Stable compositions of mrna-loaded lipid nanoparticles and processes of making

Abstract

Improved compositions and processes for preparing mRNA-loaded lipid nanoparticles (mRNA-LNPs) are provided.SOLUTION: (i) mixing a mRNA solution with a lipid solution in the presence of 0.05% to less than 3% of a poloxamer prior to removal, wherein the lipid solution comprises one or more cationic lipids, one or more non-cationic lipids, and less than 0.5% of one or more PEG-modified lipids or PEG, and (ii) removing the poloxamer, wherein upon removal less than 0.05% of the poloxamer remains.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 877,597, filed July 23, 2019, the disclosure of which is incorporated herein by reference. [Background technology]

[0002] Messenger RNA therapy (MRT) has become an increasingly important approach to the treatment of various diseases. MRT involves administering messenger RNA (mRNA) to patients in need of therapy to produce the protein encoded by the mRNA in their bodies. Lipid nanoparticles are commonly used to encapsulate mRNA for efficient in vivo delivery of mRNA.

[0003] Much effort has been devoted to developing improved methods and compositions that can enhance in vivo delivery and / or expression of mRNA using lipid nanoparticles, which can be adapted to scalable and cost-effective manufacturing processes. At the same time, it is important that any such enhancement of in vivo delivery and / or expression of mRNA also maintains or improves the safety and tolerability of compositions associated with lipid-mediated mRNA delivery. Summary of the Invention

[0004] The present invention provides, inter alia, further 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 storage stability and in vivo circulation time. On the other hand, PEG-modified lipids can induce accelerated blood clearance (ABC) and / or innate immune responses, particularly by generating anti-PEG antibodies. To address this issue, attempts have been made to prepare mRNA-LNPs free of PEG-modified lipids. However, it has been observed that mRNA-loaded LNPs formed in the absence of PEG-modified lipids or PEG tend to have large and unstable sizes or precipitate, especially after freezing and thawing, making them unsuitable for therapeutic use. The present invention is based, in part, on the surprising discovery that unexpectedly stable mRNA-loaded LNPs containing little or no PEG-modified lipids can be produced by mixing mRNA with lipids in the presence of amphiphilic block copolymers such as poloxamers. As described in more detail below, mRNA-LNPs produced according to the present invention have a size comparable to that of conventional LNPs containing typical amounts of PEG-modified lipids, and more importantly, are stable after one or more freeze-thaw cycles. Specifically, mRNA-LNPs according to the present invention maintain an average diameter within 50%, and in some cases within 10%, of their original average size after one or more freeze-thaw cycles. Furthermore, poloxamer-shielded LNPs containing low or no PEG-modified lipids (e.g., less than 0.5% PEG-modified lipids) achieved in vivo protein expression profiles similar to those of conventional LNPs (e.g., those containing 5% PEG-modified lipids). Thus, the present invention provides further improved mRNA-LNPs with exceptional stability and is particularly useful when LNPs containing low or no PEG-modified lipids are desired, for example, to avoid the generation of anti-PEG antibodies and / or ABC.

[0005] In one aspect, the present invention provides a stable composition comprising lipid nanoparticles encapsulating messenger RNA (mRNA) encoding a protein or peptide, wherein each of the lipid nanoparticles comprises one or more cationic lipids and less than 0.5% PEG-modified lipid or PEG. and are stable after one or more freeze-thaw cycles. In some embodiments, the lipid nanoparticles further comprise one or more non-cationic lipids.

[0006] In some embodiments, the lipid nanoparticles comprise a cationic lipid, dioleoylphosphatidylethanolamine (DOPE) as a non-cationic lipid, and less than about 0.5% PEG-modified lipid or PEG. In some embodiments, the lipid nanoparticles comprise a cationic lipid, 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE) as a non-cationic lipid, and less than about 0.5% PEG-modified lipid or PEG.

[0007] In some embodiments, lipid nanoparticles encapsulating mRNA maintain an average diameter within 50% of their original average size after one or more freeze-thaw cycles. In some embodiments, lipid nanoparticles encapsulating mRNA maintain an average diameter within 40% of their original average size after one or more freeze-thaw cycles. In some embodiments, lipid nanoparticles encapsulating mRNA maintain an average diameter within 30% of their original average size after one or more freeze-thaw cycles. In some embodiments, lipid nanoparticles encapsulating mRNA maintain an average diameter within 20% of their original average size after one or more freeze-thaw cycles. In some embodiments, lipid nanoparticles encapsulating mRNA maintain an average diameter within 10% of their original average size after one or more freeze-thaw cycles. In some embodiments, lipid nanoparticles encapsulating mRNA maintain an average diameter within 5% of their original average size after one or more freeze-thaw cycles.

[0008] In some embodiments, the lipid nanoparticles have an mRNA encapsulation efficiency of about 50% to 99%. In some embodiments, the lipid nanoparticles have an mRNA encapsulation efficiency of about 60% to 90%. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of about 60%. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of about 70%. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of about 80%. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of about 90%.

[0009] In some embodiments, each of the lipid nanoparticles further comprises a cholesterol-based lipid.

[0010] In some embodiments, each lipid nanoparticle contains 0.4% or less PEG-modified lipid. In some embodiments, each lipid nanoparticle contains 0.3% or less PEG-modified lipid. In some embodiments, each lipid nanoparticle contains 0.2% or less PEG-modified lipid. In some embodiments, each lipid nanoparticle contains 0.1% or less PEG-modified lipid.

[0011] In some embodiments, each of the lipid nanoparticles is substantially free of PEG-modified lipids.

[0012] In some embodiments, each of the lipid nanoparticles comprises an amphiphilic block copolymer.

[0013] In some embodiments, each of the lipid nanoparticles comprises less than 3% amphiphilic block copolymer. In some embodiments, each of the lipid nanoparticles comprises less than 3% amphiphilic block copolymer. In some embodiments, each of the lipid nanoparticles comprises less than 2.5% amphiphilic block copolymer. In some embodiments, each of the lipid nanoparticles comprises less than 2% amphiphilic block copolymer. In some embodiments, each of the lipid nanoparticles comprises less than 1.5% amphiphilic block copolymer. In some embodiments, In some embodiments, each of the lipid nanoparticles comprises less than 1% amphiphilic block copolymer. In some embodiments, each of the lipid nanoparticles comprises less than 0.5% amphiphilic block copolymer. In some embodiments, each of the lipid nanoparticles comprises less than 0.05% amphiphilic block copolymer. In some embodiments, each of the lipid nanoparticles comprises less than 0.01% amphiphilic block copolymer.

[0014] In some embodiments, the composition comprises less than 0.05% by weight of the total composition of amphiphilic block copolymer. In some embodiments, the composition comprises less than 0.04% by weight of the total composition of amphiphilic block copolymer. In some embodiments, the composition comprises less than 0.03% by weight of the total composition of amphiphilic block copolymer. In some embodiments, the composition comprises less than 0.02% by weight of the total composition of amphiphilic block copolymer. In some embodiments, the composition comprises less than 0.01% by weight of the total composition of amphiphilic block copolymer.

[0015] In some embodiments, the composition comprises a residue of an amphiphilic block copolymer.

[0016] In some embodiments, a suitable amphiphilic block copolymer is a poloxamer.

[0017] In some embodiments, a suitable poloxamer is Poloxamer 84. In some embodiments, a suitable poloxamer is Poloxamer 101. In some embodiments, a suitable poloxamer is Poloxamer 105. In some embodiments, a suitable poloxamer is Poloxamer 108. In some embodiments, a suitable poloxamer is Poloxamer 122. In some embodiments, a suitable poloxamer is Poloxamer 123. In some embodiments, a suitable poloxamer is Poloxamer 124. In some embodiments, a suitable poloxamer is Poloxamer 181. In some embodiments, a suitable poloxamer is Poloxamer 182. In some embodiments, a suitable poloxamer is Poloxamer 183. In some embodiments, a suitable poloxamer is Poloxamer 184. In some embodiments, a suitable poloxamer is Poloxamer 185. In some embodiments, a suitable poloxamer is Poloxamer 188. In some embodiments, a suitable poloxamer is Poloxamer 212. In some embodiments, a suitable poloxamer is Poloxamer 215. In some embodiments, a suitable poloxamer is Poloxamer 217. In some embodiments, a suitable poloxamer is Poloxamer 231. In some embodiments, a suitable poloxamer is Poloxamer 234. In some embodiments, a suitable poloxamer is Poloxamer 235. In some embodiments, a suitable poloxamer is Poloxamer 237. In some embodiments, a suitable poloxamer is Poloxamer 238. In some embodiments, a suitable poloxamer is Poloxamer 282. In some embodiments, a suitable poloxamer is Poloxamer 284. In some embodiments, a suitable poloxamer is Poloxamer 288. In some embodiments, a suitable poloxamer is Poloxamer 304. In some embodiments, a suitable poloxamer is Poloxamer 331. In some embodiments, a suitable poloxamer is Poloxamer 333.In some embodiments, a suitable poloxamer is Poloxamer 334. In some embodiments, a suitable poloxamer is Poloxamer 335. In some embodiments, a suitable poloxamer is Poloxamer 338. In some embodiments, a suitable poloxamer is Poloxamer 401. In some embodiments, a suitable poloxamer is Poloxamer 402. In some embodiments, a suitable poloxamer is Poloxamer 403. In some embodiments, a suitable poloxamer is Poloxamer 407. In some embodiments, a suitable poloxamer is Poloxamer 408. Loxamer is a combination of these.

[0018] In one aspect, the present invention provides a stable composition comprising lipid nanoparticles encapsulating messenger RNA (mRNA) encoding a protein or peptide, each of the lipid nanoparticles comprising one or more cationic lipids, one or more non-cationic lipids, a poloxamer, and being substantially free of PEG-modified lipids or PEG, wherein the lipid nanoparticles encapsulating the mRNA are stable after one or more freeze-thaw cycles.

[0019] In one aspect, the present invention provides a stable composition comprising lipid nanoparticles encapsulating messenger RNA (mRNA) encoding a protein or peptide, each of the lipid nanoparticles comprising one or more cationic lipids, one or more non-cationic lipids, a poloxamer, and being substantially free of PEG-modified lipids or PEG, wherein the lipid nanoparticles encapsulating the mRNA generate low or no anti-PEG antibodies and / or have reduced accelerated blood clearance (ABC).

[0020] In some embodiments, the poloxamer is present in the lipid nanoparticles in an amount of less than 0.1%. In some embodiments, the poloxamer is present in the lipid nanoparticles in an amount of less than 0.05%.

[0021] In some embodiments, a suitable non-cationic lipid is dioleoylphosphatidylethanolamine (DOPE). In some embodiments, a suitable non-cationic lipid is 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE).

[0022] In some embodiments, each of the lipid nanoparticles does not include a cholesterol-based lipid.

[0023] In some embodiments, each of the lipid nanoparticles is a two-component lipid nanoparticle.

[0024] In some embodiments, suitable poloxamers have from about 10 to about 150 ethylene oxide units.

[0025] In some embodiments, suitable poloxamers have from about 10 to about 100 propylene oxide units.

[0026] In some embodiments, suitable poloxamers have an average molecular weight of about 4,000 g / mol to about 20,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 1,000 g / mol to about 50,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 1,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 2,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 3,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 4,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 5,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 6,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 7,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 8,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 9,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 10,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 20,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 25 In some embodiments, suitable poloxamers have an average molecular weight of about 30,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 40,000 g / mol. In some embodiments, suitable poloxamers have an average molecular weight of about 50,000 g / mol.

[0027] In some embodiments, the lipid nanoparticles have an average size of less than about 250 nm. In some embodiments, the lipid nanoparticles have an average size of about 200 nm or less. In some embodiments, the lipid nanoparticles have an average size of about 180 nm or less. In some embodiments, the lipid nanoparticles have an average size of about 160 nm or less. In some embodiments, the lipid nanoparticles have an average size of about 150 nm or less. In some embodiments, the lipid nanoparticles have an average size of about 140 nm or less. In some embodiments, the lipid nanoparticles have an average size of about 130 nm or less. In some embodiments, the lipid nanoparticles have an average size of about 120 nm or less. In some embodiments, the lipid nanoparticles have an average size of about 110 nm or less. In some embodiments, the lipid nanoparticles have an average size of about 100 nm or less.

[0028] 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, the lipid nanoparticles have a polydispersity index (PDI) of 0.14 or less. In some embodiments, the 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 embodiments, the lipid nanoparticles have a polydispersity index (PDI) of 0.05 or less.

[0029] In one aspect, the present invention provides, inter alia, a method for delivering messenger RNA (mRNA) for the in vivo production of a protein or peptide, the method comprising administering to a subject a stable composition according to the present invention.

[0030] In one aspect, the present invention provides, inter alia, a method for delivering messenger RNA (mRNA) for in vivo production of a protein or peptide, the method comprising administering to a subject a stable composition according to the present invention, wherein administration of the stable composition does not result in anti-PEG antibodies and / or accelerated blood clearance (ABC) in the subject.

[0031] In one aspect, the present invention provides, inter alia, a method of treating a subject having a deficiency in a protein or peptide, the method comprising administering to a subject in need of treatment a stable composition according to the present invention.

[0032] In some embodiments, administration of the stable composition results in low levels of anti-PEG antibodies in the subject. Either it generates it or it doesn't generate it at all.

[0033] In some embodiments, administration of the stable composition reduces or avoids accelerated blood clearance (ABC) in a subject.

[0034] In some embodiments, the stable composition is administered by intravenous injection.

[0035] In some embodiments, the stable composition is administered by pulmonary delivery.

[0036] In some embodiments, the stable composition is administered by intramuscular delivery.

[0037] In some embodiments, administration of the stable composition results in expression of the protein or peptide encoded by the mRNA for at least about 6 hours after administration. In some embodiments, administration of the stable composition results in expression of the protein or peptide encoded by the mRNA for at least about 12 hours after administration. In some embodiments, administration of the stable composition results in expression of the protein or peptide encoded by the mRNA for at least about 18 hours after administration. In some embodiments, administration of the stable composition results in expression of the protein or peptide encoded by the mRNA for at least about 24 hours after administration. In some embodiments, administration of the stable composition results in expression of the protein or peptide encoded by the mRNA for at least about 30 hours after administration. In some embodiments, administration of the stable composition results in expression of the protein or peptide encoded by the mRNA for at least about 36 hours after administration. In some embodiments, administration of the stable composition results in expression of the protein or peptide encoded by the mRNA for at least about 48 hours after administration. In some embodiments, administration of the stable composition results in expression of the protein or peptide encoded by the mRNA for at least about 72 hours after administration. In some embodiments, administration of the stable composition results in expression of the protein or peptide encoded by the mRNA for at least about 5 days after administration. In some embodiments, administration of the stable composition results in expression of the protein or peptide encoded by the mRNA for at least about 1 week after administration. In some embodiments, administration of the stable composition results in expression of the protein or peptide encoded by the mRNA for at least about 2 weeks after administration. In some embodiments, administration of the stable composition results in expression of the protein or peptide encoded by the mRNA for at least about 3 weeks after administration. In some embodiments, administration of the stable composition results in expression of the protein or peptide encoded by the mRNA for at least about 4 weeks after administration.

[0038] In one aspect, the present invention provides, inter alia, a process for encapsulating messenger RNA (mRNA) in lipid nanoparticles, comprising mixing a solution of mRNA with a lipid solution in the presence of a poloxamer.

[0039] In some embodiments, the lipid solution comprises one or more cationic lipids, one or more non-cationic lipids, and less than 0.5% PEG-modified lipids or PEG.

[0040] In some embodiments, the lipid solution comprises pre-formed lipid nanoparticles.

[0041] In some embodiments, the mRNA solution and / or the lipid solution are at a predetermined temperature that is greater than ambient temperature.

[0042] In some embodiments, the poloxamer is first added to the mRNA solution.

[0043] In some embodiments, the poloxamer is present in an amount below its critical micelle concentration (CMC).

[0044] In some embodiments, the poloxamer is present in an amount about 1% below its CMC. In some embodiments, the poloxamer is present in an amount about 2% below its CMC. In some embodiments, the poloxamer is present in an amount about 3% below its CMC. In some embodiments, the poloxamer is present in an amount about 4% below its CMC. In some embodiments, the poloxamer is present in an amount about 5% below its CMC. In some embodiments, the poloxamer is present in an amount about 6% below its CMC. In some embodiments, the poloxamer is present in an amount about 7% below its CMC. In some embodiments, the poloxamer is present in an amount about 8% below its CMC. In some embodiments, the poloxamer is present in an amount about 9% below its CMC. In some embodiments, the poloxamer is present in an amount about 10% below its CMC. In some embodiments, the poloxamer is present in an amount about 15% below its CMC. In some embodiments, the poloxamer is present in an amount about 20% below its CMC. In some embodiments, the poloxamer is present in an amount about 25% below its CMC. In some embodiments, the poloxamer is present in an amount about 30% below its CMC. In some embodiments, the poloxamer is present in an amount about 35% below its CMC. In some embodiments, the poloxamer is present in an amount about 40% below its CMC. In some embodiments, the poloxamer is present in an amount about 45% below its CMC. In some embodiments, the poloxamer is present in an amount about 50% below its CMC.

[0045] In some embodiments, the process further comprises removing the poloxamer.

[0046] In some embodiments, the poloxamer is removed by dialysis.

[0047] In some embodiments, less than about 0.1% of the poloxamer remains upon removal. In some embodiments, less than about 0.05% of the poloxamer remains upon removal. In some embodiments, less than about 0.01% of the poloxamer remains upon removal.

[0048] In some embodiments, upon removal, residual amounts of poloxamer remain.

[0049] In some embodiments, the amount of poloxamer remaining after removal is undetectable.

[0050] In some embodiments, the process does not include mixing any cholesterol lipids.

[0051] In one aspect, the present invention provides, inter alia, compositions comprising lipid nanoparticles encapsulating mRNA formed according to the processes disclosed herein.

[0052] In this application, the use of "or" means "and / or" unless otherwise specified. As used in this disclosure, the term "comprise," as well as variations of this term, such as "comprising" and "comprises," are not intended to exclude other additives, components, integers, or steps. As used in this application, the terms "about" and "approximately" are used synonymously. Both terms are meant to cover any normal variations understood by one of ordinary skill in the relevant art.

[0053] Other features, objects, and advantages of the present invention are set forth in the following detailed description, drawings, and accompanying drawings. and the claims. It should be understood, however, that the detailed description, drawings, and claims, while referring to embodiments of the present invention, are given by way of example only and not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art. [Brief explanation of the drawings]

[0054] The following drawings are for illustrative purposes only and not for limitation.

[0055] [Figure 1] A schematic diagram of an exemplary LNP-mRNA encapsulation process is shown, which involves mixing an aqueous solution containing mRNA and poloxamer with a lipid solution using a pump system to produce mRNA-LNPs in an LNP-forming solution, and then exchanging the LNP-forming solution with a pharmaceutical formulation solution. [Figure 2] 1 shows an exemplary 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] 1 shows an exemplary graphical representation of the size, PDI, and encapsulation efficiency of mRNA-LNP formulations with various PEG-modified lipid % and poloxamer % as shown in Table 4. [Figure 4] 1 shows an exemplary graphical representation of the size and encapsulation efficiency of mRNA-LNP formulations with various PEG-modified lipid % and poloxamer, as shown in Table 4. [Figure 5] An exemplary graph of protein levels measured via ELISA at 6 and 24 hours post-administration is shown. The detected proteins resulted from in vivo translation of mRNA encapsulated in the LNP formulations shown in Table 7 and delivered to mice by subcutaneous or intravenous administration. [Figure 6A] Figures 6A and 6B show an exemplary method for quantifying the amount of poloxamer. Figure 6A shows the chemical reaction between poloxamer and cobalt thiocyanate, which forms a blue precipitate. Figure 6B shows a standard curve with known concentrations of poloxamer measured at 624 nm. [Figure 6B]Figures 6A and 6B show an exemplary method for quantifying the amount of poloxamer. Figure 6A shows the chemical reaction between poloxamer and cobalt thiocyanate, which forms a blue precipitate. Figure 6B shows a standard curve with known concentrations of poloxamer measured at 624 nm. [Figure 7]

[0033] Figure 1 shows an exemplary graph of OTC protein levels measured via ELISA 24 hours after administration. The detected proteins resulted from in vivo translation of mRNA encapsulated in the LNP formulations shown in Table 8 and delivered to mice via intravenous administration. DETAILED DESCRIPTION OF THE INVENTION

[0056] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for these and other terms are set forth throughout the specification.

[0057] Approximately or about: As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls 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 specified or otherwise clear from the context (except when such number exceeds 100% of the possible values).

[0058] Delivery: As used herein, the term "delivery" encompasses both local delivery and systemic delivery. For example, the delivery of mRNA encompasses the situation where mRNA is delivered to a target tissue, the encoded protein or peptide is expressed, and is retained in the target tissue (also referred to as "local distribution" or "local delivery"), and the situation where mRNA is delivered to a target tissue, the encoded protein or peptide is expressed, secreted into the patient's circulatory system (e.g., serum), distributed throughout the body, and taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery").

[0059] Efficacy: As used herein, the term "efficacy," or grammatical equivalents, refers to the improvement of a biologically relevant endpoint associated with delivery of mRNA encoding a relevant protein or peptide. In some embodiments, the biological endpoint is protection against ammonium chloride challenge at a specific time point after administration.

[0060] Encapsulation: As used herein, the term "encapsulation," or grammatical equivalents, refers to the process of confining individual mRNA molecules within nanoparticles.

[0061] Expression: As used herein, "expression" of mRNA refers to the translation of mRNA into a peptide (e.g., an antigen), polypeptide, or protein (e.g., an enzyme), and may also include post-translational modification of the peptide, polypeptide, or fully assembled protein (e.g., an enzyme), as indicated by the context. In this application, the terms "expression" and "production," and grammatical equivalents, are used interchangeably.

[0062] Improve, increase, or reduce: As used herein, the terms "improve," "increase," or "reduce," or grammatical equivalents, refer to a value relative to a baseline measurement, such as a measurement in the same individual before the initiation of a treatment described herein, or a measurement in a control sample or subject (or control samples or subjects) in the absence of a treatment described herein. A "control sample" is a sample that has been subjected to the same conditions as the test sample, except for the test article. A "control subject" is a subject that suffers from the same form of disease as the subject being treated and is approximately the same age as the subject being treated.

[0063] In vitro: As used herein, the term "in vitro" refers to events that take place not within a multicellular organism but in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc.

[0064] In vivo: As used herein, the term "in vivo" refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0065] Messenger RNA (mRNA): As used herein, the term "messenger RNA (mRNA)" refers to a polynucleotide that encodes at least one peptide, polypeptide, or protein. As used herein, mRNA encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA may be purified from natural sources, produced using recombinant expression systems, optionally purified, chemically synthesized, etc. Optionally, for example, in the case of chemically synthesized molecules, mRNA may contain nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications, etc. mRNA sequences are presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, mRNA contains natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, thiamin ... C5-methyl-cytidine, C5-propynyl-cytidine, C5-propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6 )-methylguanine, 2-thiocytidine, pseudouridine, and 5-methylcytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intervening bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0066] N / P ratio: As used herein, the term "N / P ratio" refers to the molar ratio of the positively charged molecular units in the cationic lipid in lipid nanoparticles to the negatively charged molecular units in the mRNA encapsulated in the lipid nanoparticles.Therefore, N / P ratio is typically calculated as the moles of amine groups in the cationic lipid in lipid nanoparticles to the moles of phosphate groups in the mRNA encapsulated in the lipid nanoparticles.

[0067] Nucleic Acid: As used herein, the term "nucleic acid" in its broadest sense refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, nucleic acids are compounds and / or substances that are 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. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone.

[0068] Patient: As used herein, the term "patient" or "subject" refers to any organism to which provided compositions can be administered, e.g., for experimental, diagnostic, preventative, 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 embodiments, the patient is a human. Humans include prenatal and postnatal forms.

[0069] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" refers to a material that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, inflammatory irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0070] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups 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 formed using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, etc. Oxalate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N-methyl-N ... + (C 1-4 Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, formed, where appropriate, using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates, and arylsulfonates. Further pharmaceutically acceptable salts include salts formed from the quaternization of amines using suitable electrophiles, for example, alkyl halides to form quaternized alkylated amino salts.

[0071] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, a subject is a human. A subject may be a patient, and refers to a person who sees a healthcare provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be afflicted with or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0072] Substantially: As used herein, the term "substantially" refers to the qualitative state of exhibiting all or nearly all extent or degree of a desired characteristic or property. Those skilled in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or reach completion, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0073] Treating: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who do not exhibit signs of disease and / or who exhibit only early signs of disease, with the goal of reducing the risk of developing conditions associated with the disease.

[0074] Detailed Description The present invention provides, inter alia, further 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 storage stability and in vivo circulation time. However, PEG-modified lipids have been shown to, inter alia, enhance blood clearance (ABC) and / or inhibit the proliferation of mRNA by generating anti-PEG antibodies. can induce innate immune responses. To address this issue, attempts have been made to prepare mRNA-LNPs that do not contain PEG-modified lipids. However, it has been observed that mRNA-loaded LNPs formed in the absence of PEG-modified lipids or PEG tend to have large and unstable sizes or precipitate, especially after freezing and thawing, making them unsuitable for therapeutic use. The present invention is based, in part, on the surprising discovery that unexpectedly stable mRNA-loaded LNPs containing small amounts or no PEG-modified lipids can be produced by mixing mRNA and lipids in the presence of amphiphilic block copolymers such as poloxamers. Thus, the present invention provides further improved mRNA-LNPs with exceptional stability and is particularly useful when LNPs containing small amounts or no PEG-modified lipids are desired, for example, to avoid the generation of anti-PEG antibodies and / or ABC.

[0075] The present invention provides a process for encapsulating mRNA in LNPs, thereby obtaining a stable mRNA-LNP composition. Specifically, the present invention provides a process for encapsulating mRNA in LNPs by mixing an mRNA solution with a lipid solution in the presence of an amphiphilic polymer (e.g., poloxamer). The amphiphilic polymer can be removed from the mRNA-LNPs, for example, by dialysis. The present invention is particularly useful for encapsulating mRNA in LNPs that contain little or no PEG-modified lipids.

[0076] Various aspects of the invention are described in detail in the following sections. The use of the sections is not meant to limit the invention. Each section may be applicable to any aspect of the invention.

[0077] The process of encapsulating mRNA in LNPs 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, the process for encapsulating mRNA described herein comprises mixing a lipid solution with an mRNA solution in the presence of an amphiphilic polymer (e.g., a poloxamer) to form lipid nanoparticles that encapsulate the mRNA. 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 mixing of the mRNA solution and the lipid solution.

[0078] In some embodiments, suitable mRNA solution is an aqueous solution that contains the mRNA that encodes target protein or peptide at desired concentration.Various methods can be used to prepare suitable mRNA solution.Exemplary methods are described in US2016 / 0038432, US2018 / 0153822 and US2018 / 0125989, which are incorporated herein by reference.

[0079] In some embodiments, a suitable lipid solution comprises a cationic lipid and a non-cationic lipid (also referred to as a helper lipid). In some embodiments, a suitable lipid solution comprises a cationic lipid, a non-cationic lipid (also referred to as a helper lipid), and a PEG-modified lipid or PEG. In some embodiments, a suitable lipid solution comprises a cationic lipid, a non-cationic lipid (also referred to as a helper lipid), a cholesterol-based lipid, and a PEG-modified lipid or PEG. The lipid solution used in the processes described herein can be prepared by dissolving various lipids in a suitable solvent in the desired amounts and / or ratios. Various methods can be used to prepare a suitable lipid solution. Exemplary methods are described in US2016 / 0038432, US2018 / 0153822, and US2018 / 0125989, which are incorporated herein by reference.

[0080] In some embodiments, a suitable lipid solution contains less than 1 mol%, less than 0.9 mol%, less than 0.8 mol%, less than 0.7 mol%, less than 0.6 mol%, less than 0.5 mol%, less than 0.4 mol%, less than 0.3 mol%, less than 0.2 mol%, or less than 0.1 mol% of the total lipids PEG-modified lipids or PEG. In some embodiments, a suitable lipid solution contains less than 1 wt%, less than 0.9 wt%, less than 0.8 wt%, less than 0.7 wt%, less than 0.6 wt%, less than 0.5 wt%, less than 0.4 wt%, less than 0.3 wt%, less than 0.2 wt%, or less than 0.1 wt% of the total lipids PEG-modified lipids or PEG. In some embodiments, a suitable lipid solution contains less than 0.09 mol%, less than 0.08 mol%, less than 0.07 mol%, less than 0.06 mol%, less than 0.05 mol%, less than 0.04 mol%, less than 0.03 mol%, less than 0.02 mol%, or less than 0.01 mol% of the total lipids PEG-modified lipids or PEG. In some embodiments, a suitable lipid solution contains less than 0.09 wt%, less than 0.08 wt%, less than 0.07 wt%, less than 0.06 wt%, less than 0.05 wt%, less than 0.04 wt%, less than 0.03 wt%, less than 0.02 wt%, or less than 0.01 wt% of the total lipids PEG-modified lipids or PEG.

[0081] Typically, the amphiphilic polymer (e.g., poloxamer) is present in the mixture in an amount lower than its critical micelle concentration (CMC). In some embodiments, the amphiphilic polymer (e.g., poloxamer) is present in the mixture in an amount 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% lower than its CMC. In some embodiments, the amphiphilic polymer (e.g., poloxamer) is present in the mixture in an amount about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% lower than its CMC. In some embodiments, the amphiphilic polymer (e.g., poloxamer) is present in the mixture in an amount that is about 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% below its CMC.

[0082] In some embodiments, the mRNA solution or the lipid solution, or both, can be heated to a predetermined temperature higher than ambient temperature before mixing.In some embodiments, the mRNA solution and the lipid solution are heated to a predetermined temperature separately before mixing.In some embodiments, the mRNA solution and the lipid solution are mixed at ambient temperature, and then heated to a predetermined temperature after mixing.In some embodiments, the lipid solution is heated to a predetermined temperature and mixed with the mRNA solution at ambient temperature.In some embodiments, the mRNA solution is heated to a predetermined temperature and mixed with the lipid solution at ambient temperature.

[0083] In some embodiments, the mRNA solution is heated to a predetermined temperature by adding an ambient temperature mRNA stock solution to a heated buffer solution to achieve the desired predetermined temperature.

[0084] In some embodiments, the mRNA-LNPs are heated after formation. As shown in the Examples, it has surprisingly been found that including a heating step in the process (before, during, or after formation) results in particularly high encapsulation of the mRNA-LNPs compared to otherwise identical processes that do not include a heating step.

[0085] As used herein, the term "ambient temperature" refers to the temperature in a room or the temperature surrounding a body of interest without heating or cooling. In some embodiments, the ambient temperature at which one or more of the solutions are maintained is about 35°C, 30°C, 25°C, 20°C, or 16°C or less. In some embodiments, the ambient temperature at which one or more of the solutions are maintained is about 15-35°C, about 15-30°C, about 15-25°C, about 15-20°C, about 20°C, or less. The ambient temperature ranges from 0-35° C., about 25-35° C., about 30-35° C., about 20-30° C., about 25-30° C., or about 20-25° C. In some embodiments, the ambient temperature at which one or more of the solutions is maintained is 20-25° C.

[0086] Thus, a predetermined temperature higher than ambient temperature is typically greater than about 25°C. In some embodiments, a predetermined temperature suitable for the present invention is greater than or equal to about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C. In some embodiments, a predetermined temperature suitable for the present invention is in the range of about 25-70°C, about 30-70°C, about 35-70°C, about 40-70°C, about 45-70°C, about 50-70°C, or about 60-70°C. In a specific embodiment, a predetermined temperature suitable for the present invention is about 65°C.

[0087] In some embodiments, the mRNA solution and the lipid solution are mixed using a pump.Since the encapsulation procedure using such mixing can occur at a wide range of scales, different types of pumps can be used to accommodate the desired scale.However, it is generally desirable to use a pulseless flow pump.As used herein, a pulseless flow pump refers to any pump that can establish a continuous flow with a stable flow rate.Suitable pump types can include, but are not limited to, gear pumps and centrifugal pumps.Exemplary gear pumps include, but are not limited to, Cole-Parmer or Diener gear pumps.Exemplary centrifugal pumps include, but are not limited to, those manufactured by Grainger or Cole-Parmer.

[0088] mRNA solution and lipid solution can be mixed at various flow rates.Typically, mRNA solution can be mixed at a speed faster than the speed of lipid solution.For example, mRNA solution can be mixed at a speed at least 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times or 20 times faster than the speed of lipid solution.

[0089] A suitable flow rate for mixing can be determined based on a scale. In some embodiments, the mRNA solution is mixed at a flow rate of about 40-400 ml / min, 60-500 ml / min, 70-600 ml / min, 80-700 ml / min, 90-800 ml / min, 100-900 ml / min, 110-1000 ml / min, 120-1100 ml / min, 130-1200 ml / min, 140-1300 ml / min, 150-1400 ml / min, 160-1700 ml / min, 170-1800 ml / min, 180-1900 ml / min, 190-2000 ml / min, 200-2100 ml / min, 210-2200 ml / min, 220-2300 ml / min, 230-2400 ml / min, 240-2500 ml / min, 250-2600 ml / min, 260-2700 ml / min, 270-2800 ml / min, 280-2900 ml / min, 290-3000 ml / min, 300-3100 ml / min, 320-3300 ml / min, 340-3500 ml / min, 350-3600 ml / min, 360-3700 ml / min, 370-3800 ml / min, 380-3900 ml / min, 390-4000 ml / min, 400-4100 ml / min, 420-4300 ml / min, 440-4500 ml / min, 450-4600 ml / min, 460-4700 ml / min In some embodiments, the mRNA solution is mixed at a flow rate of about 200 ml / min, about 500 ml / min, about 1000 ml / min, about 2000 ml / min, about 3000 ml / min, about 4000 ml / min, or about 5000 ml / min.

[0090] In some embodiments, the lipid solution is mixed at a flow rate in the range of about 25-75 ml / min, 20-50 ml / min, 25-75 ml / min, 30-90 ml / min, 40-100 ml / min, 50-110 ml / min, 75-200 ml / min, 200-350 ml / min, 350-500 ml / min, 500-650 ml / min, 650-850 ml / min, or 850-1000 ml / min. In some embodiments, the lipid solution is mixed at a flow rate of about 50 ml / min, about 100 ml / min, about 150 ml / min, about 200 ml / min, about 250 ml / min, about 300 ml / min, about 350 ml / min, about 400 ml / min, about 450 ml / min, about 500 ml / min, about 550 ml / min, about 600 ml / min, about 650 ml / min, about 700 ml / min, about 750 ml / min, about 800 ml / min, about 850 ml / min, about 900 ml / min, about 950 ml / min, or about 1000 ml / min.

[0091] Typically, the processes of the invention described herein include a step of removing the amphipathic polymer (e.g., poloxamer). In some embodiments, the amphipathic polymer (e.g., poloxamer) added during the process is subsequently removed after the formation of the mRNA-LNP. For example, the amphipathic polymer (e.g., poloxamer) can be removed by a buffer exchange technique such as dialysis. In some embodiments, the LNP formation solution is exchanged for a solution that constitutes the product formulation solution. For example, a mixture containing the formed mRNA-LNP can be dialyzed against one or more formulation solutions to remove the amphipathic polymer (e.g., poloxamer) present during mRNA-LNP formation. Suitable formulations are known in the art, and exemplary formulations are described in the formulation section of this application.

[0092] The exchange of the solution containing mRNA-LNPs from the LNP-forming solution to the formulation solution can be achieved by any of a variety of buffer exchange techniques known in the art. In some embodiments, the step of exchanging the LNP-forming solution with the formulation solution is accompanied by the purification and / or concentration of the mRNA-LNPs. Various methods can be used to achieve the exchange of solutions along with the purification or concentration of the mRNA-LNPs in the solution.

[0093] For example, in some embodiments, this solution exchange is accomplished by diafiltration. Diafiltration is a fractionation process whereby small, undesired particles pass through a filter while larger, desired nanoparticles are retained in the retentate without changing their concentration in the solution. Diafiltration is often used to remove salts or reaction buffers from a solution. Diafiltration can be either continuous or discontinuous. In continuous diafiltration, diafiltrate solution is added to the sample feed at the same rate as filtrate is produced. In discontinuous diafiltration, the solution is first diluted and then concentrated back to the starting concentration. Discontinuous diafiltration can be repeated until the desired nanoparticle concentration is reached.

[0094] In some embodiments, the solution is exchanged and the mRNA-LNP is purified using tangential flow filtration. Tangential flow filtration (TFF), also known as cross-flow filtration, is a type of filtration in which the material to be filtered passes tangentially across the filter, rather than through it. In TFF, the undesired permeate passes through the filter, while the desired retentate (mRNA-LNP and free mRNA) passes along the filter and is collected downstream. In some embodiments, the desired material is contained in the retentate in TFF, which is the opposite of what is typically encountered in traditional dead-end filtration.

[0095] Various TFF techniques are known and can be used to practice the present invention. Exemplary TFF purification methods are described in US2016 / 0040154 and US2015 / 0376220, which are incorporated herein by reference.

[0096] In some embodiments, encapsulation of mRNA in LNPs can be further enhanced by heating the formulation solution, which contains the mRNA-LNPs as well as any free mRNA that was not encapsulated in the LNP-forming solution, to a predetermined temperature as described herein.

[0097] In some embodiments, upon removal, less than about 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 amphiphilic polymer (e.g., poloxamer) present in the mixture remains. In some embodiments, upon removal, a residual amount of amphiphilic polymer (e.g., poloxamer) remains in the formulation. As used herein, residual amount refers to the amount remaining after substantially all of the material (e.g., amphiphilic polymer described herein, such as poloxamer) in the composition has been removed. The residual amount can be measured qualitatively or quantitatively using known techniques. Residual amounts may not be detectable using known techniques.

[0098] In some embodiments, excess mRNA is also removed along with the amphiphilic polymer (e.g., poloxamer) present during the formation of the mRNA-LNP.

[0099] Amphiphilic Block Copolymers A variety of amphiphilic block copolymers can be used to practice the present invention. In some embodiments, the amphiphilic block copolymers are also referred to as surfactants or nonionic surfactants.

[0100] In some embodiments, amphiphilic polymers suitable for the present invention are selected from poloxamers (Pluronic®), poloxamines (Tetronic®), polyoxyethylene glycol sorbitan alkyl esters (polysorbates), and polyvinylpyrrolidone (PVP).

[0101] Poloxamer In some embodiments, a suitable amphiphilic polymer is a poloxamer. For example, a suitable poloxamer is one having the following structure: [ka] In the formula, a is an integer of 10 to 150, and b is an integer of 20 to 60. For example, a is about 12 and b is about 20, or a is about 80 and b is about 27, or a is about 64 and b is about 37, or a is about 141 and b is about 44, or a is about 101 and b is about 56.

[0102] In some embodiments, poloxamers suitable for the present invention have from about 10 to about 150 ethylene oxide units. In some embodiments, poloxamers have from about 10 to about 100 ethylene oxide units.

[0103] Other amphiphilic polymers In some embodiments, the amphiphilic polymer is a poloxamine, such as tetronic 304 or tetronic 904.

[0104] In some embodiments, the amphiphilic polymer is polyvinylpyrrolidone (PVP), for example, PVP having a molecular weight of 3 kDa, 10 kDa, or 29 kDa.

[0105] In some embodiments, the amphiphilic polymer is polyethylene glycol ether (Brij), polysorbate, sorbitan, and derivatives thereof. In some embodiments, the amphiphilic polymer is a polysorbate, such as PS20.

[0106] In some embodiments, the amphiphilic polymer is a polyethylene glycol ether. In some embodiments, a suitable polyethylene glycol ether is a compound of formula (SI): [ka] or a salt or isomer thereof, wherein t is an integer from 1 to 100; 1BRIJ is independently C10-40 alkyl, C10-40 alkenyl, or C10-40 alkynyl, and optionally R 5PEG one or more methylene groups independently represent C3-10 carbocyclylene, 4- to 10-membered heterocyclylene, C6-10 arylene, 4- to 10-membered heteroarylene, -N(R N )-, -0-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR C(O)N(R)-, -C(O)0--OC(O)-, -OC(O)0--OC(O)N(R N )-, -NR N C(O)0--C(O)S--SC(O)-, -C(=NR N )-, -C(=NR)N(R)-, -NRNC(=NR N )--NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NRN C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)0--OS(O)0--OS(O)2--S(O)20--OS(O)20--N(R N )S(O)-, -S(O)N(R N )--N(R N )S(O)N(R N )--OS(O)N(R N )--N(R N )S(O)0--S(O)2--N(R N )S(O)2--S(O)2N(R N )-, -N(R N )S(O)2N(R N )--OS(O)2N(R N )-, or -N(R N )S(O)20- and R N Each instance of is independently hydrogen, C 1-6 alkyl, or nitrogen protecting groups.

[0107] In some embodiments, R 1BRIJ is Cis alkyl. For example, polyethylene glycol ethers can be prepared by the compound of formula (S-Ia): [ka] or a salt or isomer thereof, wherein s is an integer of 1 to 100.

[0108] In some embodiments, R 1BRIJ is Cis alkenyl. For example, a suitable polyethylene glycol ether is a compound of formula (S-Ib): [ka] or a salt or isomer thereof, wherein s is an integer of 1 to 100.

[0109] Stable mRNA-LNP composition In particular, the present invention provides mRNA-LNPs prepared using the process of the present invention described herein. Specifically, the present invention provides stable compositions comprising mRNA-LNPs that contain a small amount (e.g., less than 0.5% by weight or mol%) of PEG-modified lipids or PEG, or that do not contain any PEG at all. Such mRNA-LNPs are suitable for effective delivery and expression of mRNA in vivo. In this application, LNP and mRNA-LNP are used interchangeably unless otherwise specified. For example, mRNA-LNPs used herein include both mRNA-loaded LNPs and empty LNPs, unless otherwise specified.

[0110] Typically, the term "stable" in relation to an LNP composition refers to an LNP composition that can be stored at room temperature for more than 2 hours or at 4°C overnight without precipitation.

[0111] In some embodiments, the stable compositions described herein comprise LNPs that maintain an average diameter within 60% of their original average size after one or more freeze-thaw cycles.

[0112] cationic lipids As used herein, the term "cationic lipid" refers to any of a number of lipid and lipidoid species that have a net positive charge at a selected pH, such as physiological pH. Several cationic lipids have been described in the literature, and many of them are commercially available.

[0113] Suitable cationic lipids for use in the compositions and methods of the present invention include the cationic lipids described in International Patent Publication No. 2010 / 144740, which is incorporated herein by reference.In certain embodiments, the compositions and methods of the present invention comprise a cationic lipid having the following compound structure: (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate: [ka] and pharmaceutically acceptable salts thereof.

[0114] Other suitable cationic lipids for use in the compositions and methods of the present invention include the ionizable cationic lipids described in International Patent Publication No. 2013 / 149140, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid of one of the following formulas: [ka] or a pharmaceutically acceptable salt thereof, wherein R and R are each independently hydrogen, an optionally substituted variably saturated or unsaturated C-C 20 Alkyl, and optionally substituted variably saturated or unsaturated C-C 20 acyl; L and L are each independently selected from the group consisting of hydrogen, optionally substituted C-C 30 Alkyl, optionally substituted variably unsaturated C-C 30 Alkenyl, and optionally substituted C-C 30 alkynyl, wherein m and o are each independently selected from the group consisting of zero and any positive integer (e.g., m is 3), and n is zero or any positive integer (e.g., n is 1). In certain embodiments, the compositions and methods of the present invention provide a cationic lipid (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine ("HGT5000") having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine ("HGT5001") having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise a cationic lipid having the following compound structure and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine ("HGT5002"): [ka] and pharmaceutically acceptable salts thereof.

[0115] Other suitable cationic lipids for use in the compositions and methods of the present invention include the cationic lipids described as amino alcohol lipidoids in International Patent Publication No. 2010 / 053572, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention comprise cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0116] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publication No. 2016 / 118725, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0117] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publication No. 2016 / 118724, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0118] Other suitable cationic lipids for use in the compositions and methods of the present invention include cationic lipids having the formula 14,25-ditridecyl 15,18,21,24-tetraaza-octatriacontane, and pharmaceutically acceptable salts thereof.

[0119] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publications 2013 / 063468 and 2016 / 205691, each of which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise cationic lipids of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R L Each instance of is independently an optionally substituted C-C 40 In certain embodiments, the compositions and methods of the present invention provide cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0120] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publication No. 2015 / 184256, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise cationic lipids of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein each X is independently O or S, each Y is independently O or S, each m is independently 0 to 20, each n is independently 1 to 6, and each R A are independently hydrogen, optionally substituted C alkyl, optionally substituted C alkenyl, optionally substituted C alkynyl, optionally substituted C carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C aryl, optionally substituted 5-14 membered heteroaryl or halogen; and each R Bare independently hydrogen, optionally substituted C1-50 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. In certain embodiments, the compositions and methods of the invention provide a cationic lipid, "Target 23," having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0121] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publication No. 2016 / 004202, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the following compound structure: [ka] or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the following compound structure: [ka] or a pharmaceutically acceptable salt thereof.

[0122] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in U.S. Provisional Patent Application No. 62 / 758,179, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise cationic lipids of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein each R 1 and R 2 are independently H or is a C1-C6 aliphatic group, each m is independently an integer having a value of 1 to 4, each A is independently a covalent bond or arylene, and each L 1 are independently an ester, thioester, disulfide, or anhydride group, and each L 2 independently, C2-C 10 is aliphatic, and each X 1 are independently H or OH, and each R 3 independently, C6-C 20 In some embodiments, the compositions and methods of the present invention comprise a cationic lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof.

[0123] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in J. McClellan, MCKing, Cell, 1999, 14, 149-152, which are incorporated herein by reference. 2010, 141, 210-217 and Whitehead et al., Nature Communications (2014) 5:4277. In certain embodiments, the cationic lipid of the compositions and methods of the present invention is a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0124] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publication No. 2015 / 199952, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and The method includes providing a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0125] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication No. 2017 / 004143, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0126] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publication No. 2017 / 075531, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise cationic lipids of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein L 1 or L 2 One of the groups is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, -SC(=O)-, -NRa C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -, or -NR a C(=O)O-, and L 1 or L 2 The other of the groups is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O- or a direct bond, and G 1 and G 2 are each independently an unsubstituted C-C 12 Alkylene or C1-C 12 alkenylene, G 3 is C1-C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene, and R a is H or C1-C 12 alkyl, and R 1 and R 2 are each independently C6-C 24 Alkyl or C6-C 24 alkenyl, and R 3 H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 , or -NR 5 C(=O)R 4 and R 4 is C1-C 12 alkyl, and R 5 is H or C1-C6 alkyl and x is 0, 1 or 2.

[0127] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publication No. 2017 / 117528, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0128] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publication No. 2017 / 049245, which is incorporated herein by reference. In some embodiments, the cationic lipid of the compositions and methods of the present invention is a compound of one of the following formulas: [ka] and pharmaceutically acceptable salts thereof. In any one of these four formulas, R4 is independently -(CH2) n Q and -(CH2) n CHQR, where Q is -OR, -OH, -O(CH2) nIn certain embodiments, the cationic lipid is selected from the group consisting of N(R), -OC(O)R, -CX, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)R, -N(H)S(O)R, -N(R)C(O)N(R), -N(H)C(O)N(R), -N(H)C(O)N(H)(R), -N(R)C(S)N(R), -N(H)C(S)N(R), -N(H)C(S)N(H)(R), and heterocycle, wherein n is 1, 2, or 3. In certain embodiments, the compositions and methods of the present invention provide cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0129] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publications 2017 / 173054 and 2015 / 095340, each of which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention comprise cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0130] Other suitable cationic lipids for use in the compositions and methods of the present invention include cholesterol-based cationic lipids. In certain embodiments, the compositions and methods of the present invention include imidazole cholesterol esters or "ICEs," which have the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0131] Other suitable cationic lipids for use in the compositions and methods of the invention include the cleavable cationic lipids described in International Patent Publication No. 2012 / 170889, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention comprise a cationic lipid of the following formula: [ka] , wherein R1 is selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino), and pyridyl; and wherein R2 is selected from the group consisting of one of the following two formulas: [ka] wherein R and R are each independently an optionally substituted variably saturated or unsaturated C-C 20 Alkyl and optionally substituted variably saturated or unsaturated C-C 20 acyl, wherein n is 0 or any positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more). In certain embodiments, the compositions and methods of the present invention provide a cationic lipid "HGT4001" having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid "HGT4002" having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid "HGT4003" having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid "HGT4004" having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid "HGT4005" having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0132] Other suitable cationic lipids for use in the compositions and methods of the invention include the cleavable cationic lipids 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 invention comprise a cationic lipid having any of the general formulas or 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 invention comprise a cationic lipid having a structure according to formula (I'): [ka] wherein 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-; and each L4B and L5B is independently C1-C20 alkylene, C2-C20 alkenylene, or C2-C20 alkynylene. wherein each B and B' is NR4R5 or a 5-10 membered nitrogen-containing heteroaryl; each R1, R2, and R3 is independently C6-C30 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 R1 is independently hydrogen, C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl.

[0133] In certain embodiments, the compositions and methods of the present invention comprise a cationic lipid that is compound (139) of 62 / 672,194, having the following compound structure: [ka]

[0134] In some embodiments, the compositions and methods of the present invention include the cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride ("DOTMA"). (Feigner et al. Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355, incorporated herein by reference.) Other cationic lipids suitable for the compositions and methods of the present invention include, for example, 5-carboxyspermylglycine dioctadecylamide ("DOGS"), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium ("DOSPA") (Behr et al. al. Proc. Nat. Acad. Sci. 86, 6982 (1989), U.S. Pat. No. 5,171,678, U.S. Pat. No. 5,334,761), 1,2-dioleoyl-3-dimethylammonium-propane ("DODAP"), 1,2-dioleoyl-3-trimethylammonium-propane ("DOTAP").

[0135] Additionally, additional exemplary cationic lipids suitable for the compositions and methods of the present invention include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane ("DSDMA"), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane ("DODMA"), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane ("DLinDMA"), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane ("DLenDMA"), N-dioleyl-N,N-dimethylammonium chloride ("DODAC"), N, N-distearyl-N,N-dimethylammonium bromide ("DDAB"), N-(l,2-dimyrityloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-octadecadienooxy)propane ("CLinDMA"), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy]-3-dimethyl ... ,l-2'-octadecadienooxy)propane ("CpLinDMA"), N,N-dimethyl-3,4-dioleyloxybenzylamine ("DMOBA"), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane ("DOcarbDAP"), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine ("DLinDAP"), l,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane ("DLincarbDAP"), l,2-dilinoleylcarbamyl-3-dimethylaminopropane ("DLincarbDAP"). inCDAP"), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane ("DLin-K-DMA"), 2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine ("Octyl-CLinDMA"), (2R)-2-((8-[(3beta ...12-dien-1-yloxy]propan-1-amine ("Octyl-CLinDMA(2R)"), (2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N, fsl-dimethyl 3-[(9Z,12Z)-octadeca-9,12-dien-1-yl, Also included are [oxy]propan-1-amine ("Octyl-CLinDMA(2S)"), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("DLin-K-XTC2-DMA"), and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine ("DLin-KC2-DMA") (see WO 2010 / 042877, Semple et al., Nature Biotech. 28:172-176 (2010), which is incorporated herein by reference). (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 No. 2005 / 121348). In some embodiments, one or more of the cationic lipids comprises at least one of an imidazole moiety, a dialkylamino moiety, or a guanidinium moiety.

[0136] In some embodiments, the one or more cationic lipids suitable for the compositions and methods of the present invention include 2,2-dilinoleyl-1-4-dimethylaminoethyl-1-[1,3]-dioxolane ("XTC"), (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[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").

[0137] In some embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% by weight of the total lipid content in the composition, e.g., lipid nanoparticles. In some embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 80% of the total lipid content in the composition, e.g., lipid nanoparticles. In some embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute about 30-70 wt% (e.g., about 30-65 wt%, about 30-60 wt%, about 30-55 wt%, about 30-50 wt%, about 30-45 wt%, about 30-40 wt%, about 35-50 wt%, about 35-45 wt%, or about 35-40 wt%) of the total lipid content in the composition, e.g., of the lipid nanoparticles. In some embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute about 30-70 mol% (e.g., about 30-65 mol%, about 30-60 mol%, about 30-55 mol%, about 30-50 mol%, about 30-45 mol%, about 30-40 mol%, about 35-50 mol%, about 35-45 mol%, or about 35-40 mol%) of the total lipid content in the composition, e.g., of the lipid nanoparticles.

[0138] In some embodiments, sterol-based cationic lipids can be used instead of or in addition to the cationic lipids described herein.Suitable sterol-based cationic lipids are dialkylamino-containing sterol-based cationic lipids, imidazole-containing sterol-based cationic lipids, and guanidinium-containing sterol-based cationic lipids.For example, certain embodiments may use one or more sterol-based cationic lipids containing imidazole, such as imidazole cholesterol ester or "ICE" lipid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthracene, as shown in the following structure (I): The present invention relates to a composition comprising anthren-3-yl 3-(1H-imidazol-4-yl)propanoate. In certain embodiments, lipid nanoparticles for delivery of RNA (e.g., mRNA) encoding functional proteins can include one or more imidazole-based cationic lipids, such as imidazole cholesterol ester or "ICE" lipid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, as shown in the following structure: [ka]

[0139] In some embodiments, the proportion of cationic lipid in the liposome can 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 embodiments, the cationic lipid comprises about 30-50% by weight of the liposome (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%). In some embodiments, the cationic lipid (e.g., ICE lipid) comprises about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, or about 80% by molar ratio of the liposome.

[0140] Non-cationic / Helper Lipids In some embodiments, the mRNA-LNPs described herein comprise a non-cationic / helper lipid. As used herein, the phrase "non-cationic lipid" refers to any neutral lipid, zwitterionic lipid, or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of a number of lipid species that have a net negative charge at a selected pH, such as physiological pH. Non-cationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidyl These include, but are not limited to, ethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), or mixtures thereof.

[0141] In some embodiments, non-cationic lipids may comprise at least about 5% or mol%, 10% or mol%, 15% or mol%, 20% or mol%, 25% or mol%, 30% or mol%, 35% or mol%, 40% or mol%, 45% or mol%, 50% or mol%, 55% or mol%, 60% or mol%, 65% or mol%, or 70% or mol% by weight of the total lipids. In some embodiments, the non-cationic lipids constitute about 30-50 wt% or mol% (e.g., about 30-45 wt% or mol%, about 30-40 wt% or mol%, about 35-50 wt% or mol%, about 35-45 wt% or mol%, or about 35-40 wt% or mol%) of the total lipids.

[0142] Cholesterol-based lipids In some embodiments, the mRNA-LNPs described herein comprise one or more cholesterol-based lipids. For example, suitable cholesterol-based cationic lipids include DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)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 embodiments, the cholesterol-based lipid comprises at least about 5% by weight or mol%, 10% by weight or mol%, 20% by weight or mol%, 30% by weight or mol%, 40% by weight or mol%, 50% by weight or mol%, 60% by weight or mol%, or 70% by weight or mol% of the total lipids. In some embodiments, cholesterol-based lipids comprise about 30-50 wt% or mol% of the total lipids (e.g., about 30-45 wt% or mol%, about 30-40 wt% or mol%, about 35-50 wt% or mol%, about 35-45 wt% or mol%, or about 35-40 wt% or mol%). In some embodiments, cholesterol-based lipids comprise less than about 5 wt% or mol%, less than 10 wt% or mol%, less than 20 wt% or mol%, less than 30 wt% or mol%, less than 40 wt% or mol%, less than 50 wt% or mol%, less than 60 wt% or mol%, or less than 70 wt% or mol% of the total lipids. In some embodiments, the mRNA-LNPs described herein do not comprise cholesterol-based lipids.

[0143] PEG modified lipid In some embodiments, the mRNA-LNPs described herein contain small amounts (e.g., less than 0.5 mol% or wt%) 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) comprising N-octanoyl-sphingosine-l-[succinyl(methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide), is also contemplated by the present invention. Contemplated PEG-modified lipids include C6-C 20 These include, but are not limited to, polyethylene glycol chains up to 2 kDa, up to 3 kDa, up to 4 kDa, or up to 5 kDa in length covalently attached to lipids having long alkyl chains. In some embodiments, the PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. In some embodiments, particularly useful exchangeable lipids have shorter acyl chains (e.g., C 14 or C 18 ) is a PEG-ceramide.

[0144] In some embodiments, the mRNA-LNPs described herein contain less than 0.5 mol%, less than 0.4 mol%, less than 0.3 mol%, less than 0.2 mol%, or less than 0.1 mol% of the total lipids PEG-modified lipids or PEG. In some embodiments, the mRNA-LNPs described herein contain less than 0.5 wt%, less than 0.4 wt%, less than 0.3 wt%, less than 0.2 wt%, or less than 0.1 wt% of the total lipids PEG-modified lipids or PEG. In some embodiments, the mRNA-LNPs described herein contain less than 0.4 mol% or less by weight of the total lipids PEG-modified lipids or PEG, less than 0.3 mol% or less by weight of the total lipids PEG-modified lipids or PEG, less than 0.2 mol% or less by weight of the total lipids PEG-modified lipids or PEG, or less than 0.1 mol% or less by weight of the total lipids. Contains PEG-modified lipids or PEG. In some embodiments, the mRNA-LNPs described herein contain PEG-modified lipids or PEG at 0.09 mol% or less by weight of total lipids. In some embodiments, the mRNA-LNPs described herein contain PEG-modified lipids or PEG at 0.08 mol% or less by weight of total lipids. In some embodiments, the mRNA-LNPs described herein contain PEG-modified lipids or PEG at 0.07 mol% or less by weight of total lipids. In some embodiments, the mRNA-LNPs described herein contain PEG-modified lipids or PEG at 0.06 mol% or less by weight of total lipids. In some embodiments, the mRNA-LNPs described herein contain PEG-modified lipids or PEG at 0.05 mol% or less by weight of total lipids. In some embodiments, the mRNA-LNPs described herein contain PEG-modified lipids or PEG at 0.04 mol% or less by weight of total lipids. In some embodiments, the mRNA-LNPs described herein contain PEG-modified lipids or PEG at 0.03 mol% or less by weight of total lipids. In some embodiments, the mRNA-LNPs described herein contain 0.02 mol% or less by weight of total lipids, or PEG-modified lipids or PEG, hi some embodiments, the mRNA-LNPs described herein contain 0.01 mol% or less by weight of total lipids, or PEG-modified lipids or PEG.

[0145] In some embodiments, the mRNA-LNPs described herein are substantially free of PEG-modified lipids or PEG.

[0146] Amphiphilic Block Copolymers In some embodiments, the mRNA-LNPs described herein contain an amphiphilic block copolymer (e.g., poloxamer). In some embodiments, the mRNA-LNPs contain less than 5% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, the mRNA-LNPs contain less than 3% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, the mRNA-LNPs contain less than 2.5% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, the mRNA-LNPs contain less than 2% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, the mRNA-LNPs contain less than 1.5% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, the mRNA-LNPs contain less than 1% amphiphilic block copolymer (e.g., poloxamer). In some embodiments, mRNA-LNPs contain less than 0.5% (e.g., less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%) of an amphiphilic block copolymer (e.g., poloxamer). In some embodiments, mRNA-LNPs contain 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 an amphiphilic block copolymer (e.g., poloxamer). In some embodiments, mRNA-LNPs contain less than 0.01% of an amphiphilic block copolymer (e.g., poloxamer). In some embodiments, mRNA-LNPs contain a residual amount of amphiphilic polymer (e.g., poloxamer). As used herein, residual amount refers to the amount remaining after substantially all of the material (e.g., an amphiphilic polymer described herein, such as a poloxamer) in the composition has been removed. The residual amount may be detectable qualitatively or quantitatively using known techniques. The residual amount may not be detectable using known techniques.

[0147] messenger RNA (mRNA) The present invention can be used to encapsulate any mRNA. mRNA is typically thought of as a type of RNA that carries information from DNA to the ribosome. Typically, in eukaryotes, mRNA processing involves adding a "cap" on the 5' end and a "tail" on the 3' end. A typical cap is a 7-methylguanosine cap. The cap is a guanosine linked via 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 the tail is typically a polyadenylation event, whereby a polyadenylyl moiety is added to the 3' end of the mRNA molecule. The presence of this "tail" serves to protect the mRNA from exonuclease degradation. Messenger RNA is translated by ribosomes into a series of amino acids that make up proteins.

[0148] mRNA can be synthesized by any of various known methods.For example, mRNA according to the present invention can be synthesized through in vitro transcription (IVT).Briefly, IVT is typically carried out using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and an appropriate RNA polymerase (for example, T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNAse inhibitor.The exact conditions will vary depending on the specific application.

[0149] In some embodiments, in vitro synthesized mRNA can be purified prior to formulation and encapsulation to remove unwanted impurities, including various enzymes and other reagents used during mRNA synthesis.

[0150] The present invention can be used to formulate and encapsulate mRNAs of various lengths. In some embodiments, the present invention can be used to formulate and encapsulate in vitro synthesized mRNAs of lengths of about 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 or more. In some embodiments, the present invention can be used to formulate and encapsulate in vitro synthesized mRNAs of lengths ranging from about 1 to 20 kb, about 1 to 15 kb, about 1 to 10 kb, about 5 to 20 kb, about 5 to 15 kb, about 5 to 12 kb, about 5 to 10 kb, about 8 to 20 kb, or about 8 to 15 kb.

[0151] The present invention can be used to formulate and encapsulate unmodified mRNA or mRNA containing one or more modifications that typically enhance stability, in some embodiments, the modifications are selected from modified nucleotides, modified sugar-phosphate backbones, and 5' and / or 3' untranslated regions.

[0152] In some embodiments, modification of mRNA may include modification of nucleotides of RNA. Modified mRNA according to the present invention may include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, mRNA may contain naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), as well as nucleotides such as 1-methyladenine, 2-methyladenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine. N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil- They can be synthesized as modified nucleotide analogs or derivatives of purines and pyrimidines, such as 5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, beta-D-mannosyl-queosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, pseudouridine, 5-methylcytidine, and inosine. The preparation of such analogs is known to those skilled in the art from, for example, U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,262,530, and U.S. Pat. No. 5,700,642, the disclosures of which are incorporated herein by reference in their entirety.

[0153] Typically, mRNA synthesis involves adding a "cap" onto the 5' end and a "tail" onto the 3' end. The presence of the cap is important for providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" serves to protect the mRNA from exonuclease degradation.

[0154] Thus, in some embodiments, the mRNA comprises a 5' cap structure. The 5' cap is typically added as follows: First, an RNA terminal 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 phosphate via guanylyltransferase, resulting in a 5'5'5 triphosphate linkage. The 7-nitrogen of guanine is then methylated by a methyltransferase. 2'-O-methylation can also occur at the first and / or second base after the 7-methylguanosine triphosphate residue. Examples of cap structures include, but are not limited to, m7GpppNp-RNA, m7GpppNmp-RNA, and m7GpppNmpNmp-RNA (where m refers to a 2'-O methyl residue).

[0155] In some embodiments, the mRNA comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region comprises one or more elements that affect mRNA stability or translation, such as an iron-responsive element. In some embodiments, the 5' untranslated region can be about 50-500 nucleotides in length.

[0156] In some embodiments, the 3' untranslated region includes one or more of a polyadenylation signal, a binding site for a protein that affects the positional stability of the mRNA in the cell, or one or more binding sites for an miRNA. In some embodiments, the 3' untranslated region can be 50 to 500 or more nucleotides in length.

[0157] While mRNA resulting from an in vitro transcription reaction may be desirable in some embodiments, other sources of mRNA, including mRNA produced from bacteria, fungi, plants, and / or animals, are contemplated within the scope of the present invention.

[0158] The present invention can be used to formulate and encapsulate mRNAs encoding various proteins. Non-limiting examples of mRNAs suitable for the present invention include mRNAs encoding erythropoietin (EPO) and firefly luciferase (FFL).

[0159] formulation A variety of formulations may be used in connection with the present invention. In some embodiments, a suitable formulation solution may include a buffer or salt. Agents may include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. Exemplary salts may include sodium chloride, magnesium chloride, and potassium chloride.

[0160] In some embodiments, a suitable formulation solution is an aqueous solution containing a pharmaceutically acceptable excipient, including, but not limited to, a cryoprotectant. In some embodiments, a suitable formulation solution is an aqueous solution containing a pharmaceutically acceptable excipient, including, but not limited to, a sugar, such as one or more of trehalose, sucrose, mannose, lactose, and mannitol. In some embodiments, a suitable formulation solution contains trehalose. In some embodiments, a suitable formulation solution contains sucrose. In some embodiments, a suitable formulation solution contains mannose. In some embodiments, a suitable formulation solution contains lactose. In some embodiments, a suitable formulation solution contains mannitol.

[0161] In some embodiments, a suitable formulation solution is an aqueous solution comprising 5% to 20% weight by 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% weight by volume of trehalose. In some embodiments, a suitable formulation solution is an aqueous solution comprising 5% to 20% weight by volume of sucrose. In some embodiments, a suitable formulation solution is an aqueous solution comprising 5% to 20% weight by volume of mannose. In some embodiments, a suitable formulation solution is an aqueous solution comprising 5% to 20% weight by volume of lactose. In some embodiments, a suitable formulation solution is an aqueous solution comprising 5% to 20% weight by volume of mannitol.

[0162] In some embodiments, a suitable formulation solution is an aqueous solution comprising about 10% by weight of a sugar, such as trehalose, sucrose, mannose, lactose, and mannitol. In some embodiments, a suitable formulation solution is an aqueous solution comprising about 10% by weight of trehalose. In some embodiments, a suitable formulation solution is an aqueous solution comprising about 10% by weight of sucrose. In some embodiments, a suitable formulation solution is an aqueous solution comprising about 10% by weight of mannose. In some embodiments, a suitable formulation solution is an aqueous solution comprising about 10% by weight of lactose. In some embodiments, a suitable formulation solution is an aqueous solution comprising about 10% by weight of mannitol.

[0163] 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 contains only residual citrate. In some embodiments, a suitable formulation solution contains only residual non-aqueous solvent, such as ethanol. In some embodiments, a suitable formulation solution contains less than about 10 mM citrate (e.g., less than about 9 mM, about 8 mM, about 7 mM, about 6 mM, about 5 mM, about 4 mM, about 3 mM, about 2 mM, or about 1 mM). 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 a non-aqueous solvent, such as ethanol. In some embodiments, a suitable formulation solution does not require any further downstream processing (e.g., buffer exchange and / or further purification steps and / or additional excipients) before lyophilization. In some embodiments, a suitable formulation solution does not require any further downstream processing (e.g., buffer exchange and / or further purification steps and / or additional excipients) prior to administration for sterile filling into a vial, syringe, or other container. In some embodiments, a suitable formulation solution does not require any further downstream processing (e.g., buffer exchange and / or further purification steps and / or additional excipients) prior to administration to a subject.

[0164] In some embodiments, a suitable formulation solution has a pH between pH 4.5 and pH 7.5. In some embodiments, a suitable formulation solution has a pH between pH 5.0 and pH 7.0. In some embodiments, a suitable formulation solution has a pH between pH 5.5 and pH 7.0. In some embodiments, a suitable formulation solution has a pH greater than pH 4.5. In some embodiments, a suitable formulation solution has a pH greater than pH 5.0. In some embodiments, a suitable formulation solution has a pH greater than pH 5.5. In some embodiments, a suitable formulation solution has a pH greater than pH 6.0. In some embodiments, a suitable formulation solution has a pH greater than pH 6.5.

[0165] In some embodiments, the improved or enhanced amount of encapsulation of mRNA-LNP in a suitable formulation solution after heating is retained after subsequent freeze-thawing of the pharmaceutical formulation solution. In some embodiments, a suitable formulation solution is 10% trehalose and can be stably frozen.

[0166] In some embodiments, the mRNA-LNPs in a suitable formulation solution after heating can be stably frozen (e.g., to retain enhanced encapsulation) in about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% trehalose solution. In some embodiments, the suitable formulation solution can be stably stored in frozen form without the need for any downstream purification or processing.

[0167] therapeutic use In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that encode a peptide or polypeptide for delivery to or use in treating a human subject. In some embodiments, therapeutic compositions comprising the mRNA-LNPs described herein are used for delivery to the lung or lung cells of a subject. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver an endogenous protein that may be missing or non-functional in a subject.

[0168] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver peptides or polypeptides for use in treating lung diseases. In certain embodiments, the present invention is useful in methods for producing mRNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR mRNA is delivered to the lungs of a subject in need thereof as a therapeutic composition for treating cystic fibrosis. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver peptides or polypeptides for use in treating liver or metabolic diseases. Such peptides and polypeptides may include those associated with urea cycle disorders, lysosomal storage disorders, glycogen storage disorders, amino acid metabolism disorders, lipid metabolism or fibrotic disorders, methylmalonic acidemia, or any other metabolic disorder for which delivery of enriched full-length mRNA to the liver or liver cells or treatment with the same provides a therapeutic benefit.

[0169] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver proteins associated with urea cycle disorders. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver ornithine transcarbamylase (OTC) protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver argininosuccinate synthetase 1 protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver carbamoyl The present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver argininosuccinate lyase protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver arginase protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver arginase protein.

[0170] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver proteins associated with lysosomal storage disorders. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver alpha-galactosidase proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver glucocerebrosidase proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver iduronate-2-sulfatase proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver iduronidase proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver N-acetyl-alpha-D-glucosaminidase proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver heparan N-sulfatase protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver galactosamine-6 ​​sulfatase protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver beta-galactosidase protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver lysosomal lipase protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver arylsulfatase B (N-acetylgalactosamine-4-sulfatase) protein.In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver transcription factor EB (TFEB).

[0171] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver proteins associated with glycogen storage disorders. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver acid alpha-glucosidase protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver glucose-6-phosphatase (G6PC) protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver liver glycogen phosphorylase protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver muscle phosphoglycerate mutase protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver glycogen debranching enzyme.

[0172] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver proteins related to amino acid metabolism. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver a phenylalanine hydroxylase enzyme. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver a glutaryl-CoA dehydrogenase enzyme. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver a propionyl-CoA carboxylase enzyme. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver an oxalase alanine-glyoxylate aminotransferase enzyme.

[0173] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver proteins related to lipid metabolism or fibrotic disorders. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver mTOR inhibitors. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver ATPase phospholipid transport 8B1 (ATP8B1) protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver one or more NF-kappa B inhibitors, such as one or more of I-kappa B alpha, interferon-related developmental regulator 1 (IFRD1), and sirtuin 1 (SIRT1). In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver PPAR-gamma protein or an active variant.

[0174] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver proteins associated with methylmalonic acidemia. For example, in certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver methylmalonyl-CoA mutase proteins. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver methylmalonyl-CoA epimerase proteins.

[0175] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver peptides or polypeptides for delivery to or use in the treatment of the liver. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver ATP7B protein, also known as Wilson disease protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver porphobilinogen deaminase enzymes. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver one or more coagulation enzymes, such as Factor VIII, Factor IX, Factor VII, and Factor X. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver human hemochromatosis (HFE) proteins.

[0176] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver a peptide or polypeptide for delivery to or use in treating a cardiovascular condition or cardiovascular cell in a subject. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver vascular endothelial growth factor A protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver relaxin protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver bone morphogenetic protein-9 protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the p mRNA-LNPs described herein that deliver bone morphogenetic protein-2 receptor protein.

[0177] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver a peptide or polypeptide for delivery to or use in treating a subject's muscle or muscle cells. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver a dystrophin protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver a frataxin protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver a peptide or polypeptide for delivery to or use in treating a subject's myocardium or cardiomyocytes. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver a protein that modulates one or both of potassium and sodium channels in muscle tissue or muscle cells. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver a protein that modulates Kv7.1 channels in muscle tissue or muscle cells. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver proteins that modulate Nav1.5 channels in muscle tissue or muscle cells.

[0178] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver peptides or polypeptides for delivery to or use in treating the nervous system or nervous system cells of a subject. For example, in certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver survival motor neuron 1 protein. For example, in certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver survival motor neuron 2 protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver frataxin protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver ATP-binding cassette subfamily D member 1 (ABCD1) protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver CLN3 protein.

[0179] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver a peptide or polypeptide for delivery to or use in treating a subject's blood or bone marrow, or blood or bone marrow cells. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver beta-globin protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver Bruton's tyrosine kinase protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver one or more coagulation enzymes, such as factor VIII, factor IX, factor VII, and factor X.

[0180] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver a peptide or polypeptide for delivery to or use in treating a subject's kidney or kidney cells. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver type IV collagen alpha 5 chain (COL4A5) protein.

[0181] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver a peptide or polypeptide for delivery to or use in treating a subject's eye or ocular cells. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver ATP-binding cassette subfamily A member 4 (ABCA4) protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver retinoschisin protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver retinal pigment epithelium-specific 65 kDa (RPE65) protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver 290 kDa centrosomal protein (CEP290).

[0182] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver peptides or polypeptides for use in delivering vaccines or treatments to a subject or cells of a subject. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver antigens derived from infectious pathogens, such as viruses. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver antigens derived from influenza viruses. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver antigens derived from respiratory syncytial viruses. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver antigens derived from rabies viruses. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver antigens derived from cytomegaloviruses. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver antigens derived from rotaviruses. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver antigens derived from hepatitis viruses, such as hepatitis A virus, hepatitis B virus, or hepatitis C virus. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver antigens derived from human papillomaviruses. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver antigens derived from herpes simplex viruses, such as herpes simplex virus type 1 or herpes simplex virus type 2. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver antigens derived from human immunodeficiency viruses, such as human immunodeficiency virus type 1 or human immunodeficiency virus type 2.In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver antigens derived from human metapneumovirus, 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 methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver antigens derived from viruses. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver antigens derived from malaria viruses. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver antigens derived from Zika viruses. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver antigens derived from chikungunya viruses.

[0183] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver an antigen associated with a subject's cancer or an antigen identified from a subject's cancer cells. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver an antigen identified from a subject's own cancer cells, i.e., for providing a personalized cancer vaccine. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver an antigen expressed from a mutant KRAS gene.

[0184] In certain embodiments, the present invention provides methods for producing therapeutic compositions 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, the two separate mRNA-LNPs in step (b) of the process comprise mRNA encoding the light and heavy chains of the antibody. In some embodiments, the mRNA-LNP compositions of the present invention may comprise a combination of non-identical LNPs that comprise different lipid compositions and encapsulate mRNA encoding the light or heavy chain of the antibody. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver an antibody against OX40. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver an antibody against VEGF. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver an antibody against tissue necrosis factor alpha. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver antibodies against CD3. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver antibodies against CD19.

[0185] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver immunomodulatory agents. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver interleukin-12. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver interleukin-23. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver interleukin-36 gamma. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver one or more constitutively active variants of the stimulator of interferon genes (STING) protein.

[0186] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver an endonuclease. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver an RNA-guided DNA endonuclease protein, such as a Cas 9 protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver a meganuclease protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver a transcription activator-like effector nuclease protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising mRNA-LNPs described herein that deliver a zinc finger nuclease protein.

[0187] In certain embodiments, the present invention provides methods for producing therapeutic compositions comprising the mRNA-LNPs described herein that deliver peptides or proteins for treating ocular diseases. In some embodiments, the methods are used to produce therapeutic compositions comprising the mRNA-LNPs described herein that deliver retinoschisin. [Example]

[0188] While certain compounds, compositions, and methods of the present invention have been specifically described in accordance with certain embodiments, the following examples serve only to illustrate the present invention and are not intended to limit the present invention.

[0189] Example 1. Encapsulation of mRNA in lipid nanoparticles with little or no PEG-modified lipid using poloxamer This example illustrates an exemplary process for encapsulating mRNA in lipid nanoparticles containing little or no PEG-modified lipids by applying Process A. As used herein, Process A refers to the conventional method of encapsulating mRNA by, for example, mixing the mRNA with a mixture of lipids without first preforming the lipids into lipid nanoparticles, as described in U.S. Patent Application Publication No. 2018 / 0008680, which is incorporated by reference in its entirety.

[0190] An exemplary formulation process is shown in Figure 1. In this process, a lipid solution (e.g., in ethanol) and an aqueous solution containing mRNA and poloxamer were separately prepared. Specifically, the lipid solution (e.g., cationic lipid, helper lipid, zwitterionic lipid, PEG-modified lipid, etc.) was prepared by dissolving lipids in ethanol. The aqueous solution was prepared by dissolving mRNA and poloxamer in citrate buffer. These two solutions were then mixed using a pump system to obtain LNPs with encapsulated mRNA. It is noteworthy that it is desirable to maintain the amount of poloxamer in the mixture below its critical micelle concentration (CMC) to prevent precipitation. The LNP-forming solution containing mRNA-LNPs was then dialyzed against a solution containing 10% trehalose at room temperature for several hours and then overnight at 4°C to remove excess mRNA and poloxamer. After dialysis, the mRNA-loaded formulation solution was concentrated and stored for subsequent analysis.

[0191] Five different LNP formulations were made by the above encapsulation process and analyzed, as shown in Table 1 below. [Table 1]

[0192] Stable LNPs without both PEG-modified lipids and poloxamer could not be formed because the formulation solution collapsed and precipitated (Table 1, formulation 4). In the absence of poloxamer, LNPs containing small amounts (e.g., 0.4%) of PEG-modified lipids had a large particle size of 337 nm (Table 1, formulation 5). Surprisingly, when 0.5% poloxamer was used during the encapsulation process as described above, the size of the LNPs was significantly reduced by threefold (Table 1, formulations 1–3). Furthermore, stable LNPs could be formed even in the absence of PEG-modified lipids (Table 1, formulation 1).

[0193] This example demonstrates that including poloxamer during the encapsulation process resulted in stable mRNA-LNPs containing little or no PEG-modified lipids. Significantly, poloxamer shielding significantly reduced the size of the LNPs, resulting in mRNA-LNPs containing little or no PEG-modified lipids with sizes less than 200 nm, which are particularly suitable for therapeutic applications.

[0194] Example 2. Heating after LNP formation increased the encapsulation efficiency of LNPs This example illustrates that the additional step of heating the mRNA-LNPs after formation increases encapsulation efficiency.

[0195] Specifically, after mRNA was encapsulated in LNPs by Process A as described above, the resulting mRNA-LNP formulation solution was heated above ambient temperature. After heating, the mRNA-LNP solution was cooled and stored for subsequent analysis. For each formulation, the size, PDI, and encapsulation efficiency were measured before and after heating. [Table 2]

[0196] As shown in Table 2, the encapsulation efficiency (EE%) of formulations 2 and 3, containing 0.2% and 0.4% PEG-modified lipid, respectively, increased significantly after the post-formulation heating step compared to the encapsulation efficiency of the same formulations before heating. The PDI of all formulations tested decreased slightly, and particle size remained relatively constant.

[0197] Example 3. mRNA-LNPs are stable after multiple freeze / thaw cycles This example illustrates that mRNA-loaded LNPs made according to the present invention are stable after multiple freeze / thaw cycles.

[0198] Specifically, three different LNP formulations containing various PEG-modified lipids and poloxamers were produced by the encapsulation process described above. For each formulation, the size and encapsulation efficiency were measured before and after freeze / thaw cycles. [Table 3]

[0199] As shown in Figure 2, LNP formulations 7 and 8, which contained 0.4% and 0% PEG, respectively, and were formed in the presence of 2% poloxamer, maintained an average particle size of approximately 100 nm after two freeze / thaw cycles. More specifically, the increase in particle size after one and two freeze / thaw cycles appeared to be within 10% of their original average size. Formulation 6, which contained 0.4% PEG and no poloxamer, had an average particle size of approximately 370 nm before freeze / thaw, and this average size increased to over 400 nm after two freeze / thaw cycles. More surprisingly, for formulations 7 and 8, which contained 2% poloxamer during the mRNA-LNP encapsulation process, the encapsulation efficiency significantly increased after the first freeze / thaw cycle.

[0200] Example 4. Formation of mRNA-LNPs with little or no PEG-modified lipids in the presence of poloxamer This example further illustrates that mRNA-LNPs made in the presence of poloxamer, with little or no PEG-modified lipid, have an average size and size distribution suitable for therapeutic applications.

[0201] Different LNP formulations containing various amounts of PEG-modified lipid and poloxamer were produced and analyzed by the above encapsulation process, as shown in Table 4. Notably, in this example, the same cationic lipid, helper lipid, cholesterol, cholesterol, and mRNA were used to prepare the LNP formulations. [Table 4]

[0202] mRNA-loaded 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. Notably, mRNA-LNPs without PEG-modified lipids were made using a low percentage (e.g., 0.5%) of poloxamer (formulation 9). As shown in Figure 3, mRNA-loaded LNPs were prepared using various amounts of poloxamer. The average size of all LNPs containing 0.4% PEG-modified lipids made with poloxamer was less than 100 nm. The average size of all LNPs without PEG-modified lipids made with various amounts of poloxamer was less than 130 nm. The PDI of all LNPs with or without PEG-modified lipids made with various amounts of poloxamer was about 0.25 or less.

[0203] The effect of % PEG-modified lipid was also examined, as shown in Figure 4. Figure 4 shows that mRNA-LNPs were produced with very little or no PEG-modified lipid in the presence of poloxamer, with an average size of less than 100 nm and a PDI of less than 0.25. Because the mRNA-LNPs were prepared using the same lipid components (e.g., cationic lipid, helper lipid, and cholesterol) and the same mRNA, the changes observed in this example are attributable to changes in the percentage of PEG-modified lipid and / or poloxamer.

[0204] Example 5. Poloxamer stabilizes LNPs containing few component systems This example illustrates that mRNA-LNPs containing fewer than four components can be produced according to the present invention and have an average size suitable for therapeutic use.

[0205] Specifically, mRNA-LNPs containing different components (e.g., four, three, and two) were prepared and characterized in the presence of poloxamer as described above. The specific components, average particle size, PDI, and encapsulation efficiency for the different formulations are shown in Table 5. Notably, in this example, LNP formulations were prepared using the same lipid components (e.g., cationic lipid, helper lipid, and / or cholesterol) and the same mRNA. [Table 5]

[0206] As shown in Table 5, mRNA-LNPs were made with either three or two components when poloxamer was included in the encapsulation process. All had small sizes (e.g., less than 125 nm) with acceptable PDIs (e.g., less than 0.20) and encapsulation efficiencies. Notably, two-component LNPs (formulations 17 and 18) with different cationic lipid to helper lipid ratios both had small average sizes (less than 120 nm) and high encapsulation efficiencies (e.g., greater than 75%).

[0207] Example 6. Stable LNPs substantially free of PEG-modified lipids can be formed with different cationic lipids and various poloxamers This example illustrates that the present invention can be used to produce stable mRNA-LNPs containing a variety of cationic lipids and different mRNAs, and that are substantially free of PEG-modified lipids.

[0208] Specifically, as shown in Table 6, mRNA encoding EPO or FFL protein was encapsulated into LNPs containing different cationic lipids at an N / P ratio of 4 in the presence of poloxamer 407. [Table 6]

[0209] These results demonstrate that stable mRNA-LNPs substantially free of PEG-modified lipids can be formed with various cationic lipids and mRNA constructs. LNPs containing CCBene, ML-7, and MC3 exhibited particularly small sizes of less than 120 nm.

[0210] Example 7. Successful in vivo expression by delivery of mRNA-LNPs formed using poloxamers This example demonstrates that administration of mRNA-LNPs formed using poloxamer resulted in successful in vivo protein expression. Specifically, EPO mRNA-loaded LNPs were administered to CD-1 mice via subcutaneous (SC) and intravenous (IV) routes, and EPO protein expression levels were detected in the liver and serum of the mice 6 and 24 hours after administration. As shown in Table 7 below, four different LNPs containing various amounts of PEG-modified lipids and formed using 0.5% poloxamer were tested. Conventional LNPs containing 5% PEG-modified lipids were also administered as controls (Groups B and F shown in Table 7). [Table 7]

[0211] As shown in Figure 5, poloxamer-shielded LNPs containing low or no PEG-modified lipids achieved in vivo protein expression profiles similar to conventional LNPs (e.g., those containing 5% PEG-modified lipids). These data demonstrate that mRNA-LNPs made using poloxamers according to the present invention, containing low (e.g., 0.5% or less) amounts of PEG-modified lipids or PEG, or no PEG, can be successfully used for in vivo protein expression for therapeutic purposes.

[0212] Example 8. Quantification of Poloxamer in mRNA-LNP Formulations This example illustrates an exemplary method for quantifying the final concentration of poloxamer in mRNA-LNPs produced according to the present invention.

[0213] Specifically, this method takes advantage of the fact that poloxamer competes with cobalt thiocyanate to form a blue precipitate, as shown in Figure 6A. After the precipitate is formed, the blue precipitate is dissolved in acetone, and the color intensity, which is directly proportional to the poloxamer, is measured at a wavelength of 624 nm. A standard curve using known concentrations of poloxamer was plotted, as shown in Figure 6B. This standard curve can be used to determine the amount of poloxamer in a given sample.

[0214] Example 9. Successful in vivo expression of mRNA-LNPs containing various poloxamers and non-cationic lipids This example illustrates that various poloxamers and non-cationic lipids can be used to produce stable mRNA-LNPs that are substantially free of PEG-modified lipids. This example further demonstrates that administration of mRNA-LNPs formed with poloxamers resulted in successful in vivo protein expression.

[0215] Different LNP formulations containing various poloxamer and non-cationic lipids, as well as various amounts of PEG-modified lipids, were made and analyzed using the cationic lipid cDD-TE4-E12 by the encapsulation process described above, as shown in Table 8. In this particular experiment, mRNA encoding OTC (ornithine transcarbamylase) was encapsulated. [Table 8]

[0216] mRNA-loaded LNPs were formed in the absence of PEG-modified lipids or with very small amounts (e.g., 0.5%) of PEG-modified lipids (formulations A–H). 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 size of all LNPs produced in this example was approximately 130 nm or less, with a PDI of approximately 0.15 or less and an encapsulation efficiency of approximately 90% or greater. As a control, mRNA-loaded LNPs were prepared without poloxamer and with various ratios of PEG-modified lipids (formulations I–K).

[0217] The mRNA-LNP formulations containing the OTC mRNA in Table 8 were administered intravenously (IV) to mice, and OTC protein expression levels were measured. As shown in Figure 7, poloxamer-shielded LNPs containing small amounts of PEG-modified lipids achieved in vivo protein expression levels similar to or higher than the target expression levels. These data demonstrate that mRNA-LNPs made using various poloxamers and non-cationic lipids, with or without small amounts of PEG-modified lipids, can be successfully used for in vivo protein expression for therapeutic purposes. Formulations I and J achieved higher efficacy than formulations A through H containing poloxamers.

[0218] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, 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 above-described detailed description, but rather is as 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, and 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, wherein the lipid nanoparticles encapsulating the mRNA are stable after one or more freeze-thaw cycles.

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

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

4. 4. The stable composition of any one of claims 1 to 3, 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. 5. The stable composition of any one of claims 1 to 4, 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 one of claims 1 to 5, wherein the lipid nanoparticles have an mRNA encapsulation efficiency of about 50% to 99%.

7. The stable composition of any one of claims 1 to 6, wherein each of the lipid nanoparticles further comprises a cholesterol-based lipid.

8. The stable composition of any one of claims 1 to 7, wherein each of the lipid nanoparticles contains 0.4% or less of PEG-modified lipid, 0.3% or less of PEG-modified lipid, 0.2% or less of PEG-modified lipid, or 0.1% or less of PEG-modified lipid.

9. The stable composition of any one of claims 1 to 8, wherein each of the lipid nanoparticles is substantially free of PEG-modified lipids.

10. 10. The stable composition of claim 1, wherein each of the lipid nanoparticles comprises an amphiphilic block copolymer.

11. 11. The stable composition of 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. 12. The stable composition of 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% by weight of the total composition of amphiphilic block copolymer.

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

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

15. The poloxamer may be 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 236, poloxamer 237, poloxamer 238, poloxamer 239, poloxamer 240, poloxamer 241, poloxamer 242, poloxamer 243, poloxamer 244, poloxamer 245, poloxamer 246, poloxamer 247, poloxamer 248, poloxamer 249, poloxamer 250, poloxamer 251, poloxamer 252, poloxamer 253, poloxamer 254, poloxamer 255, poloxamer 256, poloxamer 257, poloxamer 258, poloxamer 259, poloxamer 260, poloxamer 261, poloxamer 262, poloxamer 263, poloxamer 264, poloxamer 265, poloxamer 266, poloxam 15. The stable composition of claim 14, wherein the poloxamer is selected from 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 a combination thereof.

16. A stable composition comprising lipid nanoparticles encapsulating 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-modified lipids or PEG, and the lipid nanoparticles encapsulating the mRNA are stable after one or more freeze-thaw cycles.

17. A stable composition comprising lipid nanoparticles encapsulating messenger RNA (mRNA) encoding a protein or peptide, each of the lipid nanoparticles comprising one or more cationic lipids, one or more non-cationic lipids, and a poloxamer, and substantially free of PEG-modified lipids or PEG, wherein the lipid nanoparticles encapsulating the mRNA produce low or no anti-PEG antibodies and / or have reduced accelerated blood clearance (ABC).

18. 18. The stable composition of claim 17, wherein the poloxamer is present in the lipid nanoparticles in an amount of less than 0.1%.

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

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

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

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

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

26. 26. The stable composition of any one of claims 16 to 25, wherein each of the lipid nanoparticles is free of cholesterol-based lipids.

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

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

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

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

31. The poloxamer may be 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, or poloxamer 23 5, 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 a combination thereof.

32. 32. The stable composition of any one of claims 1 to 31, wherein the lipid nanoparticles have an average size of less than about 200 nm.

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

34. 34. The stable composition of any one of claims 1 to 33, wherein the lipid nanoparticles have a polydispersity index (PDI) of 0.25 or less, 0.2 or less, 0.15 or less, or 0.1 or less.

35. 35. A method for delivering messenger RNA (mRNA) for the in vivo production of a protein or peptide, the method comprising administering to a subject a stable composition according to any one of claims 1 to 34.

36. 36. A method for delivering messenger RNA (mRNA) for in vivo production of a protein or peptide, comprising administering to a subject the stable composition of any one of claims 1 to 35, wherein said administration of said stable composition does not result in anti-PEG antibodies and / or accelerated blood clearance (ABC) in said subject.

37. A method for treating a subject having a protein or peptide deficiency, comprising administering to a subject in need thereof a stable composition according to any one of claims 1 to 33. ,method.

38. The method of any one of claims 35 to 37, wherein the stable composition is administered by intravenous injection.

39. 38. The method of any one of claims 35 to 37, wherein the stable composition is administered by pulmonary delivery.

40. 38. The method of any one of claims 35 to 37, wherein the stable composition is administered by intramuscular delivery.

41. 41. The method of any one of claims 35-40, wherein said administration of said stable composition results in expression of said protein or said peptide encoded by said 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 mixing an mRNA solution with a lipid solution in the presence of a poloxamer.

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

44. 44. The process of claim 42 or 43, wherein the lipid solution comprises pre-formed lipid nanoparticles.

45. 45. The process of any one of claims 42 to 44, wherein the mRNA solution and / or the lipid solution are at a predetermined temperature that is higher than ambient temperature.

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

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

48. 48. The process of claim 47, wherein the poloxamer is present in an amount 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% below its CMC.

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

50. 50. The process of any one of claims 42 to 49, wherein the process further comprises removing the poloxamer.

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

52. 52. The process of claim 50 or 51, wherein upon removal, less than about 0.05% of the poloxamer remains.

53. 53. The process of claim 52, wherein upon removal, less than about 0.01% of the poloxamer remains. 。

54. 53. The process of claim 52, wherein upon removal, a residual amount of poloxamer remains.

55. 55. The process of any one of claims 50 to 54, wherein the amount of poloxamer remaining after removal is undetectable.

56. 56. The process of any one of claims 42 to 55, wherein the poloxamer has from about 10 to about 150 ethylene oxide units.

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

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

59. The poloxamer may be 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 2 35, 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 a combination thereof.

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

61. 60. The process of any one of claims 42 to 59, wherein the process does not include mixing any cholesterol lipids.

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

63. 62. The process of claims 42-61, wherein the lipid nanoparticles have an encapsulation efficiency of about 60% to 99%.

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

65. 65. The process of 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. 64. A composition comprising lipid nanoparticles encapsulating mRNA formed according to the process of any one of claims 42 to 63.

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