Compositions and Methods

Abstract

An aqueous dispersion is described having an aqueous mobile phase and a dispersed phase, wherein the dispersed phase comprises a lipid mixture including cationically ionizable lipids; the aqueous mobile phase comprises a buffer solution having a pH of about 6.5 to about 8, and the aqueous dispersion is substantially free of inorganic cations, organic solvents, and nucleic acids. Methods for preparing the aqueous dispersion, nucleic acid-lipid particles, and methods for preparing nucleic acid-lipid particles using the aqueous dispersion, as well as their uses in medicine, are also disclosed.

Description

[Technical Field]

[0001] The present disclosure generally relates to aqueous dispersions that typically contain pre-formed lipid nanoparticles (pre-LNPs) capable of encapsulating nucleic acids, and methods for producing them, particularly such methods that do not involve the use of organic solvents. The present disclosure also relates to nucleic acid-lipid particles, such as lipid nanoparticles (LNPs) formed from such pre-LNPs and nucleic acids, and methods for producing them. [Background technology]

[0002] Lipid nanoparticles (LNPs) have demonstrated tremendous potential as a delivery technology for nucleic acid vaccines to treat a wide range of conditions, including cancer immunotherapy, gene therapy, and the treatment of infectious diseases.

[0003] Classic LNP manufacturing methods involve a one-step process in which one part nucleic acid in an aqueous buffer is mixed with a lipid excipient dissolved in an organic solvent. Experience in the LNP domain has shown that the additional processing step of removing the organic solvent is tedious, time-consuming, and expensive, resulting in long turnover times from start to finish. Therefore, an improved LNP manufacturing process is needed to alleviate the aforementioned problems.

[0004] WO 2022 / 032087 describes a method for preparing an empty lipid nanoparticle solution (empty LNP solution), comprising: i) a nanoprecipitation step comprising mixing a lipid solution containing ionizable lipids, structural lipids, and phospholipids with an aqueous buffer containing a first buffer, thereby forming an intermediate empty lipid nanoparticle solution (intermediate empty LNP solution) containing intermediate empty nanoparticles (intermediate empty LNPs); ib) holding the intermediate empty LNP solution for a residence time; and ic) adding a dilution solution to the intermediate empty LNP solution, thereby forming an empty LNP solution. The empty LNP solution may be further processed to produce an empty LNP formulation. Methods for producing encapsulated LNPs by mixing an empty LNP solution or an empty LNP formulation with nucleic acid have also been described. However, the initial step in the formation of empty LNPs described in this document uses an aqueous buffer, such as a citrate buffer. Inorganic ions present in such aqueous buffers when used in the pre-LNP manufacturing stage are believed to destabilize the colloidal properties of lipid nanoparticle formulations and are therefore detrimental to the stability of the formulation.

[0005] Thus, there remains a need in the art for methods of producing lipid particles (particularly, although not exclusively, lipid nanoparticles) without the use of buffers, for example, in the initial mixing step during the formation of the preformed lipid particles, and without the use of organic solvents during the process of encapsulating nucleic acids into the preformed lipid particles. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2022 / 032087 Brochure Summary of the Invention [Means for solving the problem]

[0007] In a first aspect, the present invention provides a method for forming an aqueous dispersion having a pH of about 6.5 to about 8.0, the aqueous dispersion being substantially free of organic solvents and nucleic acids, the method comprising: (A) (i) an organic phase comprising a lipid mixture comprising a cationically ionizable lipid dissolved in a water-soluble organic solvent; and (ii) an aqueous phase containing aqueous acid and substantially free of inorganic cations; to produce an intermediate aqueous lipid dispersion; and (B) performing a dialysis or filtration step on the intermediate aqueous lipid dispersion to produce an aqueous dispersion, wherein the dialysis or filtration step removes organic solvents and adjusts the pH to about 6.5 to about 8.0. The present invention provides a method comprising:

[0008] In a second aspect, the present invention provides a method for forming an aqueous dispersion having a pH of about 6.5 to about 8.0, the aqueous dispersion being substantially free of organic solvents and nucleic acids, the method comprising: (A)(i) an organic phase comprising a lipid mixture comprising cationically ionizable lipids dissolved in a water-soluble organic solvent, the organic phase further comprising an aqueous acid, the organic phase being substantially free of inorganic cations; and (ii) aqueous phase to produce an intermediate aqueous lipid dispersion having a pH of about 6.5 to about 8.0; and (B) performing a dialysis or filtration step on the intermediate aqueous lipid dispersion to produce an aqueous dispersion, wherein the dialysis or filtration step removes the organic solvent. The present invention provides a method comprising:

[0009] In a third aspect, the present invention provides an aqueous dispersion obtained or obtainable by the method of the first or second aspect.

[0010] In a fourth aspect, the present invention provides an aqueous dispersion having an aqueous mobile phase and a dispersed phase, the dispersed phase comprises a lipid mixture comprising a cationically ionizable lipid; the aqueous mobile phase comprises a buffer solution having a pH of about 6.5 to about 8, the buffer being 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) optionally in combination with tris(hydroxymethyl)aminomethane (Tris); An aqueous dispersion is provided, wherein the aqueous dispersion is substantially free of organic solvents and nucleic acids.

[0011] In one embodiment of this aspect, the invention provides an aqueous dispersion having an aqueous mobile phase and a dispersed phase, the dispersed phase comprises a lipid mixture comprising a cationically ionizable lipid; the aqueous mobile phase comprises a buffer solution having a pH of about 6.5 to about 8, the buffer being 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) optionally in combination with tris(hydroxymethyl)aminomethane (Tris); the aqueous dispersion is substantially free of organic solvents and nucleic acids; An aqueous dispersion is provided, wherein the aqueous dispersion contains a cryoprotectant.

[0012] In a fifth aspect, the present invention provides a method of forming a nucleic acid-lipid particle, comprising: (x) the aqueous dispersion of the third or fourth aspect, (y) an aqueous solution containing nucleic acid; mixing to produce nucleic acid-lipid particles, Either the aqueous dispersion (x) or the aqueous solution (y) is acidified, The present invention provides a method comprising:

[0013] In a sixth aspect, the present invention provides a method of forming a nucleic acid-lipid particle, comprising: (A) (i) an organic phase comprising a lipid mixture comprising a cationically ionizable lipid dissolved in a water-soluble organic solvent; and (ii) an aqueous phase containing aqueous acid and substantially free of inorganic cations; to produce an intermediate aqueous lipid dispersion; (B) performing a dialysis or filtration step on the intermediate aqueous lipid dispersion to produce an aqueous dispersion having a pH of about 6.5 to about 8.0 and substantially free of organic solvents and nucleic acids, wherein the dialysis or filtration step removes organic solvents and adjusts the pH to about 6.5 to about 8.0; and (C) (x) The aqueous dispersion produced in step (B) (y) an aqueous solution containing nucleic acid; mixing to produce nucleic acid-lipid particles, Either the aqueous dispersion (x) or the aqueous solution (y) is acidified, The present invention provides a method comprising:

[0014] In a seventh aspect, the present invention provides a method of forming a nucleic acid-lipid particle, comprising: (A) (i) an organic phase comprising a lipid mixture comprising a cationically ionizable lipid dissolved in a water-soluble organic solvent; and (ii) aqueous phase to produce an intermediate aqueous lipid dispersion having a pH of about 6.5 to about 8.0, the organic phase comprises an aqueous acid and is substantially free of inorganic cations; (B) performing a dialysis or filtration step on the intermediate aqueous lipid dispersion to produce an aqueous dispersion having a pH of about 6.5 to about 8.0 and substantially free of organic solvents and nucleic acids, wherein the dialysis or filtration step removes organic solvents; and (C) (x) The aqueous dispersion produced in step (B), (y) an aqueous solution containing nucleic acid; mixing to produce nucleic acid-lipid particles, Either the aqueous dispersion (x) or the aqueous solution (y) is acidified, The present invention provides a method comprising:

[0015] In an eighth aspect, the present invention provides nucleic acid-lipid particles obtained or obtainable by the method of the fifth, sixth, or seventh aspects.

[0016] In a ninth aspect, the present invention provides the nucleic acid-lipid particle of the eighth aspect for use in medicine, for example for use in the prophylactic and / or therapeutic treatment of a disease involving an antigen, and / or for use in inducing an immune response, and / or for use in the treatment of cancer.

[0017] In a tenth aspect, the present invention provides a method for forming an aqueous dispersion comprising lipid particles containing nucleic acids and having a pH of about 6.5 to about 8.0, the method comprising: (a)(i) a lipid mixture comprising a cationically ionizable lipid dissolved in a water-soluble organic solvent; and (ii) aqueous phase to produce a first intermediate aqueous lipid dispersion containing an aqueous acid, the lipid mixture and / or the aqueous phase comprises an aqueous acid; (b) subjecting the first intermediate aqueous lipid dispersion to a dialysis or filtration step at a pH of about 2.5 to about 5.5 to remove the organic solvent to produce a second intermediate aqueous dispersion; (c) adding a cryoprotectant to the second intermediate aqueous dispersion to produce an aqueous dispersion that is substantially free of inorganic cations, organic solvents, and nucleic acids; (d) mixing the aqueous dispersion with an aqueous solution containing nucleic acid to produce lipid particles containing nucleic acid; and (e) adding a storage matrix to the nucleic acid-containing lipid particles and adjusting the pH to about 6.5 to about 8.0; The present invention provides a method comprising:

[0018] In an eleventh aspect, the present invention provides a method for forming an aqueous dispersion having a pH of about 6.5 to about 8.0 and substantially free of organic solvents and nucleic acids, the method comprising: (A) (i) an organic phase comprising a lipid mixture comprising a cationically ionizable lipid dissolved in a water-soluble organic solvent; and (ii) an aqueous phase having a pH of about 3.0 to 5.0; to produce an intermediate aqueous lipid dispersion having a pH of about 3.5 to about 5.5; and (B) performing a dialysis or filtration step on the intermediate aqueous lipid dispersion to produce an aqueous dispersion, wherein the dialysis or filtration step removes organic solvents and adjusts the pH to about 6.5 to about 8.0. The present invention provides a method comprising:

[0019] In a twelfth aspect, the present invention provides a method for forming an aqueous dispersion having a pH of about 6.5 to about 8.0 and substantially free of organic solvents and nucleic acids, the method comprising: (A) (i) an acidified organic phase comprising a lipid mixture comprising a cationically ionizable lipid dissolved in a water-soluble organic solvent; and (ii) an aqueous phase having a pH of about 7.0 to about 8.5; to produce an intermediate aqueous lipid dispersion having a pH of about 6.5 to about 8.0; and (B) performing a dialysis or filtration step on the intermediate aqueous lipid dispersion using a buffer having a pH of about 6.5 to about 8.5 to produce an aqueous dispersion, wherein the dialysis or filtration step removes the organic solvent. The present invention provides a method comprising:

[0020] In a thirteenth aspect, the present invention provides a lyophilized composition comprising the aqueous dispersion of the fourth aspect.

[0021] Benefits and Surprising Findings The methods of the invention described and claimed herein expand the applicability of preformed LNPs (pre-LNPs) to processes in which pre-LNPs are manufactured and stored in the neutral pH range. LNPs produced according to the invention tend to exhibit good colloidal stability, RNA integrity, and broad compatibility with different primary packaging materials.

[0022] Additionally, in contrast to the methods described in WO 2022 / 032087, the methods of the present invention described and claimed herein do not use inorganic ions present in citrate and acetate buffers during pre-LNP production, thus avoiding the deleterious effects of inorganic ions on the lipid particle formulation. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 shows a general manufacturing scheme for forming pre-LNPs using either the "acidified buffer method" described in more detail in Example 1 or the "neutral buffer method" described in more detail in Example 2. [Figure 2] Schematic of an RNA-LNP manufacturing process using exemplary buffers to acidify either (A) the pre-LNP phase or (B) the RNA phase. [Figure 3] FIG. 1 shows the freeze-thaw stability of pre-LNPs (Example 1B) prepared with 5 mM acetic acid and purified with 5 mM Tris at approximately pH 7. [Figure 4] FIG. 1 shows the freeze-thaw stability of pre-LNPs (Example 1B) prepared with 5 mM acetic acid and purified with a mixture of 10 mM HEPES and 3 mM Tris at approximately pH 7. [Figure 5] FIG. 1 shows the freeze-thaw stability of pre-LNPs (Example 1C) prepared with 5 mM acetic acid and purified with 5 mM Tris at approximately pH 7. [Figure 6] FIG. 1 shows the freeze-thaw stability of pre-LNPs (Example 2A) produced in 30 mM Tris at pH 7 and purified in 5 mM Tris at approximately pH 7. [Figure 7] FIG. 1 shows the freeze-thaw stability of pre-LNPs (Example 3A) prepared with 5 mM acetic acid and purified with a mixture of 5 mM Tris and 4.5 mM acetic acid at approximately pH 7. [Figure 8] FIG. 1 shows the freeze-thaw stability of pre-LNPs (Example 3B) prepared with 5 mM acetic acid and purified with a mixture of 5 mM Tris and 2 mM malic acid at approximately pH 7. DETAILED DESCRIPTION OF THE INVENTION

[0024] The elements of the present disclosure are described in more detail below. While these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create further embodiments. The various examples and preferred embodiments described should not be construed as limiting the disclosure to only the explicitly described embodiments. The description should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application, unless the context dictates otherwise.

[0025] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)," H.G. W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995). The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the literature in the art (e.g., Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser / Heathcook, "Organische Chemie", VCH, 1990; Beyer / Walter, "Lehrbuch der Organischen Chemie", S. Hirzel Verlag Stuttgart, 1988; Carey / Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Rompp Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A 30 Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0026] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any examples or exemplary language (e.g., "such as") provided herein is intended merely to better describe the disclosure and does not pose a limitation on the scope of the otherwise claimed disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0027] Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and unless otherwise indicated herein, each separate value is incorporated herein as if it were individually referred to herein.

[0028]

[0001] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0029] definition The following provides definitions that apply to all aspects of this disclosure. The following terms have the following meanings unless otherwise indicated. Any terms not defined have their art-recognized meanings.

[0030] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated member, integer, or step, or group of members, integers, or steps, but not the exclusion of any other member, integer, or step, or group of members, integers, or steps. The term "consisting essentially of" means excluding other members, integers, or steps of any essential significance. The term "comprising" encompasses the term "consisting essentially of," which in turn encompasses the term "consisting of." Thus, wherever it appears in this application, the term "comprising" may be replaced by the term "consisting essentially of" or "consisting of." Similarly, wherever it appears in this application, the term "consisting essentially of" may be replaced by the term "consisting of."

[0031] As used in the context of describing this disclosure (particularly in the context of the claims), the terms "a," "an," and "the" and similar references are to be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0032] As used herein, "and / or" should be taken as a specific disclosure of each of the two specified features or components, with or without the other. For example, "X and / or Y" should be taken as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, just as if each were individually set forth herein.

[0033] In the context of the present disclosure, the term "about" indicates an interval of accuracy that a person skilled in the art would understand to still ensure the technical effect of the characteristic in question. This term typically indicates a deviation of ±5%, e.g., ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, e.g., ±0.01% from the indicated numerical value. For example, with respect to pH values, the term "about" may, in preferred examples, indicate a deviation of up to 0.3 from the indicated numerical value. As will be recognized by a person skilled in the art, the specific deviation of a numerical value for a given technical effect will depend on the nature of that technical effect. For example, natural or biological technical effects may generally have a greater deviation than man-made or engineered technical effects.

[0034] The phrase "substantially free of X," as used herein, means that the compositions described herein are free of X in a manner that is practically and realistically achievable. For example, if a mixture is substantially free of X, the amount of X in the mixture can be less than 1 wt. % (e.g., less than 0.5 wt. %, less than 0.4 wt. %, less than 0.3 wt. %, less than 0.2 wt. %, less than 0.1 wt. %, 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. %, less than 0.01 wt. %, less than 0.005 wt. %, or less than 0.001 wt. %) based on the total weight of the mixture. Specific meanings of the term "substantially free" in relation to certain components of the composition are as defined herein.

[0035] As used herein, "physiological pH" refers to a pH of about 7.5 or about 7.4. In some embodiments, the physiological pH is 7.3 to 7.5. In some embodiments, the physiological pH is 7.35 to 7.45. In some embodiments, the physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0036] As used herein, "physiological conditions" refers to the conditions (especially pH and temperature) in a living subject, particularly a human. Preferably, physiological conditions refer to physiological pH and / or a temperature of about 37°C.

[0037] As used in this disclosure, "mol %" is defined as the ratio of the number of moles of one component to the total number of moles of all components multiplied by 100.

[0038] As used in this disclosure, "mol % of lipid mixture" is defined as the ratio of the number of moles of a particular lipid component to the total number of moles of all lipids in the lipid mixture multiplied by 100. In this context, in some embodiments, the terms "total lipids" and / or "total lipid mixture" include lipids and lipid-like substances.

[0039] As used herein, the term "hydrocarbyl" refers to a monovalent organic group obtained by removing one H atom from a hydrocarbon molecule. In some embodiments, the hydrocarbyl group is acyclic, e.g., linear (straight-chain) or branched. Typical examples of hydrocarbyl groups include alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, aryl groups, and combinations thereof (e.g., arylalkyl (aralkyl), etc.). Specific examples of hydrocarbyl groups include C 1-40 Alkyl (e.g., C 6-40 Alkyl, C 6-30 Alkyl, C 6-20 Alkyl or C 10-20 alkyl), C with 1, 2 or 3 double bonds 2-40 Alkenyl (e.g., C 6-40 Alkenyl, C 6-30 Alkenyl, or C 6-20 alkenyl), aryl, and aryl (C 1-6 In some embodiments, the hydrocarbyl group may be substituted with one or more, such as 1, 2 or 3, such as 1 or 2, for example 1, substituents selected from List A.

[0040] The term "heterohydrocarbyl" means a hydrocarbyl group as defined above wherein 1, 2, 3 or 4 carbon atoms of the hydrocarbyl group have been replaced by an oxygen, nitrogen, silicon, selenium, phosphorus or sulfur heteroatom, preferably O, S or N. In one embodiment, the heterohydrocarbyl is substituted by one or more, for example 1, 2 or 3, for example 1 or 2, for example 1, substituents selected from List A.

[0041] The term "alkyl" refers to a saturated linear or branched hydrocarbon monoradical. Preferably, the alkyl group contains 1 to 40, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 carbon atoms, such as 1 to 30, for example 1 to 20 carbon atoms, for example 1 to 12 carbon atoms, for example 1 to 10 carbon atoms, for example 1 to 8 carbon atoms, for example 1 to 6 or 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl (also referred to as 2-propyl or 1-methylethyl), butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1,2-dimethylpropyl, isoamyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, isoheptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl, n-icosyl, n-triacontyl, n-tetracontyl, and the like. "Substituted alkyl" means that one or more (e.g., from 1 to a maximum of the number of hydrogen atoms bonded to the alkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of an alkyl group have been replaced with a non-hydrogen substituent (when more than one hydrogen atom has been replaced, the substituents may be the same or different). In one embodiment, the alkyl is substituted with one or more, e.g., 1, 2, or 3, e.g., 1 or 2, e.g., 1 substituent selected from List A. Examples of substituted alkyl include chloromethyl, dichloromethyl, fluoromethyl, and difluoromethyl.

[0042] The term "alkylene" refers to a saturated straight or branched hydrocarbon diradical. Preferably, the alkylene group contains 1 to 40, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 carbon atoms, such as 1 to 30, for example 1 to 20 carbon atoms, for example 1 to 12 carbon atoms, for example 1 to 10 carbon atoms, for example 1 to 8 carbon atoms, for example 1 to 6 or 1 to 4 carbon atoms. Exemplary alkylene groups include methylene, ethylene (i.e., 1,1-ethylene, 1,2-ethylene), propylene (i.e., 1,1-propylene, 1,2-propylene (-CH(CH)CH-), 2,2-propylene (-C(CH)-), and 1,3-propylene), butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2-butylene, 1,3-butylene, 2,3-butylene (cis or trans or mixtures thereof), 1,4-butylene, 1,1-isobutylene, 1,2-isobutylene, and 1,3-isobutylene), pentylene isomers (e.g., 1,1-pentylene, 1,2-pentylene, 1,3-pentylene, 1,4-pentylene, 1,5-pentylene, 1,1-isopentylene, , 1,1-sec-pentyl, 1,1-neopentyl), hexylene isomers (e.g., 1,1-hexylene, 1,2-hexylene, 1,3-hexylene, 1,4-hexylene, 1,5-hexylene, 1,6-hexylene, 1,1-isohexylene), heptylene isomers (e.g., 1,1-heptylene, 1,2-heptylene, 1,3-heptylene, 1,4-heptylene), These include isomers of octylene (e.g., 1,1-octylene, 1,5-heptylene, 1,6-heptylene, 1,7-heptylene, 1,1-isoheptylene), octylene isomers (e.g., 1,1-octylene, 1,2-octylene, 1,3-octylene, 1,4-octylene, 1,5-octylene, 1,6-octylene, 1,7-octylene, 1,8-octylene, 1,1-isooctylene), etc. A linear alkylene moiety having at least three carbon atoms and a free valence at each end can also be designated as multiple methylenes (e.g., 1,4-butylene may be referred to as tetramethylene).In general, instead of using the suffix "ylene" for the alkylene moiety as specified above, the suffix "diyl" can also be used (e.g., 1,2-butylene can be referred to as butane-1,2-diyl). "Substituted alkylene" means that one or more (e.g., from 1 to up to the maximum number of hydrogen atoms bonded to the alkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of the alkylene group have been replaced with non-hydrogen substituents (when more than one hydrogen atom has been replaced, the substituents can be the same or different). In one embodiment, the alkylene is substituted with one or more, e.g., 1, 2, or 3, e.g., 1 or 2, e.g., 1 substituent selected from List A.

[0043] The term "alkenyl" refers to an unsaturated, linear or branched hydrocarbon monoradical having at least one carbon-carbon double bond. Generally, the maximum number of carbon-carbon double bonds in an alkenyl group can be equal to the integer calculated by dividing the number of carbon atoms in the alkenyl group by 2, and, if the number of carbon atoms in the alkenyl group is odd, rounding the result of the division to the next lower integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds. Preferably, the alkenyl group contains 2 to 40 carbon atoms, such as 2 to 30 carbon atoms, such as 2 to 20 carbon atoms, such as 2 to 12 carbon atoms, such as 2 to 10 carbon atoms, such as 2 to 8 carbon atoms, such as 2 to 6 carbon atoms, or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenyl group contains 2 to 40, such as 2 to 30, such as 2 to 20, such as 2 to 12, for example 2 to 10 carbon atoms and 1, 2, 3, 4, 5, or 6 (for example 1, 2, 3, 4, or 5) carbon-carbon double bonds, for example 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, for example 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bonds may be in the cis (Z) or trans (E) configuration.Exemplary alkenyl groups include vinyl, 1-propenyl, 2-propenyl (i.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, and the like. , 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5 Examples include 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11-dodecenyl, etc. "Substituted alkenyl" means that one or more (e.g., from 1 to up to the maximum number of hydrogen atoms bonded to the alkenyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of an alkenyl group have been replaced with a non-hydrogen substituent (when more than one hydrogen atom has been replaced, the substituents can be the same or different). In one embodiment the alkenyl is substituted by one or more, such as 1, 2 or 3, such as 1 or 2, for example 1, substituents selected from list A.

[0044] The term "alkenylene" refers to an unsaturated linear or branched hydrocarbon diradical having at least one carbon-carbon double bond. Generally, the maximum number of carbon-carbon double bonds in an alkenylene group can be equal to the integer calculated by dividing the number of carbon atoms in the alkenylene group by 2, and, if the number of carbon atoms in the alkenylene group is odd, rounding the result of the division to the next lower integer. For example, for an alkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds. Preferably, the alkenylene group contains 2 to 12 (e.g., 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 2 to 8 carbon atoms, e.g., 2 to 6 carbon atoms, or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenylene group contains 2 to 12 (e.g., 2 to 10 carbon atoms) atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, e.g., 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bonds may be in a cis (Z) or trans (E) configuration. Exemplary alkenylene groups include ethene-1,2-diyl, vinylidene (also called ethenylidene), 1-propene-1,2-diyl, 1-propene-1,3-diyl, 1-propene-2,3-diyl, allylidene, 1-butene-1,2-diyl, 1-butene-1,3-diyl, 1-butene-1,4-diyl, 1-butene-2,3-diyl, 1-butene-2,4-diyl, 1-butene-3,4-diyl, 2-butene-1,2-diyl, 2-butene-1,3-diyl, 2-butene-1,4-diyl, 2-butene-2,3-diyl, 2-butene-2,4-diyl, 2-butene-3,4-diyl, and the like."Substituted alkenylene" means that one or more (e.g., 1 up to the maximum number of hydrogen atoms bonded to the alkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 15, up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of an alkenylene group have been replaced with a non-hydrogen substituent (when more than one hydrogen atom has been replaced, the substituents may be the same or different). In one embodiment, the alkenylene is substituted with one or more, e.g., 1, 2, or 3, e.g., 1 or 2, e.g., 1 substituent selected from List A.

[0045] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond, wherein the total number of carbon atoms therein may be 6 to 40, e.g., 6 to 30, typically 6 to 20, e.g., 6 to 18. An alkynyl group may have one or more carbon-carbon triple bonds. Generally, the maximum number of carbon-carbon triple bonds in an alkynyl group may be equal to the integer calculated by dividing the number of carbon atoms in the alkynyl group by 2, and, if the number of carbon atoms in the alkynyl group is odd, rounding the result of the division to the next lower integer. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the alkynyl group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, more preferably 1 or 2, carbon-carbon triple bonds. "Substituted alkynyl" means that one or more (e.g., from 1 up to the maximum number of hydrogen atoms bonded to the alkynyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of an alkynyl group have been replaced with a non-hydrogen substituent (when more than one hydrogen atom has been replaced, the substituents may be the same or different). In one embodiment, the alkynyl is substituted with one or more, e.g., 1, 2, or 3, e.g., 1 or 2, e.g., 1 substituent selected from List A.

[0046] The terms "cycloalkyl" and "cycloalkenyl" refer to cyclic, non-aromatic versions of "alkyl" and "alkenyl," preferably containing 3 to 40, e.g., 3 to 30, e.g., 3 to 20, e.g., 3 to 14, carbon atoms, e.g., 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, and adamantyl. Exemplary cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclononenyl, and cyclodecenyl. A cycloalkyl or cycloalkenyl group can consist of one ring (monocyclic), two rings (bicyclic), or three or more rings (polycyclic). "Substituted cycloalkyl" means that one or more (e.g., from 1 to up to the maximum number of hydrogen atoms bonded to the cycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of the cycloalkyl group have been replaced with a non-hydrogen substituent (where two or more hydrogen atoms have been replaced, the substituents may be the same or different). In one embodiment, the cycloalkyl or cycloalkenyl is substituted with one or more, e.g., one, two, or three, e.g., one or two, e.g., one substituent selected from List A.

[0047] The terms "cycloalkylene" and "cycloalkenylene" refer to cyclic, non-aromatic versions of "alkylene" and "alkenylene," preferably containing 3 to 40, e.g., 3 to 30, e.g., 3 to 20, e.g., 3 to 14, carbon atoms, e.g., 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkylene groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cycloheptylene. Exemplary cycloalkylene groups include cyclopentenylene and cyclohexenylene.

[0048] The term "aryl" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, an aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10, e.g., 5, 6, or 10) carbon atoms, which can be arranged in a single ring (e.g., phenyl) or two or more fused rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Aryl does not include fullerenes. "Substituted aryl" means that one or more (e.g., up to the maximum number of hydrogen atoms bonded to the aryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 5, or 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of an aryl group have been replaced with a non-hydrogen substituent (when more than one hydrogen atom has been replaced, the substituents may be the same or different). In one embodiment, the aryl is substituted with one or more, e.g., 1, 2, or 3, e.g., 1 or 2, e.g., 1 substituent selected from List A. Examples of substituted aryl include biphenyl, 2-fluorophenyl, 2-chloro-6-methylphenyl, anilinyl, 4-hydroxyphenyl, and methoxyphenyl (i.e., 2-, 3-, or 4-methoxyphenyl).

[0049] The term "heteroaryl" or "heteroaromatic ring" refers to an aryl group, as defined above, in which one or more carbon atoms of the aryl group are replaced by an O, S, or N heteroatom. Preferably, heteroaryl refers to a 5- or 6-membered aromatic monocyclic ring in which one, two, or three carbon atoms are replaced by the same or different O, N, or S heteroatoms. Alternatively, it refers to an aromatic bicyclic or tricyclic ring system in which one, two, three, four, or five carbon atoms are replaced by the same or different O, N, or S heteroatoms. Preferably, in each ring of a heteroaryl group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. Exemplary heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, and the like. These include zolyl, quinolinyl, isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl, pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrolothiazolyl, phenothiazinyl, isobenzofuranyl, chromenyl, xanthenyl, pyrrolidinyl, indolizinyl, indazolyl, purinyl, quinolizinyl, phthalazinyl, naphthyridinyl, cinnolinyl, pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, and phenazinyl. Exemplary 5- or 6-membered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl (e.g., 4-pyridyl), pyrimidinyl, pyrazinyl, triazinyl, and pyridazinyl."Substituted heteroaryl" means that one or more (e.g., up to a maximum of the number of hydrogen atoms bonded to the heteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of a heteroaryl group have been replaced with a non-hydrogen substituent (when more than one hydrogen atom has been replaced, the substituents may be the same or different). In one embodiment, the heteroaryl is substituted with one or more, e.g., one, two, or three, e.g., one or two, e.g., one, substituent selected from List A.

[0050] The term "heterocyclyl" or "heterocyclic ring" refers to a cycloalkyl group, as defined above, in which 1, 2, 3, or 4 carbon atoms of the cycloalkyl group have been replaced by a heteroatom of oxygen, nitrogen, silicon, selenium, phosphorus, or sulfur, preferably O, S, or N. A heterocyclyl group preferably has one or two rings containing 3 to 10, e.g., 3, 4, 5, 6, or 7, ring atoms. Preferably, in each ring of a heterocyclyl group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The term "heterocyclyl" is also meant to include partially or fully hydrogenated forms (e.g., dihydro, tetrahydro, or perhydro forms) of the heteroaryl groups mentioned above. Exemplary heterocyclyl groups include morpholinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl (also called piperidyl), piperazinyl, di- and tetra-hydrofuranyl, di- and tetra-hydrothienyl, di- and tetra-hydropyranyl, urotropinyl, lactone, lactam, cyclic imide, and cyclic anhydride. "Substituted heterocyclyl" means that one or more (e.g., from 1 to up to the maximum number of hydrogen atoms bonded to the heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heterocyclyl group have been replaced with non-hydrogen substituents (when more than one hydrogen atom has been replaced, the substituents can be the same or different). In one embodiment the heterocyclyl is substituted by one or more, such as 1, 2 or 3, such as 1 or 2, for example 1, substituents selected from list A.

[0051] The term "alkylcycloalkyl" refers to a cycloalkyl group, as defined above, substituted by an alkyl group, as defined above, wherein the cycloalkyl portion is connected to the rest of the molecule. Each of the cycloalkyl and alkyl portions of this group may have any of the broadest or preferred meanings mentioned above. "Substituted alkylcycloalkyl" means that one or more (e.g., up to the maximum number of hydrogen atoms bonded to the alkylcycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of the alkyl or cycloalkyl portion of this group have been replaced with non-hydrogen substituents (when more than one hydrogen atom has been replaced, the substituents may be the same or different). In one embodiment, the alkylcycloalkyl is substituted with one or more, e.g., 1, 2, or 3, e.g., 1 or 2, e.g., 1, 2, or 3 ...

[0052] The term "cycloalkylalkyl" refers to an alkyl group, as defined above, substituted by a cycloalkyl group, as defined above, wherein the alkyl portion is connected to the rest of the molecule. Each of the cycloalkyl and alkyl portions of this group may have any of the broadest or preferred meanings mentioned above. "Substituted cycloalkylalkyl" means that one or more (e.g., up to the maximum number of hydrogen atoms bonded to the cycloalkylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of the alkyl or cycloalkyl portion of this group have been replaced with non-hydrogen substituents (when more than one hydrogen atom has been replaced, the substituents may be the same or different). In one embodiment, the cycloalkylalkyl is substituted with one or more, e.g., 1, 2, or 3, e.g., 1 or 2, e.g., 1, 2, or 3 ...

[0053] The term "alkylcycloalkylalkyl" refers to an alkyl group, as defined above, substituted by a cycloalkyl group, as defined above, wherein the alkyl portion is connected to the remainder of the molecule and the cycloalkyl portion is in turn substituted by a further alkyl group. Each of the cycloalkyl and alkyl portions of the group can have any of the broadest or preferred meanings mentioned above. "Substituted alkylcycloalkylalkyl" means that one or more (e.g., up to the maximum number of hydrogen atoms bonded to the alkylcycloalkylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of the alkyl or cycloalkyl portion of the group have been replaced with non-hydrogen substituents (when more than one hydrogen atom has been replaced, the substituents can be the same or different). In one embodiment, the alkylcycloalkylalkyl is substituted with one or more, e.g., 1, 2, or 3, e.g., 1 or 2, e.g., 1, 2, or 3 ...

[0054] The term "alkylaryl" refers to an aryl group, as defined above, substituted by an alkyl group, as defined above, wherein the aryl moiety is connected to the rest of the molecule. Each of the aryl and alkyl moieties of the group may have any of the broadest or preferred meanings mentioned above. "Substituted alkylaryl" means that one or more (e.g., up to the maximum number of hydrogen atoms bonded to the alkylaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of the alkyl or aryl portion of the group have been replaced with a non-hydrogen substituent (when more than one hydrogen atom has been replaced, the substituents may be the same or different). In one embodiment, the alkylaryl is substituted with one or more, e.g., 1, 2, or 3, e.g., 1 or 2, e.g., 1, 2, or 3 ...

[0055] The term "arylalkyl" refers to an alkyl group, as defined above, substituted by an aryl group, as defined above, wherein the alkyl portion is connected to the rest of the molecule. Each of the aryl and alkyl portions of the group may have any of the broadest or preferred meanings mentioned above. "Substituted arylalkyl" means that one or more (e.g., up to the maximum number of hydrogen atoms bonded to the arylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of the alkyl or aryl portion of the group have been replaced with non-hydrogen substituents (when two or more hydrogen atoms have been replaced, the substituents may be the same or different). In one embodiment, the arylalkyl is substituted with one or more, e.g., one, two, or three, e.g., one or two, e.g., one, two, or three, substituents selected from List A.

[0056] The term "alkylheteroaryl" refers to a heteroaryl group, as defined above, substituted by an alkyl group, as defined above, wherein the heteroaryl moiety is connected to the rest of the molecule. Each of the heteroaryl and alkyl moieties of the group can have any of the broadest or preferred meanings mentioned above. "Substituted alkylheteroaryl" means that one or more hydrogen atoms (e.g., up to the maximum number of hydrogen atoms bonded to the alkylheteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) of the alkyl or heteroaryl moieties of the group have been replaced with non-hydrogen substituents (when more than one hydrogen atom has been replaced, the substituents can be the same or different). In one embodiment, the alkylheteroaryl is substituted with one or more, e.g., 1, 2, or 3, e.g., 1 or 2, e.g., 1, 2, or 3 ...

[0057] The term "heteroarylalkyl" refers to an alkyl group, as defined above, substituted by a heteroaryl group, as defined above, wherein the alkyl portion is connected to the rest of the molecule. Each of the aryl and alkyl portions of the group may have any of the broadest or preferred meanings mentioned above. "Substituted heteroarylalkyl" means that one or more hydrogen atoms (e.g., up to the maximum number of hydrogen atoms bonded to the heteroarylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) of the alkyl or heteroaryl portions of the group have been replaced with non-hydrogen substituents (when more than one hydrogen atom has been replaced, the substituents may be the same or different). In one embodiment, the heteroarylalkyl is substituted with one or more, e.g., 1, 2, or 3, e.g., 1 or 2, e.g., 1, 2, or 3 ...

[0058] The term "alkylheterocyclyl" refers to a heterocyclyl group, as defined above, substituted by an alkyl group, as defined above, wherein the heteroaryl moiety is connected to the rest of the molecule. Each of the heterocyclyl and alkyl moieties of this group may have any of the broadest or preferred meanings mentioned above. "Substituted alkylheterocyclyl" means that one or more (e.g., up to 1 up to the maximum number of hydrogen atoms bonded to the alkylheterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkyl or heteroaryl portion of this group have been replaced with non-hydrogen substituents (where two or more hydrogen atoms have been replaced, the substituents may be the same or different). In one embodiment, the alkylheterocyclyl is substituted with one or more, e.g., 1, 2, or 3, e.g., 1 or 2, e.g., 1, 2, or 3 ...

[0059] The term "heterocyclylalkyl" refers to an alkyl group, as defined above, substituted by a heterocyclyl group, as defined above, wherein the alkyl portion is connected to the rest of the molecule. Each of the heterocyclyl and alkyl portions of this group may have any of the broadest or preferred meanings mentioned above. "Substituted heterocyclylalkyl" means that one or more (e.g., up to the maximum number of hydrogen atoms bonded to the heterocyclylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkyl or heterocyclyl portion of this group have been replaced with non-hydrogen substituents (when more than one hydrogen atom has been replaced, the substituents may be the same or different). In one embodiment, the heterocyclylalkyl is substituted with one or more, e.g., 1, 2, or 3, e.g., 1 or 2, e.g., 1, 2, or 3 ...

[0060] The term "organic sulfuric acid" or "sulfate" refers to a compound of formula R-OSO-OH, where R is a hydrocarbyl or heterohydrocarbyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above in their broadest or preferred embodiments). The term "sulfate" is used when this group is deprotonated. Depending on the pH, the sulfate group is either protonated or deprotonated.

[0061] The term "sulfonic acid" or "sulfonate" refers to a compound of formula R-SO-OH, where R is a hydrocarbyl or heterohydrocarbyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above in their broadest or preferred embodiments). The term "sulfonate" is used when the group is deprotonated. Depending on the pH, the sulfonate group is either protonated or deprotonated.

[0062] The term "carboxylic acid" or "carboxylate" refers to a compound of formula R-COH, where R is a hydrocarbyl or heterohydrocarbyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above in their broadest or preferred embodiments). The term "carboxylate" is used when this group is deprotonated. Depending on the pH, the carboxylic acid is either protonated or deprotonated.

[0063] The term "dicarboxylic acid" or "dicarboxylate" refers to a compound of formula HO2C-R'-CO2H, where R' is an alkylene or alkenylene group (all as defined above in their broadest or preferred embodiments). The term "dicarboxylate" is used when this group is deprotonated. Depending on the pH, the dicarboxylic acid is either protonated or deprotonated.

[0064] The term "hydroxycarboxylic acid" or "hydroxycarboxylate" refers to a compound of formula R-COH, where R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above in their broadest or preferred embodiments), which is substituted with one or more (preferably 1 to 5, e.g., 1, 2, or 3) hydroxy groups. The term "hydroxycarboxylate" is used when the group is deprotonated. Depending on the pH, the hydroxycarboxylic acid may be protonated or deprotonated.

[0065] As used herein, the term "ester" refers to a compound having the structure RC(O)O-R' (including the isomeric structure R-OC(O)-R' unless otherwise specified), where R and R' are each independently a hydrocarbyl or heterohydrocarbyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above in their broadest or preferred aspects). When this term denotes a substituent connected to the remainder of the molecule, the ester moiety may have the structure RC(O)O- or R-OC(O)-, where R is as defined above. In one embodiment, each of the two ends of the ester structure is covalently linked to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of a linker).

[0066] The term "phosphate" refers to a compound of formula R-O-P(=O)(OH)2, where R is a hydrocarbyl or heterohydrocarbyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above in their broadest or preferred embodiments). Depending on the pH, the phosphate group is protonated or deprotonated.

[0067] The term "phosphonate" refers to a compound of formula R-P(=O)(OH)2, where R is a hydrocarbyl or heterohydrocarbyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above in their broadest or preferred embodiments). Depending on the pH, the phosphonate group is protonated or deprotonated.

[0068] "Halo" means fluoro (-F), chloro (-Cl), bromo (-Br), or iodo (-I).

[0069] "Amine" refers to the group -NR2, where each R is a hydrocarbyl or heterohydrocarbyl group, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above in their broadest or preferred embodiments), preferably an alkyl group, such as C1-6 It is an alkyl group. If both groups R are hydrogen, the amine group is a primary amine group. If one R is hydrogen and the other R is other than hydrogen, the amine group is a secondary amine group. If both groups R are other than hydrogen, the amine group is a tertiary amine group.

[0070] "Quaternary ammonium" salts are -N + Compounds containing an R group, each R being a hydrocarbyl or heterohydrocarbyl group such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above in their broadest or preferred embodiments), preferably an alkyl group, such as C 1-6 In contrast to some amines, as defined above, which are protonated only at certain pH levels, quaternary ammonium salts carry a constitutive positive charge (as defined above) at all pH levels.

[0071] "Hydroxyl" means the group -OH. "Sulfhydryl" means the group -SH. "Nitro" means the group -NO2.

[0072] "Ether" means an oxygen atom to which two hydrocarbyl or heterohydrocarbyl groups, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl groups, (all as defined above in their broadest or preferred embodiments) are bonded. The ether may be a cyclic ether, where the two hydrocarbyl groups together form a ring, and may include a dioxolane group.

[0073] "Thioether" means a sulfur atom to which two hydrocarbyl or heterohydrocarbyl groups, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl groups, (all as defined above in their broadest or preferred embodiments) are bonded. The ether may be a cyclic thioether, where the two hydrocarbyl groups together form a ring, and may include a dithiane group.

[0074] "Amido" refers to the group -C(=O)NR(R'), where R and R' are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above in their broadest or preferred embodiments), preferably an alkyl group, such as C 1-6 It is an alkyl group.

[0075] "Hydroxylamido" refers to the group -C(=O)O-NR(R'), where R and R' are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group, all as defined above in their broadest or preferred embodiments.

[0076] "Sulfonamido" refers to the group -S(=O)NRR', where R and R' are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above in their broadest or preferred embodiments), preferably an alkyl group, such as C 1-6 It is an alkyl group.

[0077] "Carbamate" refers to the group -OC(=O)NRR', where R and R' are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above in their broadest or preferred embodiments), preferably an alkyl group, such as C 1-6 It is an alkyl group.

[0078] "Amidine" refers to the group -C(=NR)NR'R" where R, R', and R" are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above in their broadest or preferred embodiments), preferably an alkyl group such as C 1-6 It is an alkyl group.

[0079] "Guanidine" refers to the group -NR-C(=NR')NR"R"' or =NC(NR"R"') where R, R', R", and R"' are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above in their broadest or preferred embodiments), preferably an alkyl group, such as C 1-6 It is an alkyl group.

[0080] The above definitions, when referring to any basic nitrogen atom that is protonated, may be modified by the substitution of the suffix "ium," in accordance with standard chemical nomenclature. For example, a guanidinium group is a protonated guanidine, an ammonium group is a protonated ammonia or a protonated primary, secondary, or tertiary amine, an imidazolium group is a protonated imidazole, a pyridinium group is a protonated pyridine, an amidinium group is a protonated amidine, and a piperazinium group is a protonated piperazine.

[0081] "Carbohydrates" have the empirical formula C m (H2O) nwhere m may or may not be different from n. The term "carbohydrate residue" or "carbohydrate moiety" defines a carbohydrate residue in which one hydrogen atom of a carbohydrate is replaced by a bond to another atom. The carbohydrate moiety may be a monosaccharide moiety. The monosaccharide moiety may have a D- or L-configuration. Furthermore, the monosaccharide moiety may be an aldose moiety or a ketose moiety. Suitably, the monosaccharide moiety may have 3 to 8, preferably 4 to 6, and more preferably 5 or 6 carbon atoms. In one embodiment, the monosaccharide moiety is a hexose moiety (i.e., it has 6 carbon atoms). Examples include aldohexoses such as glucose, galactose, allose, altose, mannose, gulose, idose, and talose, and ketohexoses such as fructose and sorbose. Preferably, the hexose moiety is a glucose moiety.

[0082] In another embodiment, the monosaccharide moiety is a pentose moiety (i.e., which has 5 carbon atoms), such as ribose, arabinose, xylose, or lyxose. Preferably, the pentose moiety is an arabinose or xylose moiety.

[0083] In another embodiment, the carbohydrate may be a higher sugar (i.e., a disaccharide or oligosaccharide) consisting of two or more monosaccharide moieties linked together by a glycosidic bond. When the monosaccharide moiety is a hexose moiety, the glycosidic bond may be a 1-α,1'-α glycosidic bond, a 1,2'-glycosidic bond (which may be a 1-α2' or a 1'-β2' glycosidic bond), a 1,3'-glycosidic bond (which may be a 1-α-3' or a 1-β-3' glycosidic bond), a 1,4'-glycosidic bond (which may be a 1-α-4' or a 1-β-4' glycosidic bond), a 1,6'-glycosidic bond (which may be a 1-α-6' or a 1-β-6' glycosidic bond), or any combination thereof. In one embodiment, the higher sugar is composed of two monosaccharide units (i.e., a disaccharide). Examples of suitable disaccharides include maltose, isomaltose, isomaltulose, lactose, sucrose, cellobiose, nigerose, kojibiose, trehalose, and trehalulose. In another embodiment, the higher sugars are branched or unbranched chains of 3 to 10 monosaccharide units (i.e., oligosaccharides). Preferably, oligosaccharides are 3 to 8, more preferably 3 to 6, monosaccharide units. Examples of suitable oligosaccharides include maltodextrin, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, melezitose, cellotriose, cellotetraose, cellopentaose, cellohexaose, and celloheptaose.

[0084] "List A" substituents are C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, 6- to 14-membered (e.g., 6- to 10-membered) aryl, 3- to 14-membered (e.g., 5- or 6-membered) heteroaryl, 3- to 14-membered (e.g., 3- to 7-membered) cycloalkyl, 3- to 14-membered (e.g., 3- to 7-membered) heterocyclyl, halogen, -CN, azide, -NO2, -OR', -N(R')2, -S(O) 0-2 R', -S(O) 1-2 OR', -OS(O) 1-2 R', -OS(O) 1-2 OR', -S(O) 1-2N(R')2, -OS(O) 1-2 N(R')2, -N(R')S(O) 1-2 R', -N(R')S(O) 1-2 OR', -C(=X 1 )R', -C(=X 1 )X 1 R', -X 1 C(=X 1 )R', and -X 1 C(=X 1 )X 1 R' is selected from the group consisting of X 1 are independently selected from O, S, NH, and N(CH3), and each R' is independently H, C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 and each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is selected from the group consisting of C 1-3 Alkyl, halogen, -CF3, -CN, azide, -NO2, -OH, -O(C 1-3 alkyl), -S(C 1-3 alkyl), -NH2, -NH(C 1-3 alkyl), -N(C 1-3 alkyl)2, -NHS(O)2(C 1-3 alkyl), -S(O)2NH 2-z (C 1-3 alkyl) z , -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)NH 2-z (C 1-3 alkyl) z , -NHC(=O)(C 1-3 alkyl), -NHC(=NH)NH z-2 (C 1-3 alkyl) z , and -N(C 1-3 alkyl)C(=NH)NH 2-z (C 1-3 alkyl) zwherein each z is independently 0, 1, or 2; and each C 1-3 Alkyl is independently methyl, ethyl, or propyl. In some embodiments, the substituents of List A are C 1-3 Alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH 2-z (CH3) z , —C(═O)OH, and —C(═O)OCH3, z is 0, 1, or 2, and C 1-3 Alkyl is methyl, ethyl, propyl, or isopropyl. In some embodiments, the substituents of List A are selected from List A2 consisting of methyl, ethyl, propyl, isopropyl, halogen (e.g., F, Cl, or Br), and —CF 3 .

[0085] nucleic acid The lipid particle composition of the present application contains an active ingredient. The active ingredient is a nucleic acid. Preferably, the lipid particle composition of the present application contains RNA, such as mRNA. Typically, the lipid particle composition described herein comprises lipid particles encapsulating a nucleic acid. The term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. This term includes genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is mRNA. In one embodiment, the nucleic acid is DNA.

[0086] Nucleic acids can exist as single-stranded or double-stranded, and linear or covalently linked circular closed molecules. Nucleic acids can be isolated. The term "isolated nucleic acid," according to the present disclosure, means that the nucleic acid has been (i) amplified in vitro, for example, by polymerase chain reaction (PCR) of DNA or by in vitro transcription of RNA (e.g., using RNA polymerase), (ii) recombinantly produced by cloning, (iii) purified, for example, by cleavage and separation by gel electrophoresis, or (iv) synthesized, for example, by chemical synthesis.

[0087] The term "nucleoside" refers to a compound that can be considered a nucleotide without a phosphate group. A nucleoside is a nucleic acid base linked to a sugar (e.g., ribose or deoxyribose), while a nucleotide consists of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine. A nucleic acid may contain one or more modified nucleosides or nucleotides. Examples of modified nucleosides or nucleotides that can be incorporated into nucleic acids include N7-alkylguanine, N6-alkyladenine, 5-alkylcytosine, 5-alkyluracil, and N(1)-alkyluracil, such as N7-C1-4 alkylguanine, N6-C1-4 alkyladenine, 5-C1-4 alkylcytosine, 5-C1-4 alkyluracil, and N(1)-C1-4 alkyluracil, preferably N7-methylguanine, N6-methyladenine, 5-methylcytosine, 5-methyluridine (m5U), pseudouridine (Ψ), and N1-methylpseudouridine (m1Ψ).

[0088] RNA In some embodiments of all aspects of the present disclosure, the nucleic acid is RNA. According to the present disclosure, the term "RNA" refers to a nucleic acid molecule containing ribonucleotide residues. RNA typically includes naturally occurring nucleic acids, adenosine (A), uridine (U), cytidine (C), and guanosine (G). In a preferred embodiment, RNA contains all or most ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, without limitation, double-stranded RNA, single-stranded RNA, isolated RNA, e.g., partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the termini of the RNA. As used herein, it is also contemplated that the nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For purposes of this disclosure, these modified / altered nucleotides (or modified nucleosides) can be referred to as analogs of naturally occurring nucleotides (nucleosides), and the corresponding RNA (i.e., modified / altered RNA) comprising such modified / altered nucleotides or nucleosides can be referred to as analogs of naturally occurring RNA. A molecule contains a "majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is greater than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or their analogs)."RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), trans-amplifying RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (e.g., antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (e.g., small activating RNA), and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA. The active component may be mRNA, saRNA, taRNA, or a combination thereof. The active component is preferably mRNA. In some instances, the active component is not siRNA.

[0089] In a preferred embodiment, the RNA comprises an open reading frame (ORF) encoding a peptide, polypeptide, or protein. The RNA may be capable of expressing or configured to express the encoded peptide, polypeptide, or protein. For example, the RNA may encode, express, or be configured to express a pharmaceutically active peptide or protein. In some embodiments, the RNA may interact with the cell's translation machinery to enable translation of the peptide or protein. The cell may produce the encoded peptide or protein intracellularly (e.g., in the cytoplasm), secrete the encoded peptide or protein, or produce it on its surface. Alternatively, the RNA may be non-coding RNA, such as antisense RNA, microRNA (miRNA), or siRNA.

[0090] mRNA In preferred embodiments of all aspects of the present disclosure, the nucleic acid is mRNA. According to the present disclosure, the term "mRNA" means "messenger RNA" and includes "transcripts" that can be produced by using a DNA template. Generally, mRNA encodes a peptide, polypeptide, or protein. As established in the art, RNA (e.g., mRNA) generally comprises a 5' untranslated region (5'-UTR), a peptide / polypeptide / protein coding region, and a 3' untranslated region (3'-UTR). Typically, mRNA comprises a 5' cap, a 5' UTR, a peptide / polypeptide / protein coding region, a 3' UTR, and a poly-A tail.

[0091] Although mRNA is single-stranded, it may contain self-complementary sequences that allow part of the mRNA to fold back on itself and pair with itself to form a double helix.

[0092] According to the present disclosure, "dsRNA" means double-stranded RNA, which is RNA having two strands that are partially or completely complementary.

[0093] In a preferred embodiment of the present disclosure, mRNA relates to an RNA transcript that encodes a peptide, polypeptide, or protein.

[0094] In some embodiments, preferably the RNA encoding the peptide, polypeptide, or protein has a length of at least 45 nucleotides (e.g., at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, e.g., up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides, or up to 10,000 nucleotides.

[0095] In some embodiments, RNA (e.g., mRNA) is produced by in vitro transcription or chemical synthesis. Preferably, RNA (e.g., mRNA) is produced by in vitro transcription using a DNA template. As used herein, the term "in vitro transcription" or "IVT" means that transcription (production of RNA) is carried out in a cell-free manner. That is, IVT does not use live / cultured cells, but rather uses transcription machinery extracted from cells (e.g., cell lysate or isolated components thereof containing RNA polymerase (preferably T7, T3, or SP6 polymerase)). Methodologies for in vitro transcription are known to those skilled in the art; see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. Additionally, various in vitro transcription kits are commercially available from, for example, Thermo Fisher Scientific (e.g., TranscriptAid™ T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (e.g., HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit), Promega (e.g., RiboMAX™, HeLaScribe®, Riboprobe® systems), Jena Bioscience (e.g., SP6 or T7 transcription kits), and Epicentre (e.g., AmpliScribe™).

[0096] To provide a modified RNA (e.g., mRNA), the corresponding modified nucleotides, e.g., modified naturally occurring nucleotides, non-naturally occurring nucleotides, and / or modified non-naturally occurring nucleotides, can be introduced during synthesis (preferably in vitro transcription), or modifications can be introduced and / or added to the mRNA after transcription. RNA (e.g., mRNA) may be modified. RNA (e.g., mRNA) may contain modified nucleotides or nucleosides, such as 5-methylcytosine, 5-methyluridine (m5U), pseudouridine (Ψ), or N(1)-methylpseudouridine (m1Ψ). One or more uridines in the RNA described herein may be replaced by modified nucleosides. The modified nucleoside may be a modified uridine. The RNA may contain a modified nucleoside in place of at least one uridine. Preferably, the RNA may include a modified nucleoside in place of each uridine (e.g., all of the uridines in the RNA are replaced by modified nucleosides). The modified nucleosides may be independently selected from pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), and 5-methyluridine (m5U). The modified nucleoside is preferably pseudouridine (Ψ) or N1-methylpseudouridine (m1Ψ).

[0097] In some embodiments, the RNA (e.g., mRNA) is in vitro transcribed RNA (IVT-RNA), obtained by in vitro transcription of a suitable DNA template. The promoter controlling the transcription can be any promoter for any RNA polymerase. Specific examples of RNA polymerases include T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of the RNA.

[0098] In some embodiments of the present disclosure, the RNA (e.g., mRNA) is a "replicon RNA" (e.g., a "replicon mRNA"), or simply a "replicon," particularly a "self-replicating RNA" (e.g., a "self-replicating mRNA") or a "self-amplifying RNA" (or "self-amplifying mRNA"). The RNA-containing lipid particles described herein may contain mRNA, saRNA, taRNA, or mixtures thereof. The RNA-containing lipid particles described herein may contain mRNA encoding a replicase protein and one or more RNA molecules capable of being replicated or amplified by a replicase.

[0099] inhibitory RNA In some embodiments of all aspects of the disclosure, the nucleic acid is an inhibitory RNA.

[0100] As used herein, the term "inhibitory RNA" refers to RNA that selectively hybridizes to and / or is specific for a target mRNA, thereby inhibiting (e.g., reducing) its transcription and / or translation. Inhibitory RNA includes RNA molecules having a sequence in an antisense orientation relative to the target mRNA. Suitable inhibitory oligonucleotides typically range in length from five to several hundred nucleotides, more typically about 20 to 70 nucleotides or shorter, and even more typically about 10 to 30 nucleotides in length. Examples of inhibitory RNA include antisense RNA, ribozymes, iRNA, siRNA, and miRNA. In some embodiments of all aspects of the present disclosure, the inhibitory RNA is an siRNA.

[0101] The term "antisense RNA" as used herein refers to RNA that hybridizes to DNA containing a specific gene or mRNA of the gene under physiological conditions, thereby inhibiting transcription of the gene and / or translation of the mRNA. The size of the antisense RNA can vary from 15 nucleotides to 15,000, preferably 20 to 12,000, particularly 100 to 10,000, 150 to 8,000, 200 to 7,000, 250 to 6,000, or 300 to 5,000 nucleotides, for example, 15 to 2,000, 20 to 1,000, 25 to 800, 30 to 600, 35 to 500, 40 to 400, 45 to 300, 50 to 250, 55 to 200, 60 to 150, or 65 to 100 nucleotides.

[0102] As used herein, "small interfering RNA" or "siRNA" refers to an RNA molecule capable of specifically binding to a portion of a target mRNA, preferably greater than 10 nucleotides in length, more preferably greater than 15 nucleotides in length, and most preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. This binding induces cleavage or degradation of the portion of the target mRNA, thereby inhibiting gene expression of the target mRNA. The most preferred size range for siRNAs is 19 to 25 nucleotides. Typically, siRNAs comprise a single molecule in which two complementary portions are base-paired and covalently linked by a single-stranded "hairpin" region. While not wishing to be bound by any theory, it is believed that the hairpin region of the siRNA molecule is cleaved intracellularly by the "Dicer" protein (or its equivalent) to form two individually base-paired siRNA RNA molecules.

[0103] As used herein, "target mRNA" refers to an RNA molecule that is a target for downregulation. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide as identified herein. In some embodiments, the pharmaceutically active peptide or polypeptide is a peptide or polypeptide whose expression (e.g., increased expression compared to expression in healthy subjects) is associated with a disease. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (e.g., increased expression compared to expression in healthy subjects) is associated with a disease.

[0104] According to the present disclosure, siRNAs can target any stretch of approximately 19 to 25 contiguous nucleotides in any target mRNA sequence ("target sequence"). Techniques for selecting target sequences for siRNAs are provided, for example, in Tuschl T. et al., "The siRNA User Guide," revised October 11, 2002, the entire disclosure of which is incorporated herein by reference. Further guidance on target sequence selection and / or siRNA design can be found on the Protocol Online webpage (www.protocol-online.com) using the keyword "siRNA." Thus, in some embodiments, the sense strand of an siRNA used in the present disclosure comprises a nucleotide sequence substantially identical to any contiguous stretch of approximately 19 to approximately 25 nucleotides in the target mRNA.

[0105] siRNA can be obtained using several methods known to those skilled in the art. For example, siRNA can be chemically synthesized or recombinantly produced. Preferably, siRNA is transcribed from a recombinant circular or linear DNA plasmid using any suitable promoter. The selection of other suitable promoters is within the skill of those skilled in the art. The selection of an appropriate plasmid for transcribing siRNA, the method for inserting a nucleic acid sequence for expressing siRNA into a plasmid, and the IVT method for in vitro transcription of the siRNA are within the skill of those skilled in the art.

[0106] As used herein, the term "miRNA" (microRNA) refers to a non-coding RNA that is 21-25 (e.g., 21-23, preferably 22) nucleotides in length and induces degradation of target mRNA and / or interferes with its translation. miRNAs are typically found in plants, animals, and some viruses, and are encoded by eukaryotic nuclear DNA in plants and animals, respectively, and by viral DNA (in viruses whose genomes are DNA-based). miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), typically resulting in translational repression or target degradation or gene silencing.

[0107] miRNAs can be obtained using several techniques known to those skilled in the art. For example, miRNAs can be chemically synthesized or recombinantly produced using methods known in the art (e.g., using commercially available kits, such as the miRNA cDNA Synthesis Kit sold by Applied Biological Materials Inc.). Preferably, miRNAs are transcribed from recombinant circular or linear DNA plasmids using any suitable promoter.

[0108] DNA In some embodiments of all aspects of the present disclosure, the nucleic acid is DNA. Here, the term "DNA" refers to a nucleic acid molecule containing deoxyribonucleotide residues. DNA typically includes the naturally occurring nucleic acids adenosine (dA), thymidine (dT), cytidine (dC), and guanosine (dG) ("d" stands for "deoxy"). In a preferred embodiment, DNA contains all or most deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. DNA includes, without limitation, double-stranded DNA, single-stranded DNA, isolated DNA (e.g., partially purified DNA), essentially pure DNA, synthetic DNA, recombinantly produced DNA, and modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may refer to the addition of non-nucleotide material to internal DNA nucleotides or to the termini of the DNA. It is also contemplated herein that the nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the purposes of the present disclosure, these modified DNAs are considered analogs of naturally occurring DNA. A molecule contains a "majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is greater than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (whether they are standard (i.e., naturally occurring) nucleotide residues or their analogs). DNA may be recombinant DNA and may be obtained by cloning nucleic acids, particularly cDNA. cDNA may be obtained by reverse transcription of RNA.

[0109] Pharmaceutically active peptides or polypeptides "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, e.g., a gene, cDNA, or RNA (preferably mRNA), to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of an RNA (preferably mRNA) corresponding to that gene produces the protein in a cell or other biological system. Similarly, an RNA (e.g., mRNA) encodes a protein if translation of the RNA (e.g., in a cell) produces the protein.

[0110] In some embodiments, as described herein, the active ingredient is RNA (preferably mRNA), which comprises a nucleic acid sequence (e.g., ORF) encoding one or more polypeptides, e.g., peptides or proteins, preferably pharmaceutically active peptides or proteins. In some embodiments, the RNA (preferably mRNA) described herein is capable of expressing said peptides or proteins, particularly when delivered to a cell or a subject. Thus, in some embodiments, the RNA (preferably mRNA) described herein comprises a coding region (ORF) encoding a peptide or protein, preferably a pharmaceutically active peptide or protein. In this regard, an "open reading frame" or "ORF" is a stretch of consecutive codons beginning with an initiation codon and ending with a stop codon. Such an RNA (preferably mRNA) encoding a pharmaceutically active peptide or protein is also referred to herein as a "pharmaceutically active RNA" (or "pharmaceutically active mRNA"). In some embodiments, the RNA (preferably mRNA) described herein comprises a nucleic acid sequence encoding two or more peptides or polypeptides, e.g., two, three, four, or more peptides or polypeptides. In some embodiments, the RNA (preferably mRNA) described in this disclosure comprises a nucleic acid sequence encoding one or more (e.g., 1, 2, 3, 4, 5, or more) patient-specific antigens suitable for personalized cancer therapy. In some embodiments, a lipid particle composition comprising RNA may include one or more species of RNA, each encoding a different peptide or protein.

[0111] Preferably, the RNA (i) comprises structural elements (5' cap, 5' UTR, 3' UTR, poly(A) sequence) that are optimized to maximize the effectiveness of the RNA in terms of stability and translation efficiency, (ii) is modified (e.g., by replacing naturally occurring nucleosides (particularly cytidine) with synthetic nucleosides (e.g., modified nucleosides selected from the group consisting of pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), and 5-methyluridine) (partially or completely, preferably completely) and / or by codon optimization) to optimize the effectiveness of the RNA (e.g., to increase translation efficiency, reduce immunogenicity, and / or reduce cytotoxicity), or (iii) is both (i) and (ii).

[0112] The term "pharmaceutically active peptide or protein" may be understood to mean a peptide or protein that can be used to treat an individual in which expression of the peptide or protein is beneficial, for example, in ameliorating the symptoms of a disease or disorder. Preferably, a pharmaceutically active peptide or protein has therapeutic or palliative properties and may be administered to improve, alleviate, relieve, reverse, delay the onset of, or reduce the severity of, one or more symptoms of a disease or disorder. A pharmaceutically active peptide or protein may have prophylactic properties and may be used to delay the onset of, or reduce the severity of, a disease or disorder.

[0113] Specific examples of pharmaceutically active peptides and proteins include, but are not limited to, cytokines, interferons, such as interferon-alpha (IFN-α), interferon-beta (IFNβ), or interferon-gamma (IFN-γ), interleukins, such as interleukin 2 (IL2), IL-4, IL7, IL-10, IL-11, IL12, IL15, IL-21, and IL23, colony stimulating factors (CSFs), such as granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), and granulocyte-macrophage colony stimulating factor (GM-CSF), tumor stimulating factors (TNFs), and the like. These include tumor necrosis factor (TNF), erythropoietin (EPO), and bone morphogenetic proteins (BMPs); immunoglobulin superfamily members, including antibodies (e.g., IgG), T cell receptors (TCRs), chimeric antigen receptors (CARs), major histocompatibility complex (MHC) molecules, co-receptors (e.g., CD4, CD8, CD19), antigen receptor accessory molecules (e.g., CD-3γ, CD3-δ, CD-3ε, CD79a, CD79b), costimulatory or inhibitory molecules (e.g., CD28, CD80, CD86); and other immunologically active compounds, such as tumor-associated antigens, pathogen-associated antigens (e.g., bacterial, parasitic, or viral antigens), allergens, and autoantigens.

[0114] amino acid In some embodiments, the methods and compositions of the invention, particularly further processing steps, such as dialysis or filtration steps, dilution or storage matrix addition steps, and storage steps, use amino acids.

[0115] As used herein, the term "amino acid" has its ordinary meaning in the art in its broadest sense of a compound containing an amine group (as defined and exemplified above in its broadest or preferred embodiment) and a carboxylic acid group (as defined and exemplified above in its broadest or preferred embodiment). Amino acids may contain other functional groups as defined and exemplified herein.

[0116] As is well known to those skilled in the art, depending on the pH, amino acids can exist in multiple forms. In one embodiment, the amino acid is in a zwitterionic form (i.e., a proton from the carboxylic acid group is transferred to the amino group, thus resulting in a negative carboxylate group and a positive ammonium group). In one embodiment, the amino acid is in a neutral form (i.e., both the amino and carboxylic acid groups are uncharged). In one embodiment, typically at acidic pH, the amino acid is in a cationic form (i.e., only the amine group is protonated, thereby resulting in an uncharged carboxylic acid group and a positive ammonium group). In one embodiment, typically at basic pH, the amino acid is in an anionic form (i.e., only the carboxylic acid group is deprotonated, thus resulting in a negative carboxylate group and an uncharged amine group). Amino acids are named herein according to their neutral structures, which are generally known in the art. The use of any particular amino acid name does not imply limitation to the neutral structure, but includes all neutral, protonated, deprotonated, and zwitterionic structures.

[0117] In one embodiment, the amino acid is an alpha amino acid (i.e., the amino group is on the carbon adjacent to the carbon that forms the carboxylic acid group). Typically, such alpha amino acids have the general formula (neutral structure) HN-CH(R)-COH, where the R group is referred to as the side chain. Proline and its derivatives differ from this structure in that the nitrogen atom forms part of a pyrrolidine ring.

[0118] In one embodiment, the amino acid is a proteinogenic amino acid. Examples of proteinogenic amino acids include arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

[0119] In one embodiment, the amino acid is a substituted proteinogenic amino acid, i.e., a proteinogenic amino acid selected from the amino acids listed above, substituted by one or more substituents selected from list A. Examples of such substituted proteinogenic amino acids include 3-hydroxyglutamic acid, 2-methyl-L-serine, and O-methyl-L-serine.

[0120] In one embodiment, the amino acid is a non-proteinogenic amino acid. Examples of non-proteinogenic amino acids include α-aminoadipic acid, β-alanine, α-aminoisobutyric acid, β-aminoisobutyric acid, γ-aminobutyric acid, δ-aminolevulinic acid, 4-aminobenzoic acid, dehydroalanine, norvaline, alloisoleucine, allothreonine, homocysteine, homoserine, isoserine, citrulline, ornithine, homophenylalanine, 7-azatryptophan, norleucine, homoserine, sarcosine, L-beta-homoleucine, and any substituted derivative thereof, wherein the substituent is selected from List A.

[0121] In one embodiment, the amino acid is an acidic amino acid. In one embodiment, an acidic amino acid has an isoelectric point (pI) of less than 4, i.e., a pH at which the molecule has no net charge. In one embodiment, the acidic amino acid is an amino acid having an acidic side chain. Examples of acidic side chains include carboxylic acids, sulfonic acids, organosulfuric acids, phosphonic acids, and phosphoric acids, as defined and exemplified above. Preferably, the acidic amino acid is an amino acid having a carboxylic acid side chain. Examples of acidic amino acids include aspartic acid, glutamic acid, and any substituted derivative thereof, wherein the substituent is selected from List A. More preferably, the acidic amino acid is selected from the group consisting of aspartic acid, glutamic acid, 3-hydroxyglutamic acid, and alpha-aminoadipic acid.

[0122] In one embodiment, the amino acid is a neutral amino acid. In one embodiment, the neutral amino acid has an isoelectric point (pI) of 4 to 7.8. In one embodiment, the neutral amino acid is an amino acid lacking either an acidic or a basic side chain. Examples of neutral amino acids include serine, threonine, asparagine, glutamine, cysteine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan, as well as substituted derivatives of any of these, wherein the substituent is selected from List A. More preferably, the neutral amino acid is selected from the group consisting of leucine and isoleucine.

[0123] In one embodiment, the amino acid is a basic amino acid. In one embodiment, the basic amino acid has an isoelectric point (pI) greater than 7.8, preferably greater than 8.5. In one embodiment, the basic amino acid is an amino acid having a basic side chain. Examples of basic side chains include amines, amidines, and guanidines, as well as nitrogen-containing heteroaryls and heterocyclyls, all as defined and exemplified above. Examples of basic amino acids include arginine, histidine, lysine, and any substituted derivative thereof, wherein the substituent is selected from List A. More preferably, the basic amino acid is selected from the group consisting of arginine, histidine, and lysine.

[0124] aqueous dispersion The present disclosure provides aqueous dispersions having an aqueous mobile phase and a dispersed phase, as defined herein. As used herein, the term "dispersion" has its ordinary meaning in chemistry in its broadest sense as a system in which discrete particles of one material (the "dispersed phase") are dispersed in a phase of another material (the "continuous phase" or "mobile phase").

[0125] In one embodiment, the dispersion is a solid-liquid dispersion, where the dispersed phase is a solid and the mobile phase is a liquid. In one embodiment, the dispersion is a liquid-liquid dispersion, where both the dispersed phase and the mobile phase are liquids.

[0126] In one embodiment, the dispersion is a colloid. The term "colloid" as used herein describes a stable mixture in which the dispersed particles do not settle. Typically, the dispersed particles have dimensions of approximately 1 nm to 1 μm in at least one direction, or in such systems, discontinuities are found at distances of that order.

[0127] In one embodiment, the dispersion is a suspension. The term "suspension," as used herein, is a heterogeneous dispersion of large particles in a medium. Unlike solutions and colloids, suspended particles may settle out of the mixture if left standing for an extended period of time. The use of the terms "colloid" and "suspension" sometimes overlaps or is synonymous, and colloids are, in some instances, considered to be a subtype of suspension.

[0128] In the present disclosure, the aqueous dispersion preferably contains preformed lipid nanoparticles (pre-LNPs). In the present disclosure, such preformed LNPs can be understood as oil-in-water emulsions in which the pre-LNP core material is preferably in a liquid state and thus has a melting point below body temperature. Thus, preformed LNPs typically contain a central complex embedded in an irregular, non-lamellar phase of lipids and lipids, but are substantially free of nucleic acids. This contrasts with the structure of liposomes, which contain unilamellar or multilamellar vesicular particles consisting of lipid bilayers surrounding an encapsulated aqueous cavity. Lipids used for LNP formation typically do not form a lamellar (bilayer) phase in water under physiological conditions. LNPs typically do not contain or encapsulate an aqueous core. LNPs typically contain an oily (or oily) core.

[0129] In some instances, the pre-LNPs described herein are not liposomes. In some instances, the pre-LNPs described herein are not lipoplexes.

[0130] Preformed LNPs are substantially free of nucleic acids (as defined herein). Preformed LNPs may be free of nucleic acids, i.e., no nucleic acids are present in the preformed LNPs. Typically, nucleic acids are not used or added in any of the manufacturing steps to prepare the preformed LNPs. Preformed LNPs that are substantially free of nucleic acids may alternatively be described as "empty LNPs" and / or "encapsulatable LNPs," and the step of encapsulating nucleic acids into pre-LNPs to produce encapsulated LNPs is defined below.

[0131] In some embodiments, the pre-LNPs described herein have an average diameter ranging from about 40 nm to about 1000 nm, about 40 nm to about 800 nm, about 40 nm to about 700 nm, about 40 nm to about 600 nm, about 40 nm to about 500 nm, about 40 nm to about 450 nm, about 40 nm to about 400 nm, about 40 nm to about 350 nm, about 40 nm to about 300 nm, about 40 nm to about 250 nm, about 40 nm to about 200 nm, about 40 nm to about 150 nm, about 40 nm to about 100 nm, about 40 nm to about 90 nm, about 40 nm to about 80 nm, or about 40 nm to about 70 nm. In some embodiments, the pre-LNPs described herein have an average diameter of less than 100 nm. In some embodiments, the pre-LNPs described herein have an average diameter of about 30 nm to about 100 nm. In some embodiments, the pre-LNPs described herein have an average diameter of about 30 nm to about 90 nm. In some embodiments, the pre-LNPs described herein have an average diameter of about 30 nm to about 80 nm. In some embodiments, the pre-LNPs described herein have an average diameter of about 40 nm to about 70 nm.

[0132] In some cases, the aqueous dispersion comprises a dispersed phase comprising pre-LNPs having a size (i.e., diameter) of about 20 nm to about 500 nm, about 20 nm to about 200 nm, about 30 nm to about 180 nm, about 40 nm to about 120 nm, or preferably about 40 nm to about 80 nm. In some cases, the aqueous dispersion comprises a dispersed phase comprising pre-LNPs having a size (i.e., diameter) of about 200 nm or less.

[0133] In one embodiment, the mobile phase is a solution. The term "solution," as used herein, refers to a homogeneous mixture comprising a solvent, typically water, and solutes, which may be salts, buffers, tonicity adjusters, and others, so long as these materials are molecularly dispersed within the solvent. The mobile phase may include solutes as further described herein.

[0134] The dispersed phase comprises a lipid mixture that includes a cationically ionizable lipid, as defined below.

[0135] In one embodiment, the aqueous dispersion has an aqueous mobile phase and a dispersed phase, wherein the dispersed phase comprises a lipid mixture including a cationically ionizable lipid; and the aqueous mobile phase comprises a buffer solution having a pH of about 6.5 to about 8.

[0136] The aqueous dispersion contains a buffer solution. As is well known to those skilled in the art, a buffer solution is a solution containing a mixture of a weak acid and its conjugate base, or vice versa, whose pH changes very little when small amounts of a strong acid or strong base are added to it.

[0137] In one embodiment, the buffer used in the aqueous dispersion is a neutral buffer. In one embodiment, the buffer has a pK of about 5.5 to about 8.5. a In one embodiment, the buffer has a pK of about 6.0 to about 8.0. a In one embodiment, the buffer has a pK of about 6.5 to about 7.5. a It has.

[0138] In one embodiment, the buffer used in the aqueous dispersion comprises an anionic group / moiety. In one embodiment, the buffer used in the aqueous dispersion comprises a sulfonic acid group / moiety, or a derivative thereof. In one embodiment, the buffer used in the aqueous dispersion is a zwitterionic acidic buffer. In one embodiment, the buffer used in the aqueous dispersion comprises an amino group / moiety and a sulfonic acid group / moiety, or a derivative thereof. In one embodiment, the buffer used in the aqueous dispersion is a zwitterionic acidic buffer. In one embodiment, the buffer used in the aqueous dispersion comprises an amino group / moiety and a sulfonic acid group / moiety, or a derivative thereof. In one embodiment, the buffer used in the aqueous dispersion is a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-morpholin-4-ylethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid (HEPPS), 2-(bis(2-hydroxyethyl)amino)ethanesulfonic acid and 2-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}ethane-1-sulfonic acid (TES), and mixtures of any of these.

[0139] In one embodiment, the buffer used in the aqueous dispersion comprises cationic groups / moieties. In one embodiment, the buffer used in the aqueous dispersion comprises basic groups / moieties. In one embodiment, the buffer used in the aqueous dispersion comprises amino groups / moieties. In one embodiment, the buffer used in the aqueous dispersion comprises primary, secondary, or tertiary amine groups / moieties. In one embodiment, the buffer used in the aqueous dispersion comprises amino groups / moieties (e.g., primary, secondary, or tertiary amine groups / moieties) and no anionic moieties (e.g., sulfonic acid groups / moieties). In one embodiment, the buffer used in the aqueous dispersion is selected from the group consisting of tris(hydroxymethyl)aminomethane (Tris), TAE (a buffer solution containing a mixture of Tris, acetic acid, and ethylenediaminetetraacetic acid), TBE (a buffer solution containing a mixture of Tris, boric acid, and ethylenediaminetetraacetic acid), TEA, (2-(bis(2-hydroxyethyl)amino)acetic acid) (bicine), (N-[tris(hydroxymethyl)methyl]glycine) (tricine), triethylammonium acetate, triethanolamine, and N-(2-acetamido)iminodiacetic acid (ADA), and mixtures of any of these.

[0140] In one embodiment, the buffer used in the aqueous dispersion is a mixture of a buffer containing an anionic group / moiety and a buffer containing a cationic group / moiety. In one embodiment, the buffer used in the aqueous dispersion is a mixture of a buffer containing an amino group / moiety (e.g., primary, secondary, or tertiary amine group / moiety) and a buffer containing a sulfonic acid group / moiety. In one embodiment, the buffer used in the aqueous dispersion is a mixture of a zwitterionic acidic buffer and a buffer containing an amino group / moiety (e.g., primary, secondary, or tertiary amine group / moiety).

[0141] In one embodiment, the buffer is selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)-aminomethane (Tris), 2-morpholin-4-ylethanesulfonic acid (MES), bis-(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (bis-Tris), and phosphate buffers, and mixtures of any of these.

[0142] In one embodiment, the buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). In one embodiment, the buffer is tris(hydroxymethyl)aminomethane (Tris). In one embodiment, the buffer is 2-morpholin-4-ylethanesulfonic acid (MES). In one embodiment, the buffer is bis-(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (bis-Tris). In one embodiment, the buffer is a phosphate buffer.

[0143] In one preferred embodiment, the buffer is HEPES. In another preferred embodiment, the buffer is Tris. In an even more preferred embodiment, the buffer is a mixture of HEPES and Tris.

[0144] In one embodiment, the aqueous dispersion contains about 1 to about 100 mM HEPES. In one embodiment, the aqueous dispersion contains about 5 to about 80 mM HEPES. In one embodiment, the aqueous dispersion contains about 10 to about 60 mM HEPES. In one embodiment, the aqueous dispersion contains about 50 to about 70 mM HEPES. In one embodiment, the aqueous dispersion contains about 55 to about 65 mM HEPES. In one embodiment, the aqueous dispersion contains about 60 mM HEPES.

[0145] In one embodiment, the aqueous dispersion contains about 0.05 to about 50 mM HEPES. In one embodiment, the aqueous dispersion contains about 0.1 to about 25 mM HEPES. In one embodiment, the aqueous dispersion contains about 0.5 to about 20 mM HEPES. In one embodiment, the aqueous dispersion contains about 1 to about 10 mM HEPES. In one embodiment, the aqueous dispersion contains about 2 to about 8 mM HEPES. In one embodiment, the aqueous dispersion contains about 3 to about 7 mM HEPES. In one embodiment, the aqueous dispersion contains about 4 to about 6 mM HEPES. In one embodiment, the aqueous dispersion contains about 5 mM HEPES. In one embodiment, the aqueous dispersion contains about 2 to about 20 mM HEPES. In one embodiment, the aqueous dispersion contains about 5 to about 20 mM HEPES. In one embodiment, the aqueous dispersion contains about 5 to about 15 mM HEPES. In one embodiment, the aqueous dispersion contains about 5 to about 10 mM HEPES.

[0146] In one embodiment, the aqueous dispersion contains about 0.1 to about 40 mM Tris. In one embodiment, the aqueous dispersion contains about 0.2 to about 30 mM Tris. In one embodiment, the aqueous dispersion contains about 1 to about 20 mM Tris. In one embodiment, the aqueous dispersion contains about 1 to about 10 mM Tris. In one embodiment, the aqueous dispersion contains about 1 to about 5 mM Tris. In one embodiment, the aqueous dispersion contains about 5 to about 25 mM Tris. In one embodiment, the aqueous dispersion contains about 10 to about 20 mM Tris. In one embodiment, the aqueous dispersion contains about 0.05 to about 50 mM Tris. In one embodiment, the aqueous dispersion contains about 0.1 to about 25 mM Tris. In one embodiment, the aqueous dispersion contains about 0.5 to about 20 mM Tris. In one embodiment, the aqueous dispersion contains about 1 to about 10 mM Tris. In one embodiment, the aqueous dispersion contains about 2 to about 8 mM Tris. In one embodiment, the aqueous dispersion contains about 3 to about 7 mM Tris. In one embodiment, the aqueous dispersion contains about 4 to about 6 mM Tris. In one embodiment, the aqueous dispersion contains about 3 mM Tris. In one embodiment, the aqueous dispersion contains about 5 mM Tris. In one embodiment, the aqueous dispersion contains about 10 mM Tris. In one embodiment, the aqueous dispersion contains about 20 mM Tris.

[0147] In one embodiment, the aqueous dispersion contains 10 to 60 mM HEPES and 1 to 5 mM Tris. In one embodiment, the aqueous dispersion contains about 60 mM HEPES and about 10 mM Tris. In one embodiment, the aqueous dispersion contains about 60 mM HEPES and about 20 mM Tris.

[0148] In one embodiment, the pH of the buffer solution is about 6.5 to about 8.0. In one embodiment, the pH of the buffer solution is about 7.0 to about 8.0. In one embodiment, the pH of the buffer solution is about 6.8 to about 8.0. In one embodiment, the pH of the buffer solution is about 6.5 to about 7.5. In one embodiment, the pH of the buffer solution is about 6.8 to about 7.2. In one embodiment, the pH of the buffer solution is about 6.9 to about 7.1. In one embodiment, the pH of the buffer solution is about 7.0. In one embodiment, the pH of the buffer solution is about 7.1 to about 7.7. In one embodiment, the pH of the buffer solution is about 7.2 to about 7.6. In one embodiment, the pH of the buffer solution is about 7.3 to about 7.5. In one embodiment, the pH of the buffer solution is about 7.4. In one embodiment, the pH of the buffer solution is about 6.8 to about 7.6. In one embodiment, the pH of the buffer solution is about 7.0 to about 7.4. In one embodiment, the pH of the buffer solution is about 7.1 to about 7.4.

[0149] In one embodiment, the buffer solution is HEPES, and the pH of the buffer solution is about 6.5 to about 8.0. In one embodiment, the buffer solution is HEPES, and the pH of the buffer solution is about 7.0 to about 8.0. In one embodiment, the buffer solution is HEPES, and the pH of the buffer solution is about 6.8 to about 8.0. In one embodiment, the buffer solution is HEPES, and the pH of the buffer solution is about 6.5 to about 7.5. In one embodiment, the buffer solution is HEPES, and the pH of the buffer solution is about 6.8 to about 7.2. In one embodiment, the buffer solution is HEPES, and the pH of the buffer solution is about 6.9 to about 7.1. In one embodiment, the buffer solution is HEPES, and the pH of the buffer solution is about 7.0. In one embodiment, the buffer solution is HEPES, and the pH of the buffer solution is about 7.1 to about 7.7. In one embodiment, the buffer solution is HEPES, and the pH of the buffer solution is about 7.2 to about 7.6. In one embodiment, the buffer solution is HEPES, and the pH of the buffer solution is about 7.3 to about 7.5. In one embodiment, the buffer solution is HEPES, and the pH of the buffer solution is about 7.4. In one embodiment, the buffer solution is HEPES, and the pH of the buffer solution is about 6.8 to about 7.6. In one embodiment, the buffer solution is HEPES, and the pH of the buffer solution is about 7.0 to about 7.4. In one embodiment, the buffer solution is HEPES, and the pH of the buffer solution is about 7.1 to about 7.4.

[0150] In one embodiment, the buffer solution is Tris, and the pH of the buffer solution is about 6.5 to about 8.0. In one embodiment, the buffer solution is Tris, and the pH of the buffer solution is about 7.0 to about 8.0. In one embodiment, the buffer solution is Tris, and the pH of the buffer solution is about 6.8 to about 8.0. In one embodiment, the buffer solution is Tris, and the pH of the buffer solution is about 6.5 to about 7.5. In one embodiment, the buffer solution is Tris, and the pH of the buffer solution is about 6.8 to about 7.2. In one embodiment, the buffer solution is Tris, and the pH of the buffer solution is about 6.9 to about 7.1. In one embodiment, the buffer solution is Tris, and the pH of the buffer solution is about 7.0. In one embodiment, the buffer solution is Tris, and the pH of the buffer solution is about 7.1 to about 7.7. In one embodiment, the buffer solution is Tris, and the pH of the buffer solution is about 7.2 to about 7.6. In one embodiment, the buffer solution is Tris, and the pH of the buffer solution is about 7.3 to about 7.5. In one embodiment, the buffer solution is Tris, and the pH of the buffer solution is about 7.4. In one embodiment, the buffer solution is Tris, and the pH of the buffer solution is about 6.8 to about 7.6. In one embodiment, the buffer solution is Tris, and the pH of the buffer solution is about 7.0 to about 7.4. In one embodiment, the buffer solution is Tris, and the pH of the buffer solution is about 7.1 to about 7.4.

[0151] In one embodiment, the buffer solution is a mixture of HEPES and Tris, and the pH of the buffer solution is about 6.5 to about 8.0. In one embodiment, the buffer solution is a mixture of HEPES and Tris, and the pH of the buffer solution is about 7.0 to about 8.0. In one embodiment, the buffer solution is a mixture of HEPES and Tris, and the pH of the buffer solution is about 6.8 to about 8.0. In one embodiment, the buffer solution is a mixture of HEPES and Tris, and the pH of the buffer solution is about 6.5 to about 7.5. In one embodiment, the buffer solution is a mixture of HEPES and Tris, and the pH of the buffer solution is about 6.8 to about 7.2. In one embodiment, the buffer solution is a mixture of HEPES and Tris, and the pH of the buffer solution is about 6.9 to about 7.1. In one embodiment, the buffer solution is a mixture of HEPES and Tris, and the pH of the buffer solution is about 7.0. In one embodiment, the buffer solution is a mixture of HEPES and Tris, and the pH of the buffer solution is about 7.1 to about 7.7. In one embodiment, the buffer solution is a mixture of HEPES and Tris, and the pH of the buffer solution is about 7.2 to about 7.6. In one embodiment, the buffer solution is a mixture of HEPES and Tris, and the pH of the buffer solution is about 7.3 to about 7.5. In one embodiment, the buffer solution is a mixture of HEPES and Tris, and the pH of the buffer solution is about 7.4. In one embodiment, the buffer solution is a mixture of HEPES and Tris, and the pH of the buffer solution is about 6.8 to about 7.6. In one embodiment, the buffer solution is a mixture of HEPES and Tris, and the pH of the buffer solution is about 7.0 to about 7.4. In one embodiment, the buffer solution is a mixture of HEPES and Tris, and the pH of the buffer solution is about 7.1 to about 7.4.

[0152] Storage matrix - cryoprotectants and other components In one embodiment, the aqueous dispersion (typically containing the pre-LNPs) also contains a storage matrix. As used herein, the term "storage matrix," as typically used in its broadest sense, includes any material typically used to aid in the storage and improve the shelf life of an aqueous dispersion.

[0153] In one embodiment, the preservation matrix comprises a cryoprotectant. As used herein, the term "cryoprotectant" is used in its broadest sense to mean any substance capable of protecting a composition from damage caused by freezing and / or ice formation. Examples of cryoprotectants include glycols (i.e., alcohols containing at least two hydroxy groups, such as glycerol and propylene glycol) and carbohydrates as defined and exemplified herein.

[0154] In one embodiment, the cryoprotectant is a carbohydrate. In one embodiment, the cryoprotectant is a monosaccharide or disaccharide. In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, glycerol, trehalose, lactose, and glucose, and any mixture thereof. In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose, and glucose, and any mixture thereof. In one embodiment, the cryoprotectant is sucrose or trehalose, or a mixture thereof. In a preferred embodiment, the cryoprotectant is sucrose. In a preferred embodiment, the cryoprotectant is trehalose.

[0155] When the aqueous dispersion also contains a storage matrix, it is typically present at a concentration of about 1% to about 30% (weight / volume). In one embodiment, the storage matrix is ​​present at a concentration of about 2% to about 20% (weight / volume). In one embodiment, the storage matrix is ​​present at a concentration of about 5% to about 15% (weight / volume). In one embodiment, the storage matrix is ​​present at a concentration of about 8% to about 12% (weight / volume). In one embodiment, the storage matrix is ​​present at a concentration of about 10% (weight / volume).

[0156] When the aqueous dispersion also contains a storage matrix that is a carbohydrate, it is typically present at a concentration of about 1% to about 30% (weight / volume). In one embodiment, the storage matrix is ​​a carbohydrate and is present at a concentration of about 2% to about 20% (weight / volume). In one embodiment, the storage matrix is ​​a carbohydrate and is present at a concentration of about 5% to about 15% (weight / volume). In one embodiment, the storage matrix is ​​a carbohydrate and is present at a concentration of about 8% to about 12% (weight / volume). In one embodiment, the storage matrix is ​​a carbohydrate and is present at a concentration of about 10% (weight / volume).

[0157] In one embodiment, the storage matrix is ​​sucrose and is present at a concentration of about 1% to about 30% (weight / volume). In one embodiment, the storage matrix is ​​sucrose and is present at a concentration of about 2% to about 20% (weight / volume). In one embodiment, the storage matrix is ​​sucrose and is present at a concentration of about 5% to about 15% (weight / volume). In one embodiment, the storage matrix is ​​sucrose and is present at a concentration of about 8% to about 12% (weight / volume). In one embodiment, the storage matrix is ​​sucrose and is present at a concentration of about 10% (weight / volume).

[0158] In one embodiment, the storage matrix is ​​trehalose and is present at a concentration of about 1% to about 30% (weight / volume). In one embodiment, the storage matrix is ​​trehalose and is present at a concentration of about 2% to about 20% (weight / volume). In one embodiment, the storage matrix is ​​trehalose and is present at a concentration of about 5% to about 15% (weight / volume). In one embodiment, the storage matrix is ​​trehalose and is present at a concentration of about 8% to about 12% (weight / volume). In one embodiment, the storage matrix is ​​trehalose and is present at a concentration of about 10% (weight / volume).

[0159] In one embodiment, the storage matrix is ​​glucose and is present at a concentration of about 0.5% to about 15% (weight / volume). In one embodiment, the storage matrix is ​​glucose and is present at a concentration of about 1% to about 10% (weight / volume). In one embodiment, the storage matrix is ​​glucose and is present at a concentration of about 2.5% to about 7.5% (weight / volume). In one embodiment, the storage matrix is ​​glucose and is present at a concentration of about 4% to about 6% (weight / volume). In one embodiment, the storage matrix is ​​glucose and is present at a concentration of about 5% (weight / volume).

[0160] In one embodiment, the storage matrix further comprises an amino acid as defined and exemplified above, or any mixture thereof. In one embodiment, the amino acid is an acidic amino acid as defined and exemplified above. In one embodiment, the acidic amino acid is selected from the group consisting of aspartic acid, glutamic acid, 3-hydroxyglutamic acid, and alpha-aminoadipic acid, and mixtures thereof. In one embodiment, the amino acid is a basic amino acid as defined and exemplified above. In one embodiment, the basic amino acid is selected from the group consisting of arginine, histidine, and lysine, and mixtures thereof. In one embodiment, the storage matrix further comprises a mixture of a basic amino acid and an acidic amino acid, each as defined and exemplified above. In one embodiment, the storage matrix further comprises a mixture of a basic amino acid and a neutral amino acid, each as defined and exemplified above.

[0161] When the storage matrix contains amino acids, they are typically present at a concentration of about 0.1 to about 20 mM. In one embodiment, the amino acids are present at a concentration of about 0.5 to about 10 mM. In one embodiment, the amino acids are present at a concentration of about 1 to about 5 mM. In one embodiment, the amino acids are present at a concentration of about 1 to about 1.5 mM. In one embodiment, the amino acids are present at a concentration of about 1.25 mM. In one embodiment, the amino acids are present at a concentration of about 2.5 mM. In one embodiment, the amino acids are present at a concentration of about 4 to about 6 mM. In one embodiment, the amino acids are present at a concentration of about 5 mM.

[0162] In one embodiment, the storage matrix contains an acidic amino acid present at a concentration of about 0.1 to about 20 mM. In one embodiment, the acidic amino acid is present at a concentration of about 0.5 to about 10 mM. In one embodiment, the acidic amino acid is present at a concentration of about 1 to about 5 mM. In one embodiment, the acidic amino acid is present at a concentration of about 1 to about 1.5 mM. In one embodiment, the acidic amino acid is present at a concentration of about 1.25 mM. In one embodiment, the acidic amino acid is present at a concentration of about 2.5 mM. In one embodiment, the acidic amino acid is present at a concentration of about 4 to about 6 mM. In one embodiment, the acidic amino acid is present at a concentration of about 5 mM.

[0163] In one embodiment, the storage matrix contains an acidic amino acid selected from the group consisting of aspartic acid, glutamic acid, 3-hydroxyglutamic acid, and alpha-aminoadipic acid, present at a concentration of about 0.1 to about 20 mM. In one embodiment, the acidic amino acid is selected from the group consisting of aspartic acid, glutamic acid, 3-hydroxyglutamic acid, and alpha-aminoadipic acid, and present at a concentration of about 0.5 to about 10 mM. In one embodiment, the acidic amino acid is selected from the group consisting of aspartic acid, glutamic acid, 3-hydroxyglutamic acid, and alpha-aminoadipic acid, and present at a concentration of about 1 to about 5 mM. In one embodiment, the acidic amino acid is selected from the group consisting of aspartic acid, glutamic acid, 3-hydroxyglutamic acid, and alpha-aminoadipic acid, and present at a concentration of about 1 to about 1.5 mM. In one embodiment, the acidic amino acid is selected from the group consisting of aspartic acid, glutamic acid, 3-hydroxyglutamic acid, and alpha-aminoadipic acid and is present at a concentration of about 1.25 mM. In one embodiment, the acidic amino acid is selected from the group consisting of aspartic acid, glutamic acid, 3-hydroxyglutamic acid, and alpha-aminoadipic acid and is present at a concentration of about 2.5 mM. In one embodiment, the acidic amino acid is selected from the group consisting of aspartic acid, glutamic acid, 3-hydroxyglutamic acid, and alpha-aminoadipic acid and is present at a concentration of about 4 to about 6 mM. In one embodiment, the acidic amino acid is selected from the group consisting of aspartic acid, glutamic acid, 3-hydroxyglutamic acid, and alpha-aminoadipic acid and is present at a concentration of about 5 mM.

[0164] In one embodiment, the storage matrix contains aspartic acid at a concentration of about 0.1 to about 20 mM. In one embodiment, the aspartic acid is present at a concentration of about 0.5 to about 10 mM. In one embodiment, the aspartic acid is present at a concentration of about 1 to about 5 mM. In one embodiment, the aspartic acid is present at a concentration of about 1 to about 1.5 mM. In one embodiment, the aspartic acid is present at a concentration of about 1.25 mM. In one embodiment, the aspartic acid is present at a concentration of about 2.5 mM. In one embodiment, the aspartic acid is present at a concentration of about 4 to about 6 mM. In one embodiment, the aspartic acid is present at a concentration of about 5 mM.

[0165] In one embodiment, the storage matrix contains glutamic acid at a concentration of about 0.1 to about 20 mM. In one embodiment, glutamic acid is present at a concentration of about 0.5 to about 10 mM. In one embodiment, glutamic acid is present at a concentration of about 1 to about 5 mM. In one embodiment, glutamic acid is present at a concentration of about 1 to about 1.5 mM. In one embodiment, glutamic acid is present at a concentration of about 1.25 mM. In one embodiment, glutamic acid is present at a concentration of about 2.5 mM. In one embodiment, glutamic acid is present at a concentration of about 4 to about 6 mM. In one embodiment, glutamic acid is present at a concentration of about 5 mM.

[0166] In one embodiment, the storage matrix further comprises an organic acid as defined and exemplified above. Preferably, the organic acid is a carboxylic acid as defined and exemplified above. More preferably, the organic acid is selected from the group consisting of acetic acid, malic acid, succinic acid, citric acid, and methylmalonic acid, and any mixture thereof.

[0167] When the storage matrix contains an organic acid, it is typically present at a concentration of about 0.5 to about 50 mM. In one embodiment, the organic acid is present at a concentration of about 1 to about 20 mM. In one embodiment, the organic acid is present at a concentration of about 1 to about 10 mM. In one embodiment, the organic acid is present at a concentration of about 2 to about 8 mM.

[0168] In one embodiment, the storage matrix further contains a buffer solution. As is well known to those skilled in the art, a buffer solution is a solution containing a mixture of a weak acid and its conjugate base, or vice versa, whose pH changes very little upon the addition of small amounts of a strong acid or strong base.

[0169] In one embodiment, the buffer used in the storage matrix is ​​a neutral buffer. In one embodiment, the buffer has a pK of about 5.5 to about 8.5. a In one embodiment, the buffer has a pK of about 6.0 to about 8.0. a In one embodiment, the buffer has a pK of about 6.5 to about 7.5. a It has.

[0170] In one embodiment, the buffer used in the storage matrix comprises anionic groups / moieties. In one embodiment, the buffer used in the storage matrix comprises sulfonic acid groups / moieties, or derivatives thereof. In one embodiment, the buffer used in the storage matrix is ​​a zwitterionic acidic buffer. In one embodiment, the buffer used in the storage matrix comprises amino groups / moieties and sulfonic acid groups / moieties, or derivatives thereof.

[0171] In one embodiment, the buffer used in the storage matrix comprises cationic groups / moieties. In one embodiment, the buffer used in the aqueous dispersion comprises basic groups / moieties. In one embodiment, the buffer used in the storage matrix comprises amino groups / moieties. In one embodiment, the buffer used in the storage matrix comprises primary, secondary, or tertiary amine groups / moieties. In one embodiment, the buffer used in the storage matrix comprises amino groups / moieties (e.g., primary, secondary, or tertiary amine groups / moieties) and does not comprise anionic moieties (e.g., sulfonic acid groups / moieties). In one embodiment, the buffer used in the storage matrix is ​​selected from the group consisting of tris(hydroxymethyl)aminomethane (Tris), TAE (a buffer solution containing a mixture of Tris, acetic acid, and ethylenediaminetetraacetic acid), TBE (a buffer solution containing a mixture of Tris, boric acid, and ethylenediaminetetraacetic acid), TEA, (2-(bis(2-hydroxyethyl)amino)acetic acid) (bicine), (N-[tris(hydroxymethyl)methyl]glycine) (tricine), triethylammonium acetate, triethanolamine, and N-(2-acetamido)iminodiacetic acid (ADA), and mixtures of any of these.

[0172] In one embodiment, the buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). In one embodiment, the buffer is tris(hydroxymethyl)aminomethane (Tris). In one embodiment, the buffer is 2-morpholin-4-ylethanesulfonic acid (MES). In one embodiment, the buffer is bis-(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (bis-Tris).

[0173] When the storage matrix contains a buffer, it is typically present at a concentration of about 0.5 to about 500 mM. In one embodiment, the buffer is present at a concentration of about 1 to about 250 mM. In one embodiment, the buffer is present at a concentration of about 2 to about 100 mM. In one embodiment, the buffer is present at a concentration of about 5 to about 50 mM.

[0174] In one embodiment, the storage matrix may contain a mixture of the components defined and exemplified above.

[0175] In one embodiment, the storage matrix contains a cryoprotectant (as defined and exemplified herein, e.g., sucrose or trehalose) and an acidic amino acid (as defined and exemplified herein, e.g., aspartic acid or glutamic acid, or a mixture thereof).

[0176] In one embodiment, the storage matrix contains a cryoprotectant (as defined and exemplified herein, e.g., sucrose or trehalose) and an organic acid (as defined and exemplified herein, e.g., acetic acid, malic acid, or succinic acid, or mixtures thereof).

[0177] In one embodiment, the storage matrix contains a cryoprotectant (as defined and exemplified herein, e.g., sucrose or trehalose), an organic acid (as defined and exemplified herein, e.g., acetic acid, malic acid, or succinic acid, or mixtures thereof), and an acidic amino acid (as defined and exemplified herein, e.g., aspartic acid or glutamic acid, or mixtures thereof).

[0178] In one embodiment, the storage matrix contains a cryoprotectant (as defined and exemplified herein, e.g., sucrose or trehalose) and a buffer (as defined and exemplified herein, e.g., HEPES or Tris, or a mixture thereof).

[0179] In one embodiment, the storage matrix contains a cryoprotectant (as defined and exemplified herein, e.g., sucrose or trehalose), a buffer (as defined and exemplified herein, e.g., HEPES or Tris, or mixtures thereof), and an acidic amino acid (as defined and exemplified herein, e.g., aspartic acid or glutamic acid, or mixtures thereof).

[0180] In one embodiment, the storage matrix contains a cryoprotectant (as defined and exemplified herein, e.g., sucrose or trehalose), a buffer (as defined and exemplified herein, e.g., HEPES or Tris), and an organic acid (as defined and exemplified herein, e.g., acetic acid, malic acid, or succinic acid, or a mixture thereof).

[0181] In one embodiment, the storage matrix contains a cryoprotectant (as defined and exemplified herein, e.g., sucrose or trehalose), a buffer (as defined and exemplified herein, e.g., HEPES or Tris), an organic acid (as defined and exemplified herein, e.g., acetic acid, malic acid, or succinic acid, or mixtures thereof), and an acidic amino acid (as defined and exemplified herein, e.g., aspartic acid or glutamic acid, or mixtures thereof).

[0182] In one embodiment, the storage matrix contains a cryoprotectant (as defined and exemplified herein, e.g., sucrose or trehalose), and a basic amino acid (as defined and exemplified herein, e.g., histidine, lysine, or arginine, or mixtures thereof), and optionally a neutral amino acid (as defined and exemplified herein, e.g., leucine or isoleucine, or mixtures thereof).

[0183] In one embodiment, the storage matrix contains a cryoprotectant (as defined and exemplified herein, e.g., sucrose or trehalose), a basic amino acid (as defined and exemplified herein, e.g., histidine, lysine, or arginine, or mixtures thereof), and optionally a neutral amino acid (as defined and exemplified herein, e.g., leucine or isoleucine, or mixtures thereof), and an organic acid (as defined and exemplified herein, e.g., acetic acid, malic acid, or succinic acid, or mixtures thereof).

[0184] In one embodiment, the storage matrix contains a cryoprotectant (as defined and exemplified herein, e.g., sucrose or trehalose), a basic amino acid (as defined and exemplified herein, e.g., histidine, lysine, or arginine, or mixtures thereof), and optionally a neutral amino acid (as defined and exemplified herein, e.g., leucine or isoleucine, or mixtures thereof), and an acidic amino acid (as defined and exemplified herein, e.g., aspartic acid or glutamic acid, or mixtures thereof).

[0185] In one embodiment, the storage matrix contains a cryoprotectant (as defined and exemplified herein, e.g., sucrose or trehalose), a basic amino acid (as defined and exemplified herein, e.g., histidine, lysine, or arginine, or mixtures thereof), and optionally a neutral amino acid (as defined and exemplified herein, e.g., leucine or isoleucine, or mixtures thereof), an organic acid (as defined and exemplified herein, e.g., acetic acid, malic acid, or succinic acid, or mixtures thereof), and an acidic amino acid (as defined and exemplified herein, e.g., aspartic acid or glutamic acid, or mixtures thereof).

[0186] In one embodiment, the storage matrix is ​​sucrose and the buffer is HEPES. In one embodiment, the storage matrix is ​​sucrose and the buffer is Tris. In one embodiment, the storage matrix is ​​sucrose and the buffer is a mixture of HEPES and Tris.

[0187] In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the sucrose is present at a concentration of about 1% to about 30% (weight / volume). In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the sucrose is present at a concentration of about 2% to about 20% (weight / volume). In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the sucrose is present at a concentration of about 5% to about 15% (weight / volume). In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the sucrose is present at a concentration of about 8% to about 12% (weight / volume). In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the sucrose is present at a concentration of about 10% (weight / volume).

[0188] In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the sucrose is present at a concentration of about 1% to about 30% (weight / volume). In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the sucrose is present at a concentration of about 2% to about 20% (weight / volume). In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the sucrose is present at a concentration of about 5% to about 15% (weight / volume). In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the sucrose is present at a concentration of about 8% to about 12% (weight / volume). In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the sucrose is present at a concentration of about 10% (weight / volume).

[0189] In one embodiment, the storage matrix is ​​sucrose, the buffer is a mixture of HEPES and Tris, and the sucrose is present at a concentration of about 1% to about 30% (weight / volume). In one embodiment, the storage matrix is ​​sucrose, the buffer is a mixture of HEPES and Tris, and the sucrose is present at a concentration of about 2% to about 20% (weight / volume). In one embodiment, the storage matrix is ​​sucrose, the buffer is a mixture of HEPES and Tris, and the sucrose is present at a concentration of about 5% to about 15% (weight / volume). In one embodiment, the storage matrix is ​​sucrose, the buffer is a mixture of HEPES and Tris, and the sucrose is present at a concentration of about 8% to about 12% (weight / volume). In one embodiment, the storage matrix is ​​sucrose, the buffer is a mixture of HEPES and Tris, and the sucrose is present at a concentration of about 10% (weight / volume).

[0190] In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 6.5 to about 8.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 7.0 to about 8.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 6.8 to about 8.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 6.5 to about 7.5. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 6.8 to about 7.2. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 6.9 to about 7.1. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 7.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 7.1 to about 7.7. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 7.2 to about 7.6. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 7.3 to about 7.5. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 7.4. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 6.8 to about 7.6. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 7.0 to about 7.4. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 7.1 to about 7.4.

[0191] In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 6.5 to about 8.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 7.0 to about 8.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 6.8 to about 8.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 6.5 to about 7.5. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 6.8 to about 7.2. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 6.9 to about 7.1. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 7.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 7.1 to about 7.7. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 7.2 to about 7.6. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 7.3 to about 7.5. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 7.4. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 6.8 to about 7.6. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 7.0 to about 7.4. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is from about 7.1 to about 7.4.

[0192] In one embodiment, the storage matrix is ​​sucrose, the buffer is a mixture of HEPES and Tris, and the pH of the buffer solution is about 6.5 to about 8.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is a mixture of HEPES and Tris, and the pH of the buffer solution is about 7.0 to about 8.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is a mixture of HEPES and Tris, and the pH of the buffer solution is about 6.8 to about 8.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is a mixture of HEPES and Tris, and the pH of the buffer solution is about 6.5 to about 7.5. In one embodiment, the storage matrix is ​​sucrose, the buffer is a mixture of HEPES and Tris, and the pH of the buffer solution is about 6.8 to about 7.2. In one embodiment, the storage matrix is ​​sucrose, the buffer is a mixture of HEPES and Tris, and the pH of the buffer solution is about 6.9 to about 7.1. In one embodiment, the storage matrix is ​​sucrose, the buffer is a mixture of HEPES and Tris, and the pH of the buffer solution is about 7.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is a mixture of HEPES and Tris, and the pH of the buffer solution is about 7.1 to about 7.7. In one embodiment, the storage matrix is ​​sucrose, the buffer is a mixture of HEPES and Tris, and the pH of the buffer solution is about 7.2 to about 7.6. In one embodiment, the storage matrix is ​​sucrose, the buffer is a mixture of HEPES and Tris, and the pH of the buffer solution is about 7.3 to about 7.5. In one embodiment, the storage matrix is ​​sucrose, the buffer is a mixture of HEPES and Tris, and the pH of the buffer solution is about 7.4. In one embodiment, the storage matrix is ​​sucrose, the buffer is a mixture of HEPES and Tris, and the pH of the buffer solution is about 6.8 to about 7.6. In one embodiment, the storage matrix is ​​sucrose, the buffer is a mixture of HEPES and Tris, and the pH of the buffer solution is about 7.0 to about 7.4.In one embodiment, the storage matrix is ​​sucrose, the buffer is a mixture of HEPES and Tris, and the pH of the buffer solution is from about 7.1 to about 7.4.

[0193] In one embodiment, the aqueous dispersion is substantially free of inorganic cations (as defined herein). Such inorganic cations are believed to affect the colloidal stability of the lipid dispersion and reduce the stability of the formulation. In one embodiment, the aqueous dispersion is substantially free of alkali metal ions. In one embodiment, the aqueous dispersion is substantially free of ammonium, sodium and / or potassium ions.

[0194] In one embodiment, the aqueous dispersion is substantially free of organic solvents. In one embodiment, the term "substantially free of organic solvents" means that the aqueous dispersion contains less than about 50,000 ppm by weight of organic solvent as a percentage of the total weight of the aqueous dispersion, such as less than about 40,000 ppm, for example, less than about 30,000 ppm, such as less than about 20,000 ppm, for example, less than about 10,000 ppm, such as less than about 9,000 ppm, for example, less than about 8,000 ppm, for example, less than about 7,000 ppm, for example, less than about 6,000 ppm, for example, less than about 5 This means containing less than about 1,000 ppm, for example, less than about 4,000 ppm, for example, less than about 3,000 ppm, for example, less than about 2,000 ppm, for example, less than about 1,000 ppm, for example, less than about 900 ppm, for example, less than about 800 ppm, for example, less than about 700 ppm, for example, less than about 600 ppm, for example, less than about 500 ppm, for example, less than about 400 ppm, for example, less than about 300 ppm, for example, less than about 200 ppm, for example, less than about 100 ppm.

[0195] In one embodiment, the aqueous dispersion may be substantially free of water-soluble organic solvents, such as C1-4 alcohols (e.g., isopropanol or ethanol), ketones (e.g., acetone), or mixtures thereof; and / or non-polar organic solvents, such as hydrocarbons, e.g., pentane or hexane; chlorinated hydrocarbons, e.g., dichloromethane or chloroform; or mixtures thereof. In one embodiment, the aqueous dispersion is substantially free of organic solvents, including isopropanol, methanol, ethanol, and / or acetone. In one embodiment, the aqueous dispersion is substantially free of ethanol.

[0196] In one embodiment, the aqueous dispersion is substantially free of nucleic acids. In one embodiment, the aqueous dispersion is substantially free of RNA. In one embodiment, the aqueous dispersion is substantially free of DNA.

[0197] In one embodiment, the aqueous dispersion is substantially free of acetate buffer and citrate buffer. The aqueous dispersion may be substantially free of acetate buffer. The aqueous dispersion may be substantially free of citrate buffer. The aqueous mobile phase may be substantially free of citrate buffer. The aqueous dispersion and / or the aqueous mobile phase may be substantially free of citrate buffer containing about 10 mM citric acid, about 150 mM NaCl, and a pH of about 4.5. The aqueous dispersion may be substantially free of buffers. The aqueous dispersion may be substantially free of buffers selected from the group consisting of ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. The aqueous dispersion may be substantially free of buffers selected from the group consisting of ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate.

[0198] Method for forming an aqueous dispersion In a further aspect, the present disclosure provides a method for producing the aqueous dispersion of the present invention.

[0199] In one aspect, the present disclosure provides a method for forming an aqueous dispersion having a pH of about 6.5 to about 8.0 and substantially free of organic solvents and nucleic acids, the method comprising: (A) (i) an organic phase comprising a lipid mixture comprising a cationically ionizable lipid dissolved in a water-soluble organic solvent; and (ii) an aqueous phase containing aqueous acid and substantially free of inorganic cations; to produce an intermediate aqueous lipid dispersion; and (B) performing a dialysis or filtration step on the intermediate aqueous lipid dispersion to produce an aqueous dispersion, wherein the dialysis or filtration step removes organic solvents and adjusts the pH to about 6.5 to about 8.0. The present invention provides a method comprising:

[0200] This method is referred to herein as the “first pre-LNP formation method.” In this method, the aqueous dispersion preferably contains pre-LNPs as defined above.

[0201] In one aspect, the present disclosure provides a method for forming an aqueous dispersion having a pH of about 6.5 to about 8.0 and substantially free of organic solvents and nucleic acids, the method comprising: (A)(i) an organic phase comprising a lipid mixture comprising cationically ionizable lipids dissolved in a water-soluble organic solvent, the organic phase further comprising an aqueous acid, the organic phase being substantially free of inorganic cations; and (ii) aqueous phase to produce an intermediate aqueous lipid dispersion having a pH of about 6.5 to about 8.0; and (B) performing a dialysis or filtration step on the intermediate aqueous lipid dispersion to produce an aqueous dispersion, wherein the dialysis or filtration step removes the organic solvent. The present invention provides a method comprising:

[0202] This method is referred to herein as the “second pre-LNP formation method.” In this method, the aqueous dispersion preferably contains pre-LNPs as defined above.

[0203] Mixing Step The mixing step, which comprises step (A) of both the first and second pre-LNP formation methods defined above, involves mixing an organic phase comprising a lipid mixture comprising cationically ionizable lipids dissolved in a water-soluble organic solvent with an aqueous phase to produce an intermediate aqueous lipid dispersion, which is then processed in step (B) and further steps, if necessary, to produce a final aqueous dispersion, typically containing pre-LNPs.

[0204] Both the first and second pre-LNP formation methods use an acid. In the first pre-LNP formation method, the acid is in solution in water and therefore forms part of the aqueous phase (herein termed "aqueous acid"). In the second pre-LNP formation method, the acid is in solution in an organic solvent and therefore forms part of the organic phase.

[0205] The acid can be any inorganic or organic acid that is at least partially miscible with water and can be at least partially deprotonated in water to produce the anion of the acid (i.e., the conjugate base). Thus, it will be understood by those skilled in the art that, depending on the pH and strength of the acid, the aqueous mobile phase can contain varying proportions of both the undissociated acid and its corresponding anion. Strong acids are completely or mostly deprotonated in water, so that the species in the aqueous solution are primarily (in some embodiments, completely) the anion of the acid. In contrast, weak acids are not completely deprotonated in water, so that the species in the aqueous solution comprise a mixture of the undissociated acid and its conjugate base, with the relative amounts of each depending on the pH.

[0206] Additionally, acids undergo acid-base reactions with cationically ionizable lipids to produce the cationically ionizable lipid in its charged form and the acid in its anionic form, the extent to which such reactions occur depends on factors such as the basicity of the cationically ionizable lipid (if present in its neutral form) and the pH.

[0207] In one embodiment, the acid is an inorganic acid. Examples of suitable inorganic acids include hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0208] In one embodiment, the acid is a water-soluble organic acid. Examples of suitable organic acids include sulfonic acids, carboxylic acids, dicarboxylic acids, or hydroxycarboxylic acids (all as defined herein).

[0209] In one embodiment, the water-soluble organic acid is selected from the group consisting of acetic acid, malic acid, succinic acid, and citric acid, and combinations thereof. In one embodiment, the water-soluble organic acid may be selected from the group consisting of acetic acid and malic acid, and combinations thereof.

[0210] In one embodiment, the water-soluble organic weak acid is acetic acid. In one embodiment, the water-soluble organic weak acid is malic acid. In one embodiment, the water-soluble organic weak acid is succinic acid. In one embodiment, the water-soluble organic weak acid is citric acid.

[0211] In one embodiment, the acid concentration is in the range of about 0.1 to about 20 mM. In one embodiment, the acid concentration is in the range of about 0.2 to about 15 mM. In one embodiment, the acid concentration is in the range of about 0.5 to about 10 mM. In one embodiment, the acid concentration is in the range of about 1 to about 5 mM. In one embodiment, the acid concentration is in the range of about 2 to about 10 mM. In one embodiment, the acid concentration is in the range of about 0.5 to about 5 mM. In one embodiment, the acid concentration is in the range of about 3 to about 15 mM. In one embodiment, the acid concentration is in the range of about 5 to about 8 mM. In one embodiment, the acid concentration is in the range of about 8 to about 12 mM. In this context, this concentration is understood to include both the undissociated acid and its conjugate base.

[0212] In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 0.2 to about 20 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 0.5 to about 10 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 3 to about 15 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 5 to about 8 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 8 to about 12 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 1 to about 8 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 2 to about 7 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 4 to about 6 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 4.5 to about 5.5 mM. In one embodiment, the acid is acetic acid and is present at a concentration of about 5 mM. In one embodiment, the acid is acetic acid and forms part of the aqueous phase and is present at a concentration ranging from about 4 to about 6 mM. In one embodiment, the acid is acetic acid, forms part of the aqueous phase, and is present at a concentration ranging from about 4.5 to about 5.5 mM. In one embodiment, the acid is acetic acid, forms part of the aqueous phase, and is present at a concentration of about 5 mM. In one embodiment, the acid is acetic acid, forms part of the organic phase, and is present at a concentration ranging from about 8 to about 12 mM. In one embodiment, the acid is acetic acid, forms part of the organic phase, and is present at a concentration ranging from about 9 to about 11 mM. In one embodiment, the acid is acetic acid, forms part of the organic phase, and is present at a concentration ranging from about 9.5 to about 10.5 mM. In one embodiment, the acid is acetic acid, forms part of the organic phase, and is present at a concentration of about 10 mM.

[0213] In one embodiment, the acid is malic acid and is present at a concentration ranging from about 0.1 to about 5 mM. In one embodiment, the acid is malic acid and is present at a concentration ranging from about 0.4 to about 4 mM. In one embodiment, the acid is malic acid and is present at a concentration ranging from about 0.8 to about 2 mM. In one embodiment, the acid is malic acid and is present at a concentration ranging from about 1 to about 1.5 mM. In one embodiment, the acid is malic acid and is present at a concentration of about 1.25 mM.

[0214] In one embodiment, the acid is citric acid and is present at a concentration ranging from about 0.2 to about 15 mM. In one embodiment, the acid is citric acid and is present at a concentration ranging from about 0.5 to about 10 mM. In one embodiment, the acid is citric acid and is present at a concentration ranging from about 1 to about 8 mM. In one embodiment, the acid is citric acid and is present at a concentration ranging from about 2 to about 7 mM. In one embodiment, the acid is citric acid and is present at a concentration ranging from about 4 to about 6 mM. In one embodiment, the acid is citric acid and is present at a concentration ranging from about 4.5 to about 5.5 mM. In one embodiment, the acid is citric acid and is present at a concentration of about 5 mM.

[0215] In one embodiment, the acid is succinic acid and is present at a concentration ranging from about 0.2 to about 10 mM. In one embodiment, the acid is succinic acid and is present at a concentration ranging from about 0.4 to about 5 mM. In one embodiment, the acid is succinic acid and is present at a concentration ranging from about 1 to about 3.5 mM. In one embodiment, the acid is succinic acid and is present at a concentration ranging from about 2 to about 3 mM. In one embodiment, the acid is succinic acid and is present at a concentration of about 2.5 mM.

[0216] In one embodiment of the first pre-LNP formation method, the aqueous dispersion formed by the method is substantially free of inorganic cations.

[0217] In one embodiment of the second pre-LNP formation method, the aqueous dispersion formed by the method is substantially free of inorganic cations.

[0218] In one embodiment of the first pre-LNP formation method, the intermediate aqueous dispersion formed by step (A) of the method is substantially free of inorganic cations.

[0219] In one embodiment of the second pre-LNP formation method, the intermediate aqueous dispersion formed by step (A) of the method is substantially free of inorganic cations.

[0220] In one embodiment of the first pre-LNP formation method, step (A) is carried out at a pH of about 4.0 to about 5.0.

[0221] In one embodiment of the second pre-LNP formation method, step (A) is performed at a pH of about 6.5 to about 8.0. In one embodiment of the second pre-LNP formation method, step (A) is performed at a pH of about 7.0 to about 7.5. In one embodiment of the second pre-LNP formation method, step (A) is performed at a pH of about 7.0.

[0222] In one embodiment of step (A) of the second pre-LNP formation method, the aqueous phase comprises a buffer. In one embodiment, the buffer used in the aqueous dispersion is a neutral buffer. In one embodiment, the buffer has a pK of about 6.0 to about 8.0. a In one embodiment, the buffer has a pK of about 6.5 to about 7.5. a It has.

[0223] In one embodiment, the buffer used in step (A) of the second pre-LNP formation method comprises an anionic group / moiety. In one embodiment, the buffer used in step (A) of the second pre-LNP formation method comprises a sulfonic acid group / moiety, or a derivative thereof. In one embodiment, the buffer used in step (A) of the second pre-LNP formation method is a zwitterionic acidic buffer. In one embodiment, the buffer used in step (A) of the second pre-LNP formation method comprises an amino group / moiety and a sulfonic acid group / moiety, or a derivative thereof. In one embodiment, the buffer used in step (A) of the second pre-LNP formation method is selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-morpholin-4-ylethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-cyclohexyl-3-aminopropane-sulfonic acid (CAPS), 3-[4-(2-hydroxy-ethyl)piperazin-1-yl]propane-1-sulfonic acid (HEPPS), 2-(bis(2-hydroxy-ethyl)amino)propanesulfonic acid (BIS(2-hydroxy-ethyl)amino)propanesulfonic acid), 2-(N-morpholino)propanesulfonic acid (MOPS ... )ethanesulfonic acid (BES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), piperazine-N,N'-bis(2-ethanesulfonic acid (PIPES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), 2-(bis(2-hydroxyethyl)amino)ethanesulfonic acid (BES), and 2-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}ethane-1-sulfonic acid (TES), and mixtures of any thereof.

[0224] In one embodiment, the buffer used in step (A) of the second pre-LNP formation method comprises a cationic group / moiety. In one embodiment, the buffer used in step (A) of the second pre-LNP formation method comprises a basic group / moiety. In one embodiment, the buffer used in step (A) of the second pre-LNP formation method comprises an amino group / moiety. In one embodiment, the buffer used in step (A) of the second pre-LNP formation method comprises a primary, secondary, or tertiary amine group / moiety. In one embodiment, the buffer used in step (A) of the second pre-LNP formation method comprises an amino group / moiety (e.g., a primary, secondary, or tertiary amine group / moiety) and does not comprise an anionic moiety (e.g., a sulfonic acid group / moiety). In one embodiment, the buffer used in step (A) of the second pre-LNP formation method is selected from the group consisting of tris(hydroxymethyl)aminomethane (Tris), TAE (a buffer solution containing a mixture of Tris, acetic acid, and ethylenediaminetetraacetic acid), TBE (a buffer solution containing a mixture of Tris, boric acid, and ethylenediaminetetraacetic acid), TEA, (2-(bis(2-hydroxyethyl)amino)acetic acid) (bicine), (N-[tris(hydroxymethyl)methyl]glycine) (tricine), triethylammonium acetate, triethanolamine, N-(2-acetamido)iminodiacetic acid (ADA), and mixtures of any of these.

[0225] In one embodiment, the buffer used in step (A) of the second pre-LNP formation method is a mixture of a buffer containing an anionic group / moiety and a buffer containing a cationic group / moiety. In one embodiment, the buffer used in step (A) of the second pre-LNP formation method is a mixture of a buffer containing an amino group / moiety (e.g., primary, secondary, or tertiary amine group / moiety) and a buffer containing a sulfonic acid group / moiety. In one embodiment, the buffer used in step (A) of the second pre-LNP formation method is a mixture of a zwitterionic acidic buffer and a buffer containing an amino group / moiety (e.g., primary, secondary, or tertiary amine group / moiety).

[0226] In one embodiment of step (A) of the second pre-LNP formation method, the buffer is selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)-aminomethane (Tris), 2-morpholin-4-ylethane-sulfonic acid (MES), bis-(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (bis-Tris), and phosphate buffer, and mixtures of any of these.

[0227] In one embodiment of step (A) of the second pre-LNP formation method, the buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). In one embodiment, the buffer is tris(hydroxymethyl)aminomethane (Tris). In one embodiment, the buffer is 2-morpholin-4-ylethanesulfonic acid (MES). In one embodiment, the buffer is bis-(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (bis-Tris). In one embodiment, the buffer is a phosphate buffer.

[0228] In one embodiment of step (A) of the second pre-LNP formation method, the buffer is a mixture of HEPES and Tris.

[0229] In one embodiment of step (A) of the second pre-LNP formation method, the buffer is HEPES and is present at a concentration of about 10 mM to about 1 M. In one embodiment of the second pre-LNP formation method, the buffer is HEPES and is present at a concentration of about 20 mM to about 500 mM. In one embodiment of the second pre-LNP formation method, the buffer is HEPES and is present at a concentration of about 50 mM to about 200 mM. In one embodiment of the second pre-LNP formation method, the buffer is HEPES and is present at a concentration of about 100 mM.

[0230] In one embodiment of step (A) of the second pre-LNP formation method, the buffer is Tris and is present at a concentration of about 0.5 mM to about 50 mM. In one embodiment of the second pre-LNP formation method, the buffer is Tris and is present at a concentration of about 1 mM to about 25 mM. In one embodiment of the second pre-LNP formation method, the buffer is Tris and is present at a concentration of about 2 mM to about 10 mM. In one embodiment of the second pre-LNP formation method, the buffer is Tris and is present at a concentration of about 5 mM.

[0231] In one embodiment, the intermediate aqueous dispersion formed by step (A) of the first pre-LNP formation method has a pH of less than 5.5. In one embodiment, the intermediate aqueous dispersion formed by step (A) of the first pre-LNP formation method has a pH of about 3.5 to about 5.5.

[0232] In one embodiment, the intermediate aqueous dispersion formed by step (A) of the second pre-LNP formation method has a pH of about 6.5 to about 7.0. In one embodiment, the intermediate aqueous dispersion formed by step (A) of the second pre-LNP formation method has a pH of about 7.0.

[0233] In one embodiment, the mixing step is carried out using a T-mixer or a Y-mixer.

[0234] In one embodiment, the flow rate during the mixing step can be from about 20 mL / min to about 400 mL / min, optionally from about 100 mL / min to about 300 mL / min, and optionally from about 150 mL / min to about 250 mL / min. In one embodiment, the flow rate during the mixing step is from about 200 mL / min to about 250 mL / min.

[0235] The volume ratio of the organic solvent to the aqueous phase may be about 1:1 to about 1:10 (organic phase:aqueous phase), optionally about 1:2 to about 1:6, preferably about 1:3 to about 1:5, and more preferably about 1:4.

[0236] Storage matrix / cryoprotectant in the mixing step In one embodiment, the aqueous phase further contains a storage matrix, which can be any of the storage matrices defined and exemplified above. In one embodiment, the aqueous phase further contains a cryoprotectant, as defined and exemplified above.

[0237] In one embodiment, the cryoprotectant is a carbohydrate. In one embodiment, the cryoprotectant is a monosaccharide or disaccharide. In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose, lactose, and glucose, and any mixture thereof. In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose, and glucose, and any mixture thereof. Preferably, the cryoprotectant is sucrose, trehalose, or a mixture thereof. In a preferred embodiment, the cryoprotectant is sucrose. In a preferred embodiment, the cryoprotectant is trehalose.

[0238] When the aqueous phase dispersion also contains a cryoprotectant that is a carbohydrate, it is typically present at a concentration of about 1% to about 20% (weight / volume). When the aqueous phase dispersion also contains a cryoprotectant that is a carbohydrate, it is typically present at a concentration of about 1% to about 30% (weight / volume). In one embodiment, the cryoprotectant is a carbohydrate and is present at a concentration of about 3% to about 12% (weight / volume). In one embodiment, the cryoprotectant is a carbohydrate and is present at a concentration of about 5% to about 10% (weight / volume). In one embodiment, the cryoprotectant is a carbohydrate and is present at a concentration of about 15% to about 25% (weight / volume). In one embodiment, the cryoprotectant is a carbohydrate and is present at a concentration of about 18% to about 22% (weight / volume).

[0239] In one embodiment, the cryoprotectant is sucrose and is present at a concentration of about 1% to about 20% (weight / volume). In one embodiment, the cryoprotectant is sucrose and is present at a concentration of about 3% to about 12% (weight / volume). In one embodiment, the cryoprotectant is sucrose and is present at a concentration of about 5% to about 10% (weight / volume). In one embodiment, the cryoprotectant is sucrose and is present at a concentration of about 15% to about 25% (weight / volume), for example, about 18% to about 22% (weight / volume).

[0240] In one embodiment, the cryoprotectant is trehalose and is present at a concentration of about 1% to about 20% (weight / volume). In one embodiment, the cryoprotectant is trehalose and is present at a concentration of about 3% to about 12% (weight / volume). In one embodiment, the cryoprotectant is trehalose and is present at a concentration of about 5% to about 10% (weight / volume). In one embodiment, the cryoprotectant is trehalose and is present at a concentration of about 15% to about 25% (weight / volume), for example, about 18% to about 22% (weight / volume).

[0241] In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 0.5% to about 10% (weight / volume). In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 1.5% to about 6% (weight / volume). In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 2.5% to about 5% (weight / volume). In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 8% to about 12% (weight / volume).

[0242] Dialysis / filtration step The method of the present invention further comprises performing a dialysis or filtration step on the intermediate aqueous lipid dispersion to remove the organic solvent.

[0243] Specifically, step (B) of both the first and second pre-LNP formation methods includes subjecting the intermediate aqueous lipid dispersion to a dialysis or filtration step to remove the organic solvent. In the case of the second pre-LNP formation method, the conditions used in step (B) also involve adjusting the pH from the acidic pH used in step (A) to a near-neutral pH, typically from about 6.5 to about 8.0.

[0244] In one embodiment, the dialysis or filtration step comprises tangential flow filtration (TFF).

[0245] Typically, step (B) of the dialysis or filtration process of both the first and second pre-LNP formation methods uses a buffer solution. In one embodiment of step (B) of either the first or second pre-LNP formation method, the buffer used in the aqueous dispersion is a neutral buffer. In one embodiment, the buffer has a pK of about 6.0 to about 8.0. a In one embodiment, the buffer has a pK of about 6.5 to about 7.5. a It has.

[0246] In one embodiment, the buffer used in step (B) of either the first or second pre-LNP formation method comprises an anionic group / moiety. In one embodiment, the buffer used in step (B) of either the first or second pre-LNP formation method comprises a sulfonic acid group / moiety, or a derivative thereof. In one embodiment, the buffer used in step (B) of either the first or second pre-LNP formation method is a zwitterionic acidic buffer. In one embodiment, the buffer used in step (B) of either the first or second pre-LNP formation method comprises an amino group / moiety and a sulfonic acid group / moiety, or a derivative thereof. In one embodiment, the buffer used in step (B) of either the first or second pre-LNP formation method is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-morpholin-4-ylethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 3-[4-(2-hydroxy-ethyl)piperazin-1-yl]propane-1-sulfonic acid (HEPPS), 2-(bis(2-hydroxy-ethyl)piperazine)-1-sulfonic acid), 2-(methyl-2-methyl-4-pyridinyl) ...2-(methyl-2-methyl-4-pyridinyl)ethanesulfonic acid (MES), 2-(methyl-2-methyl-4-pyridinyl)ethanesulfonic acid (MOPS), 2-(methyl-2-methyl-4-pyridinyl)ethanesulfonic acid (MOPS), 2-(methyl-2-methyl-4-pyridinyl)ethanesulfonic acid (MOPS), 2-(methyl-2-methyl-4-pyridinyl)ethanesulfonic acid (MOPS), 2-(methyl-2-methyl-4-pyridinyl)ethanesulfonic acid (MOPS), 2-(methyl-2-methyl-4-pyridinyl)ethanesulfonic acid (MOPS), 2-(methyl-2-methyl-4 2-(bis(2-hydroxyethyl)amino)ethanesulfonic acid (BES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), piperazine-N,N'-bis(2-ethanesulfonic acid (PIPES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), 2-(bis(2-hydroxyethyl)amino)ethanesulfonic acid (BES), and 2-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}ethane-1-sulfonic acid (TES), and mixtures of any of these.

[0247] In one embodiment, the buffer used in step (B) of either the first or second pre-LNP formation method comprises a cationic group / moiety. In one embodiment, the buffer used in step (B) of either the first or second pre-LNP formation method comprises a basic group / moiety. In one embodiment, the buffer used in step (B) of either the first or second pre-LNP formation method comprises an amino group / moiety. In one embodiment, the buffer used in step (B) of either the first or second pre-LNP formation method comprises a primary, secondary, or tertiary amine group / moiety. In one embodiment, the buffer used in step (B) of either the first or second pre-LNP formation method comprises an amino group / moiety (e.g., a primary, secondary, or tertiary amine group / moiety) and does not comprise an anionic moiety (e.g., a sulfonic acid group / moiety). In one embodiment, the buffer used in step (B) of either the first or second pre-LNP formation method is selected from the group consisting of tris(hydroxymethyl)aminomethane (Tris), TAE (a buffer solution containing a mixture of Tris, acetic acid, and ethylenediaminetetraacetic acid), TBE (a buffer solution containing a mixture of Tris, boric acid, and ethylenediaminetetraacetic acid), TEA, (2-(bis(2-hydroxyethyl)amino)acetic acid) (bicine), (N-[tris(hydroxymethyl)methyl]glycine) (tricine), triethylammonium acetate, triethanolamine, and N-(2-acetamido)iminodiacetic acid (ADA), and mixtures of any of these.

[0248] In one embodiment, the buffer used in step (B) of either the first or second pre-LNP formation method is a mixture of a buffer containing an anionic group / moiety and a buffer containing a cationic group / moiety. In one embodiment, the buffer used in step (B) of either the first or second pre-LNP formation method is a mixture of a buffer containing an amino group / moiety (e.g., primary, secondary, or tertiary amine group / moiety) and a buffer containing a sulfonic acid group / moiety. In one embodiment, the buffer used in step (B) of either the first or second pre-LNP formation method is a mixture of a zwitterionic acidic buffer and a buffer containing an amino group / moiety (e.g., primary, secondary, or tertiary amine group / moiety).

[0249] In one embodiment of step (B) of either the first or second pre-LNP formation method, the buffer is selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)-aminomethane (Tris), 2-morpholin-4-ylethanesulfonic acid (MES), bis-(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (bis-Tris), and phosphate buffer, and mixtures of any of these.

[0250] In one embodiment of step (B) of either the first or second pre-LNP formation method, the buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). In one embodiment of step (B) of either the first or second pre-LNP formation method, the buffer is tris(hydroxymethyl)aminomethane (Tris). In one embodiment of step (B) of either the first or second pre-LNP formation method, the buffer is 2-morpholin-4-ylethanesulfonic acid (MES). In one embodiment of step (B) of either the first or second pre-LNP formation method, the buffer is bis-(2-hydroxyethyl)-amino-tris(hydroxymethyl)methane (bis-Tris). In one embodiment of step (B) of either the first or second pre-LNP formation method, the buffer is phosphate buffer. In one embodiment of step (B) of either the first or second pre-LNP formation method, the buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). In one embodiment of step (B) of either the first or second pre-LNP formation method, the buffer is a mixture of HEPES and Tris.

[0251] In one embodiment of step (B) of the first pre-LNP formation method, the buffer is HEPES and is present at a concentration of about 2 mM to about 100 mM. In one embodiment of step (B) of the first pre-LNP formation method, the buffer is Tris and is present at a concentration of about 1 mM to about 50 mM. In one embodiment of step (B) of the first pre-LNP formation method, the buffer is a mixture of HEPES and Tris, where HEPES is present at a concentration of about 2 mM to about 100 mM and Tris is present at a concentration of about 1 mM to about 30 mM.

[0252] In one embodiment of step (B) of the second pre-LNP formation method, the buffer is HEPES and is present at a concentration of about 2 mM to about 100 mM. In one embodiment of step (B) of the second pre-LNP formation method, the buffer is Tris and is present at a concentration of about 1 mM to about 50 mM. In one embodiment of step (B) of the second pre-LNP formation method, the buffer is a mixture of HEPES and Tris, where HEPES is present at a concentration of about 2 mM to about 100 mM and Tris is present at a concentration of about 1 mM to about 30 mM.

[0253] In one embodiment, the aqueous dispersion obtained from step (B) of the first pre-LNP formation method has a pH of about 6.5 to about 8.0. In one embodiment, the aqueous dispersion obtained from step (B) of the first pre-LNP formation method has a pH of about 6.7 to about 7.5. In one embodiment, the aqueous dispersion obtained from step (B) of the first pre-LNP formation method has a pH of about 6.8 to about 7.2. In one embodiment, the aqueous dispersion obtained from step (B) of the first pre-LNP formation method has a pH of about 6.9 to about 7.1. In one embodiment, the aqueous dispersion obtained from step (B) of the first pre-LNP formation method has a pH of about 7.0.

[0254] In one embodiment, the aqueous dispersion obtained from step (B) of the second pre-LNP formation method has a pH of about 6.5 to about 8.0. In one embodiment, the aqueous dispersion obtained from step (B) of the second pre-LNP formation method has a pH of about 6.7 to about 7.5. In one embodiment, the aqueous dispersion obtained from step (B) of the second pre-LNP formation method has a pH of about 6.8 to about 7.2. In one embodiment, the aqueous dispersion obtained from step (B) of the second pre-LNP formation method has a pH of about 6.9 to about 7.1. In one embodiment, the aqueous dispersion obtained from step (B) of the second pre-LNP formation method has a pH of about 7.0.

[0255] In one embodiment, the method further comprises subjecting the aqueous dispersion obtained from step (B) to one or more further processing steps, which in one embodiment comprise dilution or addition of a storage matrix as defined and exemplified below.

[0256] Dilution / Addition of storage matrix / Cryoprotectant In one embodiment, the method may further comprise the additional step of adding a storage matrix to the aqueous dispersion, which preferably occurs after the dialysis or filtration step, although alternatively, it may occur immediately after the mixing step to form the aqueous dispersion.

[0257] The storage matrix used in this step can be any of the storage matrices defined and exemplified above. In one embodiment, the storage matrix comprises a cryoprotectant.

[0258] Thus, in one embodiment, either the first or second pre-LNP formation method further comprises the additional step of, after step (B): (B1) Adding a cryoprotectant to the aqueous dispersion may include:

[0259] The cryoprotectant dilutes the aqueous dispersion and protects the pre-LNP from freezing damage. The cryoprotectant is not particularly limited, so long as it is capable of performing this function. In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose, glucose, sorbitol, fructose, maltose, xylose, and dextran, and mixtures of any of these. In a preferred embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose, and glucose, and mixtures of any of these. In a more preferred embodiment, the cryoprotectant is selected from the group consisting of sucrose and trehalose, and mixtures thereof. In a highly preferred embodiment, the cryoprotectant is sucrose. In another highly preferred embodiment, the cryoprotectant is trehalose.

[0260] In one embodiment, the storage matrix further comprises one or more of the following classes (a) to (c): (a) the amino acids defined and exemplified above, for example: (i) acidic amino acids as defined and exemplified above, preferably selected from the group consisting of aspartic acid, glutamic acid, 3-hydroxyglutamic acid, and alpha-aminoadipic acid, and mixtures thereof; (ii) basic amino acids, as defined and exemplified above, preferably selected from the group consisting of arginine, histidine, and lysine, and mixtures thereof; or a mixture of (i) and (ii), optionally mixed with a neutral amino acid; (b) an organic acid as defined and exemplified above, preferably selected from the group consisting of acetic acid, malic acid, succinic acid, citric acid, and methylmalonic acid, and mixtures thereof; (c) a buffer solution as defined and exemplified above, preferably a buffer solution containing an amino group, a buffer solution containing a sulfonic acid group, or a mixture thereof, more preferably a buffer solution which is HEPES, Tris, MES, or a mixture of any of these; and mixtures of any of these The compound includes a compound selected from:

[0261] In one embodiment of any step (B1) of either the first or second pre-LNP formation method, the cryoprotectant is present at a concentration of about 1% to about 30% (weight / volume). In one embodiment, the cryoprotectant is present at a concentration of about 2% to about 20% (weight / volume). In one embodiment, the cryoprotectant is present at a concentration of about 5% to about 15% (weight / volume). In one embodiment, the cryoprotectant is present at a concentration of about 8% to about 12% (weight / volume). In one embodiment, the cryoprotectant is present at a concentration of about 10% (weight / volume).

[0262] In one embodiment of any step (B1) of either the first or second pre-LNP formation method, the cryoprotectant is a carbohydrate and is present at a concentration of about 2% to about 50% (weight / volume). In one embodiment, the cryoprotectant is a carbohydrate and is present at a concentration of about 3% to about 40% (weight / volume). In one embodiment, the cryoprotectant is a carbohydrate and is present at a concentration of about 10% to about 30% (weight / volume). In one embodiment, the cryoprotectant is a carbohydrate and is present at a concentration of about 15% to about 25% (weight / volume). In one embodiment, the cryoprotectant is a carbohydrate and is present at a concentration of about 20% (weight / volume).

[0263] In one embodiment of any step (B1) of either the first or second pre-LNP formation method, the cryoprotectant is sucrose or trehalose and is present at a concentration of about 1% to about 30% (weight / volume). In one embodiment, the cryoprotectant is sucrose or trehalose and is present at a concentration of about 2% to about 20% (weight / volume). In one embodiment, the cryoprotectant is sucrose or trehalose and is present at a concentration of about 5% to about 15% (weight / volume). In one embodiment, the cryoprotectant is sucrose or trehalose and is present at a concentration of about 8% to about 12% (weight / volume). In one embodiment, the cryoprotectant is sucrose or trehalose and is present at a concentration of about 10% (weight / volume). In one embodiment, the cryoprotectant is sucrose or trehalose and is present at a concentration of about 18% to about 22% (weight / volume). In one embodiment, the cryoprotectant is sucrose or trehalose and is present at a concentration of about 20% (weight / volume).

[0264] In one embodiment of any step (B1) of either the first or second pre-LNP formation method, the cryoprotectant is glucose and is present at a concentration of about 0.5% to about 15% (weight / volume). In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 1% to about 10% (weight / volume). In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 2.5% to about 7.5% (weight / volume). In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 4% to about 6% (weight / volume). In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 5% (weight / volume). In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 8% to about 12% (weight / volume). In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 10% (weight / volume).

[0265] In one embodiment of any step (B1) of either the first or second pre-LNP formation method, the cryoprotectant is present in a buffer solution. The buffer used can be any of the buffers defined and exemplified above in connection with step (A) or step (B), or a mixture of any of these. In one embodiment, the buffer is HEPES. In one embodiment, the buffer is Tris. In one embodiment, the buffer is a mixture of HEPES and Tris.

[0266] In one embodiment of any step (B1) of either the first or second pre-LNP formation method, the cryoprotectant is sucrose and the buffer is HEPES. In one embodiment, the cryoprotectant is sucrose and the buffer is Tris. In one embodiment, the cryoprotectant is sucrose and the buffer is a mixture of HEPES and Tris.

[0267] In one embodiment of either the first or second pre-LNP formation method, the cryoprotectant solution has a pH of about 6.5 to about 8.0.

[0268] In one embodiment, either the first or second pre-LNP formation method further comprises the following additional step after step (B), and if performed, after step (B1): (B2) Adding a storage matrix to the aqueous dispersion Includes:

[0269] In one embodiment, the storage matrix is ​​selected from the group consisting of sucrose, glycerol, trehalose, lactose, glucose, and mannitol, hi one embodiment, the storage matrix is ​​sucrose.

[0270] In one embodiment of any step (B2) of either the first or second pre-LNP formation method, the storage matrix is ​​typically present at a concentration of about 1% to about 30% (weight / volume). In one embodiment, the storage matrix is ​​present at a concentration of about 2% to about 20% (weight / volume). In one embodiment, the storage matrix is ​​present at a concentration of about 5% to about 15% (weight / volume). In one embodiment, the storage matrix is ​​present at a concentration of about 8% to about 12% (weight / volume). In one embodiment, the storage matrix is ​​present at a concentration of about 10% (weight / volume).

[0271] In one embodiment of any step (B2) of either the first or second pre-LNP formation method, the storage matrix is ​​sucrose and is present at a concentration of about 1% to about 30% (weight / volume). In one embodiment, the storage matrix is ​​sucrose and is present at a concentration of about 2% to about 20% (weight / volume). In one embodiment, the storage matrix is ​​sucrose and is present at a concentration of about 5% to about 15% (weight / volume). In one embodiment, the storage matrix is ​​sucrose and is present at a concentration of about 8% to about 12% (weight / volume). In one embodiment, the storage matrix is ​​sucrose and is present at a concentration of about 10% (weight / volume).

[0272] In one embodiment of any step (B2) of either the first or second pre-LNP formation method, the storage matrix is ​​present in a buffer solution. The buffer used can be any of the buffers defined and exemplified above in connection with step (B), or a mixture of any of these. In one embodiment, the buffer is HEPES. In one embodiment, the buffer is Tris. In one embodiment, the buffer is a mixture of HEPES and Tris.

[0273] In one embodiment of optional step (B2) of the first pre-LNP formation method, the storage matrix is ​​sucrose and the buffer is HEPES. In one embodiment, the storage matrix is ​​sucrose and the buffer is Tris.

[0274] In one embodiment of optional step (B2) of the first pre-LNP formation method, the storage matrix does not contain sucrose and the buffer is HEPES. In one embodiment, the storage matrix does not contain sucrose and the buffer is Tris. In one embodiment, the storage matrix does not contain sucrose and the buffer is a mixture of HEPES and Tris.

[0275] In one embodiment of optional step (B2) of the second pre-LNP formation method, the storage matrix is ​​sucrose and the buffer is a mixture of HEPES and Tris. In one embodiment, the storage matrix does not contain sucrose and the buffer is a mixture of HEPES and Tris.

[0276] In one embodiment of optional step (B2) of either the first or second pre-LNP formation method, the storage matrix is ​​present in a solution having a pH of about 6.5 to about 8.0. In one embodiment of optional step (B2) of either the first or second pre-LNP formation method, the storage matrix is ​​present in a solution having a pH of about 7.0 to about 8.0.

[0277] In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 6.5 to about 8.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 7.0 to about 8.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 6.8 to about 8.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 6.5 to about 7.5. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 6.8 to about 7.2. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 6.9 to about 7.1. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 7.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 7.1 to about 7.7. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 7.2 to about 7.6. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 7.3 to about 7.5. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 7.4. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 6.8 to about 7.6. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 7.0 to about 7.4. In one embodiment, the storage matrix is ​​sucrose, the buffer is HEPES, and the pH of the buffer solution is about 7.1 to about 7.4.

[0278] In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 6.5 to about 8.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 7.0 to about 8.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 6.8 to about 8.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 6.5 to about 7.5. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 6.8 to about 7.2. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 6.9 to about 7.1. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 7.0. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 7.1 to about 7.7. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 7.2 to about 7.6. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 7.3 to about 7.5. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 7.4. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 6.8 to about 7.6. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is about 7.0 to about 7.4. In one embodiment, the storage matrix is ​​sucrose, the buffer is Tris, and the pH of the buffer solution is from about 7.1 to about 7.4.

[0279] Further optional steps In one embodiment, the method further comprises an additional step.

[0280] In one embodiment, the method further comprises adding a peptide-conjugated lipid (as described in detail herein) to lipid particles contained in the dispersed phase of the aqueous dispersion. In some cases, the peptide-conjugated lipid may replace (i.e., displace) a corresponding portion of a steroid (e.g., cholesterol) in the lipid particles contained in the dispersed phase of the aqueous dispersion.

[0281] In one embodiment, the method further comprises storing the aqueous dispersion for 24 hours, 48 ​​hours, 72 hours, 5 days, 1 week, 2 weeks, 4 weeks, 2 months, 4 months, 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, or longer. The aqueous dispersion may be stored at about 25°C, about room temperature (e.g., 18-23°C), about 4-8°C, about 4°C, about -20°C, or about -80°C. The aqueous dispersion may be stored at about 4°C or about -20°C. In one embodiment, the method further comprises freezing the aqueous dispersion, for example, at a temperature of -15°C to -90°C, preferably about -18°C to about -25°C.

[0282] In one embodiment, the method further comprises drying the aqueous dispersion. In one embodiment, the drying step is lyophilization (freeze drying). In one embodiment, the drying step is spray drying.

[0283] In one embodiment, the method further comprises sterile filtering the aqueous dispersion. Typically, the sterile filtration uses a 0.22 μm filter. In one embodiment, the filter is a polyethersulfone (PES) filter.

[0284] In one embodiment, the method further comprises storing the aqueous dispersion at a pH of 3 to 5.5. In one embodiment, the method further comprises storing the aqueous dispersion at a pH of 7 to 9. In one embodiment, the method further comprises storing the aqueous dispersion at a pH of about 6.5 to about 9.0. In one embodiment, the method further comprises storing the aqueous dispersion at a pH of about 6.5 to about 8.5. In one embodiment, the method further comprises storing the aqueous dispersion at a pH of about 6.5 to about 8.0. In one embodiment, the method further comprises storing the aqueous dispersion at a pH of about 7.0 to about 8.0. In one embodiment, the method further comprises storing the aqueous dispersion at a pH of about 6.5 to about 7.5.

[0285] In one embodiment, the method further comprises storing the aqueous dispersion in a container, typically a bag. The container is preferably a fluoropolymer container. One example of a suitable container is the Aramus™ fluoropolymer bag available from Entegris.

[0286] Methods for forming nucleic acid-lipid particles In a further aspect, the present disclosure provides a method for producing the nucleic acid-lipid particles disclosed herein.Generally, such a method comprises adding an aqueous dispersion, for example, an aqueous dispersion containing pre-LNPs as described herein, to a composition containing nucleic acid.In one embodiment, the composition containing nucleic acid is a solution containing nucleic acid.In one embodiment, the composition containing nucleic acid is an aqueous solution containing nucleic acid.

[0287] Thus, in one aspect, the present disclosure provides a method of forming a nucleic acid-lipid particle, comprising: (x) an aqueous dispersion of any of the above embodiments (y) an aqueous solution containing nucleic acid; mixing to produce nucleic acid-lipid particles, Either the aqueous dispersion (x) or the aqueous solution (y) is acidified, The present invention provides a method comprising:

[0288] In one aspect, the method comprises: i) preparing an aqueous dispersion as defined herein according to any of the methods defined herein; and ii) mixing the (x) aqueous dispersion with (y) an aqueous solution containing nucleic acids to produce nucleic acid-lipid particles, wherein either the aqueous dispersion (x) or the aqueous solution (y) is acidified. Includes:

[0289] Mixing Step The mixing step of this aspect of the invention comprises mixing an aqueous dispersion, as defined herein, with an aqueous solution containing nucleic acids, as defined herein, to produce nucleic acid-lipid particles.

[0290] A method for forming nucleic acid-lipid particles uses aqueous acid. Because both the nucleic acid solution and the aqueous dispersion are at neutral pH, the nucleic acid solution or aqueous dispersion (typically containing pre-LNPs) must be acidified prior to complex formation to induce electrostatic interactions between the positively charged lipid phase and the negatively charged nucleic acid. In one embodiment, an aqueous solution containing nucleic acid is acidified. In one embodiment, an aqueous dispersion (typically containing pre-LNPs) is acidified. In one embodiment, an aqueous dispersion (typically containing pre-LNPs) is acidified to a pH of about 2.5 to about 5.5. In one embodiment, an aqueous dispersion (typically containing pre-LNPs) is acidified to a pH of about 3.0 to about 5.0. In one embodiment, an aqueous dispersion (typically containing pre-LNPs) is acidified to a pH of about 4.0 to about 4.5.

[0291] In one embodiment, the aqueous acid is substantially free of inorganic cations.

[0292] In one embodiment, the aqueous acid is an inorganic acid. Examples of suitable inorganic acids include hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0293] In one embodiment, the acid is a water-soluble organic acid. Examples of suitable inorganic acids include sulfonic acids, carboxylic acids, dicarboxylic acids, hydroxycarboxylic acids (all as defined herein), or amino acids.

[0294] In one embodiment, the water-soluble organic acid is selected from the group consisting of acetic acid, malic acid, succinic acid, and citric acid, and combinations thereof. In one embodiment, the water-soluble organic acid may be selected from the group consisting of acetic acid and malic acid, and combinations thereof.

[0295] In one embodiment, the water-soluble organic weak acid is acetic acid. In one embodiment, the water-soluble organic weak acid is malic acid. In one embodiment, the water-soluble organic weak acid is succinic acid. In one embodiment, the water-soluble organic weak acid is citric acid.

[0296] In one embodiment, the concentration of the acid is in the range of about 0.5 to about 50 mM. In one embodiment, the concentration of the acid is in the range of about 1 to about 25 mM. In one embodiment, the concentration of the acid is in the range of about 2.5 to about 10 mM. In this context, this concentration is understood to include both the undissociated acid and its conjugate base.

[0297] In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 0.5 to about 50 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 1 to about 25 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 2.5 to about 10 mM.

[0298] In one embodiment of the method of forming nucleic acid-lipid particles, the aqueous dispersion (typically containing pre-LNPs) may be introduced into the mixture in solution in a storage matrix, which, in its broadest or preferred aspects, may contain any of the components defined and exemplified above in connection with the aqueous dispersion and method of forming it.

[0299] Typically, the nucleic acid is provided in the form of a buffer solution. In one embodiment, the buffer is a neutral buffer. In one embodiment, the buffer has a pK of about 6.0 to about 8.0. a In one embodiment, the buffer has a pK of about 6.5 to about 7.5. a It has.

[0300] In one embodiment, the nucleic acid is provided in a buffer comprising an anionic group / moiety. In one embodiment, the nucleic acid is provided in a buffer comprising a sulfonic acid group / moiety, or a derivative thereof. In one embodiment, the nucleic acid is provided in a buffer that is a zwitterionic acidic buffer. In one embodiment, the nucleic acid is provided in a buffer comprising an amino group / moiety and a sulfonic acid group / moiety, or a derivative thereof. In one embodiment, the nucleic acid is provided in a buffer comprising an amino group / moiety and a sulfonic acid group / moiety, or a derivative thereof. In one embodiment, the nucleic acid is provided in a buffer comprising an anionic group / moiety, such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-morpholin-4-ylethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid (HEPPS), 2-(bis(2-hydroxyethyl)amino)ethanesulfonic acid (BES), N-cyclohexyl-3-aminopropanesulfonic acid (C5H5), ...C and 2-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}ethane-1-sulfonic acid (TES), and a mixture of any of these.

[0301] In one embodiment, the nucleic acid is provided in a buffer containing cationic groups / moieties. In one embodiment, the nucleic acid is provided in a buffer containing basic groups / moieties. In one embodiment, the nucleic acid is provided in a buffer containing amino groups / moieties. In one embodiment, the nucleic acid is provided in a buffer containing primary, secondary, or tertiary amine groups / moieties. In one embodiment, the nucleic acid is provided in a buffer containing amino groups / moieties (e.g., primary, secondary, or tertiary amine groups / moieties) and no anionic moieties (e.g., sulfonic acid groups / moieties). In one embodiment, the nucleic acid is provided in a buffer selected from the group consisting of tris(hydroxymethyl)aminomethane (Tris), TAE (a buffer solution containing a mixture of Tris, acetic acid, and ethylenediaminetetraacetic acid), TBE (a buffer solution containing a mixture of Tris, boric acid, and ethylenediaminetetraacetic acid), TEA, (2-(bis(2-hydroxyethyl)amino)acetic acid) (bicine), (N-[tris(hydroxymethyl)methyl]-glycine) (tricine), triethylammonium acetate, and triethanolamine, N-(2-acetamido)-iminodiacetic acid (ADA), and mixtures of any of these.

[0302] In one embodiment, the nucleic acid is provided in a buffer solution that is a mixture of a buffer containing anionic groups / moieties and a buffer containing cationic groups / moieties. In one embodiment, the nucleic acid is provided in a buffer solution that is a mixture of a buffer containing amino groups / moieties (e.g., primary, secondary, or tertiary amine groups / moieties) and a buffer containing sulfonic acid groups / moieties. In one embodiment, the nucleic acid is provided in a buffer solution that is a mixture of a zwitterionic acidic buffer and a buffer containing amino groups / moieties (e.g., primary, secondary, or tertiary amine groups / moieties).

[0303] In one embodiment, the nucleic acid is provided in a buffer solution selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)-aminomethane (Tris), 2-morpholin-4-ylethanesulfonic acid (MES), bis-(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (bis-Tris), and phosphate buffer, and mixtures of any of these. In one embodiment, the buffer is HEPES.

[0304] The buffer may be present at any concentration that allows it to perform its buffering function. In one embodiment, the buffer is present at a concentration of about 0.1 mM to about 1 M. In one embodiment, the buffer is present at a concentration of about 1 mM to about 100 mM. In one embodiment, the buffer is present at a concentration of about 5 mM to about 20 mM. In one embodiment, the buffer is present at a concentration of about 10 mM.

[0305] In one embodiment, the buffer is HEPES and is present at a concentration of about 0.1 mM to about 1 M. In one embodiment, the buffer is HEPES and is present at a concentration of about 1 mM to about 100 mM. In one embodiment, the buffer is HEPES and is present at a concentration of about 5 mM to about 20 mM. In one embodiment, the buffer is HEPES and is present at a concentration of about 10 mM.

[0306] In one embodiment, the buffer solution containing the nucleic acid also contains a chelating agent. The function of the chelating agent is to absorb divalent cations (e.g., Mg 2+ , Ca 2+ and Zn 2+ ) to protect RNA from hydrolysis and degradation by enzymes that require divalent cations as cofactors. Examples of suitable chelating agents include ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), or mixtures thereof.

[0307] In one embodiment, the chelating agent is present at a concentration of about 0.001 mM to about 10 mM. In one embodiment, the chelating agent is present at a concentration of about 0.01 mM to about 1 mM. In one embodiment, the chelating agent is present at a concentration of about 0.1 mM.

[0308] In one embodiment, the chelating agent is EDTA. In one embodiment, the chelating agent is EDTA and is present at a concentration of about 0.001 mM to about 10 mM. In one embodiment, the chelating agent is EDTA and is present at a concentration of about 0.01 mM to about 1 mM. In one embodiment, the chelating agent is EDTA and is present at a concentration of about 0.1 mM.

[0309] In one embodiment, the aqueous dispersion (typically containing pre-LNPs) is substantially free of inorganic cations.

[0310] In one embodiment, the mixing step is carried out using a T-mixer or a Y-mixer.

[0311] In one embodiment, the flow rate during the mixing step is from about 100 mL / min to about 800 mL / min, optionally from about 200 mL / min to about 500 mL / min, and optionally from about 300 mL / min to about 400 mL / min.

[0312] The volume ratio of the aqueous solution containing nucleic acids to the aqueous dispersion (typically containing pre-LNPs) can be about 1:3 to about 3:1, optionally about 1:2 to about 2:1, preferably about 1:15 to about 1.5:1, and more preferably about 1:1.

[0313] Further processing steps In one embodiment, the method further comprises the step of subjecting the nucleic acid-lipid particles to one or more additional processing steps.

[0314] In one embodiment, the method further comprises subjecting the nucleic acid-lipid particles to one or more purification steps. In one embodiment, the purification step comprises a dialysis or filtration step. In one embodiment, the dialysis or filtration step comprises tangential flow filtration. In one embodiment, the method does not comprise subjecting the nucleic acid-lipid particles to a filtration or dialysis step. In one embodiment, the method does not comprise subjecting the nucleic acid-lipid particles to a tangential flow filtration step.

[0315] In one embodiment, the method further comprises subjecting the nucleic acid-lipid particles to one or more dilution steps. In one embodiment, the one or more dilution steps comprise the addition of a storage matrix. The storage matrix may be any of the storage matrices defined and exemplified above in relation to the storage matrix of the aqueous dispersion. In one embodiment, the storage matrix contains a cryoprotectant as defined and exemplified above in relation to the storage matrix of the aqueous dispersion. In one embodiment, the storage matrix is ​​selected from the group consisting of sucrose, glycerol, trehalose, lactose, glucose, and mannitol, and mixtures of any of these. In one embodiment, the storage matrix is ​​sucrose or trehalose, or a mixture thereof.

[0316] In one embodiment, the storage matrix is ​​sucrose or trehalose and is present at a concentration of about 10% to about 50% (weight / volume). In one embodiment, the storage matrix is ​​trehalose or sucrose and is present at a concentration of about 20% to about 40% (weight / volume). In one embodiment, the storage matrix is ​​sucrose or trehalose and is present at a concentration of about 25% to about 35% (weight / volume). In one embodiment, the storage matrix is ​​sucrose or trehalose and is present at a concentration of about 30% (weight / volume).

[0317] In one embodiment, the storage matrix also contains amino acids. In one embodiment, the amino acids are present at a concentration of about 0.1 to about 20 mM. In one embodiment, the amino acids are present at a concentration of about 0.5 to about 10 mM. In one embodiment, the amino acids are present at a concentration of about 1 to about 5 mM. In one embodiment, the amino acids are present at a concentration of about 1 to about 1.5 mM. In one embodiment, the amino acids are present at a concentration of about 1.25 mM. In one embodiment, the amino acids are present at a concentration of about 2.5 mM. In one embodiment, the amino acids are present at a concentration of about 4 to about 6 mM. In one embodiment, the amino acids are present at a concentration of about 5 mM.

[0318] In one embodiment, the amino acid is an acidic amino acid and is present at a concentration of about 0.1 to about 5 mM. In one embodiment, the amino acid is an acidic amino acid and is present at a concentration of about 1 to about 5 mM.

[0319] In one embodiment, the amino acid is histidine and is present at a concentration of about 0.5 to about 10 mM. In one embodiment, the amino acid is histidine and is present at a concentration of about 2 to about 10 mM.

[0320] In one embodiment, the storage matrix comprises one or more buffers. The buffers used can be any of the buffers defined and exemplified above, or a mixture of any of these. In one embodiment, the buffer is HEPES. In one embodiment, the buffer is Tris. In one embodiment, the buffer is a mixture of HEPES and Tris.

[0321] In some embodiments, the storage matrix may contain a mixture of components as defined and exemplified above.

[0322] In one embodiment, the storage matrix contains a cryoprotectant (as defined and exemplified herein, e.g., sucrose or trehalose) and an acidic amino acid (as defined and exemplified herein, e.g., aspartic acid or glutamic acid, or a mixture thereof).

[0323] In one embodiment, the storage matrix contains a cryoprotectant (as defined and exemplified herein, e.g., sucrose or trehalose) and a buffer (as defined and exemplified herein, e.g., HEPES or Tris, or a mixture thereof).

[0324] In one embodiment, the storage matrix contains a cryoprotectant (as defined and exemplified herein, e.g., sucrose or trehalose), a buffer (as defined and exemplified herein, e.g., HEPES or Tris, or mixtures thereof), and an acidic amino acid (as defined and exemplified herein, e.g., aspartic acid or glutamic acid, or mixtures thereof).

[0325] In one embodiment, the storage matrix contains a cryoprotectant (as defined and exemplified herein, e.g., sucrose or trehalose), and a basic amino acid (as defined and exemplified herein, e.g., histidine, lysine, or arginine, or mixtures thereof), and optionally a neutral amino acid (as defined and exemplified herein, e.g., leucine or isoleucine, or mixtures thereof).

[0326] In one embodiment, the storage matrix contains a cryoprotectant (as defined and exemplified herein, e.g., sucrose or trehalose), a basic amino acid (as defined and exemplified herein, e.g., histidine, lysine, or arginine, or mixtures thereof), and optionally a neutral amino acid (as defined and exemplified herein, e.g., leucine or isoleucine, or mixtures thereof), and an acidic amino acid (as defined and exemplified herein, e.g., aspartic acid or glutamic acid, or mixtures thereof).

[0327] In one embodiment, the storage matrix has a pH of about 4.5 to about 9.0. In one embodiment, the storage matrix has a pH of about 6.0 to about 9.0. In one embodiment, the storage matrix has a pH of about 6.0 to about 7.0. In one embodiment, the storage matrix has a pH of about 8.0 to about 9.0. In one embodiment, the storage matrix has a pH of about 6.5. In one embodiment, the storage matrix has a pH of about 8.5. In one embodiment, the storage matrix has a pH of about 4.0 to about 7.0, optionally about 4.0 to about 5.0, about 5.0 to about 6.0, or about 6.0 to about 7.0. In one embodiment, the storage matrix has a pH of about 5.0 to about 5.5. In one embodiment, the storage matrix has a pH of about 6.0 to about 6.5. In one embodiment, the storage matrix has a pH of about 4.2 to about 4.8. In one embodiment, the storage matrix has a pH of about 6.8 to about 7.2.

[0328] In one embodiment, the method further comprises the step of sterile filtration of the nucleic acid-lipid particles.

[0329] In one embodiment, the one or more purification steps of the nucleic acid-lipid particles does not include a tangential flow filtration step.

[0330] In one embodiment, the nucleic acid-lipid particles are not subjected to any further filtration steps.

[0331] In another aspect, the present disclosure provides a method of forming a nucleic acid-lipid particle, comprising: (A) (i) an organic phase comprising a lipid mixture comprising a cationically ionizable lipid dissolved in a water-soluble organic solvent; and (ii) an aqueous phase containing aqueous acid and substantially free of inorganic cations; to produce an intermediate aqueous lipid dispersion; (B) performing a dialysis or filtration step on the intermediate aqueous lipid dispersion to produce an aqueous dispersion having a pH of about 6.5 to about 8.0 and substantially free of organic solvents and nucleic acids, wherein the dialysis or filtration step removes organic solvents and adjusts the pH to about 6.5 to about 8.0; and (C) (x) The aqueous dispersion produced in step (B) (y) an aqueous solution containing nucleic acid; mixing to produce nucleic acid-lipid particles, Either the aqueous dispersion (x) or the aqueous solution (y) is acidified, The present invention provides a method comprising:

[0332] This embodiment is hereinafter referred to as the "first LNP formation method."

[0333] In another aspect, the present disclosure provides a method of forming a nucleic acid-lipid particle, comprising: (A) (i) an organic phase comprising a lipid mixture comprising a cationically ionizable lipid dissolved in a water-soluble organic solvent; and (ii) mixing the aqueous phase to produce an intermediate aqueous lipid dispersion having a pH of about 6.5 to about 8.0; the organic phase comprises an aqueous acid and is substantially free of inorganic cations; (B) performing a dialysis or filtration step on the intermediate aqueous lipid dispersion to produce an aqueous dispersion having a pH of about 6.5 to about 8.0 and substantially free of organic solvents and nucleic acids, wherein the dialysis or filtration step removes organic solvents; and (C) (x) The aqueous dispersion produced in step (B) (y) an aqueous solution containing nucleic acid; mixing to produce nucleic acid-lipid particles, Either the aqueous dispersion (x) or the aqueous solution (y) is acidified, The present invention provides a method comprising:

[0334] This embodiment is hereinafter referred to as the "second LNP formation method."

[0335] In one embodiment of the first or second LNP formation method, the method further comprises the additional step after step (B) and before step (C): (B2) adding an aqueous acid (preferably substantially free of inorganic cations) to the aqueous dispersion produced in step (B); Includes:

[0336] In one embodiment of the first or second LNP formation method, the method further comprises an additional step after step (C): (C2) Adding a storage matrix to the nucleic acid-lipid particle dispersion Includes:

[0337] In one embodiment, the storage matrix has a pH of about 6.0 to about 9.0, optionally about 6.0 to about 7.0 or about 8.0 to about 9.0, and optionally about pH 6.5 or about pH 8.5. In one embodiment, the storage matrix has a pH of about 4.0 to about 7.0, optionally about 4.0 to about 5.0, about 5.0 to about 6.0, or about 6.0 to about 7.0. In one embodiment, the storage matrix has a pH of about 5.0 to about 5.5. In one embodiment, the storage matrix has a pH of about 6.0 to about 6.5. In one embodiment, the storage matrix has a pH of about 4.2 to about 4.8. In one embodiment, the storage matrix has a pH of about 6.8 to about 7.2.

[0338] In one embodiment, the storage matrix comprises Tris, HEPES, or a mixture thereof.

[0339] Nucleic acid-lipid particles The present disclosure further provides lipid particles comprising the lipid or lipid mixture defined herein and nucleic acid.In one embodiment, lipid particles are provided that are obtained or can be obtained by the method defined herein.Such particles are also referred to herein as "nucleic acid-lipid particles".When the nucleic acid is RNA, such particles are also referred to herein as "RNA-lipid particles".

[0340] In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is mRNA, saRNA, taRNA, or a mixture thereof. In one embodiment, the nucleic acid is mRNA. In one embodiment, the nucleic acid is DNA. In one embodiment, the nucleic acid is RNA encoding one or more personalized (i.e., patient-specific) cancer antigens.

[0341] In the present disclosure, the nucleic acid-lipid particle is preferably a lipid nanoparticle (LNP). The function of LNP is to stabilize and encapsulate the nucleic acid, facilitating its uptake into cells and release into the cytosol, while enabling delivery to cells. LNP and / or its lipid component may have adjuvant activity.

[0342] In the present disclosure, LNPs can be understood as oil-in-water emulsions in which the LNP core material is preferably in a liquid state and thus has a melting point below body temperature. LNPs thus typically comprise a central complex of mRNA and lipids embedded in an irregular, non-lamellar phase composed of lipids. This contrasts with the structure of liposomes, which comprise unilamellar or multilamellar vesicular particles comprising a lipid bilayer surrounding an aqueous cavity encapsulated by lamellae. In some cases, the nucleic acid-lipid particles described herein are not liposomes. In some cases, the nucleic acid-lipid particles described herein are not lipoplexes.

[0343] Lipid nanoparticles (LNPs) can be obtained by combining nucleic acids with lipids. The lipids used to form LNPs typically do not form a lamellar (bilayer) phase in water under physiological conditions. LNPs typically do not contain or encapsulate an aqueous core. LNPs typically contain an oily (or oily) core.

[0344] In some embodiments, the nucleic acid-lipid particles (e.g., lipid nanoparticles) described herein have an average diameter ranging from about 40 nm to about 1000 nm, about 40 nm to about 800 nm, about 40 nm to about 700 nm, about 40 nm to about 600 nm, about 40 nm to about 500 nm, about 40 nm to about 450 nm, about 40 nm to about 400 nm, about 40 nm to about 350 nm, about 40 nm to about 300 nm, about 40 nm to about 250 nm, about 40 nm to about 200 nm, about 40 nm to about 150 nm, or about 40 nm to about 100 nm. In some embodiments, the nucleic acid-lipid particles (e.g., lipid nanoparticles) described herein have an average diameter of less than 100 nm. In some embodiments, the nucleic acid-lipid particles (e.g., lipid nanoparticles) described herein have an average diameter of about 40 nm to about 100 nm. In some embodiments, the nucleic acid-lipid particles (e.g., lipid nanoparticles) described herein have an average diameter of about 50 nm to about 100 nm, hi some embodiments, the nucleic acid-lipid particles (e.g., lipid nanoparticles) described herein have an average diameter that is typically 15 to 20 nm larger than the average diameter of the preformed LNPs from which they are produced.

[0345] In one embodiment, the nucleic acid-lipid particles are present in a composition having a pH of 4.0 to 6.5. In one embodiment, the nucleic acid-lipid particles are present in a composition having a pH of 4.5 to 6.0.

[0346] In one embodiment, the nucleic acid-lipid particles are present in a composition having a pH of 4.6 to 5.8. In one embodiment, the nucleic acid-lipid particles are present in a composition having a pH of 5.0 to 5.5. In one embodiment, the nucleic acid-lipid particles are present in a composition having a pH of about 5.1. In one embodiment, the nucleic acid-lipid particles are present in a composition having a pH of about 5.2. In one embodiment, the nucleic acid-lipid particles are present in a composition having a pH of about 5.3. In one embodiment, the nucleic acid-lipid particles are present in a composition having a pH of about 5.4.

[0347] In one embodiment, the nucleic acid-lipid particles are present in a composition having a pH of 7.0 to 9.0. In one embodiment, the nucleic acid-lipid particles are present in a composition having a pH of 7.0 to 8.5. In one embodiment, the nucleic acid-lipid particles are present in a composition having a pH of 7.5 to 8.1. In one embodiment, the nucleic acid-lipid particles are present in a composition having a pH of about 7.8. In one embodiment, the nucleic acid-lipid particles are present in a composition having a pH of about 7.5.

[0348] Lipids and Amphiphiles The aqueous dispersions (typically containing pre-LNPs) and nucleic acid-lipid particles of the present invention also contain a mixture of lipids. The terms "lipid" and "lipid-like substance" are broadly defined herein as molecules that contain one or more hydrophobic moieties or groups and one or more hydrophilic moieties or groups.

[0349] Lipids are usually insoluble or poorly soluble in water, but are soluble in many organic solvents. In aqueous environments, their amphiphilic nature allows the molecules to self-assemble into organized structures and different phases.

[0350] Lipids can comprise polar and non-polar (or non-polar) parts. The term "amphiphilic substance" as used herein is broadly defined herein as a molecule having hydrophobic and hydrophilic portions, and / or polar and non-polar portions. Both cationic lipids and anionic lipids contain such groups, and therefore they are amphiphilic. In this specification, the term "cationic lipid" is therefore synonymous with "cationic amphiphilic substance", and the term "anionic lipid" is synonymous with "anionic amphiphilic substance".

[0351] Hydrophobicity can be imparted by the inclusion of non-polar groups, including, but not limited to, long-chain saturated and unsaturated hydrocarbyl groups (as defined and exemplified above), such as alkyl, alkenyl, and / or alkynyl groups, and such groups substituted with one or more aryl, heteroaryl, or cycloalkyl groups (as defined and exemplified above). The hydrophilic groups may include polar and / or charged groups, including at least one amine and optionally hydrophilic uncharged groups, such as hydroxyl, carbohydrate, sulfhydryl, nitro, and other groups, and may further include anionic groups such as phosphate, phosphonate, carboxylic acid, sulfate, sulfonate (all as defined and exemplified above), and other similar groups.

[0352] The term "hydrophobic," as used herein with respect to a compound, group, or moiety, means that the compound, group, or moiety is not attracted to water molecules and will exclude water molecules when present in an aqueous solution. In some embodiments, the term "hydrophobic" refers to any compound, group, or moiety that is substantially immiscible or insoluble in an aqueous solution. In some embodiments, a hydrophobic compound, group, or moiety is substantially non-polar.

[0353] Examples of hydrophobic groups are hydrocarbyl groups (as defined and exemplified above), such as alkyl, alkenyl, and / or alkynyl groups, and such groups substituted with one or more aryl, heteroaryl, or cycloalkyl groups (as defined and exemplified above). The hydrophobic group may contain functional groups (e.g., ether, thioether, ester, dioxolane, halide, amide, sulfonamide, carbamate, etc.) and atoms other than carbon and hydrogen, provided that the group is substantially immiscible or insoluble in aqueous solution.

[0354] The hydrophobic portion of the lipid may have 24 to 60 carbon atoms and may be hydrocarbyl (as described and exemplified above, typically containing an alkyl, alkenyl, or alkynyl group as described and exemplified above). The 24 to 60 carbon atoms may be divided into two or more hydrophobic segments, each of which typically has at least 6 carbon atoms. An example of a divided hydrophobic segment in which each segment is hydrocarbyl is a lipid containing a DACA moiety, as described in WO 2011 / 003834, in which each acyl or alkyl group contains 12 to 20 carbon atoms. Another example is a lipid in which the hydrophobic portion contains a steroid moiety, such as a cholesteryl moiety.

[0355] The hydrophobic portion of the lipid may be heterohydrocarbyl, preferably having 24-60 carbon atoms, where the heteroatom is selected from N, O, or S, forming one, two, three, or four uncharged groups such as ether, thioether, ester, amide, carbamate, sulfonamide, or other groups. The 24-60 carbon atoms can be divided into two or more hydrophobic segments, provided that each such segment has at least six carbon atoms. Examples of divided hydrophobic segments in which each segment is hydrocarbyl include lipids containing diacylglycerol or dialkylglycerol moieties, each of which contains 12-20 carbon atoms in the acyl or alkyl groups. Examples of hydrophobic segments in which each segment is heterohydrocarbyl include the ester-branched portions of lipids such as SM-102 or ALC-315, as defined and exemplified below.

[0356] cationically ionizable lipids The aqueous dispersion (typically containing pre-LNP) and nucleic acid-lipid particles of the present invention also contain a cationically ionizable lipid, or any mixture thereof. As used herein, "cationically ionizable lipid" refers to a lipid or lipid-like substance that has a net positive charge or is neutral depending on whether it is protonated or deprotonated, i.e., a lipid that is not permanently cationic. Thus, depending on the pH of the composition in which the cationically ionizable lipid is dissolved, the cationically ionizable lipid has a net positive charge or is neutral.

[0357] In some embodiments, the cationically ionizable lipid preferably comprises a head group that includes at least one nitrogen atom (N) that can be protonated under physiological or slightly acidic conditions.

[0358] In one embodiment, the cationic lipid or cationically ionizable lipid is a compound represented by formula (TL-I): [ka] TL-I (In the formula, L 1 and L 2 are each independently optionally substituted C-C 30 is an aliphatic group; L 3 is a bond, optionally substituted C1-C 10 an aliphatic group or an optionally substituted 2- to 10-membered heteroaliphatic group; X 1 and X 2 are each independently a bond, -OC(O)-, -C(O)O-, or -S(O)N(R 1 )-, -N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-, -OC(S)C(R 1 )2-, -C(R 1 )2C(S)O—, and —S—, wherein X 1 or X 2One or both of -S(O)N(R 1 )-, -N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-, -OC(S)C(R 1 )2-, -C(R 1 )2C(S)O—, and —S—; Each R 1 is independently, in each instance, optionally substituted C-C 20 is aliphatic or H; T 1 and T 2 are each independently optionally substituted C3-C 30 It is aliphatic; G is -N(R 2 )C(S)N(R 2 )2, -N + (R 3 )3, -OH, -N(R 2 )2, -N(R 5 )C(O)R 3 , -N(R 5 )S(O)2R 3 , -N(R 5 )C(O)N(R 3 )2, -CH(NR 2 ), or -R 4 and; Each R 2 is independently, at each instance, H, an optionally substituted C1-C6 aliphatic group, and OR 3 or R 2 two examples of which, together with the atom to which they are attached, form an optionally substituted 4- to 12-membered heterocyclic ring or an optionally substituted 4- to 12-membered heteroaryl ring; Each R 3 is independently, in each instance, H and optionally substituted C-C 10 selected from the group consisting of aliphatic; R 4 represents an optionally substituted 4- to 12-membered heterocycle, an optionally substituted 4- to 12-membered heteroaryl, -(CH2) 0-6 -OH or -(CH2) 0-6 -N(R5 )2, C6-C substituted by one or more of 12 Aryl, or oxo, -(CH2) 0-6 -OH or -(CH2) 0-6 -N(R 5 ) C3-C substituted by one or more of 2 12 is alicyclic; Each R 5 are independently selected from H and optionally substituted C1-C6 aliphatic or a pharmaceutically acceptable salt thereof.

[0359] In some embodiments of Formula (TL-I), L 1 and L 2 are each independently -(CH2) 6-10 -It is.

[0360] In some embodiments of formula (TL-I), X 1 and X 2 are each independently -S(O)N(R 1 )-, -N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-, -OC(S)C(R 1 )2-, -C(R 1 )2C(S)O—, and —S—.

[0361] In some embodiments of formula (TL-I), X 1 and X 2 are -S(O)2N(R 1 )- and each R 1 are independently R 1 is C1-C 10 It is aliphatic.

[0362] In some embodiments of Formula (TL-I), T 1 and T 2 are each independently optionally substituted C3-C 20 alkyl.

[0363] In some embodiments of Formula (TL-I), T 1 and T 2 are each independently [ka] is selected from.

[0364] In some embodiments of Formula (TL-I), G is —N(R 2 )C(S)N(R 2 )2 or -N(R 5 )S(O)2R 3 is.

[0365] In some embodiments of Formula (TL-I), G is —N(H)C(S)N(R 2 )2, where each R 2 is selected from optionally substituted C1-C6 aliphatic and OH.

[0366] In some embodiments of Formula (TL-I), G is —OH.

[0367] In some embodiments of Formula (TL-I), G is [ka] is selected from.

[0368] In some embodiments of Formula (TL-I), -L 3 -G is [ka] is selected from.

[0369] In some embodiments of Formula (TL-I), the compound has the formula (TL-IIa): [ka] TL-IIa or a pharmaceutically acceptable salt thereof.

[0370] In some embodiments of Formula (TL-I), the compound has the formula (TL-IIc): [ka] TL-IIc or a pharmaceutically acceptable salt thereof.

[0371] In some embodiments of Formula (TL-I), the compound has the formula (TL-IIIb): [ka] (TL-IIIb) or a pharmaceutically acceptable salt thereof.

[0372] In some embodiments of Formula (TL-I), the compound has the formula (TL-IIIe): [ka] TL-IIIe or a pharmaceutically acceptable salt thereof.

[0373] In some embodiments of Formula (TL-I), the compound is 7,7'-((4-hydroxybutyl)azanediyl)bis(N-hexyl-N-octylheptane-1-sulfonamide) [ka] or a pharmaceutically acceptable salt thereof.

[0374] In some embodiments of Formula (TL-I), the compound is 7,7'-((4-(3,3-dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylheptane-1-sulfonamide) [ka] or a pharmaceutically acceptable salt thereof.

[0375] In some embodiments of formula (TL-I), the compound is [ka] or a pharmaceutically acceptable salt thereof.

[0376] The thiolipid compounds of formula (TL-I) may be prepared according to PCT / EP2023 / 071270, the contents of which are incorporated herein by reference.

[0377] In one embodiment, the cationically ionizable lipid is 7,7'-((4-hydroxybutyl)azanediyl)bis(N-hexyl-N-octylheptane-1-sulfonamide) [ka] 7,7'-((4-(3,3-dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylheptane-1-sulfonamide) [ka] structure [ka] a compound having [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-315); 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA); 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA); Heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (D-Lin-MC3-DMA); 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA); Di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319); Bis-(2-butyloctyl) 10-(N-(3-(dimethylamino)propyl)nonanamido)-nonadecanedioate (A9); (heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}-octanoate) (L5); Heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate) (SM-102); O-[N-{(9Z,12Z)-octadeca-9,12-dien-1-yl)}-N-{7-pentadecylcarbonyloxyoctyl}-amino]4-(dimethylamino)butanoate (HY501); 2-(di-((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethyl 4-(dimethylamino)butanoate (EA-2); 4-((di-((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)oxy)-N,N-dimethyl-4-oxobutan-4-amine (HYAM-2); ((2-(4-(dimethylamino)butanoyl)oxy)ethyl)azanediylbis(octane 8,1-diyl)bis(2-hexyldecanoate) (EA-405); (2-(4-(dimethylamino)butanoyl)oxy)azanediylbis(octane 8,1-diyl)bis(2-hexyldecanoate) (HY-405); Di(heptadecan-9-yl)3,3'-((2-(4-methylpiperazin-1-yl)ethyl)azanediyl)dipropionate [ka] (BHD-C2C2-PipZ, as described in U.S. Patent Application Publication No. 2022 / 0218622A1) Bis(2-octyldodecyl) 3,3'-((2-(1-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate [ka] (BODD-C2C2-1Me-Pyr, as described in U.S. Patent Application Publication No. 2022 / 0218622A1) Bis(2-octyldodecyl) 3,3'-((2-(pyrrolidin-1-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-Pyr); Bis(2-octyldodecyl)3,3'-((2-(1-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-1MePyr); Bis(2-octyldodecyl) 3,3'-(((1-methylpiperidin-3-yl)methyl)azanediyl)dipropionate (BODD-C2C2-1Me-3PipD); Bis(2-octyldodecyl) 3,3'-((2-(dimethylamino)ethyl)azanediyl)dipropionate (BODD-C2C2-DMA); Bis(2-octyldodecyl) 3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (BODD-C2C4-PipZ); Bis(2-octyldodecyl) 3,3'-((4-(pyrrolidin-1-yl)butyl)azanediyl)dipropionate (BODD-C2C4-Pyr); Bis(2-hexyldecyl) 3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (BHD-C2C4-PipZ); Di(nonadecan-9-yl)3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (DND-C2-C4-PipZ); and any mixtures thereof is selected from the group consisting of:

[0378] In one embodiment, the cationically ionizable lipid is [(4-hydroxybutyl)azanediyl]-di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-315). In one embodiment, the cationically ionizable lipid is 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA). In one embodiment, the cationically ionizable lipid is 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA). In one embodiment, the cationically ionizable lipid is heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (D-Lin-MC3-DMA). In one embodiment, the cationically ionizable lipid is 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA). In one embodiment, the cationically ionizable lipid is di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319). In one embodiment, the cationically ionizable lipid is bis-(2-butyloctyl)10-(N-(3-(dimethylamino)propyl)-nonanamido)-nonadecanedioate (A9). In one embodiment, the cationically ionizable lipid is (heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}-octanoate) (L5). In one embodiment, the cationically ionizable lipid is heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate) (SM-102). In one embodiment, the cationically ionizable lipid is O-[N-{(9Z,12Z)-octadeca-9,12-dien-1-yl)}-N-{7-pentadecylcarbonyloxyoctyl}-amino]4-(dimethylamino)butanoate (HY501).In one embodiment, the cationically ionizable lipid is 2-(di-((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethyl 4-(dimethylamino)butanoate (EA-2). In one embodiment, the cationically ionizable lipid is 7,7'-((4-hydroxybutyl)azanediyl)-bis(N-hexyl-N-octylheptane-1-sulfonamide) (BNT-51). In one embodiment, the cationically ionizable lipid is 7,7'-((4-(3,3-dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylheptane-1-sulfonamide) (BNT-52). In one embodiment, the cationically ionizable lipid is BHD-C2C2-PipZ. In one embodiment, the cationically ionizable lipid is BODD-C2C2-1Me-Pyr.

[0379] In some embodiments, the cationically ionizable lipids are generally and specifically selected from those described in WO 2018 / 087753.

[0380] In some embodiments, the cationically ionizable lipid is [ka] is selected from the group consisting of:

[0381] In one embodiment, the cationically ionizable lipid is 4-((di-((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)oxy)-N,N-dimethyl-4-oxobutan-4-amine (HYAM-2). In one embodiment, the cationically ionizable lipid is ((2-(4-(dimethylamino)butanoyl)-oxy)ethyl)-azanediylbis(octane8,1-diyl)bis(2-hexyldecanoate) (EA-405). In one embodiment, the cationically ionizable lipid is (2-(4-(dimethylamino)butanoyl)-oxy)azanediylbis(octane8,1-diyl)bis(2-hexyldecanoate) (HY-405). In one embodiment, the cationically ionizable lipid is O-[N-{(9Z,12Z)-octadeca-9,12-dien-1-yl)}-N-{7-pentadecylcarbonyloxyoctyl}-amino]4-(dimethylamino)butanoate (HY501).

[0382] In one embodiment, the cationically ionizable lipids are present in an amount of 20-70 mol % of the total lipids present in the lipid mixture. In one embodiment, the cationically ionizable lipids are present in an amount of 30-60 mol % of the total lipids present in the lipid mixture. In one embodiment, the cationically ionizable lipids are present in an amount of 40-50 mol % of the total lipids present in the lipid mixture. The term "lipid mixture" in this context applies to the lipid mixture components of both the aqueous dispersion (which typically contains pre-LNPs) and the nucleic acid-lipid particles.

[0383] In one embodiment, the aqueous dispersion (typically containing pre-LNPs) and / or nucleic acid-lipid particles are substantially free of cationic lipids (as defined herein). As used herein, the term "cationic lipid" refers to a lipid or lipid-like substance that has a constitutive positive charge, as defined herein. In this context, "constitutive charge" means that the cationic lipid carries a positive charge at all physiological pH levels. Cationic lipids that carry a constitutively charged cationic moiety are typically quaternary ammonium salts (as defined above) or salts of organic bases, such as nitrogen-containing bases. Typically, such organic bases are strong bases (i.e., bases that fully protonate when dissolved in a solvent, such as, but not limited to, an aqueous solvent, such that the concentration of unprotonated species is unmeasurably low).

[0384] In one embodiment, the cationic lipid is a monovalent cationic lipid. In one embodiment, the cationic lipid contains a charged polar moiety selected from the group consisting of guanidinium, ammonium, imidazolium, pyridinium, amidinium, and piperazinium.

[0385] Examples of cationic lipids include, but are not limited to, 1,2-dialkyloxy-3-dimethylammonium propane and 1,2-dialkenyloxy-3-dimethylammonium propane (each alkyl or alkenyl moiety is as defined and exemplified above, and preferably has 12 to 20 carbon atoms), such as 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-diacyloxy-3-dimethylammonium propane (each acyl moiety is as defined and exemplified above, and preferably has 12 to 20 carbon atoms), such as 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or 1,2-dioleoyl-3-dimethylammonium propane. (DODAP); dimethyldioctadecylammonium (DDAB); dioctadecyldimethylammonium chloride (DODAC), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate (DOSPA).

[0386] Additional fats The lipid mixture in the aqueous dispersions (typically containing pre-LNPs) and nucleic acid-lipid particles of the present invention may further comprise one or more additional lipids. In one embodiment, the one or more additional lipids comprise an anionic amphiphile as defined and exemplified below. In one embodiment, the one or more additional lipids comprise a neutral or zwitterionic lipid as defined and exemplified below. In one embodiment, the one or more additional lipids comprise a steroid as defined and exemplified below. In one embodiment, the one or more additional lipids comprise a neutral lipid as defined and exemplified below. In one embodiment, the one or more additional lipids comprise a neutral lipid (e.g., a steroid) as defined and exemplified below. In one embodiment, the one or more additional lipids comprise a peptide-conjugated lipid as defined and exemplified below.

[0387] neutral lipid The lipid mixture in the aqueous dispersions (typically containing pre-LNPs) and nucleic acid-lipid particles of the present invention may also additionally contain a neutral lipid. The neutral lipid is preferably a neutral phospholipid. In one embodiment, the phospholipid may be zwitterionic (i.e., it carries both a positive and a negative charge and is therefore neutral at a pH in the near-neutral range).

[0388] In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin. The hydrocarbyl moiety of the acyl portion of the phospholipid is as defined above, but is preferably an alkyl group (as defined above) having 6 to 40, preferably 8 to 24, carbon atoms, or an alkenyl group (as defined above) having 6 to 40, preferably 14 to 22, carbon atoms and 1 to 6 carbon-carbon double bonds. The acyl moieties of the phospholipids may be the same or different. In one embodiment, the acyl moieties are saturated fatty acid moieties having 8 to 24 carbon atoms (including the acyl carbon), preferably selected from the group consisting of lignoceroyl, behenoyl, arachidoyl, stearoyl, palmitoyl, myristoyl, lauroyl, decanoyl, and octanoyl moieties. In certain embodiments, the neutral phospholipid has a T of 30° C. or higher.m and is selected from a distearoyl or dipalmitoyl or stearoyl-palmitoyl moiety. In one embodiment, the acyl moiety is an unsaturated fatty acid moiety having 14 to 22 carbon atoms (including the acyl carbon), preferably selected from the group consisting of oleoyl, linoyl, and lineoyl moieties.

[0389] Examples of such phospholipids include diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine (DLPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-10-glycero-3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), diphytanoylphosphatidylethanolamine (DP yPE), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), and additional phosphatidylethanolamine lipids with various hydrophobic chains.

[0390] In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DOPE, and SM, and mixtures of any of these.

[0391] Thus, in some embodiments, the aqueous dispersions (typically containing pre-LNPs) and / or nucleic acid-lipids described herein comprise a cationically ionizable lipid (as defined herein) and a phospholipid. In some embodiments, the lipid nanoparticle compositions described herein comprise a cationically ionizable lipid and a phospholipid selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DOPE, and SM, and mixtures of any of these.

[0392] In one embodiment, the neutral lipids are present in the lipid mixture in an amount of about 1 mol% to about 40 mol% of the total lipids present in the lipid mixture. In one embodiment, the neutral lipids are present in the lipid mixture in an amount of about 2 mol% to about 25 mol% of the total lipids present in the lipid mixture. In one embodiment, the neutral lipids are present in the lipid mixture in an amount of about 5 mol% to about 15 mol% of the total lipids present in the lipid mixture.

[0393] In one embodiment, the neutral lipid is a phospholipid and is present in the lipid mixture in an amount of about 1 mol% to about 40 mol% of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a phospholipid and is present in the lipid mixture in an amount of about 2 mol% to about 25 mol% of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is a phospholipid and is present in the lipid mixture in an amount of about 5 mol% to about 15 mol% of the total lipids present in the lipid mixture.

[0394] In one embodiment, the neutral lipid is phosphatidylcholine and is present in the lipid mixture in an amount of about 1 mol% to about 40 mol% of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is phosphatidylcholine and is present in the lipid mixture in an amount of about 2 mol% to about 25 mol% of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is phosphatidylcholine and is present in the lipid mixture in an amount of about 5 mol% to about 15 mol% of the total lipids present in the lipid mixture.

[0395] In one embodiment, the neutral lipid is DSPC and is present in the lipid mixture in an amount of about 1 mol% to about 40 mol% of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is DSPC and is present in the lipid mixture in an amount of about 2 mol% to about 25 mol% of the total lipids present in the lipid mixture. In one embodiment, the neutral lipid is DSPC and is present in the lipid mixture in an amount of about 5 mol% to about 15 mol% of the total lipids present in the lipid mixture.

[0396] In each of the above embodiments, the term "lipid mixture" in this context applies to the lipid mixture components of both the aqueous dispersion (which typically contains pre-LNPs) and the nucleic acid-lipid particles.

[0397] steroid The aqueous dispersions (typically containing pre-LNPs) and nucleic acid-lipid particles of the present invention also include a steroid. In one embodiment, the steroid includes a sterol. In one embodiment, the steroid is cholesterol.

[0398] Thus, in some embodiments, the aqueous dispersions (typically containing pre-LNPs) and nucleic acid-lipid particles described herein comprise a cationically ionizable lipid (as defined herein) and cholesterol.

[0399] In one embodiment, the steroid is present in an amount ranging from about 10 mol% to about 65 mol% of the total lipids present in the lipid mixture. In one embodiment, the steroid is present in an amount ranging from about 20 mol% to about 60 mol% of the total lipids present in the lipid mixture. In one embodiment, the steroid is present in an amount ranging from about 30 mol% to about 50 mol% of the total lipids present in the lipid mixture.

[0400] In some embodiments, the combined concentration of neutral lipids (particularly one or more phospholipids, particularly phosphatidylcholines, e.g., DSPC) and steroids (particularly cholesterol) may comprise from about 0 mol% to about 70 mol%, e.g., from about 2 mol% to about 60 mol%, from about 5 mol% to about 55 mol%, or from about 5 mol% to about 50 mol% of the total lipids present in the lipid mixture.

[0401] In each of the above embodiments, the term "lipid mixture" in this context applies to the lipid mixture components of both the aqueous dispersion (which typically contains pre-LNPs) and the nucleic acid-lipid particles.

[0402] Grafted lipids The compositions described herein may also contain grafted lipids. As used herein, the term "grafted lipid" in its broadest sense refers to a lipid or lipid-like substance, as defined above (in its broadest or preferred embodiment), conjugated to a polymer, as defined below (in its broadest or preferred embodiment).

[0403] As used herein, the term "polymer" is given its conventional meaning: a molecular structure comprising one or more repeating units (monomers) connected by covalent bonds. The repeating units may all be identical, or in some cases, more than one type of repeating unit may be present in the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties, such as targeting moieties, may be present in the polymer. If more than one type of repeating unit is present in the polymer, the polymer is referred to as a "copolymer." The repeating units forming the copolymer can be arranged in any manner. For example, the repeating units can be arranged in random order, alternating order, or as a "block" copolymer, in which one or more regions each comprise a first repeating unit (e.g., a first block) and one or more regions each comprise a second repeating unit (e.g., a second block). Block copolymers can have two (diblock copolymers), three (triblock copolymers), or a greater number of distinct repeating units.

[0404] In one embodiment, the grafted lipid can act as a stealth lipid. As used herein, the term "stealth lipid" refers to a stealth polymer (as defined below) conjugated to a lipid (as defined herein). As used herein, the term "stealth polymer" refers to a polymer (as defined above) having the following characteristics: (a) a polar (hydrophilic) functional group, (b) a hydrogen bond acceptor group, (c) no hydrogen bond donor group, and (d) no net charge. In some embodiments, the stealth polymer is designed to sterically stabilize the lipid particle by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. In some embodiments, the stealth polymer can reduce association with serum proteins and / or the resulting uptake by the reticuloendothelial system when such lipid particles are administered in vivo.

[0405] In one embodiment, the grafted lipid is a polyethylene glycol-conjugated lipid (also known as a PEG-lipid or PEGylated lipid). The term "PEGylated lipid" refers to a molecule comprising both a lipid moiety and a polyethylene glycol moiety. PEGylated lipids are known in the art. PEG-lipids can comprise 5-1000, 5-500, 5-100, 5-50, 8-1000, 8-500, 8-100, 8-50, 10-1000, 10-500, 10-100, or 10-50 ethylene glycol repeating units, which can be constitutive.

[0406] In one embodiment, the grafted lipid is 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0407] Other examples of grafted lipids include poly(sarcosine) (pSar)-conjugated lipids, poly(oxazoline) (POX)-conjugated lipids; poly(oxazine) (POZ)-conjugated lipids, poly(vinylpyrrolidone) (PVP)-conjugated lipids; poly(N-(2-hydroxypropyl)-methacrylamide) (pHPMA)-conjugated lipids; poly(dehydroalanine) (pDha)-conjugated lipids; poly(aminoethoxyethoxyacetic acid) (pAEEA)-conjugated lipids, and poly(2-methylaminoethoxyethoxyacetic acid) (pmAEEA)-conjugated lipids.

[0408] In one embodiment, the grafted lipid is a polysarcosine-conjugated lipid, also referred to herein as a sarcosinated lipid or pSar-lipid. The term "sarcosinated lipid" refers to a molecule comprising both a lipid portion and a polysarcosine (poly(N-methylglycine)) portion, wherein the polysarcosine portion comprises the repeating unit shown below: [ka] (wherein x refers to the number of sarcosine units). Polysarcosine may contain 2 to 200, 2 to 100, 5 to 200, 5 to 100, 10 to 200, 10 to 100, optionally 5 to 80, and preferably 10 to 70 sarcosine units.

[0409] In one embodiment, the grafted lipid is a polyoxazoline (POX)-conjugated and / or polyoxazine (POZ)-conjugated lipid and / or POX / POZ-conjugated lipid, also referred to herein as a conjugate of a POX and / or POZ polymer with one or more hydrophobic chains, or an oxazolinated and / or oxazinated lipid, or a POX- and / or POZ-lipid. The term "oxazolinated lipid" or "POX-lipid" refers to a molecule comprising both a lipid portion and a polyoxazoline portion, wherein the polyoxazoline portion (pOx) has the repeating unit shown below. The term "oxazinated lipid" or "POZ-lipid" refers to a molecule comprising both a lipid portion and a polyoxazine portion, wherein the polyoxazine (pOz) portion has the repeating unit shown below. The term "oxazolinylated / oxazinylated lipid," or "POX / POZ-lipid," or "POXZ-lipid," refers to a molecule that contains both a lipid portion and a copolymer portion of polyoxazoline and polyoxazine, i.e., a polymer having both pOx and pOz repeating units, as shown below: [ka] (wherein x refers to the number of pOx and / or pOz units.) The total number of pOx and / or pOz repeating units in the polymer may include 2 to 200, 2 to 100, 5 to 200, 5 to 100, 10 to 200, 10 to 100, optionally 5 to 80, and preferably 10 to 70 pOx and / or pOz units.

[0410] In one embodiment, the grafted lipid is a poly(vinylpyrrolidone) (PVP)-conjugated lipid. In one embodiment, the lipid nanoparticle composition is substantially free of lipid-conjugated poly(vinylpyrrolidone) (PVP) (as defined above in the broadest aspect of the preferred embodiment). The term "poly(vinylpyrrolidone)" or "PVP" refers to a polymer having vinylpyrrolidine repeating units, i.e., repeating units as shown below: [ka]

[0411] In one embodiment, the grafted lipid is a poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA)-conjugated lipid. In one embodiment, the lipid nanoparticle composition is substantially free of lipid-conjugated poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA) (as defined above in the broadest aspect of the preferred embodiment). The term "poly(N-(2-hydroxypropyl)-methacrylamide)" or "pHPMA" refers to a polymer having the repeating unit shown below: [ka]

[0412] In one embodiment, the grafted lipid is a poly(dehydroalanine) (pDha)-conjugated lipid. The term "pDha" refers to a polymer having the repeating unit shown below: [ka]

[0413] In one embodiment, the grafted lipid is an amphiphilic oligoethylene glycol (OEG)-conjugated lipid. Examples of amphiphilic oligoethylene glycol (OEG)-conjugated lipids include poly(aminoethylethylene glycol acetyl) (pAEEA) and / or poly(methylaminoethylethylene glycol acetyl) (pmAEEA). The terms "pAEEA" and "pmAEAA" refer to polymers having the repeating units shown below. [ka] (wherein x refers to the total number of pAEEA and / or pmAEEA units in the polymer.) The total number of pAEEA and / or pmAEEA repeat units in the polymer may comprise 1 to 100, 5 to 50, 5 to 25, preferably 7 to 14.

[0414] In one embodiment, the grafted lipid is present in the lipid mixture in an amount of 0.5-10 mol% of the total lipid present in the lipid mixture. In one embodiment, the grafted lipid is present in the lipid mixture in an amount of 0.2-5 mol% of the total lipid present in the lipid mixture. In one embodiment, the grafted lipid is present in the lipid mixture in an amount of 1-2.5 mol% of the total lipid present in the lipid mixture. The grafted lipid may comprise a mixture of (i) grafted lipids selected from the group consisting of pSar-conjugated lipids; POX-conjugated lipids; POZ-conjugated lipids, PVP-conjugated lipids; pHPMA-conjugated lipids; pDha-conjugated lipids; pAEEA-conjugated lipids, and pmAEEA-conjugated lipids, and (ii) peptide-conjugated lipids. The term "lipid mixture" in this context applies to the lipid mixture components of both the aqueous dispersion (typically containing pre-LNPs) and the nucleic acid-lipid particles.

[0415] In one embodiment, the grafted lipid is a PEG-lipid and is present in the lipid mixture in an amount of 0.5-10 mol % of the total lipid present in the lipid mixture. In one embodiment, the grafted lipid is a PEG-lipid and is present in the lipid mixture in an amount of 0.2-5 mol % of the total lipid present in the lipid mixture. In one embodiment, the grafted lipid is a PEG-lipid and is present in the lipid mixture in an amount of 1-2.5 mol % of the total lipid present in the lipid mixture. The term "lipid mixture" in this context applies to the lipid mixture components of both the aqueous dispersion (typically containing pre-LNPs) and the nucleic acid-lipid particles.

[0416] In one embodiment, the grafted lipid is ALC-0159 and is present in the lipid mixture in an amount of 0.5-10 mol % of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is ALC-0159 and is present in the lipid mixture in an amount of 0.2-5 mol % of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is ALC-0159 and is present in the lipid mixture in an amount of 1-2.5 mol % of the total lipids present in the lipid mixture. The term "lipid mixture" in this context applies to the lipid mixture components of both the aqueous dispersion (which typically contains pre-LNPs) and the nucleic acid-lipid particles.

[0417] Pharmaceutical Composition The nucleic acid-lipid particle compositions described herein are useful as, or for the preparation of, pharmaceutical compositions or medicaments for therapeutic or prophylactic treatments.

[0418] The nucleic acid-lipid particle compositions described herein may be administered in the form of any suitable pharmaceutical composition.

[0419] The term "pharmaceutical composition" relates to a composition comprising a therapeutically effective agent, preferably together with a pharmaceutically acceptable carrier, diluent, and / or excipient. The pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administering the pharmaceutical composition to a subject. In some embodiments, the therapeutically effective agent is or comprises an active ingredient described herein. In the context of the present disclosure, a pharmaceutical composition comprises a nucleic acid described herein. In some embodiments, the therapeutically effective agent is or comprises a nucleic acid described in the present disclosure, which comprises a nucleic acid sequence (e.g., ORF) encoding one or more polypeptides, e.g., peptides or proteins, preferably pharmaceutically active peptides or proteins.

[0420] In some embodiments, when the nucleic acid is mRNA, the mRNA integrity of the initial pharmaceutical composition (i.e., after its preparation but before freezing, lyophilization, or storage) is at least 50%, preferably at least 60%, more preferably at least 70%, and most preferably at least 80%, e.g., at least 90%.

[0421] In some embodiments, the particle size (Z) of the initial pharmaceutical composition (i.e., after its preparation but before freezing, lyophilization, or storage) is average ) is about 50 nm to about 500 nm, preferably about 40 nm to about 200 nm, and more preferably about 40 nm to about 120 nm.

[0422] In some embodiments, the polydispersity index (PDI) of the particles in the initial pharmaceutical composition (i.e., after preparation but before freezing, lyophilization, or storage) is less than 0.3, preferably less than 0.2, and more preferably less than 0.1.

[0423] The pharmaceutical compositions of the present disclosure may be in a frozen form or in a "ready-to-use form" (i.e., a form that can be immediately administered to a subject without processing, such as thawing, reconstitution, or dilution, particularly in liquid form). Thus, prior to administration of a pharmaceutical composition in a shelf-stable form, the shelf-stable form must be processed or transferred to a ready-to-use or administerable form. For example, a frozen pharmaceutical composition must be thawed. Ready-to-use injections may be presented in a container such as a vial, an ampoule, or a syringe, which may contain one or more doses.

[0424] In one embodiment, the pharmaceutical composition is freeze-dried. In one embodiment, the pharmaceutical composition is spray-dried. These techniques are well known to those skilled in the art.

[0425] In some embodiments, the pharmaceutical composition is in a frozen form and can be stored at about −90° C. or higher, for example, about −90° C. to about −10° C. For example, the frozen pharmaceutical compositions described herein can be stored at a temperature ranging from about −90° C. to about −10° C., for example, from about −90° C. to about −40° C., or from about −40° C. to about −25° C., or from about −25° C. to about −10° C., or at a temperature of about −20° C.

[0426] In some embodiments of the pharmaceutical composition in frozen form, the pharmaceutical composition can be stored for at least 1 week, e.g., at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months, preferably at least 4 weeks. For example, the frozen pharmaceutical composition can be stored at -20°C for at least 4 weeks, preferably at least 1 month, more preferably at least 2 months, more preferably at least 3 months, and more preferably at least 6 months.

[0427] In some embodiments of the pharmaceutical composition in frozen form, when the nucleic acid is mRNA, the mRNA integrity after thawing of the frozen pharmaceutical composition is at least 90%, at least 95%, at least 97%, at least 98%, or substantially 100% of the initial mRNA integrity, e.g., after thawing of a frozen composition that has been stored at -20°C (for at least 1 week, e.g., at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months, preferably at least 4 weeks).

[0428] In some embodiments of the pharmaceutical composition in frozen form, the size of the particles after thawing of the frozen pharmaceutical composition (Z average ) and / or size distribution and / or PDI are determined by the particle size (Z) of the initial pharmaceutical composition before freezing. average ) and / or size distribution and / or PDI. For example, if a ready-to-use pharmaceutical composition is prepared from a frozen pharmaceutical composition described herein, the size (Z average ) and / or size distribution and / or PDI are determined by measuring the initial size (Z) of particles contained in the frozen pharmaceutical composition before freezing. average ) and / or size distribution and / or PDI are preferably essentially equal.

[0429] In some embodiments, when the nucleic acid is mRNA, the size and mRNA integrity of the mRNA particles in the pharmaceutical composition after one freeze / thaw cycle, preferably after two freeze / thaw cycles, more preferably after three freeze / thaw cycles, more preferably after four freeze / thaw cycles, more preferably after five freeze / thaw cycles, or more freeze / thaw cycles, are essentially equal to the size and mRNA integrity of the mRNA particles in the initial pharmaceutical composition (i.e., before the pharmaceutical composition is frozen for the first time).

[0430] In some embodiments, the pharmaceutical compositions are in liquid form and can be stored at a temperature ranging from about 0° C. to about 20° C. For example, the liquid pharmaceutical compositions described herein can be stored at a temperature ranging from about 1° C. to about 15° C., e.g., from about 2° C. to about 10° C., or from about 2° C. to about 8° C., or at a temperature of about 5° C.

[0431] In some embodiments, when the nucleic acid is mRNA, the mRNA integrity of the pharmaceutical composition upon storage is at least 70%, preferably at least 80%, more preferably at least 90% of the initial mRNA integrity (i.e., the mRNA integrity of the initial pharmaceutical composition).

[0432] In some embodiments of the liquid form of the pharmaceutical composition, the pharmaceutical composition can be stored for at least 1 week, e.g., at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, or at least 24 months, preferably at least 4 weeks. For example, the liquid pharmaceutical composition can be stored at 5°C for at least 4 weeks, preferably at least 1 month, more preferably at least 2 months, more preferably at least 3 months, and more preferably at least 6 months.

[0433] In some embodiments of a pharmaceutical composition in liquid form, when the nucleic acid is mRNA, the mRNA integrity of the liquid composition when stored at 0°C or higher for at least one week is such that a desired effect, such as the effect of inducing an immune response, can be achieved. For example, the mRNA integrity of the liquid composition may be at least 90% compared to the mRNA integrity of the initial composition, i.e., the mRNA integrity before the composition is stored, when stored at 0°C or higher for at least one week (e.g., at least two weeks, at least three weeks, at least four weeks, at least one month, at least two months, at least three months, at least four months, or at least six months). In some embodiments, the mRNA integrity of the composition after storage at a temperature of 0°C or higher, e.g., about 2°C to about 8°C for at least four weeks (e.g., at least three months), is at least 90% compared to the mRNA integrity before storage.

[0434] In some embodiments, when the nucleic acid is mRNA, the initial mRNA integrity of the pharmaceutical composition (i.e., after its preparation but before storage) is at least 50%, and preferably the mRNA integrity of the pharmaceutical composition after storage at 0°C or higher, e.g., about 2°C to about 8°C, for at least 1 week (e.g., at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, or at least 3 months) is at least 90% of the initial mRNA integrity.

[0435] In some embodiments of the pharmaceutical composition in liquid form, the size of the particles of the pharmaceutical composition (Z average ) (and / or size distribution and / or polydispersity index (PDI)) are such that the desired effect, e.g., inducing an immune response, can be achieved when stored, e.g., at 0°C or higher, for at least one week. For example, the particle size (Z average ) (and / or size distribution and / or polydispersity index (PDI)) indicates the particle size (Z) of the initial, i.e., pre-storage, pharmaceutical composition when stored, for example, at 0° C. or higher, for at least 1 week. average) (and / or size distribution and / or PDI).

[0436] In some embodiments, the size (Z) of the particles of the pharmaceutical composition after storage at, for example, 0° C. or higher for at least 1 week is average ) is about 50 nm to about 500 nm, preferably about 40 nm to about 200 nm, and more preferably about 40 nm to about 120 nm. In some embodiments, the PDI of the particles of the pharmaceutical composition, for example, after storage at 0°C or higher for at least 1 week, is less than 0.3, preferably less than 0.2, and more preferably less than 0.1.

[0437] In some embodiments, the size (Z) of the particles of the pharmaceutical composition after storage at, for example, 0° C. or higher for at least 1 week is average ) is about 50 nm to about 500 nm, preferably about 40 nm to about 200 nm, more preferably about 40 nm to about 120 nm, and the particle size (Z) of the pharmaceutical composition after storage at, for example, 0°C or higher for at least 1 week is average ) (and / or size distribution and / or PDI) are determined by the particle size (Z average ) (and / or size distribution and / or PDI). In some embodiments, the particle size (Z) of the pharmaceutical composition after storage at, for example, 0° C. or higher for at least 1 week is essentially equal to average ) is about 50 nm to about 500 nm, preferably about 40 nm to about 200 nm, more preferably about 40 nm to about 120 nm, and the PDI of the particles of the pharmaceutical composition after storage at, for example, 0°C or higher for at least 1 week is less than 0.3 (preferably less than 0.2, more preferably less than 0.1).

[0438] Pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and a "pharmaceutically acceptable preparation."

[0439] The term "pharmaceutically acceptable" refers to the non-toxicity of a material that does not interact with the action of the active components of the pharmaceutical composition.

[0440] The term "pharmaceutically effective amount" refers to an amount that alone or together with further doses achieves the desired response or desired effect. In the case of the treatment of a particular disease, the desired response preferably relates to the inhibition of the course of the disease. This includes delaying the progression of the disease and, in particular, preventing or reversing the progression of the disease. The desired response in the treatment of a disease may also be delaying or preventing the onset of the disease or condition. The effective amount of the particles or pharmaceutical compositions described herein will depend on the condition to be treated, the severity of the disease, individual patient parameters including age, physiological condition, size, and weight, the duration of treatment, the type of concomitant therapy (if any), the specific route of administration, and similar factors. Thus, the administered dose of the particles or pharmaceutical compositions described herein may depend on such various parameters. If the patient's response is inadequate with the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used.

[0441] In certain embodiments, pharmaceutical compositions of the present disclosure (e.g., immunogenic compositions, i.e., pharmaceutical compositions that can be used to induce an immune response) are formulated as a single dose in a container, e.g., a vial. In some embodiments, the immunogenic compositions are formulated as a multi-dose formulation in a vial. In some embodiments, the multi-dose formulation includes at least two doses per vial. In some embodiments, the multi-dose formulation includes 2-20 total doses per vial, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses per vial. In some embodiments, each dose in a vial is equal in volume. In some embodiments, the first dose is a different volume from subsequent doses.

[0442] A "stable" multi-dose formulation preferably does not exhibit unacceptable levels of microbial growth and is free or substantially free of destruction or degradation of active biological molecular components. As used herein, a "stable" immunogenic composition includes a formulation that can continue to elicit a desired immune response when administered to a subject.

[0443] Pharmaceutical compositions of the present disclosure may include buffers (particularly derived from the nucleic acid (e.g., RNA) composition with which the pharmaceutical composition is prepared), preservatives, and optionally other therapeutic agents. In one embodiment, pharmaceutical compositions of the present disclosure, particularly ready-to-use pharmaceutical compositions, include one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0444] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0445] The term "excipient" as used herein refers to a substance that may be present in the pharmaceutical compositions of the present disclosure but is not an active ingredient. Examples of excipients include, without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0446] The term "diluent" refers to an agent that dilutes and / or thins. Furthermore, the term "diluent" includes any one or more of a fluid, liquid, or solid suspension and / or mixing medium. Examples of suitable diluents include ethanol and water.

[0447] The term "carrier" refers to a component, which may be natural, synthetic, organic, or inorganic, in which an active component is combined to facilitate, enhance, or enable administration of a pharmaceutical composition. As used herein, a carrier may be one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. Suitable carriers include, without limitation, sterile water, Ringer's, Ringer's lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and particularly biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers.

[0448] Pharmaceutically acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

[0449] Pharmaceutical carriers, excipients, or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0450] In one embodiment, the compositions described herein, e.g., the pharmaceutical compositions or ready-to-use pharmaceutical compositions described herein, may be administered intravenously, intraarterially, subcutaneously, intradermally, transdermally, intranodally, intramuscularly, or intratumorally. In certain embodiments, the (pharmaceutical) composition is formulated for local or systemic administration. Systemic administration includes enteral administration, including absorption via the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to any mode of administration other than via the gastrointestinal tract, for example, by intravenous injection. In a preferred embodiment, the (pharmaceutical) composition, particularly the ready-to-use pharmaceutical composition, is formulated for systemic administration. In another preferred embodiment, the systemic administration is by intravenous administration. In another preferred embodiment, the (pharmaceutical) composition, particularly the ready-to-use pharmaceutical composition, is formulated for intramuscular administration.

[0451] Medical Use and Methods of Treatment The nucleic acid-lipid particles described herein and pharmaceutical compositions comprising them may be used in the treatment of various diseases, particularly diseases for which providing a peptide or protein to a subject provides a therapeutic or preventative effect. For example, providing an antigen or epitope derived from a virus may be useful in treating or preventing viral diseases caused by the virus. Providing a tumor antigen or epitope may be useful in treating cancer diseases in which cancer cells express the tumor antigen. Providing a functional protein or enzyme may be useful in treating genetic disorders characterized by dysfunctional proteins, such as lysosomal storage diseases (e.g., mucopolysaccharidoses) or factor deficiencies. Providing a cytokine or cytokine fusion may be useful for modulating the tumor microenvironment.

[0452] Thus, in one aspect, there is disclosed a nucleic acid-lipid particle, or pharmaceutical composition, as defined herein, for use in medicine.

[0453] In one embodiment, a nucleic acid-lipid particle or pharmaceutical composition as defined herein is provided for use in delivering a nucleic acid (e.g., mRNA) to a cell. In one embodiment, a nucleic acid-lipid particle or pharmaceutical composition as defined herein is provided for use in transfecting a nucleic acid (e.g., mRNA) into a cell. In one embodiment, a use of a nucleic acid-lipid particle or pharmaceutical composition as defined herein in the manufacture of a medicament for delivering a nucleic acid (e.g., mRNA) to a cell is provided. In one embodiment, a use of a nucleic acid-lipid particle or pharmaceutical composition as defined herein in the manufacture of a medicament for transfecting a nucleic acid (e.g., mRNA) into a cell is provided. In one embodiment, a method of delivering a nucleic acid (e.g., mRNA) to a cell is provided, the method comprising administering to the cell a nucleic acid-lipid particle or pharmaceutical composition as defined herein. In one embodiment, a method of transfecting a nucleic acid (e.g., mRNA) into a cell is provided, the method comprising adding to the cell a nucleic acid-lipid particle or pharmaceutical composition as defined herein; and incubating a mixture of the composition and the cell for a sufficient period of time. In some embodiments, particularly those in which the nucleic acid (e.g., mRNA) encodes a pharmaceutically active protein, the mixture of composition and cells is incubated for a period of time sufficient to allow expression of the pharmaceutically active protein. In some embodiments, the sufficient period of time is at least 1 hour (e.g., at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours) and / or up to about 48 hours (e.g., up to about 36 or up to about 24 hours). In some embodiments, the step of incubating the mixture of composition and cells is performed in the presence of serum (e.g., human serum).

[0454] The cell can be any cell capable of receiving a nucleic acid (e.g., mRNA) and producing a therapeutic effect. In one embodiment, the cell is a liver cell. In one embodiment, the cell is a splenocyte. In one embodiment, the cell is a lung cell.

[0455] In one embodiment, a nucleic acid-lipid particle or pharmaceutical composition as defined herein is provided for use in treating a disease treatable by nucleic acid (e.g., mRNA). In one embodiment, a use of a composition as defined herein in the manufacture of a medicament for treating a disease treatable by nucleic acid (e.g., mRNA) is provided. In one embodiment, a method of treating a disease treatable by nucleic acid (e.g., mRNA) in a subject in need thereof is provided, comprising administering to the subject a nucleic acid-lipid particle or pharmaceutical composition as defined herein.

[0456] In one embodiment, a nucleic acid-lipid particle or pharmaceutical composition as defined herein is provided for use in the prophylactic and / or therapeutic treatment of an antigen-associated disease. In one embodiment, a use of a nucleic acid-lipid particle or pharmaceutical composition as defined herein in the manufacture of a medicament for the prophylactic and / or therapeutic treatment of an antigen-associated disease is provided. In one embodiment, a method of prophylactically and / or therapeutically treating an antigen-associated disease in a subject in need thereof is provided, comprising administering to the subject a nucleic acid-lipid particle or pharmaceutical composition as defined herein.

[0457] In one embodiment, there is provided a nucleic acid-lipid particle or pharmaceutical composition as defined herein for use in inducing an immune response. In one embodiment, there is provided the use of a nucleic acid-lipid particle or pharmaceutical composition as defined herein in the manufacture of a medicament for inducing an immune response.

[0458] In one embodiment, a nucleic acid-lipid particle or pharmaceutical composition as defined herein is provided for use in treating cancer. In one embodiment, a use of a nucleic acid-lipid particle or pharmaceutical composition as defined herein in the manufacture of a medicament for treating cancer is provided. In one embodiment, a method of treating cancer in a subject in need thereof is provided, comprising administering to the subject a nucleic acid-lipid particle or pharmaceutical composition as defined herein.

[0459] The term "disease" (also referred to herein as "disorder") refers to an abnormal condition that affects an individual's body. A disease is often understood as a medical condition accompanied by specific symptoms and signs. A disease may be caused by an agent of external origin, such as an infectious disease, or by an internal malfunction, such as an autoimmune disease. In humans, "disease" often refers more broadly to any condition that causes pain, disability, suffering, social problems, or death to the affected individual, or a condition that causes similar problems to those who interact with that individual. In this broad sense, "disease" can include injury, incapacity, disorder, syndrome, infection, isolated symptoms, deviant behavior, and atypical variations in structure and function, while in other contexts and for other purposes, these may be considered distinct categories.

[0460] The term "infectious disease" refers to any disease that can be transmitted from individual to individual or organism to organism and is caused by a microbial agent. Infectious diseases are known in the art and include, for example, viral diseases, bacterial diseases, or parasitic diseases, which are caused by viruses, bacteria, and parasites, respectively. In this regard, infectious diseases include, for example, sexually transmitted diseases (e.g., chlamydia, gonorrhea, or syphilis), SARS, coronavirus diseases (e.g., COVID-19), acquired immune deficiency syndrome (AIDS), measles, chickenpox, cytomegalovirus infection, herpes simplex virus (e.g., HSV-1, HSV-2), hepatitis (e.g., hepatitis B or C), influenza (flu, e.g., human flu, swine flu, dog flu, equine flu, and avian flu), HPV infection, shingles, rabies, the common cold, gastroenteritis, rubella, and The disease may be mumps, anthrax, cholera, diphtheria, food poisoning, leprosy, meningitis, peptic ulcer disease, pneumonia, sepsis, septic shock, tetanus, tuberculosis, typhoid fever, urinary tract infection, Lyme disease, Rocky Mountain spotted fever, chlamydia, whooping cough, tetanus, meningitis, scarlet fever, malaria, trypanosomiasis, Chagas disease, leishmaniasis, trichomoniasis, dienamoebiasis, giardiasis, amoebic dysentery, coccidiosis, toxoplasmosis, sarcocystosis, rhinosporidiosis, and balantidiosis.

[0461] In some embodiments, the nucleic acid-lipid particles or pharmaceutical compositions described herein can be used in the therapeutic or prophylactic treatment of infectious diseases.

[0462] In the present context, the terms "treatment", "treating" or "therapeutic intervention" relate to the management and care of a subject for the purpose of combating a condition, such as a disease or disorder. The terms are intended to include the full range of treatments for a given condition from which a subject is afflicted, such as the administration of therapeutically effective compounds to alleviate the symptoms or complications, delay the progression of the disease, disorder or condition, alleviate or relieve the symptoms and complications, and / or cure or eliminate the disease, disorder or condition, as well as to prevent the condition, where prevention is understood to be the management and care of an individual for the purpose of combating the disease, condition or disorder and includes the administration of active compounds to prevent the onset of symptoms or complications.

[0463] The term "therapeutic treatment" relates to any treatment that improves the health status and / or prolongs (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, prevent or slow the progression of the disease in an individual, inhibit or slow the progression of the disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual who currently has or has previously had the disease.

[0464] The term "prophylactic treatment" or "preventative treatment" relates to any treatment intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventative treatment" are used interchangeably herein.

[0465] The terms "individual" and "subject" are used interchangeably herein. They refer to a human or other mammal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) or any other non-mammal, including bird (chicken), fish, or any other animal species, that may or may not have a disease or disorder (e.g., cancer, infectious disease), but that may or may not have the disease or disorder, or that may be in need of preventative intervention such as vaccination, or in need of intervention such as protein supplementation. In many embodiments, the individual is a human being. Unless otherwise stated, the terms "individual" and "subject" do not denote a particular age and thus encompass adults, elderly people, children, and newborns. In embodiments of the present disclosure, an "individual" or "subject" is a "patient."

[0466] The term "patient" means an individual or subject for treatment, particularly an individual or subject with a disease.

[0467] In some embodiments of the present disclosure, the goal is to provide protection from infectious diseases by vaccination.

[0468] In some embodiments of the present disclosure, the objective is to provide a secreted therapeutic protein, such as an antibody, bispecific antibody, cytokine, cytokine fusion protein, enzyme, to a subject, particularly a subject in need thereof.

[0469] In some embodiments of the present disclosure, the objective is to provide protein replacement therapy, such as the production of erythropoietin, Factor VII, von Willebrand factor, β-galactosidase, alpha-N-acetylglucosaminidase, to a subject, particularly a subject in need thereof.

[0470] In some embodiments of the present disclosure, the goal is to modulate / reprogram immune cells in the blood.

[0471] In some embodiments, a composition described herein containing a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of a SARS-CoV-2 S protein, or mRNA encoding an immunogenic variant thereof (hereinafter simply referred to as a "SARS-CoV-2 S nucleic acid composition," which expressly includes a SARS-CoV-2 S RNA composition), upon administration to a subject, induces an antibody response, particularly a neutralizing antibody response, in the subject that targets different S protein variants, e.g., a panel of SARS-CoV-2 S protein variants, particularly naturally occurring S protein variants. In some embodiments, the panel of different S protein variants includes at least 5, at least 10, at least 15, or more S protein variants. In some embodiments, such S protein variants include variants with amino acid modifications in the RBD domain and / or variants with amino acid modifications outside the RBD domain. In some embodiments, the SARS-CoV-2 S nucleic acid compositions described herein, after administration to a subject, induce an immune response (cellular and / or antibody response, particularly a neutralizing antibody response) in the subject that targets VOC-202012 / 01.

[0472] In some embodiments, the SARS-CoV-2 S nucleic acid compositions described herein, after administration to a subject, induce an immune response (cellular and / or antibody response, particularly a neutralizing antibody response) in the subject that targets 501.V2.

[0473] In some embodiments, the SARS-CoV-2 S nucleic acid compositions described herein, after administration to a subject, induce an immune response (cellular and / or antibody response, particularly a neutralizing antibody response) in the subject that targets "Cluster 5."

[0474] In some embodiments, the SARS-CoV-2 S nucleic acid compositions described herein induce an immune response (cellular and / or antibody response, particularly a neutralizing antibody response) in the subject that targets "B.1.1.28" after administration to the subject.

[0475] In some embodiments, the SARS-CoV-2 S nucleic acid compositions described herein induce an immune response (cellular and / or antibody response, particularly a neutralizing antibody response) in the subject that targets "B.1.1.248" after administration to the subject.

[0476] In some embodiments, the SARS-CoV-2 S nucleic acid compositions described herein, after administration to a subject, induce an immune response (cellular and / or antibody response, particularly a neutralizing antibody response) in the subject that targets the Omicron (B.1.1.529) variant.

[0477] Those skilled in the art will know that one of the principles of immunotherapy and vaccination is based on the fact that an immune protective response against a disease is generated by immunizing a subject with an immunologically related antigen or epitope for the disease to be treated. Therefore, the pharmaceutical compositions described herein can be applied to induce or enhance an immune response. Therefore, the pharmaceutical compositions described herein are useful in the prophylactic and / or therapeutic treatment of diseases involving antigens or epitopes.

[0478] The terms "immunization" or "vaccination" refer to the process of administering an antigen to an individual, for example for therapeutic or prophylactic reasons, with the aim of inducing an immune response. [Example] [Example]

[0479] Formation of pre-LNPs using the acidified buffer method Formation of pre-LNPs using the acidified buffer method follows the exemplary manufacturing scheme shown in FIG. 1A) Preformed lipid nanoparticles (pre-LNPs) were prepared by flow-mixing an organic phase containing dissolved lipids with an aqueous phase (5 mM acetic acid (AcOH), pH approximately 3.5). The mixing step was performed using a syringe pump and a T-piece as a mixing element at a total flow rate of 200 mL / min and a volume ratio of 1:4 (organic phase:aqueous phase). The lipid mixture (50.0 mM total concentration) consisted of a cationically ionizable lipid (DODMA), cholesterol, DSPC, and DMG-PEG dissolved in ethanol in a molar ratio of 47.5:40.7:10:1.8, respectively. The organic solvent in the resulting raw colloidal nanoparticles was removed by dialysis against 10 mM Tris over a range of pHs (pH ranging from 6.5 to 8.0) in a 10K MWCO Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific, Waltham, MA, USA). After dialysis, the nanoparticles were diluted to a concentration of 10% sucrose and filtered through a 0.22 μm polyethersulfone (PES) filter.

[0480] Next, the physicochemical properties of the pre-LNPs were analyzed, and the results are shown in Table 1. The pre-LNPs exhibited good particle attributes after dialysis in Tris buffer at all pH values ​​tested. The optimal particle size and PDI values ​​were observed at pH approximately 7.0 to 7.5. [Table 1]

[0481] 1B) Preformed lipid nanoparticles (pre-LNPs) were prepared by flow-mixing an organic phase containing dissolved lipids with an aqueous phase (5 mM acetic acid (AcOH) at pH ∼3.5). Mixing was performed using a syringe pump and a T-piece as the mixing element at a total flow rate of 90 mL / min and a volume ratio (organic phase:aqueous phase) of 1:3. The lipid mixture (80.0 mM total concentration) consisted of the cationically ionizable lipids ALC-0315, cholesterol, DSPC, and ALC-0159 dissolved in ethanol in a molar ratio of 47.5:40.7:10:1.8, respectively. The organic solvent in the resulting raw colloidal nanoparticles was removed by dialysis against either (i) 5 mM Tris buffer at approximately pH 7.0 or (ii) a mixture of 10 mM HEPES and 3 mM Tris buffer at approximately pH 7.0 in a 10K MWCO Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific, Waltham, MA, USA). After dialysis, the nanoparticles were diluted to a 10% sucrose concentration and filtered through a 0.22 μm polyethersulfone (PES) filter.

[0482] The physicochemical properties of the pre-LNPs were then analyzed, and the results are shown in Table 2. Dialysis against Tris or a mixture of HEPES / Tris both resulted in good particle attributes. [Table 2]

[0483] For pre-LNPs prepared with 5 mM acetic acid and dialyzed against 5 mM Tris buffer, pH 7.0, freeze-thaw tests were performed by cycling the pre-LNPs from -80°C (overnight) to room temperature (25°C) for 2 hours at least three times (see Figure 3). For pre-LNPs prepared with 5 mM acetic acid and dialyzed against a mixture of 10 mM HEPES and 3 mM Tris buffer, pH 7.0, freeze-thaw tests were performed by cycling the pre-LNPs from -20°C (overnight) to room temperature (25°C) for 2 hours at least three times (see Figure 4). Between thawing and freezing cycles, the pre-LNPs were mixed by gentle up-and-down movement. The particle size and polydispersity index (PDI) of the pre-LNPs were measured after each freeze-thaw cycle. As shown in Figures 3 and 4, the particle size and PDI remained controlled over three freeze-thaw cycles, demonstrating the highly promising colloidal stability of pre-LNPs prepared and stored under both conditions.

[0484] 1C) Preformed lipid nanoparticles (pre-LNPs) were prepared by flow-mixing an organic phase containing dissolved lipids with an aqueous phase (5 mM acetic acid (AcOH) at pH 3.5). Mixing was performed using a syringe pump and a T-piece as the mixing element at a total flow rate of 90 mL / min and a volume ratio (organic phase:aqueous phase) of 1:3. The lipid mixture (80.0 mM total concentration) consisted of the cationically ionizable lipid di(heptadecan-9-yl)3,3'-((2-(4-methylpiperazin-1-yl)ethyl)azanediyl)-dipropionate (BHD-C2C2-PipZ), cholesterol, DSPC, and α-tocopherol pAEEA14 dissolved in ethanol in a molar ratio of 47.5:38.5:10:4, respectively. The organic solvent in the resulting raw colloidal nanoparticles was removed by dialysis against 5 mM Tris buffer at approximately pH 7.0 in a 10K MWCO Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific, Waltham, MA, USA). After dialysis, the nanoparticles were diluted to a 10% sucrose concentration and filtered through a 0.22 μm polyethersulfone (PES) filter.

[0485] The physicochemical properties of the pre-LNPs were analyzed and the results are shown in Table 3. For this additional cationically ionizable lipid, good lipid properties were observed after dialysis against Tris pH 7.0, demonstrating that the selected conditions are generalizable to other formulations. [Table 3]

[0486] Freeze-thaw tests were performed by cycling the pre-LNPs from -20°C (overnight) to room temperature (25°C) (2 hours) at least three times. Between thaw and freeze cycles, the pre-LNPs were mixed by gentle up and down movement. The particle size and PDI of the nanoparticles were measured after each freeze-thaw cycle.

[0487] Figure 5 shows the freeze-thaw stability of pre-LNPs prepared with 5 mM acetic acid and dialyzed against 5 mM Tris at pH 7. Although some increase was observed, overall particle size and PDI remained controlled within acceptable limits over three freeze-thaw cycles, demonstrating that the manufacturing and storage conditions were appropriate for the alternative lipid composition and provided promising colloidal stability of such pre-LNPs. [Example]

[0488] Formation of pre-LNPs using the neutral buffer method Formation of pre-LNPs using the neutral buffer method follows the exemplary manufacturing scheme shown in FIG.

[0489] 2A) Preformed lipid nanoparticles (pre-LNPs) were prepared by flow-mixing an organic phase containing dissolved lipids and acetic acid with an aqueous phase (30 mM Tris, pH approximately 7.0). Mixing was performed using a syringe pump and a T-piece as the mixing element at a total flow rate of 90 mL / min and a volume ratio of 1:3 (organic phase:aqueous phase). The lipid mixture (80.0 mM total concentration) consisted of the cationically ionizable lipids ALC-0315, cholesterol, DSPC, and ALC-0159 dissolved in ethanol acidified with acetic acid (10 mM acetic acid) in a molar ratio of 47.5:40.7:10:1.8, respectively. The organic solvent in the resulting raw colloidal nanoparticles was removed by dialysis against 5 mM Tris, pH approximately 7.0, in a 10K MWCO Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific, Waltham, MA, USA). After dialysis, the nanoparticles were diluted to a 10% sucrose concentration and filtered through a 0.22 μm polyethersulfone (PES) filter. The physicochemical properties of the pre-LNPs were analyzed, and the results are shown in Table 4. It was also observed that direct mixing of an acidified organic phase with an aqueous phase having a pH of approximately 7.0 resulted in pre-LNPs that exhibited good particle attributes. [Table 4]

[0490] Freeze-thaw tests were performed by cycling the pre-LNPs from -20°C (overnight) to room temperature (25°C) (2 hours) at least three times. Between thaw and freeze cycles, the pre-LNPs were mixed by gentle up and down movement. The particle size and PDI of the pre-LNPs were measured after each freeze-thaw cycle.

[0491] Figure 6 shows the freeze-thaw stability of pre-LNPs prepared in 30 mM Tris at pH 7 and dialyzed against 5 mM Tris at pH 7. Although some increase is observed after the first freeze-thaw cycle, particle size and PDI remain controlled within acceptable limits over three freeze-thaw cycles, demonstrating very promising colloidal stability of pre-LNPs prepared under these conditions.

[0492] 2B) Preformed lipid nanoparticles (pre-LNPs) were prepared by flow-mixing an organic phase containing dissolved lipids and acetic acid with an aqueous phase (a mixture of 10 mM HEPES and 3 mM Tris buffer at pH 7.0). Mixing was performed using a syringe pump and a T-piece as the mixing element at a total flow rate of 90 mL / min and a volume ratio of 1:3 (organic phase:aqueous phase). The lipid mixture (80.0 mM total concentration) consisted of the cationically ionizable lipids ALC-0315, cholesterol, DSPC, and ALC-0159 in a molar ratio of 47.5:40.7:10:1.8, respectively, dissolved in ethanol acidified with acetic acid (10 mM acetic acid). The organic solvent in the resulting raw colloidal nanoparticles was removed by dialysis against a mixture of 10 mM HEPES and 3 mM Tris buffer at approximately pH 7.0 in a 10K MWCO Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific, Waltham, MA, USA). After dialysis, the nanoparticles were diluted to a 10% sucrose concentration and filtered through a 0.22 μm polyethersulfone (PES) filter.

[0493] The physicochemical properties of the pre-LNPs were analyzed and the results are shown in Table 5. Buffers containing a mixture of HEPES / Tris also yielded pre-LNPs with good particle attributes when mixed directly with the acidified organic phase. [Table 5] [Example]

[0494] Formation of pre-LNPs using an acidified buffer method (5 mM AcOH) and dialysis against Tris + organic acids 3A) Pre-LNPs were prepared by flow-mixing an organic phase containing dissolved lipids with an aqueous phase (5 mM acetic acid at approximately pH 3.5). Mixing was performed at a total flow rate of 90 mL / min and a volume ratio of 1:3 (organic phase:aqueous phase) using a syringe pump and a T-piece as the mixing element. The lipid mixture (80.0 mM total concentration) consisted of ALC-0315, cholesterol, DSPC, and ALC-0159 dissolved in ethanol in a molar ratio of 47.5:40.7:10:1.8, respectively. The organic solvent in the resulting raw colloidal nanoparticles was removed by dialysis against a neutral mixture of Tris (5 mM) and acetic acid (4.5 mM) at approximately pH 7 in a 10K MWCO Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific, Waltham, MA, USA). After dialysis, the nanoparticles were diluted to a concentration of 10% sucrose and filtered through a 0.22 μm polyethersulfone (PES) filter.

[0495] Freeze-thaw tests were performed by cycling the pre-LNPs from -20°C (overnight) to room temperature (25°C) (2 hours) at least three times. Between thaw and freeze cycles, the pre-LNPs were gently mixed by inversion. The particle size and PDI of the nanoparticles were measured after each freeze-thaw cycle.

[0496] Figure 7 shows the freeze-thaw stability of pre-LNPs prepared with 5 mM acetic acid and purified with a mixture of 5 mM Tris plus 4.5 mM acetic acid at approximately pH 7. Particle size and PDI remained controlled within acceptable limits over three freeze-thaw cycles, demonstrating the promising colloidal stability of pre-LNPs prepared and stored under these conditions. Utilizing organic acids for upstream and downstream processing of the nanoparticles enhances their coll...

Claims

1. 1. A method for forming an aqueous dispersion having a pH of about 6.5 to about 8.0, the aqueous dispersion being substantially free of organic solvents and nucleic acids, comprising: (A) (i) an organic phase comprising a lipid mixture comprising a cationically ionizable lipid dissolved in a water-soluble organic solvent; and (ii) an aqueous phase comprising an aqueous acid and substantially free of inorganic cations; to produce an intermediate aqueous lipid dispersion; and (B) performing a dialysis or filtration step on the intermediate aqueous lipid dispersion to produce the aqueous dispersion, wherein the dialysis or filtration step removes the organic solvent and adjusts the pH to about 6.5 to about 8.0; The method comprising:

2. 1. A method for forming an aqueous dispersion having a pH of about 6.5 to about 8.0, the aqueous dispersion being substantially free of organic solvents and nucleic acids, comprising: (A) (i) an organic phase comprising a lipid mixture comprising cationically ionizable lipids dissolved in a water-soluble organic solvent, the organic phase further comprising an aqueous acid, the organic phase being substantially free of inorganic cations; and (ii) aqueous phase; to produce an intermediate aqueous lipid dispersion having a pH of about 6.5 to about 8.0; and (B) performing a dialysis or filtration step on the intermediate aqueous lipid dispersion to produce the aqueous dispersion, wherein the dialysis or filtration step removes the organic solvent; The method comprising:

3. 3. The method of claim 1 or 2, wherein in step (A), the aqueous acid is acetic acid or malic acid.

4. 3. The method of claim 2, wherein in step (A), the aqueous phase comprises a buffer selected from the group consisting of a buffer containing an anionic group and a buffer containing an amino group (e.g., a primary, secondary, or tertiary amine group), and mixtures thereof.

5. The method according to any one of claims 1 to 4, wherein in step (B), the dialysis or filtration step uses a buffer solution.

6. 6. The method of claim 5, wherein the buffer solution is a buffer containing an anionic group, a buffer containing an amino group (e.g., a primary, secondary, or tertiary amine group), or a mixture thereof.

7. Additional steps after step (B): (B1) Adding a cryoprotectant to the aqueous dispersion The method of any one of claims 1 to 6, further comprising:

8. 8. The method of claim 7, wherein the cryoprotectant is selected from sucrose, trehalose, glucose, and mixtures of any of these.

9. 9. The method of claim 7 or 8, wherein step (B1) is carried out at a pH of about 6.5 to about 8.

0.

10. The cationically ionizable lipid is 7,7′-((4-hydroxybutyl)azanediyl)-bis(N-hexyl-N-octylheptane-1-sulfonamide) (BNT-51); 7,7′-((4-(3,3-dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylheptane-1-sulfonamide) (BNT-52); A compound having the following structure: 【Chemistry 1】 [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-315); 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA); 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA); Heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (D-Lin-MC3-DMA); 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA); Di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319); Bis-(2-butyloctyl) 10-(N-(3-(dimethylamino)propyl)nonanamido)-nonadecanedioate (A9); (Heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}-octanoate) (L5); Heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate) (SM-102); O-[N-{(9Z,12Z)-octadeca-9,12-dien-1-yl)}-N-{7-pentadecylcarbonyloxyoctyl}-amino]4-(dimethylamino)butanoate (HY501); ((2-(4-(dimethylamino)butanoyl)oxy)ethyl)azanediylbis(octane 8,1-diyl)bis(2-hexyldecanoate) (EA-405); (2-(4-(dimethylamino)butanoyl)oxy)azanediylbis(octane 8,1-diyl)bis(2-hexyldecanoate) (HY-405); Di(heptadecan-9-yl)3,3′-((2-(4-methylpiperazin-1-yl)ethyl)azanediyl)dipropionate (BHD-C2C2-PipZ); Bis(2-octyldodecyl)3,3′-((2-(1-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-1Me-Pyr); Bis(2-octyldodecyl) 3,3′-((2-(pyrrolidin-1-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-Pyr); Bis(2-octyldodecyl) 3,3′-((2-(1-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-1MePyr); Bis(2-octyldodecyl) 3,3′-(((1-methylpiperidin-3-yl)methyl)azanediyl)dipropionate (BODD-C2C2-1Me-3PipD); Bis(2-octyldodecyl)3,3′-((2-(dimethylamino)ethyl)azanediyl)dipropionate (BODD-C2C2-DMA); Bis(2-octyldodecyl)3,3′-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (BODD-C2C4-PipZ); Bis(2-octyldodecyl) 3,3′-((4-(pyrrolidin-1-yl)butyl)azanediyl)dipropionate (BODD-C2C4-Pyr); Bis(2-hexyldecyl) 3,3′-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (BHD-C2C4-PipZ); Di(nonadecan-9-yl)3,3′-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (DND-C2-C4-PipZ); and any mixtures thereof The method of any one of claims 1 to 9, wherein the compound is selected from the group consisting of:

11. The method of any one of claims 1 to 10, wherein the lipid mixture further comprises one or more additional lipids.

12. 12. The method of claim 11, wherein the one or more additional lipids comprise a neutral or zwitterionic lipid.

13. 13. The method of claim 12, wherein the neutral or zwitterionic lipid is a neutral or zwitterionic phospholipid.

14. Neutral or zwitterionic phospholipids, Distearoylphosphatidylcholine (DSPC); Dioleoylphosphatidylcholine (DOPC); Dimyristoylphosphatidylcholine (DMPC); Dipalmitoylphosphatidylcholine (DPPC); Palmitoyloleoyl-phosphatidylcholine (POPC); Dioleoylphosphatidylethanolamine (DOPE); 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG); N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM); and any mixtures thereof 13. The method of claim 12, selected from the group consisting of:

15. 15. The method of claim 14, wherein the neutral or zwitterionic phospholipid is distearoylphosphatidylcholine (DSPC).

16. The method of any one of claims 11 to 15, wherein the one or more additional lipids comprise a steroid.

17. 17. The method of claim 16, wherein the steroid is cholesterol.

18. 16. The method of any one of claims 11 to 15, wherein the one or more additional lipids comprise a grafted lipid.

19. The grafted lipid is 19. The method of claim 18, wherein the lipid is selected from the group consisting of poly(alkylene glycol)-conjugated lipids, poly(sarcosinate)-conjugated lipids, poly(oxazoline) (POX)-conjugated lipids; poly(oxazine) (POZ)-conjugated lipids; poly(vinylpyrrolidone) (PVP)-conjugated lipids; poly(N-(2-hydroxypropyl)-methacrylamide) (pHPMA)-conjugated lipids; poly(dehydroalanine) (pDha)-conjugated lipids; poly(aminoethoxyethoxyacetic acid) (pAEEA)-conjugated lipids; and poly(2-methylaminoethoxyethoxyacetic acid) (pmAEEA)-conjugated lipids; and mixtures of any of these.

20. An aqueous dispersion obtained or obtainable by a method according to any one of claims 1 to 19.

21. 1. An aqueous dispersion having an aqueous mobile phase and a dispersed phase, the dispersed phase comprises a lipid mixture including a cationically ionizable lipid; the aqueous mobile phase comprises a buffer solution having a pH of about 6.5 to about 8, the buffer being 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) optionally in combination with tris(hydroxymethyl)aminomethane (Tris); The aqueous dispersion, wherein the aqueous dispersion is substantially free of organic solvents and nucleic acids.

22. A freeze-dried composition comprising the aqueous dispersion of claim 20 or 21.

23. 1. A method of forming nucleic acid-lipid particles, comprising: (x) the aqueous dispersion according to claim 20 or 21, (y) an aqueous solution containing nucleic acid; mixing to produce said nucleic acid-lipid particles, Either the aqueous dispersion (x) or the aqueous solution (y) is acidified. The method comprising:

24. 1. A method of forming nucleic acid-lipid particles, comprising: (A) (i) an organic phase comprising a lipid mixture comprising a cationically ionizable lipid dissolved in a water-soluble organic solvent; and (ii) an aqueous phase comprising an aqueous acid and substantially free of inorganic cations; to produce an intermediate aqueous lipid dispersion; (B) performing a dialysis or filtration step on the intermediate aqueous lipid dispersion to produce an aqueous dispersion having a pH of about 6.5 to about 8.0 and substantially free of organic solvents and nucleic acids, wherein the dialysis or filtration step removes the organic solvent and adjusts the pH to about 6.5 to about 8.0; and (C)(x) the aqueous dispersion produced in step (B), (y) an aqueous solution containing nucleic acid; mixing to produce said nucleic acid-lipid particles, either the aqueous dispersion (x) or the aqueous solution (y) is acidified; The method comprising:

25. 1. A method of forming nucleic acid-lipid particles, comprising: (A) (i) an organic phase comprising a lipid mixture comprising a cationically ionizable lipid dissolved in a water-soluble organic solvent; and (ii) aqueous phase; to produce an intermediate aqueous lipid dispersion having a pH of about 6.5 to about 8.0; the organic phase comprising an aqueous acid and being substantially free of inorganic cations; (B) subjecting the intermediate aqueous lipid dispersion to a dialysis or filtration step to produce an aqueous dispersion having a pH of about 6.5 to about 8.0 and substantially free of organic solvents and nucleic acids; the dialysis or filtration step removes the organic solvent; and (C)(x) the aqueous dispersion produced in step (B), (y) an aqueous solution containing nucleic acid; mixing to produce nucleic acid-lipid particles, either the aqueous dispersion (x) or the aqueous solution (y) is acidified; The method comprising:

26. The method of any one of claims 23 to 25, wherein the nucleic acid is RNA.

27. 27. The method of claim 26, wherein the RNA is mRNA.

28. 28. The method of claim 27, wherein the mRNA encodes one or more patient-specific antigens suitable for personalized cancer therapy.

29. A nucleic acid-lipid particle obtained or obtainable by the method according to any one of claims 23 to 28.

30. 30. The nucleic acid-lipid particle of claim 29 for use in medicine.

31. 30. The nucleic acid-lipid particle of claim 29 for use in the treatment of cancer.

Images