kit™ (Novus Biologicals NPB2-25285). Supernatants were analyzed undiluted according to the manufacturer's recommended protocol. Example 6 Characterization data after cycle #1

[0171] All lyophilized cakes showed good integrity at time=0 hours after cycle #1. The integrity of lipid carrier+RNA-SEAP in 5% glucose was partially compromised after 24 hours at 25 degrees Celsius. The integrity of all other formulations was preserved. Fe-lipid carriers described elsewhere herein include 37.5 mg / ml squalene (SEPPIC), 37 mg / ml SPAN® 60 (Millipore Sigma), 37 mg / ml TWEEN® 80 (Fisher Chemical), 30 mg / ml DOTAP chloride (LIPOID), 0.2 mg Fe / ml 12 nm oleic acid coated iron oxide nanoparticles (Imagion Biosystems) and 10 mM sodium citrate dihydrate (Fisher Chemical). Cakes from formulations of NLC+RNA-SEAP in 10% sucrose and lipid carrier+RNA-SEAP in 5% glucose shrunk significantly after 24 hours at 42 degrees Celsius. The integrity of all other formulations was preserved. Figures 2-4 illustrate the appearance of lyophilized cakes at the indicated sugar compositions after lyophilization cycle #1. Figure 2 illustrates the appearance of NLC and lipid carrier cakes at the indicated sugar compositions at time = 0 hours. Figure 3 illustrates the appearance of NLC and lipid carrier cakes at the indicated sugar compositions after 24 hours at t = 25 degrees Celsius and 42 degrees Celsius. Figure 4 illustrates the appearance of NLC and lipid carrier cakes at the indicated sugar compositions at t = 25 degrees Celsius and 42 degrees Celsius for 24 hours.

[0172] Particle size of reconstituted cakes after 1 day of storage at 25 degrees Celsius and 42 degrees Celsius at the indicated sugar compositions after lyophilization cycle #1 was measured as in Figures 4A-4B. Figure 5A shows the fold change in particle size compared to the liquid formulation at time = 0 hours at the indicated sugar compositions. Figure 5B shows the point change in PDI of reconstituted lyophilized formulations compared to their corresponding liquid formulations at the indicated sugar compositions. Mean size and size distribution values ​​are shown in Table 11. All reconstituted lyophilized formulations resulted in an increase in the size of lipid carrier + RNA complexes after reconstitution. All reconstituted formulations containing sucrose as a cryoprotectant resulted in minimal PDI change at time = 0 hours and 24 hours at 25 degrees Celsius.

[0173] Lyophilized (t=0) in Table 11 refers to lyophilized formulations that were reconstituted on the same day as the completion of the lyophilization cycle. Liquid in Table 11 refers to the equivalent liquid (non-lyophilized) formulation of the lyophilized formulation. [Table 11] RNA Integrity by Agarose Gel Electrophoresis after Lyophilization Cycle #1:

[0174] RNA integrity by agarose gel electrophoresis at the indicated sugar composition after lyophilization cycle #1 for lipid carrier + RNA and NLC + RNA formulations is illustrated in Figures 6A-6B. Figure 6A shows RNA integrity by agarose gel electrophoresis at the indicated sugar composition after lyophilization cycle #1 for lipid carrier + RNA formulations. Figure 6B shows RNA integrity by agarose gel electrophoresis at the indicated sugar composition after lyophilization cycle #1 for lipid carrier + RNA and NLC + RNA formulations.

[0175] Lyophilized cakes at 25 degrees Celsius and 42 degrees Celsius were stored for 24 hours prior to reconstitution. All lyophilized samples after reconstitution and phenol:chloroform extraction of RNA-SEAP from lipid carrier + RNA-SEAP showed the defined RNA bands that correlated with the naked RNA-SEAP positive control, as shown in Figures 6A-6B. Figure 6A shows that liquid formulations of lipid carrier + RNA-SEAP complexes in 10% sucrose stored for 24 hours at 42 degrees Celsius resulted in a significant decrease in RNA band intensity suggestive of accelerated degradation when stored as a liquid, but did not result in a significant decrease in RNA band intensity suggestive of accelerated degradation when stored in dry lyophilized form.

[0176] Lyophilization preserved RNA integrity in liquid lipid carrier + RNA formulations upon storage at 42 degrees Celsius. SEAP expression in BHK21 cells after freeze-drying cycle #1:

[0177] SEAP expression in BHK21 cells at the indicated sugar compositions after lyophilization cycle #1 for lipid carrier + repRNA and NLC + repRNA-SEAP formulations is illustrated in Figures 7A-7G. Figure 7A shows the relative light units (RLU) for lipid carrier + repRNA-SEAP in 10% trehalose. Figure 7B shows the relative light units (RLU) for lipid carrier + repRNA-SEAP in 5% mannitol. Figure 7C shows the relative light units (RLU) for lipid carrier + repRNA-SEAP in 10% sucrose. Figure 7D shows the relative light units (RLU) for lipid carrier + repRNA-SEAP in 20% sucrose. Figure 7E shows the relative light units (RLU) for lipid carrier + repRNA-SEAP in 5% glucose. Figure 7F shows the relative light units (RLU) for lipid carrier + repRNA-SEAP in 10% maltose. FIG. 7G shows relative light units (RLU) for NLC+repRNA-SEAP in 10% sucrose.

[0178] For comparison, cells were transfected with freshly prepared lipid carrier + RNA-SEAP in the indicated sugar composition. As an additional stability comparison, cells were transfected with liquid formulations of lipid carrier + RNA-SEAP in 10% sucrose stored at 25 degrees Celsius for 24 hours or at 42 degrees Celsius for 24 hours. The latter condition served as a control to measure the effect of degraded RNA on in vitro protein expression. Figures 7A-7G illustrate that all reconstituted lyophilized formulations at time = 0 hours or after 24 hours at 25 degrees Celsius did not result in changes in the RNA dose-dependent expression profile compared to the corresponding freshly prepared lipid carrier + RNA-SEAP complexes. Lipid carrier + RNA-SEAP in 10% sucrose stored at 42 degrees Celsius for 24 hours in a liquid formulation (Figure 7C) resulted in significantly reduced SEAP expression compared to the lyophilized formulation. 10% trehalose (Figure 7A), 20% sucrose (Figure 7D) and 10% maltose (Figure 7F) also preserved the potency of RNA-SEAP, as measured by SEAP expression, after 24 hours of storage at 42 degrees Celsius. Finally, lyophilized lipid carriers + RNA-SEAP containing 5% mannitol (Figure 7B) or 5% glucose (Figure 7E) at 42 degrees Celsius resulted in a significant decrease in the dose-dependent expression profile of SEAP compared to their corresponding freshly prepared liquid formulations. For comparison, NLCs were prepared with RNA-SEAP in 10% sucrose (Figure 7G). Appearance of lipid carrier + RNA-SEAP cake after lyophilization cycle #1:

[0179] Figures 8 and 9 show the appearance of lipid carrier + RNA-SEAP cakes in the indicated sugar compositions at various time points after cycle #1. All lyophilized cakes showed good integrity at time = 0 hours after cycle #1. Figure 8 illustrates the appearance of lipid carrier + RNA-SEAP cakes in the indicated sugar compositions at various time points (time = 0 hours, after 6 days at 25 degrees Celsius and after 1 month at 25 degrees Celsius) after lyophilization cycle #1. Figure 8 shows that the integrity of lipid carrier + RNA-SEAP in 5% glucose was significantly compromised after 6 days at 25 degrees Celsius and after 1 month at 25 degrees Celsius. The integrity of NLC + RNA-SEAP in 10% sucrose was partially compromised after 6 days at 25 degrees Celsius and significantly compromised after 1 month at 25 degrees Celsius. The integrity of all other formulations was preserved after 1 month at 25 degrees Celsius.

[0180] Figure 9 illustrates the appearance of lipid carrier + RNA-SEAP cake at various time points (time = 0 hours, after 24 hours at 42 degrees Celsius, and after 3 days at 42 degrees Celsius, and after 1 month at 42 degrees Celsius) for the indicated sugar compositions after lyophilization cycle #1. Figure 8 shows that all sucrose and glucose containing formulations gradually shrank after 24 hours, 3 days, and 1 month of storage at 42 degrees Celsius. The integrity of all other formulations remained after 1 month at 42 degrees Celsius. Example 7 Characterization Data After Cycle #3 Particle size after lyophilization cycle #3:

[0181] Particle size of reconstituted cakes after 1 day of storage at 25 degrees Celsius and 42 degrees Celsius at the indicated sugar compositions after lyophilization cycle #3 was measured as in Figures 10A-10B. Figures 10A-10B illustrate particle size of reconstituted cakes after 1 day of storage at the indicated sugar compositions after lyophilization cycle #3. Figure 10A shows the point change in PDI of reconstituted formulations at the indicated sugar compositions. Figure 10B shows the log10 fold change in particle size compared to fresh complexes of lipid carrier + RNA.

[0182] The mean size and size distribution values ​​are shown in Table 12. All lyophilized formulations stored at 25° C. or 42° C. for 1 month resulted in an increase in the size of the complexes after reconstitution. The lipid carrier + RNA-SEAP formulated in 20% sucrose resulted in the smallest fold change in particle size and the least change in PDI compared to the liquid formulation stored at 5° C. for 1 month, as shown in Figures 10A-10B. [Table 12] RNA Integrity by Agarose Gel Electrophoresis after Lyophilization Cycle #3:

[0183] RNA integrity by agarose gel electrophoresis at the indicated sugar compositions after lyophilization cycle #3 is illustrated in FIG. 11. Both liquid and lyophilized formulations were stored at 25 degrees Celsius and 42 degrees Celsius for one month. FIG. 11 shows that the optical density (OD) % of the bands was normalized to RNA-SEAP extracted from freshly prepared lipid carrier + RNA-SEAP in 10% sucrose. All samples, liquid or lyophilized, stored for one month at 42 degrees Celsius showed no detectable RNA bands indicating complete degradation of RNA-SEAP. With the exception of NLC + RNA-SEAP in 10% sucrose and lipid carrier + RNA-SEAP in 5% mannitol, all lyophilized lipid carrier + RNA-SEAP formulations protected RNA-SEAP better at 25 degrees Celsius for one month than lipid carrier + RNA-SEAP stored in liquid form at 5 degrees Celsius for one month. Lipid carrier + RNA-SEAP in 10% sucrose stored as a liquid for one month at 25 degrees Celsius was intact. In comparison, RNA-SEAP was at 50% OD compared to the fresh complex when stored in lyophilized form at 25 degrees Celsius and 23% OD when stored in liquid form at 5 degrees Celsius. Lipid carrier + RNA-SEAP formulated in 20% sucrose or 10% maltose significantly preserved RNA-SEAP after one month storage at 25 degrees Celsius. SEAP expression in BHK21 cells after freeze-drying cycle #3:

[0184] SEAP expression of BHK21 cells in the indicated sugar compositions after lyophilization cycle #3 is illustrated in FIG. 12. The average SEAP expression level is shown for a single RNA transfection dose of 12.5 ng / well. For comparison, cells were transfected with freshly prepared lipid carrier + RNA-SEAP in 10% sucrose (upper dotted line) as shown in FIG. 12. Cells transfected with a liquid formulation of lipid carrier + RNA-SEAP in 10% sucrose stored at 42 degrees Celsius for one month served as a comparison to measure the effect of degraded RNA on in vitro protein expression (lower dotted line). Expression from all lyophilized samples stored at 42 degrees Celsius for one month was at or below the expression level of lipid carrier + RNA-SEAP in 10% sucrose stored in liquid form at 42 degrees Celsius for one month. Lyophilized lipid carrier + RNA-SEAP complexes in 10% sucrose, 20% sucrose, 10% trehalose and 10% maltose stored at 25 degrees Celsius for one month resulted in higher SEAP expression than the same compositions stored in liquid form at 5 degrees Celsius for one month. Example 8 Macrophage immune response.

[0185] Various formulations of lipid carriers and repRNA were prepared and analyzed to assay the innate immune response of lipid carriers in macrophages. Stimulation of protein expression and TNF production in THP-1 macrophages was studied.

[0186] First, THP-1 monocytes were differentiated into macrophages using phorbol 12-myristate 13-acetate (PMA). Then, the cells were transfected with various formulations carrying Nano Luciferase-encoding replicon RNA (SEQ ID NO: 2). Then, the cell culture medium was evaluated for NanoLuc and TNF expression.

[0187] The formulations used in this assay and their characteristics such as particle size and PDI are listed in Table 13. The concentration of repRNA-encoded NanoLuc was 909 ng / μl and maintained at −80 degrees Celsius. MIGLYOL® 812 N, a triglyceride ester of saturated coconut / palm kernel oil-derived caprylic and capric fatty acids and vegetable-derived glycerol, was used in this assay. [Table 13]

[0188] Fe-lipid carrier formulation (prepared at 100 ml scale): The Fe-lipid carrier formulation contained 37.5 mg / ml squalene (SEPPIC), 37 mg / ml SPAN® 60 (Millipore Sigma), 37 mg / ml TWEEN® 80 (Fisher Chemical), 30 mg / ml DOTAP chloride (LIPOID), 0.2 mg Fe / ml 12 nm oleic acid coated iron oxide nanoparticles (Imagion Biosystems) and 10 mM sodium citrate dihydrate (Fisher Chemical). 20 mg Fe / ml 12 nm diameter oleic acid coated iron oxide nanoparticles in 1 ml chloroform (Imagion Biosystems, lot number 95-127) were washed three times by magnetic separation in a 4:1 acetone:chloroform (v / v) solvent mixture. After the third wash, the volatile solvents (acetone and chloroform) were allowed to evaporate completely in a fume hood, leaving a coating of dry iron oxide oleate nanoparticles. To this iron oxide coating, 3.75 grams of squalene, 3.7 grams of SPAN® 60, and 3 grams of DOTAP were added to produce an oil phase. The oil phase was sonicated in a 65° Celsius water bath for 45 minutes. Separately, an aqueous phase was prepared by dissolving 19.5 grams of TWEEN® 80 in 500 ml of 10 mM sodium citrate buffer prepared in nuclease-free water. 92 ml of the aqueous phase was transferred to a separate glass bottle and heated to 65° Celsius for 30 minutes. The oil phase was mixed with 92 ml of the aqueous phase by adding the warm oil phase to the warm aqueous phase. The mixture was emulsified using a VWR 200 homogenizer (VWR International), and the resulting coarse emulsion was processed by passing it through an M110P microfluidizer (Microfluidics) equipped with a F12Y 75 μm diamond interaction chamber and a supplementary H30Z-200 μm ceramic interaction chamber at 30,000 psi until the z-average hydrodynamic diameter, measured by dynamic light scattering (Malvern Zetasizer Nano S), reached 40–80 nm with a polydispersity index (PDI) of 0.1–0.25.The microfluidized formulation was finally filtered through a 200 nm pore size polyethersulfone (PES) filter and stored at 2-8 degrees Celsius. Iron concentrations were determined by ICP-OES. DOTAP and squalene concentrations were measured by RP-HPLC.

[0189] High Fe-lipid carrier formulation (prepared at 100 ml scale): The high Fe-lipid carrier formulation contains 37.5 mg / ml squalene (SEPPIC), 37 mg / ml SPAN® 60 (Millipore Sigma), 37 mg / ml TWEEN® 80 (Fisher Chemical), 30 mg / ml DOTAP chloride (LIPOID), 1 mg Fe / ml 15 nm oleic acid coated iron oxide nanoparticles (Imagion Biosystems) and 10 mM sodium citrate dihydrate (Fisher Chemical). 20 mg Fe / ml 15 nm diameter oleic acid coated iron oxide nanoparticles in 5 ml chloroform (Imagion Biosystems, lot number 95-133) were washed three times by magnetic separation in a 4:1 acetone:chloroform (v / v) solvent mixture. After the third wash, the volatile solvents (acetone and chloroform) were allowed to evaporate completely in a fume hood, leaving a coating of dry iron oxide oleate nanoparticles. To this iron oxide coating, 3.75 grams of squalene, 3.7 grams of SPAN® 60, and 3 grams of DOTAP were added to produce an oil phase. The oil phase was sonicated in a 65° Celsius water bath for 45 minutes. Separately, an aqueous phase was prepared by dissolving 19.5 grams of TWEEN® 80 in 500 ml of 10 mM sodium citrate buffer prepared in nuclease-free water. 92 ml of the aqueous phase was transferred to a separate glass bottle and heated to 65° Celsius for 30 minutes. The oil phase was mixed with 92 ml of the aqueous phase by adding the warm oil phase to the warm aqueous phase. The mixture was emulsified using a VWR 200 homogenizer (VWR International), and the resulting coarse emulsion was processed by passing it through an M110P microfluidizer (Microfluidics) equipped with a F12Y 75 μm diamond interaction chamber and a supplementary H30Z-200 μm ceramic interaction chamber at 30,000 psi until the z-average hydrodynamic diameter, measured by dynamic light scattering (Malvern Zetasizer Nano S), reached 40–80 nm with a polydispersity index (PDI) of 0.1–0.3.The microfluidized formulation was finally filtered through a 200 nm pore size polyethersulfone (PES) filter and stored at 2-8 degrees Celsius. Iron concentrations were determined by ICP-OES. DOTAP and squalene concentrations were measured by RP-HPLC.

[0190] Fe-lipid carrier miglyol formulation (prepared at 100 ml scale): The Fe-lipid carrier miglyol formulation contains 37.5 mg / ml MIGLYOL® 812 N (IOI Oleo GmbH, Hamburg, Germany), 37 mg / ml SPAN® 60 (Millipore Sigma), 37 mg / ml TWEEN® 80 (Fisher Chemical), 30 mg / ml DOTAP chloride (LIPOID), 0.2 mg Fe / ml 15 nm oleic acid coated iron oxide nanoparticles (Imagion Biosystems) and 10 mM sodium citrate dihydrate (Fisher Chemical). 20 mg Fe / ml 15 nm diameter oleic acid coated iron oxide nanoparticles in 1 ml chloroform (Imagion Biosystems, lot number 95-127) were washed three times by magnetic separation in a 4:1 acetone:chloroform (v / v) solvent mixture. After the third wash, the volatile solvents (acetone and chloroform) were allowed to evaporate completely in a fume hood, leaving a coating of dry iron oxide oleate nanoparticles. To this iron oxide coating, 3.75 grams of squalene, 3.7 grams of SPAN® 60, and 3 grams of DOTAP were added to produce an oil phase. The oil phase was sonicated in a 65° Celsius water bath for 45 minutes. Separately, an aqueous phase was prepared by dissolving 19.5 grams of TWEEN® 80 in 500 ml of 10 mM sodium citrate buffer prepared in nuclease-free water. 92 ml of the aqueous phase was transferred to a separate glass bottle and heated to 65° Celsius for 30 minutes. The oil phase was mixed with 92 ml of the aqueous phase by adding the warm oil phase to the warm aqueous phase.The mixture was emulsified using a VWR 200 homogenizer (VWR International) and the resulting crude emulsion was processed by passing it through an M110P microfluidizer (Microfluidics) equipped with a F12Y 75 μm diamond interaction chamber and an auxiliary H30Z-200 μm ceramic interaction chamber at 30,000 psi until the z-average hydrodynamic diameter, measured by dynamic light scattering (Malvern Zetasizer Nano S), reached 40-80 nm with a polydispersity index (PDI) of 0.1-0.3. The microfluidized formulation was finally filtered through a 200 nm pore size polyethersulfone (PES) filter and stored at 2-8 degrees Celsius. Iron concentrations were determined by ICP-OES. DOTAP concentrations were measured by RP-HPLC.

[0191] High Fe-lipid carrier miglyol formulation (prepared at 100 ml scale): The high Fe-lipid carrier miglyol formulation contains 37.5 mg / ml MIGLYOL® 812 N (IOI Oleo GmbH), 37 mg / ml SPAN® 60 (Millipore Sigma), 37 mg / ml TWEEN® 80 (Fisher Chemical), 30 mg / ml DOTAP chloride (LIPOID), 1 mg / ml 15 nm oleic acid coated iron oxide nanoparticles (Imagion Biosystems) and 10 mM sodium citrate dihydrate (Fisher Chemical). 20 mg Fe / ml 15 nm diameter oleic acid coated iron oxide nanoparticles in 5 ml chloroform (Imagion Biosystems, lot no. 95-127) were washed three times by magnetic separation in a 4:1 acetone:chloroform (v / v) solvent mixture. After the third wash, the volatile solvents (acetone and chloroform) were allowed to evaporate completely in a fume hood, leaving a coating of dry iron oxide oleate nanoparticles. To this iron oxide coating, 3.75 grams of squalene, 3.7 grams of SPAN® 60, and 3 grams of DOTAP were added to produce an oil phase. The oil phase was sonicated in a 65° Celsius water bath for 45 minutes. Separately, an aqueous phase was prepared by dissolving 19.5 grams of TWEEN® 80 in 500 ml of 10 mM sodium citrate buffer prepared in nuclease-free water. 92 ml of the aqueous phase was transferred to a separate glass bottle and heated to 65° Celsius for 30 minutes. The oil phase was mixed with 92 ml of the aqueous phase by adding the warm oil phase to the warm aqueous phase.The mixture was emulsified using a VWR 200 homogenizer (VWR International) and the resulting crude emulsion was processed by passing it through an M110P microfluidizer (Microfluidics) equipped with a F12Y 75 μm diamond interaction chamber and an auxiliary H30Z-200 μm ceramic interaction chamber at 30,000 psi until the z-average hydrodynamic diameter, measured by dynamic light scattering (Malvern Zetasizer Nano S), reached 40-80 nm with a polydispersity index (PDI) of 0.1-0.3. The microfluidized formulation was finally filtered through a 200 nm pore size polyethersulfone (PES) filter and stored at 2-8 degrees Celsius. Iron concentrations were determined by ICP-OES. DOTAP concentrations were measured by RP-HPLC.

[0192] Alum-lipid carrier formulation (prepared at 100ml scale): The alum-lipid carrier formulation contains 37.5mg / ml squalene (SEPPIC), 37mg / ml SPAN® 60 (Millipore Sigma), 37mg / ml TWEEN® 80 (Fisher Chemical), 30mg / ml DOTAP chloride (LIPOID), 1mg Al / ml TOPO coated Alhydrogel® (aluminum oxyhydroxide) particles (Croda) and 10mM sodium citrate. 10ml of Alhydrogel was washed three times in methanol by centrifugation at 1000rpm for 20 minutes. After the third wash, the Alhydrogel was dispersed in 10ml of methanol and 1ml of 250mg / ml trioctylphosphine oxide (TOPO) was added to the dispersion and incubated overnight at 37 degrees Celsius on an orbital shaker. Excess TOPO was removed by additional methanol washes and then dispersed in 11 ml of methanol. The methanol was allowed to evaporate overnight in a fume hood leaving a dry layer of TOPO-Alhydrogel. To this dry TOPO-Alhydrogel layer, 3.75 grams of squalene, 3.7 grams of SPAN® 60, and 3 grams of DOTAP were added to produce an oil phase. The oil phase was sonicated in a 65° Celsius water bath for 45 minutes. Separately, an aqueous phase was prepared by dissolving 19.5 grams of TWEEN® 80 in 500 ml of 10 mM sodium citrate buffer prepared in nuclease-free water. 92 ml of the aqueous phase was transferred to a separate glass bottle and heated to 65° Celsius for 30 minutes. The oil phase was mixed with 92 ml of the aqueous phase by adding the warm oil phase to the warm aqueous phase.The mixture was emulsified using a VWR 200 homogenizer (VWR International) and the resulting crude emulsion was processed by passing it through an M110P microfluidizer (Microfluidics) equipped with a F12Y 75 μm diamond interaction chamber and an auxiliary H30Z-200 μm ceramic interaction chamber at 30,000 psi until the z-average hydrodynamic diameter, measured by dynamic light scattering (Malvern Zetasizer Nano S), reached 40-80 nm with a polydispersity index (PDI) of 0.1-0.3. The microfluidized formulation was finally filtered through a 200 nm pore size polyethersulfone (PES) filter and stored at 2-8 degrees Celsius. Aluminum concentrations were determined by ICP-OES. DOTAP and squalene concentrations were measured by RP-HPLC.

[0193] Fe-lipid carrier solanesol formulation (prepared at 100 ml scale): The Fe-lipid carrier solanesol formulation contains 37.5 mg / ml solanesol (Cayman chemicals), 37 mg / ml SPAN® 60 (Millipore Sigma), 37 mg / ml TWEEN® 80 (Fisher Chemical), 30 mg / ml DOTAP chloride (LIPOID), 0.2 mg Fe / ml oleic acid coated iron oxide nanoparticles (Imagion Biosystems) and 10 mM sodium citrate. 20 mg Fe / ml oleic acid coated iron oxide nanoparticles with a diameter of 15 nm in 1 ml chloroform (Imagion Biosystems, lot number 95-133) were washed three times by magnetic separation in a 4:1 acetone:chloroform (v / v) solvent mixture. After the third wash, the volatile solvents (acetone and chloroform) were allowed to evaporate completely in a fume hood, leaving a coating of dry iron oxide oleate nanoparticles. To this iron oxide coating, 3.75 grams of solanesol, 3.7 grams of SPAN® 60, and 3 grams of DOTAP were added to produce an oil phase. The oil phase was sonicated for 45 minutes in a 65° Celsius water bath. Separately, an aqueous phase was prepared by dissolving 19.5 grams of TWEEN® 80 in 500 ml of 10 mM sodium citrate buffer prepared in nuclease-free water. 92 ml of the aqueous phase was transferred to a separate glass bottle and heated to 65° Celsius for 30 minutes. The oil phase was mixed with 92 ml of the aqueous phase by adding the warm oil phase to the warm aqueous phase. The mixture was emulsified using a VWR 200 homogenizer (VWR International) and the resulting crude emulsion was processed by passing it through an M110P microfluidizer (Microfluidics) equipped with a F12Y 75 μm diamond interaction chamber and an auxiliary H30Z-200 μm ceramic interaction chamber at 30,000 psi. The microfluidized formulation was finally filtered through a 200 nm pore size polyethersulfone (PES) filter and stored at 2-8 degrees Celsius. Iron concentrations were determined by ICP-OES.DOTAP and solanesol concentrations were measured by RP-HPLC.

[0194] NLC formulation (prepared at 100 ml scale): The NLC formulation contains 37.5 mg / ml squalene (SEPPIC), 37 mg / ml SPAN® 60 (Millipore Sigma), 37 mg / ml TWEEN® 80 (Fisher Chemical), 30 mg / ml DOTAP chloride (LIPOID), 2.4 mg / ml Dynasan 114 (IOI Oleo GmbH) and 10 mM sodium citrate. To a 200 ml beaker, 3.75 grams of squalene, 3.7 grams of SPAN® 60, 0.24 grams of Dynasan 114 and 3 grams of DOTAP were added to generate the oil phase. The oil phase was sonicated in a 65 degree Celsius water bath for 45 minutes. Separately, the aqueous phase was prepared by dissolving 19.5 grams of TWEEN® 80 in 500 ml of 10 mM sodium citrate buffer prepared in nuclease-free water. 92 ml of the aqueous phase was transferred to a separate glass bottle and heated to 65 degrees Celsius for 30 minutes. The oil phase was mixed with 92 ml of the aqueous phase by adding the warm oil phase to the warm aqueous phase. The mixture was emulsified using a VWR 200 homogenizer (VWR International) and the resulting crude emulsion was processed by passing it through an M110P microfluidizer (Microfluidics) equipped with a F12Y 75 μm diamond interaction chamber and an auxiliary H30Z-200 μm ceramic interaction chamber at 30,000 psi until the z-average hydrodynamic diameter reached 40-80 nm with a polydispersity index (PDI) of 0.1-0.3 as measured by dynamic light scattering (Malvern Zetasizer Nano S). The microfluidized formulation was finally filtered through a 200 nm pore size polyethersulfone (PES) filter and stored at 2-8 degrees C. DOTAP and squalene concentrations were measured by RP-HPLC.

[0195] CNE formulation (prepared at 100 ml scale): The CNE formulation contains 43 mg / ml squalene (SEPPIC), 5 mg / ml SPAN® 85 (Millipore Sigma), 5 mg / ml TWEEN® 80 (Fisher Chemical), 4 mg / ml DOTAP chloride (LIPOID) and 10 mM sodium citrate. In a 200 ml beaker, 4.3 grams of squalene, 0.5 grams of SPAN® 85, and 0.4 grams of DOTAP were added to generate the oil phase. The oil phase was sonicated for 45 minutes in a 65 degree Celsius water bath. Separately, the aqueous phase was prepared by dissolving 2.6 grams of TWEEN® 80 in 500 ml of 10 mM sodium citrate buffer prepared in nuclease-free water. 95 ml of the aqueous phase was transferred to a separate glass bottle and heated to 65 degrees Celsius for 30 minutes. The oil phase was mixed with 95 ml of the aqueous phase by adding the warm oil phase to the warm aqueous phase. The mixture was emulsified using a VWR 200 homogenizer (VWR International) and the resulting coarse emulsion was processed by passing it through an M110P microfluidizer (Microfluidics) equipped with a F12Y 75 μm diamond interaction chamber and an auxiliary H30Z-200 μm ceramic interaction chamber at 30,000 psi until the z-average hydrodynamic diameter, measured by dynamic light scattering (Malvern Zetasizer Nano S), reached 100 ± 10 nm with a polydispersity index (PDI) of 0.05-0.1. The microfluidized formulation was finally filtered through a 200 nm pore size polyethersulfone (PES) filter and stored at 2-8 degrees Celsius. DOTAP and squalene concentrations were measured by RP-HPLC.

[0196] Treatment groups were prepared. Eight of these groups were NanoLuc repRNA groups, with 600ng dose per well prepared using Fe-lipid carrier, high Fe-lipid carrier, Fe-lipid carrier miglyol, high Fe-lipid carrier miglyol, alum-lipid carrier, Fe-lipid carrier solanesol (SLN), NLC, and CNE formulations. The untreated group did not have NanoLuc.

[0197] Various formulations were prepared by diluting NanoLuc repRNA to 8 ng / μL in 2.2 mL of RNAse-free water. The lipid carrier formulation and RNA master mix were complexed by adding 250 μL of each diluted formulation and 250 μL of diluted RNA and mixed by pipetting up and down.

[0198] Cell transfections were performed at 7 × 10 cells per well in 24-well plates. 5 The transfection was performed by seeding 100 THP-1s per well. 80 μM PMA was added and incubated at 37 degrees Celsius. The following day, 1 hour prior to transfection, the PMA-containing medium was removed and replaced with cRPMI. The samples were then serially diluted in Opti-MEM™ to generate a 10-point 1.5-fold dilution series starting at 0.45 ng / μL. The culture medium was then removed from the plate by pipetting. 450 μL of Opti-MEM™ and 150 μL of the complexed formulation were added to the plate in duplicate. Empty wells received 450 μL of Opti-MEM™ only. After 4 hours, the samples were removed from the plate by pipetting and replaced with 500 μL of growth medium. The plate was then incubated overnight at 37 degrees Celsius. The growth medium was collected the next day and stored at -80 degrees Celsius. Downstream assays were performed.

[0199] Luciferase assays were performed by first diluting Nano-Glo luciferase assay reagent 1:50 in buffer. 25 μL of supernatant was removed and mixed with 25 μL of Nano-Glo reagent in a 96-well plate. This was incubated at room temperature for 3 minutes. Luminescence was read.

[0200] ELISA assay was performed to evaluate TNF-alpha protein levels in culture medium using Human TNF-alpha DuoSet ELISA from R&D Systems according to the manufacturer's protocol. 96-well microplates were coated with anti-TNF capture antibody. Plates were blocked and then culture medium samples were added directly without dilution. After addition of biotinylated detection antibody SA-HRP and substrate, absorbance was read at 450 nm in a SPECTRAMAX® i3 (Molecular Devices, LLC, San Jose CA, USA) plate reader.

[0201] All studies in this example were performed in duplicate, and the results from the duplicates are represented as the first experiment and the second experiment, respectively.

[0202] The assays demonstrate that liquid formulations composed of lipid carriers and Miglyol induced higher protein production from the replicon, as shown in the first assay in Figure 13A and the second assay in Figure 13B. A reduction in the innate immune response, as measured by TNF-alpha secretion, was detected, as shown in the first assay in Figure 14A and the second assay in Figure 14B.

[0203] A correlation between enhanced protein production and low TNF-alpha stimulation was observed with the Miglyol lipid carrier formulation, as shown in the first assay in Figure 15A and the second assay in Figure 15B. Solanesol induced slightly lower protein production, but potentially higher TNF production, as shown in the first assay in Figure 15A and the second assay in Figure 15B.

[0204] 16A-16F illustrate SEAP levels in BALB / c mice injected intramuscularly with various replicates of lipid carrier-formulated DNA SEAP. FIG. 16A shows the relative light units (RLU) on day 4. FIG. 16B shows the relative light units (RLU) on day 6. FIG. 16C shows the relative light units (RLU) on day 8. FIG. 16D is a copy of the relative light units (RLU) on day 4. FIG. 16E is a copy of the relative light units (RLU) on day 6. FIG. 16F is a copy of the relative light units (RLU) on day 8.

[0205] FIG. 17 is a bar graph with Z-average and polydispersity index (PDI) measurements on the Y-axis and group number on the X-axis for conditions 1-14.

[0206] 18A-18B are dot charts with IgG (μg / ml) on the Y-axis and group number on the X-axis for conditions 1-14, with traces showing measurements with anti-D614G (1:40 dilution) on day 14 and anti-D614G (1:200 dilution) on day 28, respectively.

[0207] FIG. 19 shows a dot chart at day 28 with IgG (μg / ml) measurements of anti-D614G (1:200 dilution) on the Y-axis and indications of storage conditions on the X-axis.

[0208] No other major differences between the formulations were observed, suggesting that the formulation composed of lipid carrier and miglyol (Fe-lipid carrier miglyol) is a promising formulation in therapeutic applications, such as, but not limited to, antiviral and cancer therapy, where high protein production and low immune stimulation are desired, such as, but not limited to, in vivo production of therapeutic proteins or antibodies. Example 9 DNA(SEAP) bioactivity in mice.

[0209] This example studies the effect of injected dose on the biological activity of lipid carrier and DNA (SEAP) in BALB / c mice. Kinetics, duration and magnitude were studied. Materials: DNA encoding SEAP (supplier: Aldevron; catalog number: gWiz-SEAP, lot number: 38611), 5 mg / mL, SEAP encoding repRNA (SEQ ID NO: 1), 2217 μg / mL, at -20 degrees Celsius, and lipid carrier formulation (30 mg DOTAP / ml) at 4 degrees Celsius were used in this example. C57BL / 6 mice were inoculated as described, with treatment groups listed in Table 14, and SEAP levels were then measured in serum. [Table 14]

[0210] Seven different formulations were prepared and administered intramuscularly across seven treatment groups (Groups 1-7). DNA-SEAP or RNA-SEAP was diluted according to the volumes set out in Table 15 to prepare the formulations for Groups 1-7. [Table 15]

[0211] The concentrations of the diluted DNA or RNA prior to complexation with lipid carriers were as follows (measured by NanoDrop spec): Groups 1, 4 and 5 contained approximately 820 μg / ml DNA; Groups 2 and 3 contained approximately 480 μg / ml DNA; Groups 6 and 7 contained approximately 43 μg / ml RNA. Formulations for Groups 1-6 were diluted with 100 mM citrate as shown in Table 16 below. [Table 16]

[0212] The above formulations were complexed by adding 250μl of diluted lipid carrier to 250μl of diluted DNA or RNA. The resulting complexed formulation was incubated on ice for at least 30 minutes. Table 17 shows the schedule for this assay. [Table 17]

[0213] Mice were bled at regular intervals and serum was immediately prepared and stored at -80 degrees Celsius until analysis for SEAP activity.

[0214] To evaluate SEAP levels in serum, all serum samples were thawed at the same time and SEAP detection was performed. Figures 16A-16F illustrate SEAP levels in BALB / c mice intramuscularly injected with various replicates of lipid carrier-formulated DNA SEAP. Mice were bled at regular intervals and serum was prepared and stored until analysis by SEAP assay. Data are presented as mean and SE (n=5 per group).

[0215] As can be seen from Figures 16A-16F, the lipid carrier formulation supports target protein production more than delivery of DNA alone, especially after 6 days post-injection. In addition, this example shows that the inclusion of miglyol enhances protein production from an RNA replicon more than lipid carrier formulations lacking miglyol. Example 10 Formulation of lipid carriers without inorganic core.

[0216] A formulation of lipid carrier without inorganic core was prepared at 100ml scale. The lipid carrier without inorganic core contains 37.5mg / ml squalene (SEPPIC), 37mg / ml SPAN® 60 (Millipore Sigma), 37mg / ml TWEEN® 80 (Fisher Chemical), 30mg / ml DOTAP chloride (LIPOID) and 10mM sodium citrate. 3.75 grams of squalene, 3.7 grams of SPAN® 60 and 3.0 grams of DOTAP were added to a 200ml beaker to generate an oil phase. The oil phase was sonicated in a water bath at 65 degrees Celsius for 45 minutes. Separately, an aqueous phase was prepared by dissolving 19.5 grams of TWEEN® 80 in 500ml of 10mM sodium citrate buffer prepared in nuclease-free water. 96 ml of the aqueous phase was transferred to a separate glass bottle and heated to 65 degrees Celsius for 30 minutes. The oil phase was mixed with 96 ml of the aqueous phase by adding the warm oil phase to the warm aqueous phase. The mixture was emulsified using a VWR 200 homogenizer (VWR International) and the resulting coarse emulsion was processed by passing it through an M110P microfluidizer (Microfluidics) equipped with a F12Y 75 μm diamond interaction chamber and a supplementary H30Z-200 μm ceramic interaction chamber at 30,000 psi until the z-average hydrodynamic diameter, measured by dynamic light scattering (Malvern Zetasizer Nano S), reached 40-80 nm with a polydispersity index (PDI) of 0.1-0.3. The microfluidized formulation of the inorganic core-free formulation was finally filtered through a 200 nm pore size polyethersulfone (PES) filter and stored at 2-8 degrees Celsius. DOTAP and squalene concentrations were measured by RP-HPLC. Example 11 Additional nanoparticle formulations

[0217] Additional nanoparticle formulations are produced according to the following tables (Tables 18 and 19). [Table 18] [Table 19] Example 16 Evaluation of a freeze-dried COVID vaccine in mice

[0218] The following was performed to assay the activity of lyophilized NP-1 with replicon RNA encoding the SARS-CoV-2 spike antigen sequence, the physicochemical properties, potency, and immunogenicity of the reconstituted vaccine. Briefly, the materials in Table 20 were used. [Table 20]

[0219] Preparation of formulation complexes. The lipid nanoparticle / RNA complex compositions were prepared in this assay as shown in Table 21 below. NP-1 or NP-7 and repRNA were complexed at an N to P ratio of 15 to obtain a final repRNA concentration of 50 mg / ml or 100 mg / ml ("2x" material), and a sucrose content of 10% or 20% w / v, respectively. The complexed material with 10% sucrose (50 mg / ml repRNA) contained 5 mM sodium citrate, while the complexed material with 20% sucrose (100 mg / ml repRNA) contained 10 mM citrate. The complexes were filled into 2 ml sterile depyrogenated baked vials. The complexes with 10% sucrose were filled at 0.7 ml per vial and the complexes with 20% sucrose were filled at 0.35 ml per vial. The vials were then either lyophilized and stored or stored in liquid form. Storage temperatures were 25 degrees Celsius or 42 degrees Celsius for one week. The amounts of lyophilized and liquid vials per composition are summarized in Table 21. [Table 21]

[0220] Lyophilization Cycle. A batch lyophilizer filled with SP VirTis Advantage Pro trays and inert gas and stopper capacity was used. The lyophilization cycle is outlined in Table 22 below. After completion of the cycle, the vials were filled with 48 torr of nitrogen, stoppered, and then equilibrated to room pressure. [Table 22]

[0221] Condition Groups. A summary of the 14 groups analyzed in this assay is provided below in Table 23. Groups 1 and 4 were freshly prepared to serve as positive controls for comparison with the standard protocol for vaccine preparation, as indicated in the Storage column. [Table 23]

[0222] Immunogenicity assay. Induction of anti-spike IgG responses was evaluated in 6-8 week old female C57Bl / 6 mice. Group sizes of 5 mice were used. The schedule is shown in Table 24. [Table 24]

[0223] After 1 week of storage in a stability chamber at 25 degrees Celsius or 42 degrees Celsius. The lyophilized nanoparticle / RNA complexes were reconstituted in 0.7 ml of sterile milliQ water and gently swirled until particles were no longer visible to the naked eye. The particle size (z-average) and size distribution (PDI) of the complexes were measured and summarized in Figure 17, with group designation shown in Table 25. The particle size and PDI of the freshly prepared NP-1 / WT-S complex (Group 1) were 76.8 nm and 0.223, respectively. After reconstitution, the lyophilized samples (Groups 7-14) grew on average by 45% (+ / - 11%). A summary of the % change in z-average compared to Group 1 is included in Table 25. [Table 25]

[0224] Agarose gel electrophoresis of phenol-chloroform extracted repRNA. Liquid formulations of NP-1 / repRNA and NP-7+repRNA in 10% or 20% sucrose were stored for 1 week in NP-1 / repRNA and NP-7+repRNA, respectively, resulting in partial or complete degradation of the repRNA product (data not shown). Lyophilization of NP-1 / repRNA and NP-7+repRNA in 10% or 20% sucrose preserved repRNA integrity after 1 week of storage in NP-1 / repRNA and NP-7+repRNA (data not shown).

[0225] Potency assay. Lyophilized NP-1 / WT-S in 10% sucrose stored at 25 degrees Celsius for 1 week produced dose-dependent expression of spike protein in transfected BHK cells. The expression profile was similar to freshly complexed NP-1 / WT-S. Storage of lyophilized NP-1 / WT-S in 10% sucrose at 42 degrees Celsius for 1 week significantly reduced in vitro protein expression. Liquid NP-1 / WT-S in 10% sucrose stored at 25 degrees Celsius or 42 degrees Celsius for 1 week did not produce spike protein in BHK cells (data not shown).

[0226] Anti-D614G spiked IgG responses by ELISA. Serum anti-D614G spiked IgG levels were assessed 14 and 28 days after prime, as shown below in Figures 18A and 18B, respectively. Mouse sera were assayed in anti-D614G spiked ELISA at 1:40 (day 14) or 1:200 (day 28) dilutions. Serum IgG levels (μg / ml) were interpolated from a 4PL standard curve generated by mouse IgG standards of known concentrations.

[0227] Anti-D614G IgG responses 28 days after prime. After 1 week at 25 degrees Celsius, liquid NP-1 / WT-S in 10% sucrose resulted in a statistically significant reduction in anti-spike IgG compared to the freshly prepared NP-1 / WT-S positive control. There was no significant difference in mean IgG levels between freshly prepared NP-1 / WT-S and lyophilized NP-1 / WT-S in 10% sucrose stored at 25 degrees Celsius for 1 week.

[0228] After one week at 42 degrees Celsius, lyophilized NP-1 / WT-S in 10% sucrose induced 100% seroconversion, but mean IgG levels were significantly reduced compared to freshly prepared NP-1 / WT-S. A summary of the mean + / - standard deviation IgG concentration data from day 28 post-immunization, including p-values ​​determined by ordinary one-way ANOVA comparing against the freshly prepared NP-1 / WT-S positive control, is shown in Table 26. P<0.05 was considered statistically significant. [Table 26]

[0229] Comparison of fresh vs. lyophilized formulations. Anti-D614G spiked IgG concentrations in serum 28 days after prime are shown in FIG. 19. Statistical differences between mean IgG values ​​were determined by ordinary one-way ANOVA with Dunnett's multiple comparison test. All groups were compared to freshly prepared NP-1 / RNA in 10% sucrose. No significant differences were noted between freshly prepared NP-1 / RNA and lyophilized NP-1 / RNA in 10% sucrose stored at 25 degrees Celsius for 7 days. At 42 degrees Celsius, lyophilized NP-1 / RNA in 10% or 20% sucrose induced significantly lower anti-spiked IgG compared to freshly prepared NP-1 / RNA. Lyophilized NP-1 / RNA in 20% sucrose stored at 25 degrees Celsius or 42 degrees Celsius induced significantly lower IgG than freshly prepared NP-1 / WT-S. Lyophilized NP-1 / Delta-S in 10% or 20% sucrose stored at 25 degrees Celsius induced similar mean IgG (not statistically significant) as freshly prepared NP-1 / WT-S.

[0230] All references disclosed herein, including patent literature and non-patent literature, are hereby incorporated by reference in their entirety as if each was individually incorporated.It should be understood that the terminology used herein is merely for the purpose of describing specific embodiments and is not intended to be limiting.It should be further understood that unless specifically defined herein, the terminology used herein should be given its conventional meaning known in the relevant technical field. [ka]

Claims

1. 1. A dry composition comprising: a) a lipid carrier, said lipid carrier being a nanoemulsion comprising a hydrophobic core and one or more lipids; b) optionally one or more nucleic acids; and c) at least one sugar present in an amount of at least about 50% by weight of said dry composition; A dry composition comprising:

2. The dry composition described in claim 1, wherein the at least one sugar is present in an amount of 80 to 98 weight percent of the dry composition.

3. The dry composition of claim 1 which is freeze-dried.

4. The dry composition of claim 1, which is thermally stable at approximately -20 degrees Celsius.

5. The dry composition described in claim 1, which is thermally stable at about 2 degrees Celsius to about 8 degrees Celsius.

6. 10. The dry composition of claim 1, which is thermally stable at about 25 degrees Celsius.

7. 10. The dry composition of claim 1, which is thermally stable at about 45 degrees Celsius.

8. 10. The dry composition of claim 1, which is thermally stable for at least one week.

9. The dry composition of claim 1 , wherein the hydrophobic core comprises an oil.

10. 10. The dry composition of claim 9, wherein the oil comprises at least one of alpha-tocopherol, lauroyl polyoxylglycerides, monoacylglycerol, propolis, squalene, mineral oil, grape seed oil, olive oil, paraffin oil, peanut oil, soybean oil, sunflower oil, soy lecithin, triglycerides, vitamin E, caprylic / capric triglyceride, triglyceride esters of saturated coconut / palm kernel oil-derived caprylic and capric fatty acids and vegetable-derived glycerol, dihydroisosqualene (DHIS), farnesene, and squalane.

11. 2. The dry composition of claim 1, wherein the one or more lipids are selected from the group consisting of cationic lipids, anionic lipids, neutral lipids, and any combination thereof.

12. The one or more lipids may be selected from the group consisting of 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP); 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol); dimethyldioctadecylammonium (DDA); 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP), dipalmitoyl (C16:0)trimethylammoniumpropane (DPTAP); distearoyltrimethylammoniumpropane (DSTAP); N-[1-(2,3-dioleyloxy)propyl]-N,N,N trimethylammonium chloride (DOTMA); N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dioleoyl-3-dimethylammonium 1,2-Dilinoleyloxy-3-dimethylaminopropane (DLin-DMA); 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), 1,2-dioleoyl-sn-glycero-3-phosphoethanol 12. The dry composition of claim 11, comprising a cationic lipid selected from the group consisting of: amine (DOPE); N-decyl-N,N-dimethyldecane-1-aminium bromide (DDAB); 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA); ethylphosphatidylcholine (ePC); and any combination thereof.

13. The dry composition of claim 1 , wherein the lipid carrier comprises at least one surfactant.

14. 14. The dry composition of claim 13, wherein the at least one surfactant is selected from the group consisting of a hydrophobic surfactant, a hydrophilic surfactant, and any combination thereof.

15. 15. The dry composition of claim 14, wherein the hydrophobic surfactant comprises a sorbitan ester selected from the group consisting of sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, and sorbitan trioleate; and the hydrophilic surfactant comprises a polysorbate.

16. 2. The dry composition of claim 1, wherein the lipid carrier has a z-average hydrodynamic diameter in the range of about 40 nm to about 150 nm with an average polydispersity index in the range of about 0.1 to about 0.

4.

17. The dry composition of claim 1 , wherein the one or more nucleic acids comprise DNA or RNA.

18. 20. The dry composition of claim 17, wherein the RNA is a self-replicating RNA.

19. 2. The dry composition of claim 1, wherein the one or more nucleic acids are incorporated into or complexed with the lipid carrier to form a lipid carrier-nucleic acid complex.

20. 20. The dry composition of claim 19, wherein the lipid carrier-nucleic acid complex is formed through non-covalent interactions or through reversible covalent interactions.

21. 2. The dry composition of claim 1, wherein the molar ratio of the lipid carrier to the one or more nucleic acids, characterized by the nitrogen to phosphate (N:P) molar ratio, ranges from about 1:1 to about 150:

1.

22. 2. The dry composition of claim 1, wherein the at least one sugar is selected from the group consisting of sucrose, maltose, trehalose, mannitol, glucose, and any combination thereof.

23. The dry composition of claim 1 , wherein the hydrophobic core comprises one or more inorganic nanoparticles.

24. 25. The dry composition of claim 24, wherein the one or more inorganic nanoparticles are selected from the group consisting of a metal salt, a metal oxide, a metal hydroxide, a metal phosphate, and any combination thereof.

25. The one or more inorganic nanoparticles are aluminum oxide (Al ２ O ３ ), aluminum oxyhydroxide, iron oxide, titanium dioxide, silicon dioxide (SiO ２ ), aluminum hydroxyphosphate (Al(OH) ｘ (P.O. ４ ) ｙ ), calcium phosphate (Ca ３ (P.O. ４ ) ２ ), calcium hydroxyapatite (Ca １０ (P.O. ４ ) ６ (OH) ２ ), iron gluconate, iron sulfate, magnetite (Fe 3 O 4 ), maghemite (y-Fe 2 O 3 ), wustite (FeO), hematite (alpha (α)-Fe 2 O 3 ), or any combination thereof.

26. 10. The dry composition of claim 1, wherein the at least one sugar is present in an amount of at least about 50 mg.

27. Squalene at a concentration of up to about 1.88 mg / ml; Sorbitan monostearate at a concentration up to about 1.86 mg / ml; Polysorbate 80 at a concentration of up to about 1.86 mg / ml; and Optionally, iron oxide at a concentration of up to about 0.01 mg / ml wherein the nucleic acid is present in an amount of up to about 200 micrograms (μg); the lipid is a cationic lipid present at a concentration of up to about 1.5 mg / ml; The sugar is sucrose present at a concentration of up to about 50 mg / ml; The dry composition of claim 1.

28. The dry composition of claim 27, further comprising citric acid monohydrate present at a concentration of up to about 2.1 mg / ml.

29. 10. A pharmaceutical composition comprising the dry composition of claim 1 reconstituted in a suitable diluent and a pharma- ceutically acceptable carrier.

30. 13. Use of the dry composition of claim 1 in the preparation of a medicament for generating an immune response in a subject, characterized in that when the medicament is used, a therapeutically effective amount of the dry composition reconstituted in a suitable diluent is administered to the subject.

31. A dry composition as described in claim 1 for generating an immune response in a subject, characterized in that a therapeutically effective amount of the dry composition reconstituted in a suitable diluent is administered to the subject.

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