STABLE COMPOSITIONS OF mRNA-LOADED LIPID NANOPARTICLES AND MANUFACTURING PROCESSES

Abstract

The present invention provides improved compositions and processes for preparing mRNA-loaded lipid nanoparticles (mRNA-LNPs); in some embodiments, the present invention provides mRNA-LNPs with exceptional stability and is particularly useful in cases where LNPs comprising low or zero PEG-modified lipid content are desired.

Description

STABLE COMPOSITIONS OF mRNA-LOADED LIPID NANOPARTICLES AND MANUFACTURING PROCESSES CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority over the provisional application of the United States with serial number 62 / 877,597 filed on July 23, 2019, the description of which is incorporated herein by reference. BACKGROUND OF THE INVENTION Messenger RNA therapy (MRT) is becoming an increasingly important approach for treating a variety of diseases. MRT involves administering messenger RNA (mRNA) to a patient in need of therapy to stimulate the production of the protein encoded by the mRNA within the patient's body. Lipid nanoparticles are commonly used to encapsulate the mRNA for efficient in vivo mRNA delivery. Significant effort has been invested in developing improved methods and compositions that can enhance in vivo mRNA delivery and / or expression using lipid nanoparticles, and that can be adapted to a scalable and cost-effective manufacturing process. At the same time, it is important that such improvements in in vivo mRNA delivery and / or expression also maintain or improve the safety and tolerability of the compositions associated with lipid-mediated mRNA delivery. BRIEF DESCRIPTION OF THE INVENTION The present invention provides, among other things, improved compositions and processes for preparing mRNA-loaded lipid nanoparticles (mRNA-LNPs). Prior to the present invention, PEG-modified lipids were typically included in lipid nanoparticle (LNP) formulations because they were known to enhance stability during storage and in vivo circulation time. On the other hand, PEG-modified lipids can induce accelerated blood clearance (ABC) and / or an innate immune response, among other things, by producing anti-PEG antibodies. To address this issue, attempts have been made to prepare mRNA-LNPs without PEG-modified lipids.However, it has been observed that mRNA-laden LNPs formed in the absence of PEG-modified lipids or PEG are large and unstable, particularly after freezing and thawing, or tend to precipitate, rendering them unsuitable for therapeutic use. The present invention is based, in part, on the surprising discovery that unexpectedly stable mRNA-laden LNPs with a low or zero content of PEG-modified lipids can be prepared by mixing the mRNA and lipids in the presence of an amphiphilic block copolymer such as a poloxamer. As described in more detail below, the mRNA-LNPs manufactured according to the present invention are comparable in size to conventional LNPs containing a typical amount of PEG-modified lipids and, more importantly, are stable after one or more freeze-thaw cycles.In particular, the mRNA-LNPs according to the present invention maintain an average diameter within 50%, and in some cases within 10%, of the original average size after one or more freeze-thaw cycles. Furthermore, poloxamer-protected LNPs with a low or zero content of PEG-modified lipids (e.g., <0.5% PEG-modified lipids) achieved an in vivo protein expression profile similar to conventional LNPs (e.g., those with 5% PEG-modified lipids). Therefore, the present invention provides other enhanced mRNA-LNPs with exceptional stability and is particularly useful in cases where LNPs comprising a low or zero content of PEG-modified lipids are desired, for example, to prevent the generation of anti-PEG and / or ABC antibodies. In one aspect, the present invention provides a stable composition comprising lipid nanoparticles encapsulating messenger RNA (mRNA) encoding a protein or peptide, wherein each lipid nanoparticle comprises one or more cationic lipids and less than 0.5% PEG- or PEG-modified lipids and is stable after one or more freeze-thaw cycles. In some embodiments, the lipid nanoparticles further comprise one or more non-cationic lipids. In some embodiments, the lipid nanoparticles comprise a cationic lipid, dioleoylphosphatidylethanolamine (DOPE), as the non-cationic lipid, and less than approximately 0.5% of PEG-modified lipids. In some embodiments, the lipid nanoparticles comprise a cationic lipid, L,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), as the non-cationic lipid, and less than approximately 0.5% of PEG-modified lipids. In some embodiments, the lipid nanoparticles encapsulating the mRNA maintain an average diameter within 50% of the original average size after one or more freeze-thaw cycles. In some embodiments, the lipid nanoparticles encapsulating the mRNA maintain an average diameter within 40% of the original average size after one or more freeze-thaw cycles. In some embodiments, the lipid nanoparticles encapsulating the mRNA maintain an average diameter within 30% of the original average size after one or more freeze-thaw cycles. In some embodiments, the lipid nanoparticles encapsulating the mRNA maintain an average diameter within 20% of the original average size after one or more freeze-thaw cycles.In some embodiments, the lipid nanoparticles encapsulating the mRNA maintain an average diameter within 10% of the original average size after one or more freeze-thaw cycles. In some embodiments, the lipid nanoparticles encapsulating the mRNA maintain an average diameter within 5% of the original average size after one or more freeze-thaw cycles. In some embodiments, lipid nanoparticles have an mRNA encapsulation efficiency between approximately 50% and 99%. In some embodiments, lipid nanoparticles have an mRNA encapsulation efficiency between approximately 60% and 90%. In some embodiments, lipid nanoparticles have an mRNA encapsulation efficiency of approximately 60%. In some embodiments, lipid nanoparticles have an mRNA encapsulation efficiency of approximately 70%. In some embodiments, lipid nanoparticles have an mRNA encapsulation efficiency of approximately 80%. In some embodiments, lipid nanoparticles have an mRNA encapsulation efficiency of approximately 90%. In some forms, each of the lipid nanoparticles also comprises a cholesterol-based lipid. In some embodiments, each lipid nanoparticle comprises 0.4% PEG-modified lipids or less. In some embodiments, each lipid nanoparticle comprises 0.3% PEG-modified lipids or less. In some embodiments, each lipid nanoparticle comprises 0.2% PEG-modified lipids or less. In some embodiments, each lipid nanoparticle comprises 0.1% PEG-modified lipids or less. In some forms, each of the lipid nanoparticles is substantially free of PEG-modified lipids. In some embodiments, each of the lipid nanoparticles comprises an amphiphilic block copolymer. In some embodiments, each lipid nanoparticle comprises less than 3% amphiphilic block copolymer. In some embodiments, each lipid nanoparticle comprises less than 3% amphiphilic block copolymer. In some embodiments, each lipid nanoparticle comprises less than 2.5% amphiphilic block copolymer. In some embodiments, each lipid nanoparticle comprises less than 2% amphiphilic block copolymer. In some embodiments, each lipid nanoparticle comprises less than 1.5% amphiphilic block copolymer. In some embodiments, each lipid nanoparticle comprises less than 1% amphiphilic block copolymer. In some embodiments, each lipid nanoparticle comprises less than 0.5% amphiphilic block copolymer. In some embodiments, each lipid nanoparticle comprises less than 0.0.5% amphiphilic block copolymer. In some embodiments, each of the lipid nanoparticles comprises less than 0.01% amphiphilic block copolymer. In some embodiments, the composition comprises less than 0.05% amphiphilic block copolymer of the total composition by weight. In some embodiments, the composition comprises less than 0.04% amphiphilic block copolymer of the total composition by weight. In some embodiments, the composition comprises less than 0.03% amphiphilic block copolymer of the total composition by weight. In some embodiments, the composition comprises less than 0.02% amphiphilic block copolymer of the total composition by weight. In some embodiments, the composition comprises less than 0.01% amphiphilic block copolymer of the total composition by weight. In some embodiments, the composition comprises an amphiphilic block copolymer residue. In some embodiments, a suitable amphiphilic block copolymer is a poloxamer. In some forms, a suitable poloxamer is poloxamer 84. In some forms, a suitable poloxamer is poloxamer 101. In some forms, a suitable poloxamer is poloxamer 105. In some forms, a suitable poloxamer is poloxamer 108. In some forms, a suitable poloxamer is poloxamer 122. In some forms, a suitable poloxamer is poloxamer 123. In some forms, a suitable poloxamer is poloxamer 124. In some forms, a suitable poloxamer is poloxamer 181. In some forms, a suitable poloxamer is poloxamer 182. In some forms, a suitable poloxamer is poloxamer 183. In some forms, a suitable poloxamer is poloxamer 184. In some In some modalities, a suitable poloxamer is poloxamer 185. In some modalities, a suitable poloxamer is poloxamer 188. In some modalities, a suitable poloxamer is poloxamer 212.In some forms, a suitable poloxamer is poloxamer 215. In some forms, a suitable poloxamer is poloxamer 217. In some forms, a suitable poloxamer is poloxamer 231. In some forms, a suitable poloxamer is poloxamer 234. In some forms, a suitable poloxamer is poloxamer 235. In some forms, a suitable poloxamer is poloxamer 237. In some forms, a suitable poloxamer is poloxamer 238. In some forms, a suitable poloxamer is poloxamer 282. In some forms, a suitable poloxamer is poloxamer 284. In some forms, a suitable poloxamer is poloxamer 288. In some forms, a poloxamer The appropriate poloxamer is 304. In some forms, a suitable poloxamer is 331. In some forms, a suitable poloxamer is 333. In some forms, a suitable poloxamer is 334.In some forms, a suitable poloxamer is poloxamer 335. In some forms, a suitable poloxamer is poloxamer 338. In some forms, a suitable poloxamer is poloxamer 401. In some forms, a suitable poloxamer is poloxamer 402. In some forms, a suitable poloxamer is poloxamer 403. In some forms, a suitable poloxamer is poloxamer 407. In some forms, a suitable poloxamer is a combination of these. In one aspect, the present invention provides a stable composition comprising lipid nanoparticles encapsulating a messenger RNA (mRNA) encoding a protein or peptide, wherein each of the lipid nanoparticles comprises one or more cationic lipids, one or more non-cationic lipids, a poloxamer and is substantially free of PEG or PEG-modified lipids, and wherein the mRNA-encapsulating lipid nanoparticles are stable after one or more freeze-thaw cycles. In one aspect, the present invention provides a stable composition comprising lipid nanoparticles encapsulating a messenger RNA (mRNA) encoding a protein or peptide, wherein each of the lipid nanoparticles comprises one or more cationic lipids, one or more non-cationic lipids, a poloxamer and is substantially free of PEG or PEG-modified lipids, and wherein the mRNA-encapsulating lipid nanoparticles generate few or no anti-PEG antibodies and / or reduce accelerated blood clearance (ABC). In some formulations, the poloxamer is present in the lipid nanoparticles in an amount less than 0.1%. In some formulations, the poloxamer is present in the lipid nanoparticles in an amount less than 0.05%. In some forms, a suitable non-cationic lipid is dioleoylphosphatidylethanolamine (DOPE). In some forms, a suitable non-cationic lipid is 1,2-diurecoyl-sn-glycero-3-phosphoethanolamine (DEPE). In some forms, each of the lipid nanoparticles does not comprise a cholesterol-based lipid. In some forms, each of the lipid nanoparticles is a two-component lipid nanoparticle. In some forms, a suitable poloxamer has ethylene oxide units from around 10 to around 150. frPRnnn / zznz / E / YiAi In some forms, a suitable poloxamer has propylene oxide units from around 10 to around 100. In some embodiments, a suitable poloxamer has an average molecular weight of approximately 4,000 g / mol to approximately 20,000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of approximately 1,000 g / mol to approximately 50,000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of approximately 1,000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of approximately 2,000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of approximately 3,000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of approximately 4,000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of approximately 5,000 g / mol. In some forms, a suitable poloxamer has an average molecular weight of around 6,000 g / mol.In some embodiments, a suitable poloxamer has an average molecular weight of approximately 7,000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of approximately 8,000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of approximately 9,000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of approximately 10,000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of approximately 20,000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of approximately 25,000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of approximately 30,000 g / mol. In some embodiments, a suitable poloxamer has an average molecular weight of approximately 40,000 g / mol. In some forms, a suitable poloxamer has an average molecular weight of around 50,000 g / mol. In some embodiments, the lipid nanoparticles have an average size smaller than 250 nm. In some embodiments, the lipid nanoparticles have an average size of approximately 200 nm or less. In some embodiments, the lipid nanoparticles have an average size of approximately 180 nm or less. In some embodiments, the lipid nanoparticles have an average size of approximately 160 nm or less. In some embodiments, the lipid nanoparticles have an average size of approximately 150 nm or less. In some embodiments, the lipid nanoparticles have an average size of approximately 140 nm or less. In some embodiments, the lipid nanoparticles have an average size of approximately 130 nm or less. In some embodiments, the lipid nanoparticles have an average size of approximately 120 nm or less.In some embodiments, the lipid nanoparticles frFRnnn / zznz / E / YiAi have an average size of around 110 nm or less. In some embodiments, the lipid nanoparticles have an average size of around 100 nm or less. In some embodiments, the lipid nanoparticles have a polydispersity index (PDI) of 0.3 or less. In some embodiments, the lipid nanoparticles have a polydispersity index (PDI) of 0.25 or less. In some embodiments, the lipid nanoparticles have a polydispersity index (PDI) of 0.20 or less. In some embodiments, the lipid nanoparticles have a polydispersity index (PDI) of 0.18 or less. In some embodiments, the lipid nanoparticles have a polydispersity index (PDI) of 0.17 or less. In some embodiments, the lipid nanoparticles have a polydispersity index (PDI) of 0.16 or less. In some embodiments, the lipid nanoparticles have a polydispersity index (PDI) of 0.15 or less. In some embodiments, lipid nanoparticles have a polydispersity index (PDI) of 0.14 or less. In some embodiments, lipid nanoparticles have a polydispersity index (PDI) of 0.13 or less.In some embodiments, the lipid nanoparticles have a polydispersity index (PDI) of 0.12 or less. In some embodiments, the lipid nanoparticles have a polydispersity index (PDI) of 0.11 or less. In some embodiments, the lipid nanoparticles have a polydispersity index (PDI) of 0.10 or less. In some embodiments, the lipid nanoparticles have a polydispersity index (PDI) of 0.09 or less. In some embodiments, the lipid nanoparticles have a polydispersity index (PDI) of 0.08 or less. In some embodiments, the lipid nanoparticles have a polydispersity index (PDI) of 0.07 or less. In some embodiments, the lipid nanoparticles have a polydispersity index (PDI) of 0.06 or less. In some forms, lipid nanoparticles have a polydispersity index (PDI) of 0.05 or less. In one aspect, the present invention provides, among other things, a method for supplying messenger RNA (mRNA) for in vivo production of a protein or peptide, comprising administering to a subject a stable composition according to the present invention. In one aspect, the present invention provides, among other things, a method for supplying messenger RNA (mRNA) for the in vivo production of a protein or peptide, comprising administering to a subject a stable composition according to the present invention, wherein the administration of the stable composition does not result in anti-PEG antibodies and / or accelerated blood clearance (ABC) in the subject. In one aspect, the present invention provides, among other things, a method for treating a subject who has a deficiency in a protein or peptide, comprising administering to a subject in need of treatment a stable composition according to the present invention. frFRnnn / zznz / E / YiAi In some modalities, administration of the stable composition generates little or no anti-PEG antibodies in the subject. In some modalities, administration of the stable composition reduces or prevents accelerated blood clearance (AUC) in the subject. In some forms, the stable composition is administered by intravenous injection. In some modalities, the stable composition is administered via pulmonary delivery. In some modalities, the stable composition is administered via intramuscular delivery. In some formulations, administration of the stable composition results in the expression of the protein or peptide encoded by the mRNA for at least approximately 6 hours after administration. In some formulations, administration of the stable composition results in the expression of the protein or peptide encoded by the mRNA for at least approximately 12 hours after administration. In some formulations, administration of the stable composition results in the expression of the protein or peptide encoded by the mRNA for at least approximately 18 hours after administration. In some formulations, administration of the stable composition results in the expression of the protein or peptide encoded by the mRNA for at least approximately 24 hours after administration.In some formulations, administration of the stable composition results in the expression of the protein or peptide encoded by the mRNA for at least approximately 30 hours after administration. In some formulations, administration of the stable composition results in the expression of the protein or peptide encoded by the mRNA for at least approximately 36 hours after administration. In some formulations, administration of the stable composition results in the expression of the protein or peptide encoded by the mRNA for at least approximately 48 hours after administration. In some formulations, administration of the stable composition results in the expression of the protein or peptide encoded by the mRNA for at least approximately 72 hours after administration.In some formulations, administration of the stable composition results in the expression of the protein or peptide encoded by the mRNA for at least approximately 5 days after administration. In some formulations, administration of the stable composition results in the expression of the protein or peptide encoded by the mRNA for at least approximately 1 week after administration. In some formulations, administration of the stable composition results in the expression of the protein or peptide encoded by the frPRnnn / zznz / E / YiAi. mRNA expression for at least approximately 2 weeks after administration. In some formulations, administration of the stable formulation results in expression of the protein or peptide encoded by the mRNA for at least approximately 3 weeks after administration. In some formulations, administration of the stable formulation results in expression of the protein or peptide encoded by the mRNA for at least approximately 4 weeks after administration. In one aspect, the present invention provides, among other things, a process for encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising a step of mixing an mRNA solution and a lipid solution in the presence of a poloxamer. In some forms, the lipid solution comprises one or more cationic lipids, one or more non-cationic lipids, and less than 0.5% PEG-modified or PEG-modified lipids. In some forms, the lipid solution comprises preformed lipid nanoparticles. In some modalities, the mRNA solution and / or the lipid solution are at a predetermined temperature higher than room temperature. In some forms, the poloxamer is first added to the mRNA solution. In some forms, the poloxamer is present in a lower amount than its critical micelle concentration (CMC). In some formulations, the poloxamer is present in an amount approximately 1% lower than its CMC. In some formulations, the poloxamer is present in an amount approximately 2% lower than its CMC. In some formulations, the poloxamer is present in an amount approximately 3% lower than its CMC. In some formulations, the poloxamer is present in an amount approximately 4% lower than its CMC. In some formulations, the poloxamer is present in an amount approximately 5% lower than its CMC. In some formulations, the poloxamer is present in an amount approximately 6% lower than its CMC. In some formulations, the poloxamer is present in an amount approximately 7% lower than its CMC. In some formulations, the poloxamer is present in an amount approximately 8% lower than its CMC. In some forms, the poloxamer is present in an amount approximately 9% lower than its CMC.In some formulations, the poloxamer is present in an amount approximately 10% lower than its CMC. In some formulations, the poloxamer is present in an amount approximately 15% lower than its CMC. In some formulations, the poloxamer is present in an amount approximately 20% lower than its CMC. In some formulations, the poloxamer is present in an amount approximately 25% lower than its CMC. In some formulations, the poloxamer is present in an amount approximately 30% lower than its CMC. In some formulations, the poloxamer is present in an amount approximately 35% lower than its CMC. In some formulations, the poloxamer is present in an amount approximately 40% lower than its CMC. In some forms, the poloxamer is present in an amount approximately 45% lower than its CMC.In some forms, the poloxamer is present in an amount about 50% less than its CMC. In some forms, the process also includes a poloxamer removal stage. In some forms, the poloxamer is removed by dialysis. In some formulations, less than approximately 0.1% of poloxamer remains after elimination. In some formulations, less than approximately 0.05% of poloxamer remains after elimination. In some formulations, less than approximately 0.01% of poloxamer remains after elimination. In some forms, a residual amount of poloxamer remains after elimination. In some forms, the amount of poloxamer remaining after elimination is undetectable. In some forms, the process does not include mixing cholesterol lipids. In one aspect, the present invention provides, among other things, a composition comprising lipid nanoparticles that encapsulate mRNA formed according to a process described herein. In this application, the use of "or" means "and / or" unless otherwise indicated. As used herein, the term "comprises" and variations thereof, such as "comprising" and "comprising," are not intended to exclude any other additives, components, whole numbers, or stages. As used herein, the expressions "around" and "approximately" are used interchangeably. Both terms are intended to cover any normal fluctuations appreciated by a person skilled in the relevant art. Other features, objects, and advantages of the present invention are evident from the detailed description, figures, and claims that follow. It should be understood, however, that the detailed description, figures, and claims, while indicating embodiments of the present invention, are provided only by way of illustration and without limitation. Various changes and modifications within the scope of the invention will be apparent to those skilled in the art. frFRnnn / zznz / E / YiAi BRIEF DESCRIPTION OF THE FIGURES The following figures are for illustrative purposes only, and are not exhaustive. Figure 1 shows a schematic of an illustrative LNP-mRNA encapsulation process that involves mixing an aqueous solution comprising mRNA and poloxamer with a lipids solution by using a pump system to generate LNP-mRNA in an LNP forming solution and then swapping the LNP forming solution for a pharmaceutical formulation solution. Figure 2 represents an illustrative graphical representation of the size and encapsulation efficiency of the mRNA-LNP formulations shown in Table 3 before and after one or two freeze / thaw cycles. Figure 3 represents an illustrative graphical representation of the size, PDI, and encapsulation efficiency of mRNA-LNP formulations with varying PEG-modified lipid percentages and poloxamer percentages, as shown in Table 4. Figure 4 represents an illustrative graphical representation of the size and encapsulation efficiency of mRNA-LNP formulations with various % of PEG-modified lipids and poloxamer, as shown in Table 4. Figure 5 shows an illustrative graph of protein levels measured by ELISA at 6 and 24 hours after administration. The detected protein is the result of in vivo translation of mRNA encapsulated in the LNP formulations shown in Table 7, which were administered subcutaneously or intravenously to mice. Figures 6A and 6B show an illustrative method for quantifying the amount of poloxamer. Figure 6A depicts a chemical reaction between poloxamer and cobalt thiocyanate to form a blue precipitate. Figure 6B shows a standard curve with known poloxamer concentrations, measured at 624 nm. Figure 7 shows an illustrative graph of OTC protein levels measured by ELISA, 24 hours after administration. The detected protein is the result of in vivo translation of mRNA encapsulated in the LNP formulations shown in Table 8, which were administered intravenously to mice. DEFINITIONS To facilitate understanding of the present invention, certain terms are defined below. Further definitions for the following terms and other terms are set forth throughout the description. frPRnnn / zznz / E / YiAi Approximately or around. As used herein, the term approximately or around, as applied to one or more values ​​of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term approximately or around refers to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such a number exceeds 100% of a possible value). Delivery: As used in this description, the term delivery encompasses both local and systemic delivery. For example, mRNA delivery encompasses situations where an mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and maintained within the target tissue (also referred to as local distribution or local delivery) and situations where an mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into the patient's circulatory system (e.g., serum) and is distributed and absorbed systemically by other tissues (also referred to as systemic distribution or systemic delivery). Efficacy: As used in this description, the term efficacy, or grammatical equivalents, refers to an improvement in a biologically relevant endpoint relative to the delivery of mRNA encoding a relevant protein or peptide. In some modalities, the biological endpoint protects against an ammonium chloride challenge at certain time points after administration. Encapsulation: As used in the present description, the term encapsulation, or the grammatical equivalent, refers to the process of confining an individual mRNA molecule within a nanoparticle. Expression: As used in this description, mRNA expression refers to the translation of an mRNA into a peptide (e.g., an antigen), polypeptide, or protein (e.g., an enzyme) and may also include, as indicated in the context, the post-translational modification of the fully assembled peptide, polypeptide, or protein (e.g., an enzyme). In this application, the terms expression and production, and their grammatical equivalents, are used interchangeably. Improve, increase, or decrease. As used herein, the terms improve, increase, or decrease, or their grammatical equivalents, indicate values ​​relative to a measurement of baseline values, such as a measurement in the same individual before starting the treatment described herein, or a measurement in a control subject (or multiple control subjects) in the absence of the treatment described herein. A control sample is a sample subjected to the same conditions as a test sample, except for the test item. A control subject is a subject who has the same form of the disease as the subject receiving the treatment and is approximately the same age as the subject receiving the treatment. In vitro: As used in the present description, the term 'in vitro' refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in a cell culture, etc., rather than within a multicellular organism. In vivo: As used in this description, the term 'in vivd' refers to events occurring within a multicellular organism, such as a human or non-human animal. In the context of cell-based systems, the term may be used to refer to events occurring within a living cell (as opposed to, for example, in vitrd systems). Messenger RNA (mRNA): As used herein, the term messenger RNA (mRNA) refers to a polynucleotide that encodes at least one peptide, polypeptide, or protein. mRNA as used herein comprises both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA may be purified from natural sources, produced by the use of recombinant expression systems, and optionally purified, chemically synthesized, etc. Where appropriate, for example, in the case of chemically synthesized molecules, mRNA may comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5' to 3' direction unless otherwise stated.In some forms, an mRNA is or comprises natural nucleosides {e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogues {e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine, pseudouridine, and 5-methylcytidine}; chemically modified bases; biologically modified bases {e.g., mediated bases}; intercalated bases; modified sugars {e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose and hexose); and / or modified phosphate groups {e.g., phosphorothioate linkages and 5'- / V-phosphoramidite). N / P Ratio: As used herein, the term N / P ratio refers to the molar ratio of positively charged molecular units in the cationic lipids within a lipid nanoparticle to the negatively charged molecular units in the mRNA encapsulated within that lipid nanoparticle. As such, the ratio frPRnnn / zznz / E / YiAi N / P is typically calculated as the ratio of moles of amine groups in cationic lipids in a lipid nanoparticle to the moles of phosphate groups in the mRNA encapsulated within that lipid nanoparticle. Nucleic acid: As used in this description, the term nucleic acid, in its broadest sense, refers to any compound and / or substance that is found in or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is found in or can be incorporated into a polynucleotide chain via a phosphodiester bond. In some embodiments, nucleic acid refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, nucleic acid refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, nucleic acid encompasses RNA as well as single- and / or double-stranded DNA and / or cDNA. In addition, the terms nucleic acid, DNA, RNA, and / or similar terms include nucleic acid analogs, i.e., analogs having a backbone other than a phosphodiester bond. Patient: As used herein, the term patient or subject refers to any organism to which a provided composition may be administered, for example, for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some modalities, a patient is a human being. A human being includes both prenatal and postnatal forms. Pharmaceutically acceptable. The term pharmaceutically acceptable, as used in this description, refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, consistent with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceuticai Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art, such as ion exchange.Other pharmaceutically acceptable salts include salts of adipate, alginate, frPRnnn / zznz / E / YiAi ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from appropriate bases include salts of alkali metal, alkaline earth metal, ammonium, and N+(alkyl Ci-4)4.Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and similar metals. Other pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate, and aryl sulfonate. Further pharmaceutically acceptable salts include salts formed from the quaternization of an amine using an appropriate electrophile, for example, an alkyl halide, to form a quaternized alkylated amino salt. Subject. As used in this description, the term subject refers to a human animal or any nonhuman animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse, or primate). A human being includes both prenatal and postnatal forms. In many contexts, a subject is a human being. A subject may be a patient, which refers to a human being who seeks medical care from a provider for diagnosis or treatment of a disease. The term subject is used in this description interchangeably with individual and patient. A subject may have or be susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder. Substantially. As used herein, the term substantially refers to the qualitative condition of exhibiting a total or near-total extent or degree of a characteristic or property of interest. A person skilled in biological technique will understand that biological and chemical phenomena rarely, if ever, reach termination and / or proceed to complete, achieve, or avoid an absolute result. Therefore, the term substantially is used herein to capture the potential lack of totality inherent in many biological and chemical phenomena. Treatment. As used in this description, the term treat, treatment, or that treats refers to any method used to relieve, improve, mitigate, inhibit, prevent, delay the onset, reduce the severity, and / or reduce the incidence, in whole or in part, of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and / or only exhibits early signs of the disease in order to decrease the risk of developing the pathology associated with the disease. DETAILED DESCRIPTION OF THE INVENTION The present invention provides, among other things, improved compositions and processes for preparing mRNA-loaded lipid nanoparticles (mRNA-LNPs). Prior to the present invention, PEG-modified lipids were typically included in lipid nanoparticle (LNP) formulations because they were known to increase stability during storage and in vivo circulation time. On the other hand, PEG-modified lipids can induce accelerated blood clearance (ABC) and / or an innate immune response, among other things, by producing anti-PEG antibodies. To address this problem, attempts have been made to prepare mRNA-LNPs without PEG-modified lipids.However, it has been observed that mRNA-laden LNPs formed in the absence of PEG-modified lipids or PEG-modified lipids are large and unstable, particularly after freezing and thawing, or tend to precipitate, rendering them unsuitable for therapeutic use. The present invention is based, in part, on the surprising discovery that unexpectedly stable mRNA-laden LNPs with a low or zero content of PEG-modified lipids can be prepared by mixing the mRNA and lipids in the presence of an amphiphilic block copolymer such as a poloxamer. Therefore, the present invention provides other enhanced mRNA-LNPs with exceptional stability and is particularly useful in cases where LNPs comprising a low or zero content of PEG-modified lipids are desired, for example, to prevent the generation of anti-PEG and / or ABC antibodies. The present invention provides novel processes for encapsulating mRNA in LNPs and the resulting stable mRNA-LNP compositions. In particular, the present invention provides a process for encapsulating mRNA in LNPs by mixing an mRNA solution and a lipid solution in the presence of an amphiphilic polymer (e.g., a poloxamer). The amphiphilic polymer can be removed from the mRNA-LNPs by, for example, dialysis. The present invention is particularly useful for encapsulating mRNA in LNPs with a low or zero content of PEG-modified lipids. Various aspects of the invention are described in detail in the following sections. The use of sections does not limit the invention. Each section may be applied to any aspect of the invention. frFRnnn / zznz / E / YiAi mRNA encapsulation processes in LNP The present invention provides a process for encapsulating mRNA in LNPs in the presence of an amphiphilic polymer (e.g., a poloxamer). In some embodiments, an mRNA encapsulation process described herein comprises a step of mixing a lipid solution with an mRNA solution in the presence of an amphiphilic polymer (e.g., a poloxamer) such that lipid nanoparticles encapsulating the mRNA are formed. In some embodiments, the amphiphilic polymer (e.g., a poloxamer) is present in the mRNA solution before mixing. In some embodiments, the amphiphilic polymer (e.g., a poloxamer) is present in the lipid solution before mixing. In some embodiments, the amphiphilic polymer (e.g., a poloxamer) is added during the mixing of an mRNA solution and a lipid solution. In some embodiments, a suitable mRNA solution is an aqueous solution comprising mRNA encoding a protein or peptide of interest at a desired concentration. Various methods may be used to prepare a suitable mRNA solution. Illustrative methods are described in documents Nos. US 2016 / 0038432, US 2018 / 0153822, and US 2018 / 0125989, which are incorporated herein by reference. In some embodiments, a suitable lipid solution comprises cationic lipids and non-cationic lipids (also called auxiliary lipids). In some embodiments, a suitable lipid solution comprises cationic lipids, non-cationic lipids (also called auxiliary lipids), and PEG-modified lipids. In some embodiments, a suitable lipid solution comprises cationic lipids, non-cationic lipids (also called auxiliary lipids), cholesterol-based lipids, and PEG-modified lipids. Various lipids can be dissolved in a suitable solvent in the respective amounts and / or ratios desired to prepare a lipid solution for use in a process described herein. Several methods can be used to prepare a suitable lipid solution. Illustrative methods are described in documents Nos.US 2016 / 0038432, US 2018 / 0153822 and US 2018 / 0125989, which are incorporated in this description by reference. In some formulations, a suitable lipid solution contains less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of PEG-modified or PEG-modified lipids of the total lipids by molar. In some formulations, a suitable lipid solution contains less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% of PEG-modified or PEG-modified lipids of the total lipids by molarity. In some formulations, a suitable lipid solution contains less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02% or less than 0.01% of PEG-modified lipids or PEG of total lipids by weight. Typically, an amphiphilic polymer (e.g., a poloxamer) is present in the mixture at a lower concentration than its critical micelle concentration (CMC). In some embodiments, an amphiphilic polymer (e.g., a poloxamer) is present in the mixture at a concentration approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% lower than its CMC. In some embodiments, an amphiphilic polymer (e.g., a poloxamer) is present in the mixture in an amount of about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% less than its CMC.In some embodiments, an amphiphilic polymer (e.g., a poloxamer) is present in the mixture at an amount of about 55%, 60%, 65%, 70%, 75%, 80%, 90% or 95% less than its CMC. In some embodiments, the mRNA solution or the lipid solution, or both, may be heated to a predetermined temperature above room temperature before mixing. In some embodiments, the mRNA solution and the lipid solution are heated separately to the predetermined temperature before mixing. In some embodiments, the mRNA solution and the lipid solution are mixed at room temperature but then heated to the predetermined temperature after mixing. In some embodiments, the lipid solution is heated to the predetermined temperature and mixed with the mRNA solution at room temperature. In some embodiments, the mRNA solution is heated to the predetermined temperature and mixed with the lipid solution at room temperature. In some modalities, the mRNA solution is heated to the predetermined temperature by adding a room temperature mRNA stock solution to a heated buffer solution to achieve the desired predetermined temperature. In some methods, LNP-mRNAs are heated after formation. As the examples show, it was surprisingly found that including a heating step during the process (before, during, or after formation) provides significantly greater LNP-mRNA encapsulation compared to an identical process without the heating step. frPRnnn / zznz / E / YiAi As used in this description, the term room temperature refers to the temperature in a room, or the temperature surrounding an object of interest without heating or cooling. In some embodiments, the room temperature at which one or more of the solutions are maintained is or is less than about 35°C, 30°C, 25°C, 20°C, or 16°C. In some embodiments, the room temperature at which one or more of the solutions are maintained varies from about 15–35°C, about 15–30°C, about 15–25°C, about 15–20°C, about 20–35°C, about 25–35°C, about 30–35°C, about 20–30°C, about 25–30°C, or about 20–25°C. In some forms, the ambient temperature at which one or more of the solutions are heated is 20-25 °C. Therefore, a predetermined temperature higher than ambient temperature is typically greater than approximately 25°C. In some embodiments, a suitable predetermined temperature for the present invention is or is greater than approximately 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C. In some embodiments, a suitable predetermined temperature for the present invention varies from approximately 25-70°C, approximately 30-70°C, approximately 35-70°C, approximately 40-70°C, approximately 45-70°C, approximately 50-70°C, or approximately 60-70°C. In particular embodiments, a suitable predetermined temperature for the present invention is approximately 65°C. In some formulations, the mRNA solution and the lipid solution are mixed using a pump. Because the encapsulation procedure with such a mixture can occur across a wide range of scales, different types of pumps can be used to suit the desired scale. However, a pulseless flow pump is generally preferred. As used herein, a pulseless flow pump refers to any pump capable of establishing continuous flow at a steady rate. Suitable pump types may include, but are not limited to, gear pumps and centrifugal pumps. Illustrative gear pumps include, but are not limited to, Cole-Parmer or Diener gear pumps. Illustrative centrifugal pumps include, but are not limited to, those manufactured by Grainger or Cole-Parmer. The mRNA solution and the lipid solution can be mixed at different flow rates. Typically, the mRNA solution can be mixed at a higher rate than the lipid solution. For example, the mRNA solution can be mixed at a rate at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x higher than the rate of the lipid solution. Appropriate flow rates for mixing can be determined based on the scale. In some modalities, an mRNA solution is mixed at a flow rate that varies from approximately 40–400 mL / min, 60–500 mL / min, 70–600 mL / min, 80–700 mL / min, 90–800 mL / min, 100–900 mL / min, 110–1,000 mL / min, 120–1,100 mL / min, 130 mL / min. 1,200 ml / minute, 140-1,300 ml / minute, 150-1,400 ml / minute, 160-1,500 ml / minute, 1701,600 ml / minute, 180-1,700 ml / minute, 150-250 ml / minute, 250-500 ml / minute, 5001,000 ml / minute, 1,000-2,000 ml / minute, 2,000-3,000 ml / minute, 3,000-4,000 ml / minute or 4,0005,000 ml / minute. In some modalities, mRNA stock solution is mixed at a flow rate that varies from about 200 ml / minute, about 500 ml / minute, about 1,000 ml / minute, about 2,000 ml / minute, about 3,000 ml / minute, about 4,000 ml / minute, or about 5,000 ml / minute. In some modalities, a lipid solution is mixed at a flow rate that varies from about 25-75 ml / minute, 20-50 ml / minute, 25-75 ml / minute, 30-90 ml / minute, 40-100 ml / minute, 50-110 ml / minute, 75-200 ml / minute, 200-350 ml / minute, 350-500 ml / minute, 500-650 ml / minute, 650-850 ml / minute or 850-1,000 ml / minute. In some modalities, the lipid solution is mixed at a flow rate of about 50 ml / minute, about 100 ml / minute, about 150 ml / minute, about 200 ml / minute, about 250 ml / minute, about 300 ml / minute, about 350 ml / minute, about 400 ml / minute, about 450 ml / minute, about 500 ml / minute, about 550 ml / minute, about 600 ml / minute, about 650 ml / minute, about 700 ml / minute, about 750 ml / minute, about 800 ml / minute, about 850 ml / minute, about 900 ml / minute, about 950 ml / minute or about 1,000 ml / minute. Typically, a novel process described herein includes a step to remove the amphiphilic polymer (e.g., poloxamer). In some embodiments, the amphiphilic polymer (e.g., poloxamer) added during the process is subsequently removed after the formation of the LNP-mRNAs. For example, amphiphilic polymers (e.g., poloxamers) can be removed by buffer exchange techniques such as dialysis. In some embodiments, the LNP-forming solution is exchanged for a solution that constitutes the product formulation solution. For example, the mixture containing the formed LNP-mRNAs can be dialyzed in one or more formulation solutions to remove the amphiphilic polymer (e.g., poloxamer) present during LNP-mRNA formation. Suitable formulations are known in the art, and illustrative formulations are described in the Formulations section of this application. The exchange of the LNP-mRNA solution from the LNP formation solution to the formulation solutions can be achieved using any of a variety of buffer exchange techniques known in the art. In some embodiments, the step of exchanging the LNP formation solution for a formulation solution is accompanied by purification and / or concentration of the LNP-mRNAs. Several methods can be used to achieve the solution exchange along with the purification or concentration of the LNP-mRNAs in the solution. For example, in some methods, this solution exchange is achieved through diafiltration. Diafiltration is a fractionation process in which small, unwanted particles pass through a filter while larger, desired nanoparticles are retained in the retentate without changing the concentration of those nanoparticles in solution. Diafiltration is frequently used to remove salts or reaction buffers from a solution. Diafiltration can be continuous or batch. In continuous diafiltration, a diafiltration solution is added to the sample feed at the same rate as the filtrate is generated. In batch diafiltration, the solution is first diluted and then concentrated after the initial concentration. Batch diafiltration can be repeated until a desired nanoparticle concentration is reached. In some methods, the solution is swapped, and the LNP-mRNAs are purified using tangential flow filtration. Tangential flow filtration (TFF), also called cross-flow filtration, is a type of filtration in which the material to be filtered passes tangentially through a filter rather than directly through it. In TFF, the unwanted permeate passes through the filter, while the desired retentate (LNP-mRNA and free mRNA) passes along the filter and is collected downstream. In some methods, the desired material is contained within the retentate in the TFF, which is the opposite of what is typically found in traditional one-way filtration. Several TFF techniques are known and can be used to practice the present invention. Illustrative TFF purification methods are described in documents Nos. US 2016 / 0040154 and US 2015 / 0376220, which are incorporated herein by reference. In some embodiments, the encapsulation of mRNA in LNPs can be further enhanced by heating the formulation solution comprising the mRNA-LNPs, as well as some free mRNA that was not encapsulated in the LNP forming solution, to a predetermined temperature as described in the present description. In some embodiments, less than approximately 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of the original amount of the amphiphilic polymer (e.g., poloxamer) present in the mixture remains after removal. In some embodiments, a residual amount of the amphiphilic polymer (e.g., poloxamer) remains in a formulation after its removal. As used herein, a residual amount means an amount remaining after substantially all of the substance (an amphiphilic polymer described herein, such as a poloxamer) has been removed from a composition. A residual amount may be detected qualitatively or quantitatively using a known technique. It is possible that a residual amount may not be detectable using a known technique. In some modalities, the excess mRNA is also removed, along with the amphiphilic polymer (e.g., poloxamer) present during the formation of mRNA-LNPs. frPRnnn / zznz / E / YiAi Amphiphilic block copolymers Various amphiphilic block copolymers can be used to implement the present invention. In some embodiments, an amphiphilic block copolymer is also referred to as a surfactant or a nonionic surfactant. In some embodiments, a suitable amphiphilic polymer for the invention is selected from poloxamers (Pluronic®), poloxamines (Tetronic®), polyoxyethylene glycol sorbitan alkyl esters (polysorbates) and polyvinylpyrrolidones (PVP). Poloxamers In some embodiments, a suitable amphiphilic polymer is a poloxamer. For example, a suitable poloxamer has the following structure: where a is an integer between 10 and 150 and b is an integer between 20 and 60. For example, a is around 12 and b is around 20, a is around 80 and b is around 27, a is around 64 and b is around 37, a is around 141 and b is around 44, a is around 101 and b is around 56. In some embodiments, a poloxamer suitable for the invention has ethylene oxide units from about 10 to about 150. In some embodiments, a poloxamer has ethylene oxide units from about 10 to about 100. Other amphiphilic polymers In some forms, an amphiphilic polymer is a poloxamine, for example, tetronic 304 or tetronic 904. In some embodiments, an amphiphilic polymer is a polyvinylpyrrolidone (PVP), such as PVP with a molecular weight of 3 kDa, 10 kDa, or 29 kDa. In some embodiments, an amphiphilic polymer is a polyethylene glycol ether (Brij), polysorbate, sorbitan, and derivatives thereof. In some embodiments, an amphiphilic polymer is a polysorbate, such as PS 20. In some embodiments, an amphiphilic polymer is a polyethylene glycol ether. In some embodiments, a suitable polyethylene glycol ether is a compound of Formula (Sl): (Sl), or a salt or isomer thereof, wherein t is an integer between 1 and 100; R1BRIJindependently is alkyl Cio-40, alkenyl Cio-40 or alkynyl Cio-40; and optionally one or more methylene groups of R5PEG are independently replaced with C3-10 carbocyclylene, 4-10 membered heterocyclylene, C6-10 arylene, 4-10 membered heteroarylene, -N(RN)-, -0-, S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -NR C(O)N(R)-, -C(O)0- -OC(O)-, -OC(O)0- - OC(O)N(RN)-, NRNC(O)0- -C(O)S- -SC(O)-, -C(=NRN)-,— C(=NR)N(R)—, - NRNC(=NRn)- -NRnC(=NRn)N(Rn)-, C(S)-, -C(S)N(RN)-, -NRNC(S)-, -NRnC(S)N(Rn)-, -S(O)-, -OS(O)-, -S(O)0- -OS(O)0- -OS(O)2- -S(O)20-OS(O)20- -N(RN)S(O)-, - S(O)N(RN)- -N(Rn)S(O)N(Rn)- -OS(O)N(RN)- -N(RN)S(O)0- -S(O)2- N(Rn)S(O)2- - S(O)2N(Rn)-, -N(Rn)S(O)2N(Rn)- -OS(O)2N(Rn)- or -N(Rn)S(O)20-; and each case of RNis independently hydrogen, Ci-6 alkyl, or a nitrogen protecting group. In some forms, R1BRIJ is cis alkyl. For example, polyethylene glycol ether is a compound of the formula (S-la): frFRnnn / zznz / E / YiAi or a salt or isomer thereof, where s is an integer between 1 and 100. In some embodiments, R1BRIJ is a cis alkenyl. For example, a suitable polyethylene glycol ether is a compound of the formula (S-lb): or a salt or isomer thereof, where s is an integer between 1 and 100. Stable compositions of LNP-mRNA Among other things, the invention provides LNP-mRNAs prepared using a novel process described herein. In particular, the invention provides stable compositions comprising LNP-mRNAs with a low (e.g., <0.5 wt% or mol%) or zero content of PEG- or PEG-modified lipids. Such LNP-mRNAs are suitable for the efficient delivery and expression of mRNA in vivo. In this application, LNP and LNP-mRNA are used interchangeably unless specifically indicated. For example, the LNP-mRNAs used herein include both mRNA-loaded and empty LNPs unless specifically identified. Typically, the term stable in relation to an LNP composition means an LNP composition that can be stored at room temperature for more than 2 hours or at 4°C overnight without precipitation. In some forms, a stable composition described herein comprises LNPs that maintain an average diameter within 60% of the original average size after one or more freeze-thaw cycles. Canonical lipids As used in this description, the phrase cationic lipids refers to any of a number of lipid or lipoid species that have a net positive charge at a selected pH, such as physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available. Cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2010 / 144740, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include a cationic lipid, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, having a structure composed of: frFRnnn / zznz / E / YiAi and pharmaceutically acceptable salts thereof. Other cationic lipids suitable for use in the compositions and methods of the present invention include ionizable cationic lipids as described in International Patent Publication WO 2013 / 149140, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid of one of the following formulas: R2 r2 or a pharmaceutically acceptable salt thereof, wherein each of Ri and R2 is independently selected from the group consisting of hydrogen, a variably saturated or optionally substituted C1-C20 alkyl and an optionally substituted C6-C20 variably saturated or optionally substituted acyl; wherein each of Li and L2 is independently selected from the group consisting of hydrogen, an optionally substituted C1-C30 alkyl, an optionally substituted C1-C30 variably unsaturated alkenyl, and an optionally substituted C1-C30 alkynyl; wherein each of myo is independently selected from the group consisting of zero and any positive integer (e.g., where m is three); and wherein n is zero or any positive integer (e.g., where n is one).In certain embodiments, the compositions and methods of the present invention include the cationic lipid (15Z, 18Z)-N,Ndimethyl-6-(9Z,12Z)-octadec-9,12-dien-1-1) tetracosa-15,18-dien-lamine (HGT5000), which has a structure composed of:. (HGT-5000) and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include the cationic lipid (15Z, 18Z)-N,N-dimethyl-6-((9Z,12Z)-octadecaca-9,12-den-1-1)tetracosa-4,15,18-tren-1-amine (HGT5001), which has a structure composed of: frFRnnn / zznz / E / YiAi and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include the cationic lipid and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadecaca-9,12-dien-l-yl)tetracosa-5,15,18-trien-1-amine (HGT5002), which has a structure composed of: (HGT-5002) and pharmaceutically acceptable salts thereof. Other cationic lipids suitable for use in the compositions and methods of the invention include cationic lipids described as amino alcohol lipidoids in International Patent Publication WO 2010 / 053572, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a structure composed of: C10H2i HO C10H2i and pharmaceutically acceptable salts thereof. Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2016 / 118725, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a structure composed of: frFRnnn / zznz / E / YiAi and pharmaceutically acceptable salts thereof. Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2016 / 118724, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a structure composed of: and pharmaceutically acceptable salts thereof. Other cationic lipids suitable for use in the compositions and methods of the invention include a cationic lipid having the formula 14,25-ditridecyl 15,18,21,24-tetraazaoctatriacontane and pharmaceutically acceptable salts thereof. Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publications WO 2013 / 063468 and WO 2016 / 205691, each of which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid of the following formula: or pharmaceutically acceptable salts thereof, wherein each instance of RLs is independently C6-C40 alkenyl optionally substituted. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a structure composed of: (CKK-E12) frPRnnn / zznz / E / YiAi and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a structure composed of: (OF-02) and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a structure composed of: frFRnnn / zznz / E / YiAi and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a structure composed of: and pharmaceutically acceptable salts thereof. Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2015 / 184256, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid of the following formula: frFRnnn / zznz / E / YiAi H3C-(CH2)m^OH H3C-(CH2)mγ N OH | (CRaRb), (CRARB)n| OH A '(CH2)m-CH3HO7CH2)m-CH3o a pharmaceutically acceptable salt thereof, wherein each X is independently OS; each Y is independently OS; each m is independently from 0 to 20; each n is independently from 1 to 6; each of Ra is independently hydrogen, optionally substituted Cl-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl or halogen; and each Rb is independently hydrogen, optionally substituted Cl-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted heteroaryl or 5-14 membered halogen.In certain embodiments, the compositions and methods of the present invention include a cationic lipid, Diana 23, which has a structure composed of:. OH (Diana 23) frPRnnn / zznz / E / YiAi and pharmaceutically acceptable salts thereof. Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2016 / 004202, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following structure: or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following structure: or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following structure: frPRnnn / zznz / E / YiAi or a pharmaceutically acceptable salt thereof. Other cationic lipids suitable for use in the compositions and methods of the present invention include cationic lipids as described in U.S. Provisional Patent Application Serial No. 62 / 758,179, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid of the following formula: or a pharmaceutically acceptable salt thereof, wherein each R1 and R2 is independently H or a C1-C2 aliphatic group; each M is independently an integer having a value from 1 to 4; each A is independently a covalent bond or arylene; each L1 is independently an ester, thioester, disulfide, or anhydride group; each L2 is independently a C2-C10 aliphatic group; each X1 is independently H or OH; and each R3 is independently a C6-C20 aliphatic group. In some embodiments, the compositions and methods of the present invention include a cationic lipid of the following formula: frPRnnn / zznz / E / YiAi (Compound 1) or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid of the following formula: or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid of the following formula: or a pharmaceutically acceptable salt thereof. Other cationic lipids suitable for use in the compositions and methods of the present invention include the cationic lipids as described in McCIellan, MC King, Cell 2010, 141, 210-217 and in Whitehead et al., Nature Communications (2014) 5:4277, which are incorporated herein by reference. In certain embodiments, the cationic lipids of the compositions and methods of the present invention include a cationic lipid having a structure composed of: Ci3H27 C13H27 frFRnnn / zznz / E / YiAi and pharmaceutically acceptable salts thereof. Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2015 / 199952, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following structure: and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following structure: bPRnnn / zznz / B / YiAi and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following composite structure: N. and pharmaceutically acceptable salts thereof, compositions and methods of the present invention include a composite structure: In some forms, the cationic lipid that has the N. and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following structure: frFRnnn / zznz / E / YiAi and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following composite structure: and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following structure: and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following composite structure: frFRnnn / zznz / E / YiAi and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following composite structure: and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following composite structure: and pharmaceutically acceptable salts thereof. Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2017 / 004143, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid that frFRnnn / zznz / E / YiAi and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following composite structure: and pharmaceutically acceptable salts thereof, compositions and methods of the present invention include a composite structure: In some forms, the cationic lipid that has the and pharmaceutically acceptable salts thereof, compositions and methods of the present invention include a composite structure: In some forms, the cationic lipid that has the and pharmaceutically acceptable salts thereof, compositions and methods of the present invention include a composite structure: In some modalities, the cationic lipid that has the frFRnnn / zznz / E / YiAi and pharmaceutically acceptable salts thereof, compositions and methods of the present invention include a composite structure: In some forms, the cationic lipid that has the and pharmaceutically acceptable salts thereof, compositions and methods of the present invention include a composite structure: In some forms, the cationic lipid that has the and pharmaceutically acceptable salts thereof, compositions and methods of the present invention include a composite structure: In some forms, the cationic lipid that has the and pharmaceutically acceptable salts thereof, compositions and methods of the present invention include a composite structure: In some forms, the cationic lipid that has the and pharmaceutically acceptable salts thereof, compositions and methods of the present invention include a composite structure: In some forms, the cationic lipid that has the and pharmaceutically acceptable salts thereof, compositions and methods of the present invention include a composite structure: In some forms, the cationic lipid that has the frPRnnn / zznz / E / YiAi and pharmaceutically acceptable salts thereof, compositions and methods of the present invention include a composite structure: In some forms, the cationic lipid that has the and pharmaceutically acceptable salts thereof, compositions and methods of the present invention include a composite structure: In some forms, the cationic lipid that has the and pharmaceutically acceptable salts thereof, compositions and methods of the present invention include a composite structure: In some forms, the cationic lipid that has the frPRnnn / zznz / E / YiAi and pharmaceutically acceptable salts thereof, compositions and methods of the present invention include a composite structure: In some forms, the cationic lipid that has the and pharmaceutically acceptable salts thereof, compositions and methods of the present invention include a composite structure: In some forms, the cationic lipid that has the and pharmaceutically acceptable salts thereof, compositions and methods of the present invention include a composite structure: In some forms, the cationic lipid that has the frPRnnn / zznz / E / YiAi and pharmaceutically acceptable salts thereof. Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2017 / 075531, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid of the following formula: Rk ^G3,|2 / L2 R1\G1 \G2 \r2 or a pharmaceutically acceptable salt thereof, wherein one of L1o L2s is O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x, -SS-, -C(=O)S-C-,-, -SC(-=O -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa-, o -NRaC(=O)O-; and the other of L1o L2es -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x, -SS-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=)NRO)NRa(=„- -OC(=O)NRa- or NRaC(=O)O- or a direct G1y G2bond each is independently alkylene C1-C12 or unsubstituted alkenylene C1-C24, alkenylene C1-C24; Ca-Cs; Raes H or alkyl C1-C12; alkyl Ci-Ce;yx is 0, 1, or 2. Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2017 / 117528, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following structure: frPRnnn / zznz / E / YiAi and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following composite structure: and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following composite structure: and pharmaceutically acceptable salts thereof. Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2017 / 049245, which is incorporated herein by reference. In some embodiments, the cationic lipids of the compositions and methods of the present invention include a compound of one of the following formulas: frPRnnn / zznz / E / YiAi and pharmaceutically acceptable salts thereof. For any of these four formulas, R4 is independently selected from -(CH2)nQ and -(CH2)nCHQR; Q; Q is selected from the group consisting of -OR, -OH, -O(CH2)nN(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, 10 N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), and a heterocycle; yn is 1, 2, or 3. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a structure composed of: EITHER and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a structure composed of: EITHER frFRnnn / zznz / E / YiAi and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a structure composed of: and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a structure composed of: and pharmaceutically acceptable salts thereof. Other cationic lipids suitable for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publications WO 2017 / 173054 and WO 2015 / 095340, each of which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a structure composed of: and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a structure composed of: and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a structure composed of: and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a structure composed of: frFRnnn / zznz / E / YiAi and pharmaceutically acceptable salts thereof. Other cationic lipids suitable for use in the compositions and methods of the invention include cholesterol-based cationic lipids. In certain embodiments, the compositions and methods of the present invention include an imidazole cholesterol ester or ICE, and pharmaceutically acceptable salts thereof. Other cationic lipids suitable for use in the compositions and methods of the present invention include scindiol cationic lipids as described in International Patent Publication WO 2012 / 170889, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid of the following formula: wherein Ri is selected from the group consisting of imidazole, guanidinium, amino, imimine, enamine, an optionally substituted alkylamino (for example, an alkylamino such as dimethylamino), and pyridyl; wherein R2 is selected from the group consisting of one of the following two formulas: bPRnnn / zznz / B / YiAi and wherein each of FU and R4 is independently selected from the group consisting of a variably saturated or optionally substituted C6-C20 alkyl and a variably saturated or optionally substituted C6-C20 acyl; and wherein n is zero or any positive integer (for example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more). In certain embodiments, the compositions and methods of the present invention include a cationic lipid, HGT4001, having a structure composed of: (HGT4001) and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid, HGT4002, having a structure composed of: NH2(HGT4002) and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid, HGT4003, having a structure composed of: H.H frFRnnn / zznz / E / YiAi (HGT4003) and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid, HGT4004, having a structure composed of: (HGT4004) and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid HGT4005, which has a structure composed of: (HGT4005) and pharmaceutically acceptable salts thereof. Other cationic lipids suitable for use in the compositions and methods of the present invention include scindiol cationic lipids as described in U.S. Provisional Application No. 62 / 672,194, filed May 16, 2018, and incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having any of the general formulas or any of the structures (1a)-(21a), (1b)-(21b), and (22)-(237) described in U.S. Provisional Application No. 62 / 672,194. In certain embodiments, the compositions and methods of the present invention include a cationic lipid having a structure according to Formula (Γ), b-14B-14A-o O Οχ R3_L3 L2-R2(Oíen where RX is independently -H, -L1-R1 or -L5A-L5B-B'; each of L1, L2 and L3 is independently a covalent bond, -C(O) -C(O)O-, -C(O)S- or -C(O)NRL-; each L4A and L5A is independently -C(O) -C(O)O- or -C(O)NRL-; each L4B and L5B is independently a C1-C20 alkylene; a C2-C20 alkenylene; or a C2-C20 alkynylene; each B and B' is NR4R5 or a 5- to 10-membered nitrogen-containing heteroaryl; each R1, R2 and R3 is independently a C6C30 alkyl, C6-C30 alkenyl or C6-C30 alkynyl; each R4 and R5 is independently hydrogen, C1-C10 alkyl; C2-C10 alkenyl; or C2-C10 alkynyl; and each RL is independently hydrogen, C1-C20 alkyl, C2-C20 alkenyl or C2-C20 alkynyl. In certain embodiments, the compositions and methods of the present invention include a cationic lipid that is Compound (139) of document no. 62 / 672,194, having a compound structure of: frPRnnn / zznz / E / YiAi (18:1 carbon tail ribose lipid). In some embodiments, the compositions and methods of the present invention include the cationic lipid, N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). (Feigner et al. (Proc. Nat. Acad. Sci. 84, 7413 (1987); U.S. Patent No. 4,897,355, which is incorporated herein by reference). Other cationic lipids suitable for the compositions and methods of the present invention include, for example, 5-carboxyspermylglycinedioctadecylamide (DOGS); 2,3-dioleyloxy-N-[2-(spermine-carboxamido)ethyl]N,N-dimethyl-1-propanaminium (DOSPA) (Behr et al., Proc. Nat. Acad. Sci. 86, 6982 (1989), U.S. Patent No. 5,171,678; U.S. Patent No. 5,334,761), l,2-dioleoyl-3dimethylammonium-propane (DODAP); l,2-dioleol-3-trimethylammonium-propane (DOTAP). Additional illustrative cationic lipids suitable for the compositions and methods of the present invention also include: l,2-d¡steanlox¡-N,N-dimethyl-3-aminopropane (DSDMA); l,2-d¡oleyloxy¡-N,Nd¡methyl-3-aminopropane (DODMA); l,2-dilinoleyloxy-N,N-dimet¡l-3aminopropane (DLinDMA); l,2-dil¡nolen¡lox¡-N,Nd¡methyl-3-am¡nopropane (DLenDMA); Nd¡oleyl-N,Nd¡methlamon¡o chloride (DODAC); N,Nd¡stear¡lN,Nd¡methylammonium bromide (DDAB); N-(l,2-d¡myristyloxyprop-3-¡l)-N,N-dimethyl-Nh¡drox¡et¡l ammonium bromide (DMRIE); 3-dimethylamino2-(colest-5-en-3-beta-ox¡butan-4-oxy)-l-(cis,ds-9,12-octadecadienox¡)propane (CLinDMA); 2-[5'(cholest-5-en-3-beta-oxy)-3'-oxapentox¡)-3-d¡methyl-l-(c¡s,c¡s-9', l-2'-octadecadienoxy)propane (CpLinDMA); N,N-dimeth¡l-3,4-diole¡lox¡bench¡lamine (DMOBA); l,2-N,N'-dioleylcarbamyl-3dimethylaminopropane (DOcarbDAP); 2,3-D¡l¡noleo¡loxi-N,Nd¡meth¡lpropylam¡ne (DLinDAP); 1,2NjN'-Dilinoleylcarbamyl-S-dimethylaminopropane (DLincarbDAP); l,2-Dilinoleoylcarbamyl-3dimethylaminopropane (DLinCDAP); 2,2-dyl¡nole¡l-4-dimeth¡lam¡nomethyl-[l,3]-d¡oxolane (DLin-KDMA); 2-((8-[(3P)-colest-5-en-3-yloxy]oct¡l)ox¡)-N, N-dimeth¡l-3-[(9Z, 12Z)-octadeca-9, 12-dien-l yloxy]propano-l-amine (Octyl-CLinDMA); (2R)-2-((8-[(3beta)-colest-5-en-3-yloxy]octyl)oxy)-N, Ndimethyl-3-[(9Z, 12Z)-octadeca-9, 12-dien-l-yloxy]propan-l -amine (Octyl-CLinDMA (2R));(2S)-2-((8[(3P)-cholest-5-en-3-yloxy]oct¡l)ox¡)-N, fsl-dimethih3-[(9Z, 12Z)-octadeca-9, 12-dien-l -yloxy]propan-l amine (Octyl-CLinDMA (2S)); 2,2-dil¡nole¡l-4-dimet¡lam¡noethyl¡l-[l,3]-dioxolane (DLin-K-XTC2-DMA); and 2-(2,2-di((9Z,12Z)-octadeca-9,1 2-dien- 1-1)-1,3-dioxolan-4-11)-N,N-dimethyletanamyl (DLin-KC2DMA) (see document WO 2010 / 042877, which is incorporated herein by reference; Semple et al., Nature Biotech. 28: 172-176 (2010)). (Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, DV., et al., Nat. Biotechnol. 23(8): 1003-1007 (2005); International Patent Publication WO 2005 / 121348). In some forms, one or more of the cationic lipids comprise at least one imidazole, dialkylamino, or guanidinium residue. In some embodiments, one or more cationic lipids suitable for the compositions and methods of the present invention include 2,2-Dilinole1-4-dimethylaminoethyl-[1,3]dioxolane (XTC); (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadecaca-9,12-dienyl)tetrahydro-3aHcyclopenta[d][1,3]dioxol-5-amine (ALNY-100) and / or 4,7,13-tris(3-oxo-3-(undecylamino)propyl)N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide (NC98-5). In some embodiments, the compositions of the present invention include one or more cationic lipids constituting at least approximately 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, measured by weight, of the total lipid content in the composition, for example, a lipid nanoparticle. In some embodiments, the compositions of the present invention include one or more cationic lipids constituting at least approximately 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 80%, measured as a mole percent, of the total lipid content in the composition, for example, a lipid nanoparticle.In some embodiments, the compositions of the present invention include one or more cationic lipids that constitute about 30-70% (for example, about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%), measured by weight, of the total lipid content in the composition, for example, a lipid nanoparticle. In some embodiments, the compositions of the present invention include one or more cationic lipids that constitute about 30-70% (for example, about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%), measured as mol%, of the total lipid content in the composition, for example, a lipid nanoparticle. In some embodiments, sterol-based cationic lipids may be used instead of or in addition to the cationic lipids described herein. Suitable sterol-based cationic lipids are sterol-based cationic lipids containing dialkylamino, imidazole, and guanidinium. For example, certain embodiments are directed to a composition comprising one or more sterol-based cationic lipids comprising an imidazole, for example, cholesterol and imidazole ester or lipid ICE (3S, 10R, 13R, 17R)-10, 13dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1Hcyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, as represented by structure (I) below.In certain embodiments, a lipid nanoparticle for the delivery of RNA (e.g., mRNA) encoding a functional protein may comprise one or more imidazole-based cationic lipids, e.g., the cholesterol imidazole ester or lipid ICE (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17tetradecahydro-1H-cyclopenta[a]phenanthren-3-11 3-(1H-imidazol-4-yl)propanoate, as represented by the following structure:. frPRnnn / zznz / E / YiAi In some formulations, the percentage of cationic lipid in a liposome may be greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70%. In some formulations, the cationic lipid(s) constitute approximately 30–50% (e.g., approximately 30–45%, approximately 30–40%, approximately 35–50%, approximately 35–45%, or approximately 35–40%) of the liposome by weight. In some formulations, the cationic lipid (e.g., ICE lipid) constitutes approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 60%, approximately 70%, or approximately 80% of the liposome in molar ratio. Non-cationic / auxiliary lipids In some forms, the LNP-mRNAs described herein include non-cationic / auxiliary lipids. As used herein, the term non-cationic lipid refers to any neutral, bipolar, or anionic lipid. As used herein, the term anionic lipid refers to any of a number of lipid species that carries a net negative charge at a selected pH, such as physiological pH.Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylleoylphosphatidylcholine (POPC), palmitoylleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-1), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) or a mixture of these. In some formulations, non-cationic lipids may constitute at least approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65%, or 70% of the total lipids by weight or molar mass. In some formulations, the non-cationic lipid(s) constitute(s) approximately 30–50% (e.g., approximately 30–45%, approximately 30–40%, approximately 35–50%, approximately 35–45%, or approximately 35–40%) of the total lipids by weight or molar mass. Choysteroid-based lipids In some embodiments, the LNP-mRNAs described herein include one or more cholesterol-based lipids. Suitable cholesterol-based cationic lipids include, for example, DC-Cho (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleaminopropyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al., BioTechniques 23, 139 (1997); U.S. patent no. 5,744,335), or ICE. In some forms, the coiesteroi-based lipid(s) constitute(s) at least approximately 5, 10, 20, 30, 40, 50, 60%, or 70% of the total lipids by weight or molarity. In some forms, the coiesteroi-based lipid(s) constitute(s) approximately 30–50% (e.g., approximately 30–45%, approximately 30–40%, approximately 35–50%, approximately 35–45%, or approximately 35–40%) of the total lipids by weight or molarity.In some embodiments, the coiesteroi-based lipid(s) constitute less than approximately 5, 10, 20, 30, 40, 50, 60%, or 70% of the total lipids by weight or molarity. In some embodiments, the LNP-mRNAs described herein do not include coiesteroi-based lipids. frFRnnn / zznz / E / YiAi PEG-modified lipids In some embodiments, the LNP-mRNAs described herein include a low amount (e.g., < 0.5 wt% or mol%) of one or more PEG-modified lipids (also known as PEGylated lipids) or PEG. For example, the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-octanyl-sphingosine-1-[succinyl(methoxy polyethylene glycol)-2000] (C8 PEG2000 ceramide), is also contemplated by the present invention. The PEG-modified lipids considered include, but are not limited to, a polyethylene glycol chain up to 2 kDa, up to 3 kDa, up to 4 kDa, or up to 5 kDa in length that is covalently linked to a lipid with alkyl chain(s) of Ce-Czo length. In some embodiments, a PEGylated or PEG-modified lipid is PEGylated cholesterol or PEG-2K.In some modalities, particularly useful exchangeable lipids are PEG-ceramides that have shorter acyl chains (e.g., Ci4 or Cie). In some embodiments, the LNP-mRNAs described herein contain less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of PEG-modified lipids or PEG of the total lipids by molar. In some embodiments, the LNP-mRNAs described herein contain 0.4% or less of PEG-modified lipids, 0.3% or less of PEG-modified lipids, 0.2% or less of PEG-modified lipids, or 0.1% or less of PEG-modified lipids of the total lipids on a molar or weight basis. In some embodiments, the LNP-mRNAs described herein contain 0.09% or less of PEG-modified lipids of the total lipids on a molar or weight basis.In some embodiments, the LNP-mRNAs described herein contain 0.08% or less of PEG-modified lipids or PEG-modified lipids of the total lipids on a molar or weight basis. In some embodiments, the LNP-mRNAs described herein contain 0.07% or less of PEG-modified lipids or PEG-modified lipids of the total lipids on a molar or weight basis. In some embodiments, the LNP-mRNAs described herein contain 0.06% or less of PEG-modified lipids or PEG-modified lipids of the total lipids on a molar or weight basis. In some embodiments, the LNP-mRNAs described herein contain 0.05% or less of PEG-modified lipids or PEG-modified lipids of the total lipids on a molar or weight basis. In some embodiments, the LNP-mRNAs described herein contain 0.04% or less of PEG-modified lipids or PEG of the total lipids by molar or weight. In some embodiments, the LNP-mRNAs described herein contain 0.0.03% or less of PEG-modified lipids or PEG-modified lipids of the total lipids on a molar or weight basis. In some embodiments, the LNP-mRNAs described herein contain 0.02% or less of PEG-modified lipids or PEG-modified lipids of the total lipids on a molar or weight basis. In some embodiments, the LNP-mRNAs described herein contain 0.01% or less of PEG-modified lipids or PEG-modified lipids of the total lipids on a molar or weight basis. In some forms, the LNP-mRNAs described herein are substantially free of PEG or PEG-modified lipids. Flake!amphiphilic block groupers In some embodiments, the LNP-mRNAs described herein contain amphiphilic block copolymers (e.g., poloxamers). In some embodiments, the LNP-mRNAs comprise less than 5% amphiphilic block copolymers (e.g., poloxamers). In some embodiments, the LNP-mRNAs comprise less than 3% amphiphilic block copolymers (e.g., poloxamers). In some embodiments, the LNP-mRNAs comprise less than 2.5% amphiphilic block copolymers (e.g., poloxamers). In some embodiments, the LNP-mRNAs comprise less than 2% amphiphilic block copolymers (e.g., poloxamers). In some embodiments, the LNP-mRNAs comprise less than 1.5% amphiphilic block copolymers (e.g., poloxamers). In some embodiments, LNP-mRNAs comprise less than 1% amphiphilic block copolymers (e.g., poloxamers). In some embodiments, LNP-mRNAs comprise less than 0.5% (e.g., less than 0.4%, 0.3%, 0.2%, 0.1%) of amphiphilic block copolymers (e.g., poloxamers). In some embodiments, LNP-mRNAs comprise less than 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of amphiphilic block copolymers (e.g., poloxamers). In some embodiments, LNP-mRNAs comprise less than 0.01% of amphiphilic block copolymers (e.g., poloxamers). In some embodiments, LNP-mRNAs contain a residual amount of amphiphilic polymers (e.g., poloxamers). As used herein, a residual amount means an amount remaining after substantially all of the substance (an amphiphilic polymer described herein, such as a poloxamer) has been removed from a composition. A residual amount can be detected qualitatively or quantitatively using a known technique.A residual amount may not be detected using a known technique. messenger RNA (mRNA) The present invention can be used to encapsulate any mRNA. Typically, mRNA is considered to be the type of RNA that carries information from DNA to the ribosome. Typically, in eukaryotic organisms, mRNA processing involves the addition of a 5' cap and a 3' tail. A typical cap is a methylguanosine cap, which is a guanosine linked by a 5'-5'-triphosphate bond to the first transcribed nucleotide. The presence of the cap is important for providing resistance to nucleases found in most eukaryotic cells. The addition of a tail is typically a polyadenylation event whereby a polyadenyl group is added to the 3' end of the mRNA molecule. The presence of this tail serves to protect the mRNA from degradation by exonucleases. Messenger RNA is translated by ribosomes into a series of amino acids that make up a protein. mRNAs can be synthesized according to any of a variety of known methods. For example, the mRNAs according to the present invention can be synthesized via in vitro transcription (IVT). In summary, IVT is typically performed using a linear or circular DNA template containing a promoter, a group of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and a suitable RNA polymerase (e.g., RNA polymerase T3, T7, or SP6), DNase I, pyrophosphatase, and / or RNase inhibitor. The exact conditions will vary according to the specific application. In some modalities, the in vitro synthesized mRNA can be purified prior to formulation and encapsulation to remove undesirable impurities, including various enzymes and other reagents used during mRNA synthesis. The present invention can be used to formulate and encapsulate mRNA of a variety of lengths. In some embodiments, the present invention can be used to formulate and encapsulate in vitro synthesized mRNA equal to or greater than approximately 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or 20 kb in length. In some embodiments, the present invention can be used to formulate and encapsulate in vitro synthesized mRNA that varies from about 1-20 kb, about 1-15 kb, about 110 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-15 kb in length. The present invention can be used to formulate and encapsulate unmodified mRNA or mRNA containing one or more modifications that typically improve stability. In some embodiments, the modifications are selected from modified nucleotides, modified sugar phosphate backbones, and 5' and / or 3' untranslated regions. In some embodiments, the modifications to mRNAs may include modifications to the RNA nucleotides. An mRNA modified according to the invention may include, for example, modifications to the backbone, modifications to the sugar, or modifications to the bases. In some forms, mRNAs can be synthesized from naturally occurring nucleotides and / or nucleotide analogues (modified nucleotides) that include, but are not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)) and as modified nucleotide analogues or derivatives of purines and pyrimidines, such as, for example, 1-methyladenine, 2-methyladenine, 2-methylthio-N-6-isopentenyladenine, N-6-methyladenine, N-6-isopentenyladenine, 2-thiocytosine, 3-methylcytosine, 4-acetylcytosine, 5-methylcytosine, 2,6-diaminopurine, 1-methylguanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine,pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromouracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, methyl ester of N-uracil-5-oxyacetic acid, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, methyl ester of uracil-5-oxyacetic acid, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosin, .beta-D-mannosyl-queosine, wibutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, pseudouridine, 5-methylcytidine, and inosine. The preparation of such analogues is known to a person skilled in the art, for example, from U.S. Patent No. 4,373,071, U.S. Patent No. 4,401,796, U.S. Patent No. 4,415,732, and U.S. Patent No. 4,458,066.United States patent no. 4,500,707, United States patent no. 4,668,777, United States patent no. 4,973,679, United States patent no. 5,047,524, United States patent no. 5,132,418, United States patent no. 5,153,319, United States patents nos. 5,262,530 and 5,700,642, the description of which is included herein in its full scope by reference. Typically, mRNA synthesis includes the addition of a 5' cap and a 3' tail. The cap is important for providing resistance to nucleases found in most eukaryotic cells. The tail protects the mRNA from degradation by exonucleases. Therefore, in some forms, mRNAs include a 5' cap structure. A 5' cap is typically added as follows: first, a terminal RNA phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphates via a guanylyl transferase, producing a 5'5'5 triphosphate linkage; and finally, the nitrogen 7 of guanine is methylated by a methyltransferase. 2'-O-methylation can also occur at the first and / or second base after the 7-methylguanosine triphosphate residues. Examples of cap structures include, but are not limited to, m7GpppNp-RNA, m7GpppNmp-RNA, and m7GpppNmpNmp-RNA (where m denotes 2'-o-methyl residues). In some forms, mRNAs include a 5' and / or 3' untranslated region. In some forms, a 5' untranslated region includes one or more elements that affect the stability or translation of an mRNA, for example, an iron-sensitive element. In some forms, a 5' untranslated region can be between approximately 50 and 500 nucleotides in length. In some forms, a 3' untranslated region includes one or more polyadenylation signals, a binding site for proteins that affect the stability of mRNA localization in a cell, or one or more binding sites for miRNAs. In some forms, a 3' untranslated region can be 50 to 500 nucleotides or longer. While mRNA from in vitro transcription reactions may be convenient in some modalities, other sources of mRNA are contemplated within the scope of the invention, including mRNA produced from bacteria, fungi, plants and / or animals. The present invention can be used to formulate and encapsulate mRNAs encoding a variety of proteins. Non-limiting examples of mRNAs suitable for the present invention include mRNAs encoding erythropoietin (EPO) and firefly luciferase (FFL). Formulations Several formulations may be used in connection with the present invention. In some formulations, a suitable solution may include a buffering agent or a salt. Examples of buffering agents include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. Examples of salts include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, a suitable formulation solution is an aqueous solution comprising pharmaceutically acceptable excipients, including, but not limited to, a cryoprotectant. In some embodiments, a suitable formulation solution is an aqueous solution comprising pharmaceutically acceptable excipients, including, but not limited to, sugar, such as one or more of trehalose, sucrose, mannose, lactose, and mannitol. In some embodiments, a suitable formulation solution comprises trehalose. In some embodiments, a suitable formulation solution comprises sucrose. In some embodiments, a suitable formulation solution comprises mannose. In some embodiments, a suitable formulation solution comprises lactose. In some embodiments, a suitable formulation solution comprises mannitol. In some embodiments, a suitable formulation solution is an aqueous solution comprising 5% to 20% by weight per volume of a sugar, such as trehalose, sucrose, mannose, lactose, and mannitol. In some embodiments, a suitable formulation solution is an aqueous solution comprising 5% to 20% by weight per volume of trehalose. In some embodiments, a suitable formulation solution is an aqueous solution comprising 5% to 20% by weight per volume of sucrose. In some embodiments, a suitable formulation solution is an aqueous solution comprising 5% to 20% by weight per volume of mannose. In some embodiments, a suitable formulation solution is an aqueous solution comprising 5% to 20% by weight per volume of lactose. In some embodiments, a suitable formulation solution is an aqueous solution comprising 5% to 20% by weight per volume of mannitol. In some embodiments, a suitable formulation solution is an aqueous solution comprising approximately 10% by weight per volume of a sugar, such as trehalose, sucrose, mannose, lactose, and mannitol. In some embodiments, a suitable formulation solution is an aqueous solution comprising approximately 10% by weight per volume of trehalose. In some embodiments, a suitable formulation solution is an aqueous solution comprising approximately 10% by weight per volume of sucrose. In some embodiments, a suitable formulation solution is an aqueous solution comprising approximately 10% by weight per volume of mannose. In some embodiments, a suitable formulation solution is an aqueous solution comprising approximately 10% by weight per volume of lactose. In some embodiments, a suitable formulation solution is an aqueous solution comprising approximately 10% by weight per volume of mannitol. In some embodiments, one or both of a non-aqueous solvent, such as ethanol, and citrate are absent from the pharmaceutical formulation solution. In some embodiments, a suitable formulation solution includes only residual citrate. In some embodiments, a suitable formulation solution includes only residual non-aqueous solvent, such as ethanol. In some embodiments, a suitable formulation solution contains less than approximately 10 mM (e.g., less than approximately 9 mM, approximately 8 mM, approximately 7 mM, approximately 6 mM, approximately 5 mM, approximately 4 mM, approximately 3 mM, approximately 2 mM, or approximately 1 mM) of citrate. In some embodiments, a suitable formulation solution contains less than about 25% (e.g., less than about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%) of non-aqueous solvents, such as ethanol.In some modalities, a suitable formulation solution does not require any further processing (e.g., buffer exchange and / or additional purification steps and / or additional excipients) prior to lyophilization. In some modalities, a suitable formulation solution does not require any further processing (e.g., buffer exchange and / or additional purification steps and / or additional excipients) prior to administration to a sterile refill in a vial, syringe, or other container. In some modalities, a suitable formulation solution does not require any further processing (e.g., buffer exchange and / or additional purification steps and / or additional excipients) prior to administration to a subject. In some forms, a suitable formulation solution has a pH between 4.5 and 7.5. In some forms, a suitable formulation solution has a pH between 5.0 and 7.0. In some forms, a suitable formulation solution has a pH between 5.5 and 7.0. In some forms, a suitable formulation solution has a pH higher than 4.5. In some forms, a suitable formulation solution has a pH higher than 5.0. In some forms, a suitable formulation solution has a pH higher than 5.5. In some forms, a suitable formulation solution has a pH higher than 6.0. In some forms, a suitable formulation solution has a pH higher than 6.5. In some embodiments, the enhanced or increased amount of LNP-mRNA encapsulation in a suitable formulation solution after heating is retained after subsequent freeze-thaw cycles of the pharmaceutical formulation solution. In some embodiments, a suitable formulation solution is 10% trehalose and can be stably frozen. In some formulations, LNP-mRNAs in a suitable formulation solution can be stably frozen (e.g., retaining enhanced encapsulation) in a trehalose solution at approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some formulations, a suitable formulation solution requires no further purification or processing and can be stably stored in frozen form. Therapeutic uses In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising LNP-mRNAs described herein that encode a peptide or polypeptide for use in delivery to or treatment of a human subject. In some embodiments, a therapeutic composition comprising LNP-mRNAs described herein is used for delivery to the lung of a subject or to a lung cell. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising LNP-mRNAs described herein that deliver an endogenous protein that may be deficient or nonfunctional in a subject. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising LNP-mRNAs described herein that deliver a peptide or polypeptide for use in the treatment of a pulmonary disease. In certain embodiments, the present invention is useful in a method for manufacturing mRNA encoding the cystic fibrosis transmembrane conductance regulator, CFTR. The CFTR mRNA is delivered to the lung of a subject in need in a therapeutic composition to treat cystic fibrosis. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising LNP-mRNAs described herein that deliver a peptide or polypeptide for use in the treatment of a hepatic or metabolic disease.Such peptides and polypeptides may include those associated with a urea cycle disorder, associated with a lysosomal storage disorder, with a glycogen storage disorder, associated with an amino acid metabolism disorder, associated with a lipid metabolism disorder or fibrotic disorder, associated with methylmalonic acidemia, or associated with any other metabolic disorder for which delivery to the liver or a liver cell, or treatment of these, with full-length enriched mRNA provides a therapeutic benefit. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a protein associated with a urea cycle disorder. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver an ornithine transcarbamylase (OTC) protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver an arginosuccinate synthetase 1 protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a carbamoyl phosphate synthetase I protein.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that supply an arginosuccinate lyase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that supply an arginase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a protein associated with a lysosomal storage disorder. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver an alpha-galactosidase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a glucocerebrosidase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver an iduronate-2-sulfatase protein.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that supply the iduronidase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that supply the N-acetyl-alpha-D-glucosaminidase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that supply the heparan N-sulfatase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that supply the galactosamine-6-sulfatase protein.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that supply the beta-galactosidase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that supply the lysosomal lipase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that supply an arylsulfatase B protein (N-acetylgalactosamine-4-sulfatase). In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that supply the EB transcription factor (TFEB). In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a protein associated with a glycogen storage disorder. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver the acid alpha-glucosidase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver the glucose-6-phosphatase (G6PC) protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver the hepatic glycogen phosphorylase protein.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that supply the muscle phosphoglycerate mutase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that supply the glycogen debranching enzyme. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that supply a protein associated with amino acid metabolism. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that supply the enzyme phenylalanine hydroxylase. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that supply the enzyme glutaryl-CoA dehydrogenase. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that supply the enzyme propionyl-CoA carboxylase.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that supply the enzyme alanine-glyoxylate aminotransferase oxalate. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a protein associated with a lipid metabolism disorder or a fibrotic disorder. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver an mTOR inhibitor. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver the ATPase 8B1 phospholipid transporter protein (ATP8B1).In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that deliver one or more NF-kappa B inhibitors, such as one or more I-kappa B alpha, interferon-related developmental regulator 1 (IFRD1), and Sirtuin 1 (SIRT1). In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that deliver the PPAR-gamma protein or an active variant thereof. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a protein associated with methylmalonic acidemia. For example, in certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver the methylmalonyl CoA mutase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver the methylmalonyl CoA epimerase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a peptide or polypeptide for use in delivery to, or treatment of, the liver. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver the ATP7B protein, also known as Wilson's disease protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver the porphobilinogen deaminase enzyme.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that supply one of the coagulation enzymes, such as factor VIII, factor IX, factor VII, and factor X. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that supply the human hemochromatosis protein (HFE). In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a peptide or polypeptide for use in delivering to, or treating, the cardiovascular system of a subject or cardiovascular cell. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver vascular endothelial growth factor A protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver relaxin protein. frFRnnn / zznz / E / YiAi In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver bone morphogenetic protein 9. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver the receptor protein for bone morphogenetic protein 2. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a peptide or polypeptide for use in delivering or treating muscle in a subject or muscle cell. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver the dystrophin protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver the frataxin protein.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a peptide or polypeptide for use in delivering or treating cardiac muscle in a subject or cardiac muscle cell. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a protein modulating one or both of a potassium channel and a sodium channel in muscle tissue or a muscle cell. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a protein modulating a Kv7.1 channel in muscle tissue or a muscle cell.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a protein modulating a Navl.5 channel in muscle tissue or in a muscle cell. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a peptide or polypeptide for use in delivering to, or treating, the nervous system of a subject or a nervous system cell. For example, in certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver motor neuron survival protein 1. For example, in certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver motor neuron survival protein 2.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that deliver the frataxin protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that deliver the ATP-binding cassette subfamily D member 1 (ABCD1) protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that deliver the CLN3 protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a peptide or polypeptide for use in delivering to, or treating, the blood or bone marrow of a subject or a blood or bone marrow cell. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver beta-globin protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver Bruton's tyrosine kinase protein.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that supply one of the coagulation enzymes, such as factor VIII, factor IX, factor VII, and factor X. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a peptide or polypeptide for use in the delivery to, or treatment of, a kidney in a subject or a kidney cell. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver the type IV collagen alpha chain 5 protein (COL4A5). In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a peptide or polypeptide for use in delivering to, or treating, the eye of a subject or an eye cell. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver the protein of ATP-binding cassette subfamily A member 4 (ABCA4). In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver the protein retinoschisin.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that deliver the 65 kDa retinal pigment epithelium-specific protein (RPE65). In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that deliver the 290 kDa centrosomal protein (CEP290). In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the mRNA-LNPs described herein that deliver a peptide or polypeptide for use in delivering a vaccine, or in a treatment with a vaccine, to a subject or a cell of a subject. For example, in certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the mRNA-LNPs described herein that deliver an antigen of an infectious agent, such as a virus. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the mRNA-LNPs described herein that deliver an antigen of the influenza virus.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a respiratory syncytial virus antigen. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a rabies virus antigen. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a cytomegalovirus antigen. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a rotavirus antigen.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver an antigen from a hepatitis virus, such as hepatitis A virus, hepatitis B virus, or hepatitis C virus. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver an antigen from a human papillomavirus. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver an antigen from a herpes simplex virus, such as herpes simplex virus 1 or herpes simplex virus 2.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver an antigen of a human immunodeficiency virus, such as human immunodeficiency virus type 1 or human immunodeficiency virus type 2. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver an antigen of a human metapneumovirus. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver an antigen of a human parainfluenza virus, such as human parainfluenza virus type 1, human parainfluenza virus type 2, or human parainfluenza virus type 3.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a malaria virus antigen. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a Zika virus antigen. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a chikungunya virus antigen. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver an antigen associated with a cancer of a subject or identified from a cancer cell of a subject. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a specific antigen from the subject's own cancer cell, i.e., to provide a personalized cancer vaccine. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver an antigen expressed from a mutant KRAS gene. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the mRNA-LNPs described herein that deliver an antibody. In certain embodiments, the antibody may be a bispecific antibody. In certain embodiments, the antibody may be part of a fusion protein. In some embodiments, two mRNA-LNPs separated in step (b) of the process comprise mRNA encoding a light chain and a heavy chain of an antibody. In some embodiments, the mRNA-LNP composition of the invention may comprise a combination of non-identical LNPs comprising different lipid compositions and encapsulating mRNA encoding either a light chain or a heavy chain of an antibody.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that deliver an antibody against OX40. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that deliver an antibody against VEGF. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that deliver an antibody against tissue necrosis factor alpha. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that deliver an antibody against CD3.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver an antibody against CD19. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver an immunomodulator. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver interleukin-12. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver interleukin-23. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver interleukin-36-gamma.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that supply a constitutively active variant of one or more interferon gene-stimulating proteins (STING). In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that supply an endonuclease. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that supply an RNA-guided DNA endonuclease protein, such as a Cas9 protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that supply a meganuclease protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP mRNAs described herein that supply an effector nuclease protein similar to a transcription activator.In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that supply a zinc-finger nuclease protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition comprising the LNP-mRNAs described herein that deliver a peptide or protein for treating an eye disease. In some embodiments, the method is used to produce a therapeutic composition comprising the LNP-mRNAs described herein that deliver retinoschisin. EXAMPLES While certain compounds, compositions, and methods of the present invention have been described specifically according to certain embodiments, the following examples serve only to illustrate the invention and are not intended to limit it. Example 1. Encapsulation of mRNA within lipid nanoparticles comprising a low or zero content of PEG-modified lipids by using poloxamer This example illustrates an illustrative process for encapsulating mRNA within lipid nanoparticles with a low or zero content of PEG-modified lipids by applying Process A. As used herein, Process A refers to a conventional method of encapsulating mRNA by mixing the mRNA with a lipid mixture, e.g., without first forming the lipids into lipid nanoparticles, as described in U.S. patent application serial number US2018 / 0008680, which is incorporated herein in full by reference. An illustrative formulation process is shown in Figure 1. In this process, a lipid solution (e.g., in ethanol) and an aqueous solution comprising mRNA and poloxamer were prepared separately. Specifically, a lipid solution (containing cationic lipids, auxiliary lipids, zwitterionic lipids, PEG-modified lipids, etc.) was prepared by dissolving the lipids in ethanol. The aqueous solution was prepared by dissolving the mRNA and poloxamer in citrate buffer. These two solutions were then mixed using a pump system to provide the LNPs with encapsulated mRNA. It is worth noting that it is advisable to keep the amount of poloxamer in the mixture below its critical micelle concentration (CMC) to avoid precipitation.The LNP-forming solution comprising mRNA-LNP was then dialyzed against a 10% trehalose solution to remove extra mRNA and the poloxamer at room temperature for a few hours and then at 4 °C overnight. After dialysis, the mRNA-loaded formulation solution was concentrated and stored for further analysis. Five different LNP formulations were prepared using the encapsulation process described above and analyzed as shown in Table 1 below. frPRnnn / zznz / E / YiAi Table 1 LNP-mRNA with varying amounts of PEG- and poloxamer-modified lipids Lipid modified formulation with PEG poloxamer included during the encapsulation process Size (nm) PDI % Encapsulation efficiency 1 0.0% 0.5% 124 0.177 54 2 0.2% 0.5% 123 0.186 58 3 0.4% 0.5% 109 0.165 59 4 0.0% 0.0% n / an / an / a 5 0.4% 0.0% 337 0.069 92 Stable LNPs without PEG-modified lipids or poloxamer could not form because the formulation solution collapsed and precipitated (Table 1, Formulation 4). In the absence of poloxamer, the LNP with a low content of PEG-modified lipids (e.g., 0.4%) had a large particle size of 337 nm (Table 1, Formulation 5). When 0.5% poloxamer was used during the encapsulation process as described above, surprisingly, the LNP size decreased significantly, by a factor of 3 (Table 1, Formulations 1–3). Furthermore, stable LNPs could form even in the absence of PEG-modified lipids (Table 1, Formulation 1). This example demonstrates that the inclusion of poloxamer during an encapsulation process resulted in stable LNP-mRNAs containing low or no PEG-modified lipids. Importantly, poloxamer protection significantly reduced LNP sizes, resulting in LNP-mRNAs with low or no PEG-modified lipids smaller than 200 nm, particularly suitable for therapeutic use. Example 2. After LNP formation, heating increased the encapsulation efficiency of the LNPs This example illustrates that an additional heating step after the formation of mRNA-LNPs increases encapsulation efficiency. Specifically, after mRNA encapsulation in LNPs using Process A as described above, the resulting mRNA-LNP formulation solution was heated above room temperature. After heating, the mRNA-LNP solution was cooled and stored for later analysis. For each formulation, size, PDI, and encapsulation efficiency were measured before and after heating. frPRnnn / zznz / E / YiAi Table 2. Effect of the heating step after LNP-mRNA formation Lipid-modified formulation with PEG. Poloxamer included during the encapsulation process. No heating after LNP-mRNA formation. Heating after LNP-mRNA formation. Size (nm) PDI EE% Size (nm) PDI EE% 1 0.0% 0.5% 121 0.202 66 124 0.177 54 2 0.2% 0.5% 122 0.195 32 123 0.186 58 3 0.4% 0.5% 110 0.180 47 109 0.165 59 As shown in Table 2, the encapsulation efficiency (EE%) of formulations 2 and 3, comprising 0.2% and 0.4% PEG-modified lipids, respectively, increased significantly after a post-forming heating step compared to the encapsulation efficiency of the same formulation before heating. The PDI of all tested formulations decreased slightly, and the particle size remained relatively constant. Example 3. LNP-mRNAs are stable after multiple freeze / thaw cycles This example illustrates that mRNA-loaded LNPs prepared according to the present invention are stable after multiple freeze / thaw cycles. Specifically, three different LNP formulations were prepared with different PEG-modified lipids and poloxamer using the encapsulation process described above. For each formulation, the size and encapsulation efficiency were measured before and after freeze-thaw cycles. frPRnnn / zznz / E / YiAi Table 3. Stable LNP-mRNAs after freeze / thaw cycles Lipid modified formulation with PEG poloxamer included during the encapsulation process 6 0.4% 0.0% 7 0.4% 2% 8 0.0% 2% As shown in Figure 2, LNP formulations 7 and 8, containing 0.4% and 0% PEG, respectively, and formed in the presence of 2% poloxamer, maintained their average particle sizes of approximately 100 nm after two freeze-thaw cycles. More specifically, the increases in particle sizes after one and two freeze-thaw cycles appeared to be within 10% of their respective original average sizes. Formulation 6, containing 0.4% PEG, was formed without poloxamer. It had an average particle size of approximately 370 nm before freezing / thawing, and the average size increased to over 400 nm after two freeze-thaw cycles. More surprisingly, the encapsulation efficiency increased significantly after the first freeze / thaw cycle for formulations 7 and 8, which included 2% poloxamer during the mRNA-LNP encapsulation process. Example 4. Formation of LNP-mRNA with a low or zero content of PEG-modified lipids in the presence of poloxamer This example further illustrates that PEG-modified LNP-mRNAs with low or zero lipid content prepared in the presence of poloxamer have average sizes and size distributions suitable for therapeutic uses. Different LNP formulations with varying amounts of PEG-modified lipids and poloxamers, as shown in Table 4, were prepared using the encapsulation process described above and analyzed. Specifically, the same cationic lipids, auxiliary lipids, cholesterol, and mRNA were used to prepare the LNP formulations in this example. Table 4. Examples of LNP-mRNA with varying amounts of PEG-modified lipids v poloxamer frPRnnn / zznz / E / YiAi Lipid modified formulation with PEG poloxamer included during the encapsulation process 9 0.0% 0.5% 10 0.0% 1.0% 11 0.0% 2.0% 12 0.4% 0.5% 13 0.4% 1.0% 14 0.4% 20% mRNA-laden LNPs were formed in the absence of PEG-modified lipids (formulations 9–11). This was achieved by adding poloxamer during the encapsulation process as described above. Specifically, PEG-lipid-free mRNA-LNPs were prepared with a low percentage (e.g., 0.5%) of poloxamer (formulation 9). As shown in Figure 3, the average sizes of all LNPs containing 0.4% PEG-modified lipids prepared with varying amounts of poloxamer were below 100 nm. The average sizes of all PEG-lipid-free LNPs prepared with varying amounts of poloxamer were below 130 nm. The PDIs for all LNPs, with or without PEG-modified lipids, prepared with varying amounts of poloxamer were at or below 0.25. The effect of the percentage of PEG-modified lipids was also analyzed, as shown in Figure 4. Figure 4 shows that the LNP mRNAs were prepared with very low PEG-modified lipid content or without PEG in the presence of poloxamer, with average sizes below 100 nm and PDI below 0.25. Since the same lipid components (e.g., cationic lipids, auxiliary lipids, and cholesterol) and the same mRNA were used to prepare the LNP mRNAs, the changes observed in this example are due to changes in the percentage of PEG-modified lipids and / or poloxamer. Example 5. Poloxamer stabilizes LNPs with systems of fewer components This example illustrates that LNP-mRNAs with fewer than four components can be prepared according to the present invention and have average sizes suitable for therapeutic uses. Specifically, LNP-mRNAs with different components (e.g., four, three, and two) were prepared in the presence of poloxamer as previously described and characterized. Table 5 shows the specific components, average particle sizes, PDI, and encapsulation efficiencies for the different formulations. Notably, the same lipid components (e.g., cationic lipid, auxiliary lipid, and / or cholesterol) and the same mRNA were used to prepare the LNP formulations in this example. frFRnnn / zznz / E / YiAi Table 5. LNP with different components Formulation Number of components Components Size (nm) PDI % Encapsulation efficiency 16 Four Cationic lipid, auxiliary lipid, cholesterol, PEG-modified lipid (without poloxamer) 87 0.184 87 1 Three Cationic lipid, auxiliary lipid, cholesterol (+ poloxamer) 124 0.177 54 17 Two Cationic lipid, auxiliary lipid (50:50) (+ poloxamer) 113 0.185 75 18 Two Cationic lipid, auxiliary lipid (25:75) (+ poloxamer) 112 0.188 95 As shown in Table 5, the LNP mRNAs were prepared with three or two components when poloxamer was included during the encapsulation process. All had small sizes (e.g., below 125 nm) with acceptable PDI (e.g., below 0.20) and encapsulation efficiency. Surprisingly, the two-component LNPs (formulations 17 and 18), with different ratios of cationic to auxiliary lipids, had small average sizes (less than 120 nm) and high encapsulation efficiency (e.g., >75%). Example 6. Substantially stable LNPs can be formed from PEG-modified lipids with different cationic lipids and various poloxamers This example illustrates that the present invention can be used to produce stable LNP mRNAs containing various cationic lipids and different mRNAs and substantially free of PEG-modified lipids. Specifically, as shown in Table 6, mRNA encoding an EPO or FFL protein was encapsulated in LNPs containing different cationic lipids in an N / P ratio of 4 in the presence of poloxamer 407. frFRnnn / zznz / E / YiAi Table 6 LNP-mRNAs comprising poloxamer 407 are formed with different cationic lipids v mRNA constructs Cationic lipid mRNA Size (nm) PDI EE% CCBene EPO 97 0.143 60 FFL 92 0.164 41 ML-7 EPO 114 0.166 58 FFL 108 0.155 94 MC-3 EPO 93 0.156 32 FFL 92 0.178 ML-2 EPO 140 0.094 86 FFL 280 0.091 The results show that substantially lipid-free, stable PEG-modified mRNA-LNPs can be formed with various cationic lipids and mRNA constructs. The LNPs comprising CCBene, ML-7, and MC3 showed particularly small sizes of less than 120 nm. Example 7. Successful in vivo expression by supplying LNP-mRNAs formed using poloxamer This example demonstrates that administration of poloxamer-formed mRNA-LNPs resulted in successful protein expression in vivo. Specifically, EPO mRNA-laden LNPs were administered subcutaneously (SC) and intravenously (IV) to CD-I mice, and the level of EPO protein expression was detected in the liver and serum of mice at 6 and 24 hours post-administration. Four different LNPs with varying amounts of PEG-modified lipids and formed with 0.5% poloxamer were tested, as shown in Table 7 below. Conventional LNPs with 5% PEG-modified lipids (Groups B and F shown in Table 7) were also administered as controls. frFRnnn / zznz / E / YiAi Table 7 Animal study of mRNA-LNPs comprising poloxamer ROA Group Number of Animals Formulation PEG-modified lipid Poloxamer included during the encapsulation process Dose level (mg / kg) A SC 4 Saline solution 0.0 B SC 4 16 5.0% 0.0% 0.8 C SC 4 3 0.4% 0.5% 0.8 D SC 4 2 0.2% 0.5% 0.8 E SC 4 1 0.0% 0.5% 0.8 F IV 4 16 5.0% 0.0% 0.4 G IV 4 3 0.4% 0.5% 0.4 H IV 4 2 0.2% 0.5% 0.4 I IV 4 1 0.0% 0.5% 0.4 As shown in Figure 5, poloxamer-protected LNPs with low or no PEG-modified lipid content achieved an in vivo protein expression profile similar to conventional LNPs (e.g., those with 5% PEG-modified lipids). These data demonstrate that mRNA-LNPs prepared using poloxamer according to the invention, comprising low (e.g., <0.5%) or no PEG-modified lipid or PEG content, can be successfully used for in vivo protein expression for therapeutic purposes. Example 8. Quantification of poloxamer in mRNA-LNP formulations This example illustrates an illustrative method for quantifying the final poloxamer concentration in mRNA-LNPs prepared according to the present invention. Specifically, this method takes advantage of the fact that poloxamer competes with cobalt thiocyanate and forms a blue precipitate, as shown in Figure 6A. Once the precipitate forms, it is dissolved in acetone, and the color intensity, which is directly proportional to the poloxamer concentration, is measured at a wavelength of 624 nm. A standard curve was plotted using known poloxamer concentrations, as shown in Figure 6B. This standard curve can be used to determine the amount of poloxamer in a given sample. Example 9. Successful in vivo expression of LNP-mRNA with various poloxamers and non-cationic lipids This example illustrates that various poloxamers and non-cationic lipids can be used to produce stable LNP-mRNAs that are substantially free of PEG-modified lipids. This example further demonstrates that administration of poloxamer-formed LNP-mRNAs resulted in protein expression in vivo. Different LNP formulations were prepared with various poloxamers and non-cationic lipids, and varying amounts of PEG-modified lipids, as shown in Table 8, with the cationic lipid cDD-TE4-E12 using the encapsulation process described above, and were analyzed. In this particular experiment, the mRNA encoding OTC (ornithine transcarbamylase) was encapsulated. ΐτΓβηηη / ζζηζ / Β / γίΛΐ Table 8. Examples of LNP-mRNAs with multiple components Formulation Poloxamer (Pluronic) Ratio (PEG: cationic lipid: cholesterol: noncationic lipid) Noncationic lipid Size (nm) PDI EE% A P-234 (P-84) 0.5:40:27.5:32 DOPE 95 0.075 100 B P-407 (F127) 0.5:40:27.5:32 DOPE 87 0.134 87 C P-234 (P-84) 0:40:28:32 DOPE Collapse D P-407 (F127) 0:40:28:32 DOPE 92 0.152 89 E P-338 (P108) . 0:40:28:32 DOPE 97 0.146 Low F P-234 (P-84) 0.5:40:27.5:32 DEPE 125 0.080 100 G P-407 (F127) 0.5:40:27.5:32 DEPE 133 0.140 99 H P-338 (P108) 0.5:40:27.5:32 DEPE 126 0.090 99 I Sin 3:40:25:32 DEPE poloxamer J Without poloxamer 2:40:26:32 DEPE K Without poloxamer 1.5:40:26.5:32 DOPE frFRnnn / zznz / E / YiAi mRNA-laden LNPs were formed in the absence of, or with very little (e.g., 0.5%) PEG-modified lipids (AH formulations). This was achieved by adding poloxamer during the encapsulation process as described above. Various poloxamers and non-cationic lipids can be used to optimize the encapsulation process. The average sizes of all LNPs prepared in this example were approximately 130 nm or smaller, with a PDI of approximately 0.15 or less and an encapsulation efficiency of approximately 90% or more. As controls, mRNA-laden LNPs without poloxamers were prepared with varying proportions of PEG-modified lipids (IK formulations). The OTC mRNA-LNP formulations listed in Table 8 were administered intravenously (IV) to mice, and the OTC protein expression level was measured. As shown in Figure 7, the PEG-modified, low-lipid poloxamer-protected LNPs achieved in vivo protein expression at or above the target expression level. These data demonstrate that PEG-modified or non-PEG-modified, low-lipid LNP mRNAs prepared using various poloxamers and non-cationic lipids can be successfully used for in vivo protein expression for therapeutic purposes. Formulations I and J achieved higher potency than the poloxamer-protected AH formulations. EQUIVALENTS Skilled individuals will recognize, or will be able to determine through ordinary experimentation alone, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the foregoing description, but is set forth in the following claims:

Claims

1. A stable composition comprising lipid nanoparticles encapsulating messenger RNA (mRNA), wherein the mRNA encodes a protein or peptide, wherein each of the lipid nanoparticles comprises one or more cationic lipids, one or more non-cationic lipids and less than 0.5% PEG-modified lipids or PEG, and wherein the mRNA-encapsulating lipid nanoparticles are stable after one or more freeze-thaw cycles.

2. The stable composition according to claim 1, wherein each of the lipid nanoparticles comprises a cationic lipid, dioleoylphosphatidylethanolamine (DOPE), and less than about 0.5% of PEG or PEG-modified lipids.

3. The stable composition according to claim 1 or 2, wherein the lipid nanoparticles encapsulating the mRNA maintain an average diameter within 50% of the original average size after one or more freeze-thaw cycles.

4. The stable composition of any of the preceding claims, wherein the lipid nanoparticles encapsulating the mRNA maintain an average diameter within 10% of the original average size after one or more freeze-thaw cycles.

5. The stable composition of any of the preceding claims, wherein the lipid nanoparticles encapsulating the mRNA maintain an average diameter within 5% of the original average size after one or more freeze-thaw cycles.

6. The stable composition of any of the preceding claims, wherein the lipid nanoparticles have an mRNA encapsulation efficiency of between about 50% and 99%.

7. The stable composition of any of the preceding claims, wherein each of the lipid nanoparticles further comprises a cholesterol-based lipid.

8. The stable composition of any of the preceding claims, wherein each of the lipid nanoparticles comprises 0.4% PEG-modified lipids or less, 0.3% PEG-modified lipids or less, 0.2% PEG-modified lipids or less, or 0.1% PEG-modified lipids or less.

9. The stable composition of any of the preceding claims, wherein each of the lipid nanoparticles is substantially free of PEG-modified lipids. frPRnnn / zznz / E / YiAi 10. The stable composition of any of the preceding claims, wherein each of the lipid nanoparticles comprises an amphiphilic block copolymer.

11. The stable composition according to claim 10, wherein each of the lipid nanoparticles comprises less than 3% amphiphilic block copolymer, less than 2.5% amphiphilic block copolymer, less than 2% amphiphilic block copolymer, less than 1.5% amphiphilic block copolymer, less than 1% amphiphilic block copolymer, less than 0.5% amphiphilic block copolymer, less than 0.05% amphiphilic block copolymer, or less than 0.01% amphiphilic block copolymer.

12. The stable composition according to claim 11, wherein the composition comprises less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02% or less than 0.01% of amphiphilic block copolymer of the total composition by weight.

13. The stable composition according to claim 12, wherein the composition comprises an amphiphilic block copolymer residue.

14. The stable composition of any of claims 10-13, wherein the amphiphilic block copolymer is a poloxamer.

15. The stable composition according to claim 14, wherein the poloxamer is selected from poloxamer 84, poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, poloxamer 231, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer 282, poloxamer 284, poloxamer 288, poloxamer 304, poloxamer 331, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer 401, poloxamer 402, poloxamer 403, poloxamer 407 or combinations thereof.

16. A stable composition comprising lipid nanoparticles encapsulating a messenger RNA (mRNA) encoding a protein or peptide, wherein each of the lipid nanoparticles comprises one or more cationic lipids, one or more non-cationic lipids, a poloxamer and is substantially free of PEG or PEG-modified lipids, and wherein the mRNA-encapsulating lipid nanoparticles are stable after one or more freeze-thaw cycles.

17. A stable composition comprising lipid nanoparticles encapsulating a messenger RNA (mRNA) encoding a protein or peptide, wherein each lipid nanoparticle comprises one or more cationic lipids, one or more non-cationic lipids, a poloxamer, and is substantially free of PEG or PEG-modified lipids, and wherein the mRNA-encapsulating lipid nanoparticles generate few or no anti-PEG antibodies and / or reduce accelerated blood clearance (ABC). frPRnnn / zznz / E / YiAi 18. The stable composition according to claim 17, wherein the poloxamer is present in the lipid nanoparticles in an amount less than 0.1%.

19. The stable composition of any of claims 16-18, wherein the poloxamer is present in the lipid nanoparticles in an amount less than 0.05%.

20. The stable composition of any of claims 16-19, wherein the non-cationic lipid is dioleoylphosphatidylethanolamine (DOPE).

21. The stable composition of any of claims 16-20, wherein the lipid nanoparticle maintains an average diameter within 50% of the original average size after one or more freeze-thaw cycles.

22. The stable composition according to claim 21, wherein the lipid nanoparticles maintain an average diameter within 10% of the original average size after one or more freeze-thaw cycles.

23. The stable composition according to claim 22, wherein the lipid nanoparticles maintain an average diameter within 5% of the original average size after one or more freeze-thaw cycles.

24. The stable composition of any of claims 16-23, wherein the lipid nanoparticles have an mRNA encapsulation efficiency of between about 50% and 99%.

25. The stable composition of any of claims 16-24, wherein each of the lipid nanoparticles further comprises cholesterol-based lipids.

26. The stable composition of any of claims 16-25, wherein each of the lipid nanoparticles does not comprise cholesterol-based lipids.

21. The stable composition of any of claims 16-26, wherein each of the lipid nanoparticles is a two-component lipid nanoparticle.

28. The stable composition of any of claims 16-27, wherein the poloxamer has ethylene oxide units from about 10 to about 150.

29. The stable composition according to claim 28, wherein the poloxamer has propylene oxide units from about 10 to about 100.

30. The stable composition of any of claims 16-29, wherein the poloxamer has an average molecular weight of about 4,000 g / mol to about 20,000 g / mol.

31. The stable composition of any of claims 16-30, wherein the poloxamer is selected from poloxamer 84, poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, poloxamer 231, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer 282, poloxamer 284, poloxamer 288, poloxamer 304, poloxamer 331, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer 401, poloxamer 402, poloxamer 403, poloxamer 407 or combinations thereof.

32. The stable composition of any of the preceding claims, wherein the lipid nanoparticles have an average size of less than about 200 nm.

33. The stable composition according to claim 32, wherein the average size is about 150 nm, 140 nm, 130 nm, 120 nm, 110 nm, 100 nm or less.

34. The stable composition of any of the preceding claims, wherein the lipid nanoparticles have a polydispersity index (PDI) of 0.25 or less, 0.2 or less, 0.15 or less, 0.1 or less.

35. A method for supplying messenger RNA (mRNA) for in vivo production of a protein or peptide, comprising administering to a subject a stable composition according to any of the preceding claims.

36. A method for supplying messenger RNA (mRNA) for in vivo production of a protein or peptide, comprising administering to a subject a stable composition according to any of the preceding claims, wherein administration of the stable composition does not result in anti-PEG antibodies and / or accelerated blood clearance (ABC) in the subject.

37. A method for treating a subject having a deficiency in a protein or peptide, comprising administering to a subject in need of treatment a stable composition according to any of claims 1-33. frPRnnn / zznz / E / YiAi 38. The method of any of the claims, the stable composition is administered by intravenous injection.

39. The method of any of the claims, the stable composition is administered by pulmonary delivery.

40. The method of any of the claims, the stable composition is administered by intramuscular delivery.

41. The method of any of claims 35-37, 35-37, 35-37, 35-40, wherein the administration of the stable composition results in the expression of the protein or peptide encoded by the mRNA for at least about 12, 24, 36, 48, 60 or 72 hours after administration.

42. A process for encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising a step of mixing an mRNA solution and a lipid solution in the presence of a poloxamer.

43. The process according to claim 42, wherein the lipid solution comprises one or more cationic lipids, one or more non-cationic lipids and less than 0.5% of PEG or PEG-modified lipids.

44. The process according to claim 42 or 43, wherein the lipid solution comprises preformed lipid nanoparticles.

45. The process of any of claims 42-44, wherein the mRNA solution and / or the lipid solution are at a predetermined temperature above ambient temperature.

46. ​​The process of any of claims 42-45, wherein the poloxamer is first added to the mRNA solution.

47. The process of any of claims 42-46, wherein the poloxamer is present in an amount less than its critical micelle concentration (CMC).

48. The process according to claim 47, wherein the poloxamer is present in an amount of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50% less than its CMC.

49. The process according to claim 47, wherein the poloxamer is present in an amount less than about 50% of its CMC.

50. The process of any of claims 42-49, wherein the process further comprises a poloxamer removal step.

51. The process according to claim 50, wherein the poloxamer is removed by dialysis.

52. The process according to claim 50 or 51, wherein less than about 0.05% of poloxamer remains after removal.

53. The process according to claim 52, wherein less than about 0.01% of poloxamer remains after removal.

54. The process according to claim 52, wherein a residual amount of poloxamer remains after removal.

55. The process of any of claims 50-54, wherein the amount of poloxamer remaining after elimination is undetectable. frFRnnn / zznz / E / YiAi 56. The process of any of claims 42-55, wherein the poloxamer has ethylene oxide units from about 10 to about 150.

57. The process according to claim 56, wherein the poloxamer has propylene oxide units from about 10 to about 100.

58. The process of any of claims 42-57, wherein the poloxamer has an average molecular weight of about 4,000 g / mol to about 20,000 g / mol.

59. The process of any of claims 42-58, wherein the poloxamer is selected from poloxamer 84, poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, poloxamer 231, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer 282, poloxamer 284, poloxamer 288, poloxamer 304, poloxamer 331, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer 401, poloxamer 402, poloxamer 403, poloxamer 407 or combinations thereof.

60. The process of any of claims 42-59, wherein the non-cationic lipid is dioleoylphosphatidylethanolamine (DOPE).

61. The process of any of claims 42-59, wherein the process does not include mixing coiesteroi lipids.

62. The process of any of claims 42-61, wherein the lipid nanoparticles have an encapsulation efficiency of at least 50%.

63. The process according to claim 42-61, wherein the lipid nanoparticles have an encapsulation efficiency of between about 60% and 99%.

64. The process of any of claims 42-63, wherein the lipid nanoparticles have an average size of about 200 nm or less.

65. The process according to claim 64, wherein the lipid nanoparticles have an average size of about 150 nm or less, 140 nm or less, 130 nm or less, 120 nm or less, 110 nm or less, or about 100 nm or less.

66. A composition comprising lipid nanoparticles encapsulating mRNA formed according to a process of any of claims 42-63.