Compositions and Methods

Abstract

An aqueous dispersion is described, which has an aqueous mobile phase and a dispersed phase, wherein the dispersed phase comprises a lipid mixture containing cationically ionizable lipids; the aqueous mobile phase comprises anions of aqueous acids; and the aqueous dispersion is substantially free of inorganic cations, organic solvents, and RNA. A method for preparing the aqueous dispersion, nucleic acid-lipid particles, and a method for preparing nucleic acid-lipid particles using the aqueous dispersion, as well as their use in medical treatment, are also disclosed.

Description

[Technical Field]

[0001] The present disclosure relates generally to lipid-based formulations suitable for delivering nucleic acids, particularly RNA, lipid particles containing such nucleic acids, aqueous lipid dispersions capable of receiving nucleic acids, and methods for producing them, particularly such methods that do not involve the use of organic solvents. [Background technology]

[0002] The traditional manufacturing route for preparing nucleic acid-containing lipid nanoparticles proceeds via a one-step process that combines one part nucleic acid (e.g., RNA) in an aqueous buffer with three parts lipid mixture dissolved in an organic solvent. While this route has demonstrated considerable success, established processes typically involve manufacturing process complexities, such as the need for a subsequent tangential flow filtration (TFF) step to remove the organic solvent used to dissolve the lipids. This additional processing increases process complexity and ultimately increases manufacturing lead time and associated costs. Furthermore, this approach is typically unfavorable for personalized patient treatment due to the need to manufacture multiple batches with very short turnaround times. The use of organic solvents in the production of RNA-lipid nanoparticle formulations requires longer processing times, which can also be a disadvantage when working with compounds with limited chemical stability.

[0003] WO 2018 / 089801 describes a method for preparing empty lipid nanoparticles by mixing lipids dissolved in ethanol with a citrate buffer at pH 4.5, followed by purification using tangential flow filtration, which involves buffer exchange. These preformed empty lipid nanoparticles can then be mixed with mRNA to produce encapsulated lipid nanoparticles in which the mRNA is encapsulated. WO 2020 / 047061 similarly describes a method for preparing empty lipid nanoparticles by mixing lipids dissolved in ethanol with a citrate buffer at pH 4.5, followed by buffer exchange (e.g., tangential flow filtration), which produces empty lipid nanoparticles in a 10% wt / vol trehalose buffer. These preformed empty lipid nanoparticles can then be mixed with mRNA to produce encapsulated lipid nanoparticles in which the mRNA is encapsulated.

[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. A method for producing encapsulated LNPs by mixing the empty LNP solution or empty LNP formulation with nucleic acid is also described.

[0005] However, although ethanol, citrate buffer, and other destabilizing agents may be absent upon addition of mRNA, all of these documents use buffers, particularly citrate or acetate buffers, to form empty lipid nanoparticles. The inorganic ions present in such buffers (e.g., citrate buffers) are thought to destabilize the colloidal properties of lipid nanoparticle formulations and are therefore detrimental to the stability of the formulations.

[0006] WO 2022 / 069632 describes a method for preparing RNA lipoplex particles for delivering RNA to target tissues. The method described in this document uses only cationic lipids as defined herein. Therefore, this document does not disclose a method for producing aqueous dispersions such as pre-LNPs or nucleic acid-lipid particles in which the lipid is a cationically ionizable lipid as defined herein.

[0007] WO 2011 / 144745 describes a method for preparing liposomes capable of encapsulating pharmaceutically and / or diagnostically active agents and / or cosmetics substantially solubilized by the liposome membrane. The method described in this document uses only cationic lipids as defined herein. Therefore, this document does not disclose a method for producing aqueous dispersions of pre-LNPs or nucleic acid-lipid particles, etc., in which the lipid is a cationically ionizable lipid as defined herein.

[0008] WO 2022 / 101471 and WO 2022 / 101486 describe pharmaceutical compositions containing lipid nanoparticles and mRNA, as well as methods for preparing and storing them. Specifically, these documents describe a method for preparing a pharmaceutical composition containing DODMA, DOPE, cholesterol, and C in ethanol. 16 -PEG 2000

[0003] The literature describes a lipid mix with a molar ratio of 40:10:48:2 between α- and α-ceramides. These documents describe mixing liposomes composed of this lipid mix in a 5 mM aqueous acetate buffer with RNA (10 mM HEPES, 0.1 mM EDTA, pH 7.0) to produce RNA-lipoplexes, which can then be diluted in a sucrose-containing buffer to a final sucrose concentration of 10%. These documents do not describe the lipid mix in aqueous solution in the absence of RNA, including a cryoprotectant such as sucrose. Furthermore, these documents do not teach or suggest the steps of freezing or lyophilizing the resulting aqueous dispersion and the subsequent benefits for producing personalized therapeutics.

[0009] WO 2021 / 155274 describes a method for preparing an empty lipid nanoparticle (empty LNP) solution containing empty lipid nanoparticles, comprising mixing a lipid solution with a solution containing a first buffer, thereby forming an empty LNP solution containing empty LNPs, the empty LNP solution comprising an acetate buffer and having a pH ranging from about 4.6 to about 6.0. The empty LNP solution may then be mixed with RNA or other nucleic acids to produce encapsulated LNPs in which the lipids encapsulate the RNA. However, for similar reasons outlined above in connection with WO 2020 / 047061 and WO 2018 / 089801, inorganic ions present in the acetate buffer used in the method described therein have been found to be detrimental to the stability of lipid nanoparticle formulations. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2018 / 089801 Brochure [Patent Document 2] International Publication No. 2020 / 047061 Brochure [Patent Document 3] International Publication No. 2022 / 032087 Brochure [Patent Document 4] International Publication No. 2022 / 069632 Brochure [Patent Document 5] International Publication No. 2011 / 144745 Brochure [Patent Document 6] International Publication No. 2022 / 101471 Brochure [Patent Document 7] International Publication No. 2022 / 101486 Brochure [Patent Document 8] International Publication No. 2021 / 155274 Brochure Summary of the Invention [Means for solving the problem]

[0011] In a first aspect, the present disclosure provides an aqueous dispersion having an aqueous mobile phase and a dispersed phase, the dispersed phase comprises a lipid mixture comprising a cationic lipid or a cationically ionizable lipid; the aqueous mobile phase comprises an anion of an aqueous acid; the aqueous dispersion is substantially free of inorganic cations, organic solvents, and RNA; An aqueous dispersion is provided, wherein the aqueous mobile phase comprises a cryoprotectant.

[0012] In one embodiment of this aspect, the disclosure 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 an anion of an aqueous acid; the aqueous dispersion is substantially free of inorganic cations, organic solvents, and RNA; An aqueous dispersion is provided, wherein the aqueous mobile phase comprises a cryoprotectant.

[0013] In a second aspect, the present disclosure provides an aqueous dispersion having an aqueous mobile phase and a dispersed phase, the dispersed phase comprises a cationic lipid or a cationically ionizable lipid; the aqueous mobile phase comprises an anion of an aqueous acid; the concentration of the aqueous acid is at least 6 mM; An aqueous dispersion is provided in which the aqueous mobile phase is substantially free of inorganic cations, organic solvents, and RNA.

[0014] In a third aspect, the present disclosure provides an aqueous dispersion having an aqueous mobile phase and a dispersed phase, the dispersed phase comprises a cationic lipid or a cationically ionizable lipid; the aqueous mobile phase comprises malate or succinate anions; An aqueous dispersion is provided, wherein the aqueous dispersion is substantially free of inorganic cations, organic solvents, and RNA.

[0015] In a fourth aspect, the present disclosure provides a method of forming the aqueous dispersion of the first aspect, comprising: (i) a lipid mixture comprising a cationic lipid or a cationically ionizable lipid; (ii) an aqueous phase containing an aqueous acid and a cryoprotectant; to produce an aqueous dispersion comprising an anion of an aqueous acid.

[0016] In one embodiment of the fourth aspect, the present disclosure provides a method of forming the aqueous dispersion of the first aspect, comprising: (i) a lipid mixture containing cationically ionizable lipids; (ii) an aqueous phase containing an aqueous acid and a cryoprotectant; to produce an aqueous dispersion comprising an anion of an aqueous acid.

[0017] In a fifth aspect, the present disclosure provides a method of forming the aqueous dispersion of the first aspect, comprising: (a)(i) a lipid mixture comprising a cationic lipid or a cationically ionizable lipid; and (ii) an aqueous phase containing an aqueous acid to produce a first intermediate aqueous dispersion containing anions of an aqueous acid; and (b) adding a cryoprotectant to the first intermediate aqueous dispersion to produce an aqueous dispersion; The present invention provides a method comprising:

[0018] In one embodiment of the fifth aspect, the present disclosure provides a method of forming the aqueous dispersion of the first aspect, comprising: (a)(i) a lipid mixture comprising a cationically ionizable lipid; and (ii) an aqueous phase containing an aqueous acid to produce a first intermediate aqueous dispersion containing anions of an aqueous acid; and (b) adding a cryoprotectant to the first intermediate aqueous dispersion to produce an aqueous dispersion; The present invention provides a method comprising:

[0019] In a sixth aspect, the present disclosure provides a method of forming an aqueous dispersion comprising an anion of an aqueous acid, the method comprising: (a)(i) a lipid mixture comprising a cationic lipid or a cationically ionizable lipid dissolved in a water-soluble organic solvent; and (ii) an aqueous phase containing an aqueous phase, a lipid mixture, and / or an aqueous acid; to produce a first intermediate acidified aqueous lipid dispersion comprising an anion of an aqueous acid; (b) performing a dialysis or filtration step on the first intermediate acidified aqueous lipid dispersion at a pH of about 2.5 to about 5.5 to remove the organic solvent to produce a second intermediate aqueous dispersion; and (c) adding a cryoprotectant to the second intermediate aqueous dispersion; Producing an aqueous dispersion containing anions of an aqueous acid. The present invention provides a method comprising:

[0020] In a seventh aspect, the present disclosure provides a method of forming an aqueous dispersion comprising an anion of an aqueous acid, the method comprising: i) mixing a lipid mixture comprising cationic lipids or cationically ionizable lipids dissolved in a water-soluble organic solvent with an aqueous phase to produce a first intermediate acidified aqueous lipid dispersion comprising anions of an aqueous acid; the lipid solution and / or the aqueous phase comprises an aqueous acid; ii) performing a dialysis or filtration step on the first intermediate acidified aqueous lipid dispersion at a pH of about 2.5 to about 5.5, or at a pH of 6.5 to 8.5, to remove the organic solvent and produce a second intermediate aqueous dispersion; and iii) adding a cryoprotectant to the second intermediate aqueous dispersion to produce an aqueous dispersion. Including, The method is provided wherein the aqueous dispersion is substantially free of inorganic cations, organic solvents, and RNA.

[0021] In an eighth aspect, the present disclosure provides a method of forming the aqueous dispersion of the second aspect, comprising: (i) a lipid mixture comprising a cationic lipid or a cationically ionizable lipid; (ii) an aqueous phase containing an aqueous acid to produce an aqueous dispersion comprising anions of an aqueous acid, the concentration of the aqueous acid is at least 6 mM The present invention provides a method comprising:

[0022] In a ninth aspect, the present disclosure provides a method of forming the aqueous dispersion of the third aspect, comprising: (i) a lipid mixture comprising a cationic lipid or a cationically ionizable lipid; (ii) an aqueous phase containing malic acid or succinic acid to produce an aqueous dispersion containing malate or succinate anions. The present invention provides a method comprising:

[0023] In a tenth aspect, the present disclosure provides a method of forming lipid particles containing nucleic acid (e.g., RNA such as mRNA), the method comprising: i) preparing an aqueous dispersion according to the method of any one of the first to ninth aspects; and ii) mixing the aqueous dispersion with an aqueous solution containing nucleic acids to produce lipid particles containing nucleic acids; The present invention provides a method comprising:

[0024] In an eleventh aspect, the present disclosure provides a method of forming lipid particles containing nucleic acid (e.g., RNA such as mRNA), the method comprising: i) mixing a lipid mixture comprising cationic lipids or cationically ionizable lipids dissolved in a water-soluble organic solvent with an aqueous phase to produce a first intermediate dispersion comprising anions of an aqueous acid; the lipid solution and / or the aqueous phase comprises an aqueous acid; ii) performing a dialysis or filtration step on the first intermediate dispersion at a pH of about 2.5 to about 5.5 or at a pH of 6.5 to 8.5 to remove the organic solvent to produce a second intermediate aqueous dispersion; iii) adding a cryoprotectant to the second intermediate aqueous dispersion to produce an aqueous dispersion, wherein the aqueous dispersion is substantially free of inorganic cations, organic solvents, and nucleic acids; and iv) mixing the aqueous dispersion with an aqueous solution containing nucleic acids to produce lipid particles containing nucleic acids. The present invention provides a method comprising:

[0025] In a twelfth aspect, the present disclosure provides a lipid-nucleic acid particle (e.g., a lipid-RNA particle), such as a lipid nanoparticle obtained or obtainable by the method of the tenth or eleventh aspect.

[0026] In a thirteenth aspect, the present disclosure provides a pharmaceutical composition comprising the lipid particle of the twelfth aspect and a pharmaceutical carrier.

[0027] In a fourteenth aspect, the present disclosure provides a lipid particle of the twelfth aspect for use in medicine.

[0028] In a fifteenth aspect, the present disclosure provides a lipid particle of the twelfth aspect for use in the prophylactic and / or therapeutic treatment of a disease involving an antigen and / or for use in inducing an immune response.

[0029] In a sixteenth aspect, the present disclosure provides the lipid particle of the twelfth aspect for use in treating cancer.

[0030] In a seventeenth aspect, the present disclosure provides the use of the lipid particle of the twelfth aspect in the manufacture of a medicament for use in the prophylactic and / or therapeutic treatment of a disease involving an antigen and / or for use in inducing an immune response.

[0031] In an eighteenth aspect, the present disclosure provides the use of the lipid particle of the twelfth aspect in the manufacture of a medicament for use in the treatment of cancer.

[0032] In a nineteenth aspect, the present disclosure provides a method for prophylactic and / or therapeutic treatment of an antigen-associated disease and / or a method of inducing an immune response in a subject in need thereof, comprising administering to the subject a lipid particle of the twelfth aspect.

[0033] In a twentieth aspect, the present disclosure provides a method for the prophylactic and / or therapeutic treatment of cancer in a subject in need thereof, comprising administering to the subject a lipid particle of the twelfth aspect.

[0034] In a twenty-first aspect, the present disclosure provides a lyophilized composition comprising the aqueous dispersion of any one of the first to third aspects.

[0035] In a twenty-second aspect, the present disclosure provides a frozen composition comprising the aqueous dispersion of any one of the first to third aspects, the frozen composition being at a temperature of -15°C to -90°C.

[0036] Benefits and Surprising Findings The present inventors have surprisingly found that the method described herein allows both aqueous dispersion compositions (lacking nucleic acid) and nucleic acid-lipid particles to be prepared without using organic solvents.This avoids both the risk of organic solvents degrading chemically unstable lipids and the need for complex purification processes to remove organic solvents.One large batch of preformed aqueous dispersion can be used to produce several batches of nucleic acid-lipid particle (e.g., patient-specific mRNA lipid nanoparticle) formulations, which may be particularly interesting in areas such as personalized immunotherapy platforms, where small batches of final product are required.

[0037] Furthermore, the method disclosed herein offers significant manufacturing advantages compatible with Class D manufacturing environments. Additionally, nucleic acid-lipid particles formed by this novel process have been demonstrated to have improved colloidal stability, lipid stability, and RNA integrity while maintaining biological efficacy under both frozen and liquid conditions, compared to other classical lipid nanoparticle manufacturing routes. Furthermore, the method disclosed herein allows for great flexibility in modifying formulation properties, such as particle size, surface charge, and functionalization, without affecting process robustness. Additionally, nucleic acid-lipid particles produced by the disclosed method can also be functionalized with ligands to target specific cells, organs, etc.

[0038] Additionally, in contrast to the methods described in WO 2020 / 047061, WO 2018 / 089801, WO 2021 / 155274, and WO 2022 / 032087, the methods described herein do not use inorganic ions present in citrate and acetate buffers, thus avoiding the deleterious effects of inorganic ions on nucleic acid-lipid particle formulations. Specifically, the use of malic acid or succinic acid is believed to further improve colloidal stability and / or RNA integrity during, for example, RNA-lipid particle formation and subsequent storage. Similarly, it has also been surprisingly found that higher concentrations of acid, such as acetic acid at 6 mM or higher, provide improved colloidal properties and / or do not negatively affect particle stability and / or RNA integrity. Adding a cryoprotectant, such as sucrose, to the aqueous dispersion enhances the long-term stability of the lipid particles and facilitates storage under frozen conditions.

[0039] Furthermore, the inventors have found that adding the cryoprotectant during the initial mixing step in the method of forming the aqueous dispersion offers the advantage that an HPLC step between the tangential flow filtration (TFF) and dilution steps is not required, since it is no longer necessary to measure the lipid concentration to determine the amount of cryoprotectant to add upon dilution, which streamlines and shortens the manufacturing process.

[0040] In addition, the present inventors have found that, in contrast to the methods described in WO 2022 / 101486 and WO 2022 / 101471, adding a cryoprotectant during the initial mixing step in the method of forming an aqueous dispersion offers the advantage of enhanced colloidal stability. Without wishing to be bound by theory, it is believed that including a cryoprotectant during the initial mixing step avoids osmotic pressure changes during filtration (e.g., TFF) / dialysis or dilution steps, thereby enhancing colloidal stability. Surprisingly, it has also been found that encapsulating nucleic acids, such as RNA, in aqueous dispersions containing a cryoprotectant results in viable nucleic acid-lipid particle compositions; however, this was not thought to be possible due to the expected difficulty of encapsulating nucleic acids in more viscous aqueous dispersions. In particular, it has been surprisingly discovered that nucleic acids (e.g., RNA) can be encapsulated in aqueous dispersions containing high concentrations of sucrose (e.g., 20% sucrose), allowing the preparation of nucleic acid-lipid particles in a one-step procedure and eliminating the need for an additional dilution step for adding a cryoprotectant. This streamlines and shortens this second step of the manufacturing process, which is beneficial for RNA integrity and particle stability. Furthermore, the publication dates of WO2022 / 101486 and WO2022 / 101471 did not foresee the possibility of freezing or lyophilizing aqueous dispersions, nor did they suggest the benefits of using a two-step method for the manufacture of personalized therapeutics. [Brief explanation of the drawings]

[0041] [Figure 1(a)] ~ [Figure 1(b)] (a) shows a general manufacturing scheme for aqueous dispersions of the present invention (also referred to herein as "pre-formed lipid nanoparticles" ("pre-LNPs")); (b) shows a manufacturing scheme for exemplary aqueous dispersions of the present invention (also referred to herein as "pre-formed lipid nanoparticles", i.e., prior to the introduction of nucleic acids) (IPA means isopropyl alcohol). [Figure 2] FIG. 1 shows an exemplary manufacturing scheme for RNA-lipid particles according to the present invention. [Figure 3] FIG. 1 shows the stability of an aqueous dispersion according to the invention with grafted lipids (Example 1) when stored both in liquid and frozen conditions, in terms of particle size (a) and polydispersity index (b). [Figure 4] FIG. 1 shows the long-term stability of RNA-lipid particles according to the present invention (Example 1) produced according to the two-step process described herein as a function of particle size (a), polydispersity index (b), and RNA integrity (c) at −80° C. and −20° C. [Figure 5] FIG. 1 shows an INF-γ ELISpot showing a high T cell response for a drug product (designated LNP2) manufactured by a process according to the present invention (Example 2), all formulations tested have similar lipid and N / P ratios and are administered at the same dose. [Figure 6] FIG. 1 shows the stability of an aqueous dispersion of the present invention (Example 2) containing DODMA with C14-Psar(23)-Ac when stored in liquid (4° C. and 25° C.) conditions in terms of particle size (a) and polydispersity index (b). [Figure 7] FIG. 1 shows the stability of an aqueous dispersion according to the invention (Example 3) with DODMA and DMG-PEG2k at different pH with 5 mM acetic acid (a), 40 mM acetate buffer (b), and 10 mM HEPES buffer (c). [Figure 8] FIG. 1 shows the colloidal stability of ungrafted RNA-lipid particles according to the present invention (Example 4) (the formulation was prepared with an N / P ratio of 6, pH 5.5, and an RNA content of 0.1 mg / mL, and stored in HEPES buffer containing 10% (wt / vol) sucrose; the lipid mixture consisted of the ionizable lipid HY-501, cholesterol, and DSPC in a molar ratio of 47.5:42.5:10) monitored over a 3-month period in both frozen and liquid conditions, with both particle size (a) and polydispersity (b) remaining within specifications over the tested time periods. [Figure 9]FIG. 1 shows the stability of aqueous dispersions according to the invention (Example 6) containing Alfa-tag lipids when stored in liquid (4° C. and 25° C.) conditions, in terms of particle size (a) and polydispersity index (b). [Figure 10] FIG. 1 shows particle size (a) and polydispersity index (b) analysis of functionalized RNA-lipid particles according to the present invention (Example 6) subjected to two freeze-thaw (FT) cycles from −20° C. to room temperature and from −80° C. to room temperature. [Figure 11] FIG. 1 shows particle size and polydispersity index (PDI) of starting RNA-lipid particles (Example 7), alfa-tagged RNA-lipid particles by a post-insertion approach, and functionalized RNA-lipid particles according to the present invention. [Figure 12] FIG. 10 shows the freeze-thaw stability of pre-LNPs produced and purified with various concentrations of acetic acid (1.25, 2.5, and 5 mM) in accordance with the present invention (Example 10A). [Figure 13A] ~ [Figure 13B] FIG. 10B shows the freeze-thaw stability of pre-LNPs produced and purified with 5 mM acetic acid according to the present invention, diluted and stored in (A) 8% w / v sucrose and (B) 12% w / v sucrose (Example 10B). [Figure 14] FIG. 10 shows the freeze-thaw stability of pre-LNPs produced and purified with 5 mM acetic acid, diluted and stored in 10% trehalose according to the present invention (Example 10C). [Figure 15] FIG. 10 shows the freeze-thaw stability of pre-LNPs produced and purified with 5 mM acetic acid, diluted and stored in 5% w / v glucose in accordance with the present invention (Example 10D). [Figure 16A] ~ [Figure 16B] 16A and 16B show the freeze-thaw stability of pre-LNPs prepared with 2.5 mM acetic acid (FIG. 16A) or 5 mM acetic acid (FIG. 16B) in accordance with the present invention (Example 11). [Figure 17]FIG. 12 shows the freeze-thaw stability of pre-LNPs produced and purified with various concentrations of malic acid (2.5, 5, and 10 mM) in accordance with the present invention (Example 12). [Figure 18] FIG. 12 shows the freeze-thaw stability of pre-LNPs produced and purified in accordance with the present invention with a mixture of 5 mM acetic acid plus malic acid at various concentrations as indicated (Example 12). [Figure 19A] ~ [Figure 19B] FIG. 14 shows the freeze-thaw stability of pre-LNPs produced and purified in accordance with the present invention with a mixture of a) 5 mM acetic acid, 5% w / v sucrose, or b) 5 mM acetic acid, 10% w / v sucrose (Example 14). [Figure 20A] ~ [Figure 20D] (Comparative) Freeze-thaw stability of pre-LNPs produced and purified according to Example 15 (comparative) in (A) 5 mM acetate buffer, pH 5.0 or 5.5 mM, (B) citrate buffer of various concentrations (2.5, 5, 10, and 20 mM); (C) 30 mM succinate buffer, approximately pH 4, and (D) 30 mM malate buffer, approximately pH 4. [Figure 21] FIG. 1 shows the colloidal stability of RNA-LNPs according to the present invention over five freeze-thaw cycles (Example 17). [Figure 22] FIG. 1 shows the long-term stability of RNA-LNP according to the present invention (Example 17). [Figure 23] FIG. 1 shows the particle size and PDI of freeze-thawed RNA-LNPs according to the present invention (Example 18). [Figure 24] FIG. 1 shows the long-term stability of RNA-LNP according to the present invention (Example 18). [Figure 25] FIG. 1 shows the particle size and PDI of freeze-thawed RNA-LNP according to the present invention (Example 19). [Figure 26]FIG. 1 shows particle size and PDI of liquid-state RNA-LNPs according to the present invention (Example 20) in 2.5 mM acetic acid, shown in different storage matrices: (a) 60 mM HEPES, 3 mM Tris, 30% sucrose at pH 6.3; (b) 50 mM Tris, 30% sucrose at pH 8.5. [Figure 27] FIG. 1 shows the particle size and PDI of freeze-thawed RNA-LNPs according to the present invention (Example 21). [Figure 28] FIG. 1 shows the particle size and PDI of freeze-thawed RNA-LNPs according to the present invention (Example 22). [Figure 29] FIG. 1 shows the long-term stability of concentrated LNPs according to the present invention (Example 23) (RNA concentration (mg / mL): GrA=0.1; GrB=0.2; GrC=0.3; GrD=0.6; GrD=1.3). [Figure 30] FIG. 1 shows the particle size and PDI of RNA-LNPs according to the present invention prepared from lyophilized and reconstituted pre-LNPs (Example 25). [Figure 31] FIG. 1 shows particle size and PDI of freeze-thawed LNPs according to the present invention (Example 26). [Figure 32] FIG. 1 shows an exemplary simplified manufacturing scheme for RNA-LNPs according to the present invention, as described in Example 27. [Figure 33] FIG. 1 shows the particle size and PDI of RNA-LNP according to the present invention (Example 27). DETAILED DESCRIPTION OF THE INVENTION

[0042] 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.

[0043] 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).

[0044] 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.

[0045] 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.

[0046]

[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.

[0047] 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.

[0048] 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."

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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).

[0067] 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.

[0068] 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.

[0069] 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 ...

[0070] 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 ...

[0071] 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 ...

[0072] 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 ...

[0073] 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.

[0074] 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 ...

[0075] 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 ...

[0076] 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 ...

[0077] 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 ...

[0078] The term "organosulfuric 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 protonated or deprotonated (in anionic amphiphiles, as defined below, sulfonic acid groups are typically deprotonated at physiological pH).

[0079] 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 this group is deprotonated. Depending on the pH, the sulfonate group is protonated or deprotonated (in anionic amphiphiles, as defined below, the sulfonate group is typically deprotonated at physiological pH).

[0080] 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 (in anionic amphiphiles, as defined below, the carboxylic acid group is typically protonated at acidic pH and deprotonated at neutral or alkaline pH).

[0081] 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 (in anionic amphiphiles, as defined below, the dicarboxylic acid group is typically protonated at acidic or neutral pH and deprotonated at alkaline pH).

[0082] The term "hydroxycarboxylic acid" or "hydroxycarboxylate" 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), 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 can be protonated or deprotonated (in anionic amphiphiles, as defined below, the carboxylic acid group is typically protonated at acidic pH and deprotonated at neutral or alkaline pH).

[0083] 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).

[0084] The term "hemiester" as used herein in reference to a functional moiety refers to an ester of a dicarboxylic acid, as defined above, in which one of the carboxylic acid groups forms an ester bond with the rest of the molecule and the other carboxylic acid group is free. Depending on the pH, the free carboxylic acid group is protonated or deprotonated (in anionic amphiphiles, as defined below, the free carboxylic acid group is typically protonated at acidic pH and deprotonated at neutral or alkaline pH).

[0085] The term "phosphate" or "organophosphate" refers to a compound of formula R-O-P(=O)(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). Depending on the pH, the phosphate group is protonated or deprotonated (in anionic amphiphiles, as defined below, the phosphate group is typically deprotonated at physiological pH).

[0086] The term "phosphonate" or "organophosphonic acid" refers to a compound of formula R-P(=O)(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). Depending on the pH, the phosphonate group is protonated or deprotonated (in anionic amphiphiles, as defined below, the phosphonate group is typically deprotonated at physiological pH).

[0087] "Halo" means fluoro (-F), chloro (-Cl), bromo (-Br), or iodo (-I).

[0088] "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 C 1-6 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.

[0089] "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.

[0090] "Hydroxyl" means the group -OH. "Sulfhydryl" means the group -SH. "Nitro" means the group -NO2.

[0091] "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.

[0092] "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.

[0093] "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.

[0094] "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.

[0095] "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.

[0096] "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.

[0097] "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.

[0098] "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.

[0099] 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.

[0100] "Carbohydrates" have the empirical formula C m (H2O) n where 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. In another embodiment, the monosaccharide moiety is a pentose moiety (i.e., it has 5 carbon atoms), such as ribose, arabinose, xylose, or lyxose. Preferably, the pentose moiety is an arabinose or xylose moiety.

[0101] 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.

[0102] "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', and 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, C 1-3 Alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH 2-z (CH3) z , —C(═O)OH, and —C(═O)OCH3, where z is 0, 1, or 2; 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 .

[0103] amino acid In some embodiments, the methods and compositions of the invention, particularly further processing steps, such as dialysis or filtration steps, dilution or addition of storage matrix steps, and storage steps, use amino acids.

[0104] "Amino acid," in its broadest sense, has its ordinary meaning in the art 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] In one embodiment, the amino acid is a non-proteinogenic amino acid. Examples of 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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).

[0116] 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.

[0117] 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.

[0118] mRNA In a preferred embodiment 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).

[0119] 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.

[0120] According to the present disclosure, "dsRNA" means double-stranded RNA, which is RNA having two strands that are partially or completely complementary.

[0121] In a preferred embodiment of the present disclosure, mRNA relates to an RNA transcript that encodes a peptide, polypeptide, or protein.

[0122] 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.

[0123] 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™).

[0124] 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).

[0125] 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.

[0126] 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.

[0127] inhibitory RNA In some embodiments of all aspects of the disclosure, the nucleic acid is an inhibitory RNA.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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).

[0140] 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.

[0141] 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). CSF), tumor necrosis factor (TNF), erythropoietin (EPO), and bone morphogenetic proteins (BMPs); immunoglobulin superfamily members, including antibodies (e.g., IgG), T cell receptors (TCRs), major histocompatibility complex (MHC) molecules, co-receptors (e.g., CD4, CD8, CD19), antigen receptor accessory molecules (e.g., CD3-γ, CD3-δ, CD3-ε, CD79a, CD79b), costimulatory or inhibitory molecules (e.g., CD28, CD80, CD86); other immunologically active compounds, such as tumor-associated antigens, pathogen-associated antigens (e.g., bacterial, parasitic, or viral antigens), allergens, and autoantigens.

[0142] aqueous dispersion The present disclosure provides aqueous dispersions having an aqueous mobile phase and a dispersed phase. 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").

[0143] 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.

[0144] 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.

[0145] 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.

[0146] In one embodiment, the mobile phase is a solution. The term "solution," as used herein, refers to a homogeneous mixture containing 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 described herein.

[0147] The dispersed phase comprises a lipid mixture comprising a cationic lipid or a cationically ionizable lipid as defined herein. In one embodiment, the dispersed phase comprises a lipid mixture comprising a cationically ionizable lipid as defined herein. In one embodiment, the dispersed phase comprises a lipid mixture comprising a cationically ionizable lipid as defined herein, wherein the lipid mixture does not comprise a cationic lipid as defined herein.

[0148] In one embodiment, the aqueous dispersion is substantially free of inorganic cations.

[0149] In one embodiment, the aqueous dispersion is substantially free of organic solvents.

[0150] In one embodiment, the aqueous dispersion is substantially free of nucleic acids. In one embodiment, the aqueous dispersion is substantially free of RNA.

[0151] 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. Preformed LNPs thus typically contain a central, complex, irregular, non-lamellar layer composed of lipids, but are substantially free of nucleic acids. This contrasts with the structure of liposomes, which comprise unilamellar or multilamellar vesicular particles composed 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.

[0152] In some cases, the pre-LNPs described herein are not liposomes. In some cases, the pre-LNPs or nucleic acid-lipid particles formed from the pre-LNPs described herein are not lipoplexes.

[0153] 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.

[0154] 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 40 nm to about 100 nm. In some embodiments, the pre-LNPs described herein have an average diameter of about 40 nm to about 70 nm.

[0155] In some examples, 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 100 nm, or preferably about 60 nm to about 100 nm.

[0156] In one embodiment, the aqueous mobile phase includes a cryoprotectant, described in more detail below, which may be introduced in the mixing step or in further processing steps, described in more detail below.

[0157] In one embodiment, the dispersed phase comprises a lipid mixture comprising cationic lipids or cationically ionizable lipids; the aqueous mobile phase comprises anions of aqueous acids; the aqueous dispersion is substantially free of inorganic cations, organic solvents, and RNA; The aqueous mobile phase contains a cryoprotectant.

[0158] In one embodiment, the dispersed phase comprises a cationic lipid or a cationically ionizable lipid; the aqueous mobile phase comprises an anion of an aqueous acid; The concentration of the aqueous acid is at least 6 mM; The aqueous mobile phase is substantially free of inorganic cations, organic solvents, and RNA.

[0159] In one embodiment, the dispersed phase comprises a cationic lipid or a cationically ionizable lipid; the aqueous mobile phase comprises a malate anion or a succinate anion; The aqueous dispersion is substantially free of inorganic cations, organic solvents, and RNA.

[0160] In one embodiment, the aqueous dispersion has a maximum pH of about 4.5, for example, a maximum pH of 4.2 to 4.8. In one embodiment, the aqueous dispersion has a maximum pH of 4.5. The aqueous dispersion may have a pH less than 4.5.

[0161] In one embodiment, the aqueous dispersion has a pH of about 2.5 to about 4.5. The aqueous dispersion may have a pH of 2.5 to 4.5. The aqueous dispersion may have a pH of 2.5 to 3.5 or 3.5 to 4.5. The aqueous dispersion may have a pH of 4.0 to 4.5. The aqueous dispersion may have a pH of about 4.5.

[0162] The aqueous mobile phase comprises the anion of an aqueous acid. The acid can be any inorganic or organic acid that is at least partially miscible with water and capable of being 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, and the species in aqueous solution are primarily (in some embodiments, completely) the anion of the acid. In contrast, weak acids do not completely deprotonate in water, and the species in aqueous solution comprise a mixture of the undissociated acid and its conjugate base, the relative amounts of each depending on the pH.

[0163] In one embodiment, the anion is acetate. In one embodiment, the anion is malate. In one embodiment, the anion is succinate.

[0164] Additionally, aqueous 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.

[0165] In addition, the anion of an aqueous acid can interact with the constitutively charged head group of a cationic lipid to form a lipid salt.The interaction of an anion of an aqueous acid with a cationic lipid or a cationically ionizable lipid is expected to promote the formation of stable lipid particles.For example, the formation of a lipid salt of an anion of an aqueous acid with a cationic lipid can affect the shape factor kappa (κ) of the cationic lipid (i.e., the volume ratio of the polar section to the non-polar section of the lipid; κ = molecular volume (head, polar) / molecular volume (tail, non-polar)), promoting the formation of lipid nanoparticle structure (see, for example, WO2008 / 043575, WO2009 / 047006, Siepi et al., Biophys J. 2011, 100, 2412-2421).For example, the lipid salt can have a shape factor κ of less than 0.25, optionally less than 0.15.

[0166] 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.

[0167] In one embodiment, the aqueous acid is a water-soluble organic acid. Examples of suitable inorganic acids include a sulfonic acid, a carboxylic acid, a dicarboxylic acid, a hydroxycarboxylic acid (all as defined herein), or an amino acid.

[0168] In one embodiment, the water-soluble organic acid is selected from the group consisting of acetic acid, malic acid, maleic acid, succinic acid, ascorbic acid, oxalic acid, and citric acid, or a combination thereof. The water-soluble organic acid may be selected from the group consisting of acetic acid, malic acid, maleic acid, succinic acid, ascorbic acid, oxalic acid, and citric acid. The water-soluble organic acid may be selected from the group consisting of acetic acid, malic acid, and succinic acid. The water-soluble organic acid may be selected from the group consisting of acetic acid, malic acid, maleic acid, succinic acid, ascorbic acid, and oxalic acid. The water-soluble organic acid may be selected from the group consisting of malic acid, maleic acid, succinic acid, ascorbic acid, oxalic acid, and citric acid. The water-soluble organic acid may be selected from the group consisting of malic acid, maleic acid, succinic acid, ascorbic acid, oxalic acid, and citric acid.

[0169] In one embodiment, the water-soluble weak organic acid is acetic acid. In one embodiment, the water-soluble weak organic acid is malic acid. In one embodiment, the water-soluble weak organic acid is succinic acid.

[0170] In one embodiment, the aqueous mobile phase further comprises a cryoprotectant as described and exemplified herein. In one embodiment, the aqueous dispersion comprises a cryoprotectant as described and exemplified herein. In one embodiment, the cryoprotectant is a carbohydrate, such as a monosaccharide or disaccharide. In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose, and glucose, and mixtures of any of these. Preferably, the cryoprotectant is sucrose.

[0171] In one embodiment, the cryoprotectant is sucrose or trehalose and is present in the aqueous dispersion at a concentration of about 1% to about 30% (wt / vol), about 2% to about 20% (wt / vol), or about 5% to about 15% (wt / vol). In one embodiment, the cryoprotectant is sucrose or trehalose and is present at a concentration of about 8% to about 12% (wt / vol), optionally about 10% (wt / vol). In one embodiment, the cryoprotectant is sucrose or trehalose and is present at a concentration of about 15% to about 25% (wt / vol), optionally about 18% to about 22% (wt / vol). In one embodiment, the cryoprotectant is sucrose or trehalose and is present at a concentration of about 20% (wt / vol). In one embodiment, the cryoprotectant is glucose and is present in the aqueous dispersion at a concentration of about 1% to about 15% (wt / vol), optionally about 2% to about 10% (wt / vol). In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 4% to about 8% (wt / vol), optionally about 5% (wt / vol). In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 8% to about 12% (wt / vol), optionally about 10% (wt / vol).

[0172] 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, at a pH of about 4.5. The aqueous dispersion may be substantially free of buffers. The aqueous dispersion may be substantially free of acetate buffer, citrate buffer, phosphate buffer, and / or tris buffer. The aqueous dispersion may be substantially free of a buffer 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 a buffer selected from the group consisting of ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, tris(hydroxymethyl)aminomethane (tris), sodium phosphate, and HEPES.

[0173] In one embodiment, the aqueous dispersion is substantially free of inorganic cations.It is believed that such inorganic cations affect the colloidal stability of lipid dispersions and reduce the stability of formulations.In one embodiment, the aqueous dispersion is substantially free of alkali metal ions (as defined herein).In one embodiment, the aqueous dispersion is substantially free of inorganic cations, such as ammonium, sodium, and / or potassium ions.

[0174] In one embodiment, the aqueous dispersion is substantially free of organic solvents (as defined herein). In one embodiment, the term "substantially free of organic solvents" means that the aqueous dispersion contains less than about 5,000 ppm by weight of organic solvent as a percentage of the total weight of the aqueous dispersion, such as less than about 4,000 ppm, for example less than about 3,000 ppm, such as less than about 2,000 ppm, for example less than about 1,000 ppm, such as less than about 900 ppm, for example less than about 800 ppm, for example less than about 700 ppm, such as 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, such as less than about 200 ppm, for example less than about 100 ppm.

[0175] For example, 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, ethanol, and / or acetone.

[0176] In one embodiment, the concentration of the aqueous acid is at least 6 mM. In one embodiment, the concentration of the aqueous acid is in the range of 1 to 20 mM. In one embodiment, the concentration of the aqueous acid is in the range of 5.5 to 20 mM. In one embodiment, the concentration of the aqueous acid is in the range of 6 to 20 mM. In one embodiment, the concentration of the aqueous acid is in the range of 2.5 to 10 mM. In one embodiment, the concentration of the aqueous acid is in the range of 5.5 to 10 mM. In one embodiment, the concentration of the aqueous acid is in the range of 6 to 10 mM. In this context, this concentration is understood to include both the undissociated acid and its conjugate base.

[0177] In one embodiment, the cationic lipid or cationically ionizable lipid and the anion of the aqueous acid are present in a molar ratio of about 20:1 to about 1:20. In one embodiment, the cationic lipid or cationically ionizable lipid and the anion of the aqueous acid are present in a molar ratio of about 10:1 to about 1:10. In one embodiment, the cationic lipid or cationically ionizable lipid and the anion of the aqueous acid are present in a molar ratio of about 5:1 to about 1:5. In one embodiment, the cationic lipid or cationically ionizable lipid and the anion of the aqueous acid are present in a molar ratio of about 3:1 to about 1:3. In this context, it is understood that the number of moles of cationic lipid or cationically ionizable lipid includes both the non-ionized lipid and its conjugate acid, and the number of moles of aqueous acid includes both the undissociated acid and its conjugate base.

[0178] When the acid is a strong acid, in one embodiment, the cationic lipid or cationically ionizable lipid and the anion of the aqueous acid are present in a molar ratio of 1:10 to 10:1. In one embodiment, the cationic lipid or cationically ionizable lipid and the anion of the aqueous acid are present in a molar ratio of 1:5 to 5:1. In one embodiment, the cationic lipid or cationically ionizable lipid and the anion of the aqueous acid are present in a molar ratio of 1:3 to 3:1. In one embodiment, the cationic lipid or cationically ionizable lipid and the anion of the aqueous acid are present in a molar ratio of 1:2 to 2:1. In one embodiment, the cationic lipid or cationically ionizable lipid and the anion of the aqueous acid are present in a molar ratio of 1:1.5 to 1.5:1. In one embodiment, the cationic lipid or cationically ionizable lipid and the anion of the aqueous acid are present in a molar ratio of 1:1.2 to 1.2:1. In one embodiment, the cationic lipid or cationically ionizable lipid and the anion of the aqueous acid are present in a 1:1 molar ratio.

[0179] When the acid is a weak acid, in one embodiment, the cationic lipid or cationically ionizable lipid and the anion of the aqueous acid are present in a molar ratio of 1:20 to 5:1. In one embodiment, the cationic lipid or cationically ionizable lipid and the anion of the aqueous acid are present in a molar ratio of 1:10 to 2.5:1. In one embodiment, the cationic lipid or cationically ionizable lipid and the anion of the aqueous acid are present in a molar ratio of 1:6 to 1.5:1. In one embodiment, the cationic lipid or cationically ionizable lipid and the anion of the aqueous acid are present in a molar ratio of 1:4 to 1.25:1. In one embodiment, the cationic lipid or cationically ionizable lipid and the anion of the aqueous acid are present in a molar ratio of 1:3 to 1:1.33. In one embodiment, the cationic lipid or cationically ionizable lipid and the anion of the aqueous acid are present in a molar ratio of 1:2.

[0180] In some cases, the aqueous dispersion comprises a dispersed phase containing lipid particles. In some cases, the lipid particles in the dispersed phase are lipid nanoparticles. In some cases, the lipid particles in the dispersed phase are not liposomes. In some cases, the aqueous dispersion comprises a dispersed phase containing lipid particles 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 containing lipid particles having a size (i.e., diameter) of about 200 nm or less.

[0181] 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," when used in its broadest sense, typically includes any substance typically used to aid in the storage and improve the shelf life of an aqueous dispersion. The storage matrix is ​​typically added to the aqueous dispersion after a filtration (e.g., TFF) / dialysis step.

[0182] 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.

[0183] 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 mixtures of any of these. In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose, and glucose, and mixtures of any of these. Preferably, the cryoprotectant is sucrose.

[0184] 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).

[0185] In one embodiment, the storage matrix is ​​sucrose or trehalose and is present at a concentration of about 1% to about 30% (weight / volume). In one embodiment, the storage matrix is ​​sucrose or trehalose and is present at a concentration of about 2% to about 20% (weight / volume). In one embodiment, the storage matrix is ​​sucrose or trehalose and is present at a concentration of about 5% to about 15% (weight / volume). In one embodiment, the storage matrix is ​​sucrose or trehalose and is present at a concentration of about 8% to about 12% (weight / volume). In one embodiment, the storage matrix is ​​sucrose or trehalose and is present at a concentration of about 10% (weight / volume). In one embodiment, the storage matrix is ​​sucrose or trehalose and is present at a concentration of about 15% to about 25% (weight / volume). In one embodiment, the storage matrix is ​​sucrose or trehalose and is present at a concentration of about 18% to about 22% (weight / volume). In one embodiment, the storage matrix is ​​sucrose or trehalose and is present at a concentration of about 20% (weight / volume).

[0186] In one embodiment, the storage matrix is ​​glucose and is present at a concentration of about 1% to about 15% (weight / volume). In one embodiment, the storage matrix is ​​glucose and is present at a concentration of about 2% to about 10% (weight / volume). In one embodiment, the storage matrix is ​​glucose and is present at a concentration of about 4% to about 8% (weight / volume). In one embodiment, the storage matrix is ​​glucose and is present at a concentration of about 5% (weight / volume). In one embodiment, the storage matrix is ​​glucose and is present at a concentration of about 8% to about 12% (weight / volume). In one embodiment, the storage matrix is ​​glucose and is present at a concentration of about 10% (weight / volume).

[0187] 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.

[0188] Several methods for making dispersions are known in the art, and one skilled in the art would be readily able to select and apply an appropriate method to make the dispersion composition of the present invention.

[0189] In one embodiment, the method comprises: (i) a lipid mixture comprising a cationic lipid or a cationically ionizable lipid; (ii) an aqueous phase containing an aqueous acid and a cryoprotectant; to produce an aqueous dispersion containing anions of the aqueous acid.

[0190] In one embodiment, the method comprises: (a)(i) a lipid mixture comprising a cationic lipid or a cationically ionizable lipid; and (ii) an aqueous phase containing an aqueous acid to produce a first intermediate aqueous dispersion containing anions of an aqueous acid; and (b) adding a cryoprotectant to the first intermediate aqueous dispersion to produce an aqueous dispersion; Includes.

[0191] In one embodiment, the method comprises: (a)(i) a lipid mixture comprising a cationic lipid or a cationically ionizable lipid dissolved in a water-soluble organic solvent; and (ii) aqueous phase to produce a first intermediate acidified aqueous lipid dispersion comprising an anion of an aqueous acid, the lipid mixture and / or the aqueous phase comprises an aqueous acid; (b) performing a dialysis or filtration step on the first intermediate acidified aqueous lipid dispersion at a pH of about 2.5 to about 5.5 to remove the organic solvent and produce a second intermediate aqueous dispersion; and (c) adding a cryoprotectant to the second intermediate aqueous dispersion to produce an aqueous dispersion containing anions of an aqueous acid. The addition of the cryoprotectant typically does not affect the pH of the aqueous dispersion containing anions of an aqueous acid, such that the pH of the aqueous dispersion containing anions of an aqueous acid is essentially the same as the pH of the second intermediate aqueous dispersion, i.e., from about 2.5 to about 5.5.

[0192] In one embodiment, the method comprises: i) mixing a lipid mixture comprising cationic lipids or cationically ionizable lipids dissolved in a water-soluble organic solvent with an aqueous phase to produce a first intermediate acidified aqueous lipid dispersion comprising anions of an aqueous acid; the lipid solution and / or the aqueous phase comprises an aqueous acid; ii) performing a dialysis or filtration step on the first intermediate acidified aqueous lipid dispersion at a pH of about 2.5 to about 5.5, or about 6.5 to about 8.5, to remove the organic solvent and produce a second intermediate aqueous dispersion; and iii) adding a cryoprotectant to the second intermediate aqueous dispersion to produce an aqueous dispersion; The aqueous dispersion is substantially free of inorganic cations, organic solvents, and RNA. The addition of a cryoprotectant typically does not affect the pH of the aqueous dispersion, such that the pH is essentially the same as the pH of the second intermediate aqueous dispersion, i.e., about 2.5 to about 5.5 or about 6.5 to about 8.5.

[0193] In one embodiment, the method comprises: i) preparing a solution of a lipid mixture comprising cationic lipids or cationically ionizable lipids dissolved in a water-soluble and / or non-polar organic solvent (preferably, such solvent is volatile); ii) evaporating the organic solvent at sub-atmospheric pressure to provide the lipid mixture in the form of a lipid film (or layer), optionally a thin film, typically a uniform thin film; iii) adding an aqueous acid to a coating (e.g., a thin film) of the lipid mixture to produce an aqueous dispersion; and iv) diluting the aqueous dispersion with a cryoprotectant, The aqueous dispersion is substantially free of inorganic cations, organic solvents, and RNA. An exemplary suitable solvent for dissolving lipid mixture in step i) can be, for example, a 1:1 mixture of methanol and dichloromethane. This method is referred to herein as "thin film method".

[0194] In one embodiment of the above thin film method, the method further comprises, after step ii), the following step ii'): ii') Reducing the particle size of the aqueous dispersion by standard unit operations such as extrusion, sonication, homogenization, preferably pore size extrusion. Further includes:

[0195] In one embodiment, the method comprises: i) preparing a solution of a lipid mixture comprising cationic lipids or cationically ionizable lipids dissolved in a non-polar, water-immiscible organic solvent, or, if necessary for dissolving the lipids, a mixture of a non-polar, water-immiscible organic solvent and a polar organic solvent; ii) adding an aqueous phase to produce a first intermediate composition comprising a lipid mixture, wherein the lipid mixture and / or aqueous phase solution is acidified; iv) removing the organic solvent at subatmospheric pressure by standard unit operations, such as evaporation or filtration (preferably evaporation), to produce a second intermediate composition comprising a lipid mixture; v) sonicating the second intermediate composition to produce an aqueous dispersion; and vi) diluting the aqueous dispersion with a cryoprotectant, the aqueous dispersion being substantially free of inorganic cations, organic solvents, and RNA; Includes.

[0196] This method is referred to herein as the "emulsification method."

[0197] In one embodiment, the emulsification method comprises, after step v), the following step v'): v') Reducing the particle size of the aqueous dispersion by standard unit operations such as extrusion, sonication, homogenization, preferably pore size extrusion. Further includes:

[0198] In one embodiment, the method further comprises storing the aqueous dispersion at a pH of 2.5 to 5.5. In one embodiment, the method further comprises storing the aqueous dispersion at a pH of 3.0 to 5.5. In one embodiment, the method further comprises storing the aqueous dispersion at a pH of 3.5 to 5.0. In one embodiment, the method further comprises storing the aqueous dispersion at a pH of 3.5 to 4.5.

[0199] In one embodiment, the method of the invention for producing an aqueous dispersion is carried out at about 0° C. to about 25° C., optionally about 4° C. to about 25° C., preferably about 15° C. to about 25° C. In one embodiment, the method of the invention for producing an aqueous dispersion is carried out at about room temperature (e.g., 18-25° C.).

[0200] Mixing Step The mixing step of the method of forming the aqueous dispersion of the present invention comprises mixing (i) an organic phase comprising a lipid mixture comprising cationically ionizable lipids dissolved in a water-soluble organic solvent; and (ii) an aqueous phase comprising anions of an aqueous acid, wherein the aqueous dispersion is substantially free of inorganic cations, organic solvents, and RNA.

[0201] In the method of the present invention, the organic solvent (e.g., a water-soluble organic solvent) can be selected from the list of Class 2 and Class 3 solvents listed in FDA's "Q3C - Tables and List Guidance for Industry", June 2017, Revision 3 (see, for example, https: / / www.fda.gov / media / 71737 / download). When the organic solvent is a water-soluble organic solvent, examples include C1-4 alcohols (e.g., isopropanol or ethanol), ketones (e.g., acetone), or mixtures thereof. When the organic solvent is a non-polar organic solvent, examples include hydrocarbons such as pentane or hexane; chlorinated hydrocarbons such as dichloromethane or chloroform; or mixtures thereof. The organic solvent (e.g., a water-soluble organic solvent) is preferably ethanol or isopropanol.

[0202] In one embodiment, the lipid mixture does not include phosphatidylserine.

[0203] In a preferred embodiment, the acid is a water-soluble organic acid as generally defined above. Examples of suitable organic acids include sulfonic acids, phosphoric acids, phosphonic acids, carboxylic acids, dicarboxylic acids, or hydroxycarboxylic acids (all as defined herein).

[0204] In one embodiment, the water-soluble organic acid is selected from the group consisting of acetic acid, malic acid, succinic acid, and citric acid, or a combination thereof. In one embodiment, the water-soluble organic acid may be selected from the group consisting of acetic acid and malic acid, or a combination thereof.

[0205] In one embodiment, the water-soluble organic acid is acetic acid. In one embodiment, the water-soluble organic acid is malic acid. In one embodiment, the water-soluble organic acid is succinic acid. In one embodiment, the water-soluble organic acid is citric acid.

[0206] 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.

[0207] 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 0.2 to about 3 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 0.5 to about 2 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 1 to about 1.5 mM. In one embodiment, the acid is acetic acid and is present at a concentration of about 1.25 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 0.5 to about 4 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 1 to about 3.5 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 2 to about 3 mM. In one embodiment, the acid is acetic acid and is present at a concentration of about 2.5 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 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.

[0208] 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. In one embodiment, the acid is malic acid and is present at a concentration ranging from about 0.5 to about 4 mM. In one embodiment, the acid is malic acid and is present at a concentration ranging from about 1 to about 3.5 mM. In one embodiment, the acid is malic acid and is present at a concentration ranging from about 2 to about 3 mM. In one embodiment, the acid is malic acid and is present at a concentration of about 2.5 mM. In one embodiment, the acid is malic acid and is present at a concentration ranging from about 1 to about 8 mM. In one embodiment, the acid is malic acid and is present at a concentration ranging from about 1 to about 8 mM. In one embodiment, the acid is malic acid and is present at a concentration ranging from about 2 to about 7 mM. In one embodiment, the acid is malic acid and is present at a concentration ranging from about 4 to about 6 mM. In one embodiment, the acid is malic acid and is present at a concentration ranging from about 4.5 to about 5.5 mM. In one embodiment, the acid is malic acid and is present at a concentration of about 5 mM.

[0209] In one embodiment, the acid is citric acid and the concentration of the acid is greater than 0.3 mM. 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.

[0210] 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.

[0211] In one embodiment, the mixing step is carried out using a T-mixer or a Y-mixer.

[0212] In one embodiment, the flow rate during the mixing step is at least 50 mL / min. The flow rate during the mixing step may be from about 50 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. The volume ratio of organic solvent to aqueous phase may be from about 1:6 to about 6:1, optionally from about 1:2 to about 1:6, and optionally about 1:4.

[0213] The pH of the aqueous dispersion produced according to any of the above methods may be about 2.5 to about 5.5, optionally about 2.5 to about 4.5. The pH of the aqueous dispersion produced according to any of the above methods may be about 2.5 to about 3.5. The pH of the aqueous dispersion produced according to any of the above methods may be about 3.5 to about 4.5. The pH of the aqueous dispersion produced according to any of the above methods may be about 6.5 to about 8.5, optionally 6.8 to 8.5, and optionally about 7.0 to about 8.0.

[0214] Cryoprotectant in the mixing step In one embodiment, the aqueous phase further contains a cryoprotectant as defined and exemplified herein. In this embodiment, the aqueous phase therefore contains an anion of an aqueous acid as defined and exemplified above, as well as a cryoprotectant as defined and exemplified above.

[0215] 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 mixtures of any of these. In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose, and glucose, and mixtures of any of these. Preferably, the cryoprotectant is sucrose.

[0216] If the aqueous phase also contains a cryoprotectant that is a carbohydrate, it is typically present at a concentration of about 1% to about 50% (weight / volume).

[0217] In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose, and lactose and is present at a concentration of about 2% to about 20% (weight / volume). In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose, and lactose and is present at a concentration of about 3% to about 25% (weight / volume). In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose, and lactose and is present at a concentration of about 5% to about 20% (weight / volume). In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose, and lactose and is present at a concentration of about 8% to about 12% (weight / volume). In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose, and lactose and is present at a concentration of about 18% to about 22% (weight / volume).

[0218] In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 1% to about 15% (weight / volume). In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 3% to about 12% (weight / volume). In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 5% to about 10% (weight / volume).

[0219] Further processing steps In one embodiment, the method further comprises subjecting the aqueous dispersion to one or more further processing steps. In one embodiment, the method further comprises subjecting the aqueous dispersion to one or more further dilution or purification steps.

[0220] Dialysis / filtration step In one embodiment, the purification step comprises a dialysis or filtration step, the purpose of which is typically to remove organic solvents. In one embodiment, the dialysis or filtration step is carried out at a pH of about 4.0 to about 5.0. In one embodiment, the dialysis or filtration step comprises tangential flow filtration.

[0221] In one embodiment, the dialysis or filtration step comprises the steps of: (a) an amino acid or a mixture thereof, preferably: (i) an acidic amino acid preferably selected from the group consisting of aspartic acid, glutamic acid, 3-hydroxyglutamic acid, and alpha-aminoadipic acid, and mixtures thereof; (ii) a basic amino acid, preferably selected from the group consisting of arginine, histidine, and lysine, and mixtures thereof; or a mixture of (i) and (ii), or a mixture of (i) and (ii) and either or both of a neutral amino acid; (b) an organic acid preferably selected from the group consisting of acetic acid, malic acid, succinic acid, citric acid, and methylmalonic acid, and mixtures thereof; (c) a cryoprotectant, optionally a carbohydrate, e.g., a monosaccharide or disaccharide, preferably selected from the group consisting of sucrose, trehalose, lactose, and glucose, and mixtures of any of these; or a mixture of any of these The present invention uses a composition comprising a compound selected from any one of the following:

[0222] In one embodiment, the dialysis or filtration step is carried out using one or more weak water-soluble organic acids. In one embodiment, the weak water-soluble organic acids are selected from the group consisting of acetic acid, malic acid, maleic acid, and succinic acid. In one embodiment, the weak water-soluble organic acid is acetic acid.

[0223] In one embodiment, the dialysis or filtration step is carried out using an amino acid. In one embodiment, the composition used for dialysis or filtration is an acidic amino acid, as defined and exemplified above, or a mixture thereof. In one embodiment, the composition used for dialysis or filtration is selected from the group consisting of aspartic acid, glutamic acid, 3-hydroxyglutamic acid, and alpha-aminoadipic acid, and mixtures thereof.

[0224] In one embodiment, the composition used for dialysis or filtration is a mixture of amino acids as defined and exemplified above, or a mixture thereof, and a water-soluble organic acid as defined and exemplified above, or a mixture thereof.

[0225] 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. This preferably occurs after the dialysis or filtration step, although alternatively, it may occur immediately after the mixing step to form the aqueous dispersion.

[0226] The storage matrix used in this step may be any of the storage matrices defined and exemplified above. In one embodiment, the storage matrix includes a cryoprotectant such that the dilution step includes the addition of a cryoprotectant. The cryoprotectant dilutes the aqueous dispersion to protect the pre-LNP from freezing damage. The cryoprotectant is not particularly limited as 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 thereof. In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, glycerol, trehalose, lactose, glucose, and mannitol. In a preferred embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose, and glucose, and mixtures thereof. In a more preferred embodiment, the cryoprotectant is selected from the group consisting of sucrose and trehalose, and mixtures thereof. In one embodiment, the cryoprotectant is sucrose.

[0227] In one embodiment, the storage matrix 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; or a mixture of any of these The compound further includes a compound selected from:

[0228] 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.

[0229] Thus, the composition of lipid particles contained in the dispersed phase of the aqueous dispersion prior to the addition of peptide-conjugated lipids may comprise a cationic lipid or cationically ionizable lipid described herein, a neutral or zwitterionic phospholipid described herein, a steroid described herein, and optionally a grafted lipid described herein in a molar ratio of 20-70 mol%, 5-15 mol%, 20-60 mol%, and optionally 0.5-10 mol%, respectively; preferably, a molar ratio of 40-60 mol%, 8-12 mol%, 30-50 mol%, and optionally, 1.0-5 mol%, respectively. After the addition of peptide-conjugated lipids, the peptide-conjugated lipids may comprise 0.05-1.0 mol%, optionally 0.1-0.5 mol%, and preferably 0.1-0.3 mol%, of the lipid particles contained in the dispersed phase of the aqueous dispersion, with a corresponding reduction in the molar percentage of steroid.

[0230] In one embodiment, the purification is carried out using an aqueous phase that is essentially free of buffers, hi one embodiment, the purification is carried out using an aqueous phase that is essentially free of buffers other than amino acids.

[0231] Further optional steps 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.

[0232] In one embodiment, the purification comprises sterile filtration of the aqueous dispersion. Typically, the sterile filtration uses a 0.22 μm filter. In one embodiment, the filter is a polyethersulfone (PES) filter.

[0233] 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. In one embodiment, the method further comprises drying the aqueous dispersion. In one embodiment, the drying step is freeze-drying or spray-drying.

[0234] In one embodiment, the aqueous dispersion is stable at 4°C for at least 3 months. In one embodiment, the aqueous dispersion is stable at -20°C for at least 6 months. Thus, in one embodiment, an aqueous dispersion is provided that is stable at 4°C for at least 3 months. In one embodiment, an aqueous dispersion is provided that is stable at -20°C for at least 6 months. In this context, "stable" refers to the size (Z) of the particles in the aqueous dispersion after storage at the specified temperature for the specified period. average ) and / or size distribution and / or PDI of the particles immediately after preparation and before storage (Z average ) and / or size distribution and / or PDI. For example, the size (Z average ) and / or size distribution and / or PDI do not change by more than 20%, optionally by more than 10%, preferably by more than 5% during storage as described.

[0235] 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".

[0236] 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 cancer antigens.

[0237] 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.

[0238] 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.

[0239] 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.

[0240] In some embodiments, the lipid nanoparticles described herein may be about 50 nm to about 1000 nm, about 50 nm to about 800 nm, about 50 nm to about 700 nm, about 50 nm to about 600 nm, about 50 nm to about 500 nm, about 50 nm to about 450 nm, about 50 nm to about 400 nm, about 50 nm to about 350 nm, about 50 nm to about 300 nm, about 50 nm ~ about 250 nm, about 50 nm - about 200 nm, about 100 nm - about 1000 nm, about 100 nm - about 800 nm, about 100 nm - about 700 nm, about 100 nm - about 600 nm, about 100nm to about 500nm, about 100nm to about 450nm, about 100nm to about 400nm, about 100nm to about 350nm, about 100nm to about 300nm, about 100nm to about 250nm , about 100nm to about 200nm, about 150nm to about 1000nm, about 150nm to about 800nm, about 150nm to about 700nm, about 150nm to about 600nm, about 150nm to about 5 00nm, about 150nm to about 450nm, about 150nm to about 400nm, about 150nm to about 350nm, about 150nm to about 300nm, about 150nm to about 250nm, about 150nm In some embodiments, the lipid nanoparticles described herein have an average diameter ranging from about 60 nm to about 100 nm.

[0241] In one embodiment, the nucleic acid-lipid particles are stable for at least 3 months at 4° C. In one embodiment, the nucleic acid-lipid particles are stable for at least 6 months at −20° C.

[0242] 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. 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.

[0243] 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.

[0244] Thus, in one embodiment, nucleic acid-lipid particles are provided that are stable for at least 3 months at 4° C. In one embodiment, nucleic acid-lipid particles are provided that are stable for at least 6 months at −20° C.

[0245] In one embodiment, the integrity of the nucleic acid (preferably RNA) in the nucleic acid-lipid particles is not reduced by more than 20% after storage of the nucleic acid-lipid particles for at least 3 months at 4° C. In one embodiment, the integrity of the nucleic acid (preferably RNA) in the nucleic acid-lipid particles is not reduced by more than 20% after storage of the nucleic acid-lipid particles for at least 6 months at −20° C.

[0246] Thus, in one embodiment, nucleic acid-lipid particles (preferably RNA-lipid particles) are provided, in which the integrity of the nucleic acid (preferably RNA) in the nucleic acid-lipid particles is not reduced by more than 20% after storage of the nucleic acid-lipid particles for at least 3 months at 4° C. In one embodiment, nucleic acid-lipid particles (preferably RNA-lipid particles) are provided, in which the integrity of the nucleic acid (preferably RNA) in the nucleic acid-lipid particles is not reduced by more than 20% after storage of the nucleic acid-lipid particles for at least 6 months at −20° C.

[0247] In one embodiment, the nucleic acid-lipid particles are capable of inducing a comparable or higher (e.g., 0.5-fold, 2-fold, 5-fold, 100-fold) antibody and / or T cell response following administration in vivo compared to nucleic acid-lipid particles produced using standard processes.

[0248] Thus, in one embodiment, nucleic acid-lipid particles (preferably RNA-lipid particles) are provided that are capable of inducing equivalent or higher (e.g., 0.5-fold, 2-fold, 5-fold, 100-fold) antibody and / or T cell responses following in vivo administration compared to nucleic acid-lipid particles produced using standard processes.

[0249] 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 the aqueous dispersion (typically containing pre-LNP) 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.

[0250] 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 aqueous dispersion with an aqueous solution containing nucleic acids to produce nucleic acid-lipid particles; Includes.

[0251] In one embodiment, the method of forming an RNA-lipid particle comprises: i) preparing an aqueous dispersion as defined herein according to any of the methods defined herein; and ii) mixing the aqueous dispersion with an aqueous solution containing RNA to produce RNA-lipid particles; Includes.

[0252] In one embodiment, the method of forming nucleic acid-lipid particles comprises: i) mixing a lipid mixture comprising cationic lipids or cationically ionizable lipids dissolved in a water-soluble organic solvent with an aqueous phase to produce an intermediate acidified aqueous lipid dispersion; the lipid solution and / or the aqueous phase is acidified; ii) performing a dialysis or filtration step on the intermediate dispersion at a pH of about 2.5 to about 5.5 (preferably about 2.5 to about 4.5), or about 6.5 to about 8.5 (preferably about 7.5 or about 8.5) to remove the organic solvent and produce an aqueous dispersion; the aqueous dispersion is substantially free of acetate buffer, citrate buffer, organic solvent, and RNA; the aqueous dispersion comprising a cryoprotectant; iii) mixing the aqueous dispersion with an aqueous solution containing nucleic acids to produce nucleic acid-lipid particles; Includes.

[0253] Mixing Step The mixing step of this aspect of the invention involves mixing an aqueous dispersion as defined herein (typically containing pre-LNPs) with an aqueous solution containing a nucleic acid as defined herein to produce nucleic acid-lipid particles.

[0254] In one embodiment, the aqueous dispersion is provided at a neutral pH, and either the aqueous dispersion or the aqueous solution is acidified.

[0255] In one embodiment, the aqueous dispersion is provided at an acidic pH, and neither the aqueous dispersion nor the aqueous solution is acidified.

[0256] In one embodiment, the volume ratio of the aqueous dispersion to the aqueous solution containing nucleic acid is 1.5:1 to 1:1.5. In one embodiment, the volume ratio of the aqueous dispersion to the aqueous solution containing nucleic acid is 1.2:1 to 1:1.2. In one embodiment, the volume ratio of the aqueous dispersion to the aqueous solution containing nucleic acid is 1.1:1 to 1:1.1. In one embodiment, the volume ratio of the aqueous dispersion to the aqueous solution containing nucleic acid is 1.05:1 to 1:1.05. In one embodiment, the volume ratio of the aqueous dispersion to the aqueous solution containing nucleic acid is 1:1.

[0257] In a preferred embodiment, the acid is a water-soluble organic acid as generally defined above. Examples of suitable organic acids include sulfonic acids, phosphoric acids, phosphonic acids, carboxylic acids, dicarboxylic acids, or hydroxycarboxylic acids (all as defined above).

[0258] In one embodiment, the water-soluble organic acid is selected from the group consisting of acetic acid, malic acid, succinic acid, and citric acid, or a combination thereof. In one embodiment, the water-soluble organic acid may be selected from the group consisting of acetic acid and malic acid, or a combination thereof.

[0259] In one embodiment, the water-soluble organic acid is acetic acid. In one embodiment, the water-soluble organic acid is malic acid. In one embodiment, the water-soluble organic acid is succinic acid. In one embodiment, the water-soluble organic acid is citric acid.

[0260] 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.

[0261] 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 0.5 to about 4 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 1 to about 3.5 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 2 to about 3 mM. In one embodiment, the acid is acetic acid and is present at a concentration of about 2.5 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 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 is present at a concentration ranging from about 5.5 to about 9 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 6 to about 8.5 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 7 to about 8 mM. In one embodiment, the acid is acetic acid and is present at a concentration of about 7.5 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 6 to about 14 mM. In one embodiment, the acid is acetic acid and is present at a concentration ranging from about 7 to about 13 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 9 to about 11 mM. In one embodiment, the acid is acetic acid and is present at a concentration of about 10 mM.

[0262] 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. In one embodiment, the acid is malic acid and is present at a concentration ranging from about 0.5 to about 4 mM. In one embodiment, the acid is malic acid and is present at a concentration ranging from about 1 to about 3.5 mM. In one embodiment, the acid is malic acid and is present at a concentration ranging from about 2 to about 3 mM. In one embodiment, the acid is malic acid and is present at a concentration of about 2.5 mM.

[0263] In one embodiment, the acid is citric acid and the concentration of the acid is greater than 0.3 mM. 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.

[0264] 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.

[0265] In a preferred embodiment of any of the above methods for forming nucleic acid-lipid particles, the aqueous dispersion comprises a cryoprotectant as defined and exemplified herein. 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 or trehalose.

[0266] When the aqueous dispersion includes a cryoprotectant that is a carbohydrate, it is typically present at a concentration of about 1% to about 30% (weight / volume).

[0267] In one embodiment, the cryoprotectant is sucrose or trehalose and is present at a concentration of about 1% to about 30% (wt / vol), optionally about 3% to about 25% (wt / vol), and preferably about 5% to about 20% (wt / vol). In one embodiment, the cryoprotectant is sucrose or trehalose and is present at a concentration of about 8% to about 12% (wt / vol), for example, about 10% (wt / vol). In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 1% to about 15% (wt / vol), optionally about 3% to about 12% (wt / vol), and preferably about 5% to about 10% (wt / vol).

[0268] In one embodiment, the cryoprotectant is sucrose or trehalose and is present at a concentration of about 1% to about 30% (wt / vol), optionally about 10% to about 25% (wt / vol), and preferably about 15% to about 25% (wt / vol). In one embodiment, the cryoprotectant is sucrose or trehalose and is present at a concentration of about 18% to about 22% (wt / vol), e.g., about 20% (wt / vol). In one embodiment, the cryoprotectant is glucose and is present at a concentration of about 1% to about 15% (wt / vol), optionally about 5% to about 15% (wt / vol), and preferably about 8% to about 12% (wt / vol). In such embodiments, the nucleic acid-lipid particles are preferably not subjected to further dilution and / or cryoprotectant addition steps. For example, the nucleic acid-lipid particles may not require any further processing steps.

[0269] In one embodiment, the method of the invention for forming nucleic acid-lipid particles is carried out at about 0° C. to about 25° C., optionally about 4° C. to about 25° C., and preferably about 15° C. to about 25° C. In one embodiment, the method of the invention for producing aqueous dispersions is carried out at about room temperature (e.g., 18-25° C.).

[0270] In one embodiment, the aqueous solution containing nucleic acids also contains one or more buffers. In one embodiment, the buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) optionally in combination with ethylenediaminetetraacetic acid (EDTA) or an acceptable salt thereof. The aqueous solution containing nucleic acids may have a pH of about 6.5 to about 8.5, optionally about 6.8 to about 7.5. The aqueous solution containing nucleic acids may have a pH of about 7.0.

[0271] Optional 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.

[0272] In one embodiment, the method further includes adding a peptide-conjugated lipid (described in detail herein) to the nucleic acid-lipid particle. In some cases, the peptide-conjugated lipid may replace (i.e., replace) the corresponding portion of the steroid (e.g., cholesterol) in the nucleic acid-lipid particle. The composition of the nucleic acid-lipid particle prior to the addition of the peptide-conjugated lipid may contain the cationic lipid or cationically ionizable lipid described herein, the neutral or zwitterionic phospholipid described herein, the steroid described herein, and optionally the grafted lipid described herein in the following molar ratios: 20-70 mol%, 5-15 mol%, 20-60 mol%, and optionally 0.5-10 mol%; or optionally, 40-60 mol%, 8-12 mol%, 30-50 mol%, and optionally 1.0-5 mol%, respectively. After addition of the peptide-conjugated lipid, the peptide-conjugated lipid may comprise 0.05-1.0 mol %, optionally 0.1-0.5 mol %, preferably 0.1-0.3 mol % of lipid in the nucleic acid-lipid particle, with a corresponding reduction in the mol % of steroid.

[0273] 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.

[0274] 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 cryoprotectant. In a preferred embodiment, the method does not comprise subjecting the nucleic acid-lipid particles to any of the following: (i) a dialysis or filtration step (e.g., a TFF step), (ii) a dilution step, and (iii) a dilution step comprising the addition of a cryoprotectant. In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, glycerol, trehalose, lactose, glucose, and mannitol. In one embodiment, the cryoprotectant is sucrose.

[0275] In one embodiment, the purification step is carried out using an aqueous phase that is essentially free of buffer.

[0276] In one embodiment, the method further comprises a step of sterile filtration of the nucleic acid-lipid particles. Typically, the sterile filtration uses a 0.22 μm filter. In one embodiment, the filter is a polyethersulfone (PES) filter.

[0277] In one embodiment, the method further comprises drying the nucleic acid-lipid particles. In one embodiment, the drying step is freeze-drying. In one embodiment, the drying step is spray-drying.

[0278] In one embodiment, one or more purification steps of the lipid-nucleic acid particles does not include a tangential flow filtration step.

[0279] In one embodiment, the nucleic acid-lipid particles are not subjected to any further purification steps.

[0280] In one embodiment, the method further comprises diluting the lipid particles with a storage matrix. In one embodiment, the storage matrix comprises one or more buffers. In one embodiment, the buffer or mixture thereof has a pH of 4.5 to 8.5. In one embodiment, the buffer is selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris-(hydroxymethyl)aminomethane (Tris), histidine, triethanolamine, and any mixture thereof. In one embodiment, the buffer is a mixture of HEPES and Tris. The preferred molar ratio of HEPES:Tris is 100:1 to 1:100, preferably 10:1 to 1:10.

[0281] Lipids and Amphiphiles The compositions of the present invention also include lipid mixtures. 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.

[0282] 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.

[0283] 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".

[0284] 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.

[0285] 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.

[0286] 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.

[0287] 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.

[0288] 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.

[0289] Cationic Lipids and Cationically Ionizable Lipids The aqueous dispersions and lipid particles of the present invention also contain cationic lipids or cationically ionizable lipids, or mixtures of any of these. In one embodiment, the aqueous dispersions and lipid particles of the present invention contain cationically ionizable lipids, and preferably do not contain cationic lipids.

[0290] 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.The cationic lipid that carries a constitutively charged cationic moiety is typically a quaternary ammonium salt (as defined above) or an organic base, such as a salt of a nitrogenous base.Typically, such an organic base is a strong base (i.e., a base that is completely protonated when dissolved in a solvent, such as, but not limited to, an aqueous solvent, so that the concentration of unprotonated species is so low that it cannot be measured).

[0291] In one embodiment, the cationic lipid is a monovalent cationic lipid.

[0292] In one embodiment, the cationic lipid contains a charged polar moiety selected from the group consisting of guanidinium, ammonium, imidazolium, pyridinium, amidinium, and piperazinium.

[0293] 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).

[0294] The structures of DODMA and DODAP are shown below. [ka]

[0295] The structures of DOTMA, DOTAP, and their analogs are shown below. [ka]

[0296] The structures of DOTAP and further suitable homologues are shown below. [ka]

[0297] The structures of DOTMA, DORIE, and further suitable homologs are shown below. [ka]

[0298] Further suitable cationic lipids are described in Sun and Lu, Pharmaceutical Research, 2023, https: / / doi.org / 10.1007 / s11095-022-03460-2.

[0299] In one embodiment, lipid is cationically ionizable lipid.As used herein, " cationically ionizable lipid " refers to the lipid or lipid-like substance that has net positive charge or neutral depending on whether it is protonated or deprotonated, that is, the lipid that is not permanently cationic.Therefore, depending on the pH of the composition that cationically ionizable lipid is dissolved in, cationically ionizable lipid has net positive charge or neutral.

[0300] In some embodiments, the cationically ionizable lipid comprises a head group that includes at least one nitrogen atom (N) that can be protonated, preferably under physiological or slightly acidic conditions.

[0301] In one embodiment, the 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 30is 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 2 One 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 2is 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(R 5 )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.

[0302] In some embodiments of Formula (TL-I), L 1 and L 2 are each independently -(CH2) 6-10 -It is.

[0303] 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—.

[0304] 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.

[0305] In some embodiments of Formula (TL-I), T 1 and T 2 are each independently optionally substituted C3-C 20 alkyl.

[0306] In some embodiments of Formula (TL-I), T 1 and T 2 are each independently [ka] is selected from.

[0307] 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.

[0308] 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.

[0309] In some embodiments of Formula (TL-I), G is —OH.

[0310] In some embodiments of Formula (TL-I), G is [ka] is selected from.

[0311] In some embodiments of Formula (TL-I), -L 3 -G is [ka] is selected from.

[0312] In some embodiments of Formula (TL-I), the compound has the formula (TL-IIa): [ka] TL-IIa or a pharmaceutically acceptable salt thereof.

[0313] In some embodiments of Formula (TL-I), the compound has the formula (TL-IIc): [ka] TL-IIc or a pharmaceutically acceptable salt thereof.

[0314] In some embodiments of Formula (TL-I), the compound has the formula (TL-IIIb): [ka] (TL-IIIb) or a pharmaceutically acceptable salt thereof.

[0315] In some embodiments of Formula (TL-I), the compound has the formula (TL-IIIe): [ka] TL-IIIe or a pharmaceutically acceptable salt thereof.

[0316] 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.

[0317] 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.

[0318] In some embodiments of formula (TL-I), the compound is [ka] or a pharmaceutically acceptable salt thereof.

[0319] The thiolipid compounds of formula (TL-I) may be prepared according to PCT / EP2023 / 071270, the contents of which are incorporated herein by reference.

[0320] In one embodiment, the cationic lipid or cationically ionizable lipid is [(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); Palmitoyl-oleoyl-nor-arginine (PONA); Guanidino-di[(heptadecyl)methyl]carboxylic acid (GUADACA); 4-methylpyridinium-di(heptadecyl)methylcarboxylic acid (MPDACA); 1,2-dioleoyl-3 trimethylammonium propane (DOTAP); 1,2-dioleoyl-3-dimethylammonium propane (DODAP); 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA); and any mixtures thereof is selected from the group consisting of:

[0321] 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); and any mixtures thereof is selected from the group consisting of:

[0322] In one embodiment, 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); structure [ka] a compound having [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); 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 is selected from the group consisting of:

[0323] In one embodiment, the cationic lipid is palmitoyl-oleoyl-nor-arginine (PONA). In one embodiment, the cationic lipid is 4-methylpyridinium-di(heptadecyl)-methylcarboxylic acid (MPDACA). In one embodiment, the cationic lipid is 1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP). In one embodiment, the cationic lipid is 1,2-dioleoyl-3-dimethylammonium propane (DODAP).

[0324] 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 has the structure: [ka] In one embodiment, the cationically ionizable lipid is BHD-C2C2-PipZ. In one embodiment, the cationically ionizable lipid is BODD-C2C2-1Me-Pyr.

[0325] In some embodiments, the cationically ionizable lipids are generally and specifically selected from those described in WO 2018 / 087753.

[0326] In some embodiments, the cationically ionizable lipid is [ka] is selected from the group consisting of:

[0327] 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).

[0328] In one embodiment, the cationic lipids or 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 cationic lipids or 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 cationic lipids or 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 and the nucleic acid-lipid particles.

[0329] Additional fats The lipid mixture in the aqueous dispersions and 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.

[0330] neutral lipid The composition may further comprise a neutral lipid.The neutral lipid is preferably a neutral phospholipid.In one embodiment, the phospholipid may be zwitterionic (i.e., it carries both positive and negative charges, and therefore is neutral in the pH range around neutral).

[0331] 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. mand 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.

[0332] 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.

[0333] In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DOPE, and SM, or a mixture of any of these.

[0334] Thus, in some embodiments, the lipid nanoparticle compositions described herein comprise a cationic lipid or cationically ionizable lipid (as defined herein) and a phospholipid, hi some embodiments, the lipid nanoparticle compositions described herein comprise a cationic lipid or 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.

[0335] 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.

[0336] 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.

[0337] 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.

[0338] 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.

[0339] In each of the above embodiments, the term "lipid mixture" in this context applies to the lipid mixture components of both the aqueous dispersion and the nucleic acid-lipid particles.

[0340] steroid The lipid nanoparticle compositions of the present invention also include a steroid. In one embodiment, the steroid comprises a sterol. In one embodiment, the steroid is cholesterol.

[0341] Thus, in some embodiments, the lipid nanoparticle compositions described herein comprise a cationically ionizable lipid (as defined herein) and cholesterol.

[0342] 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.

[0343] 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.

[0344] In each of the above embodiments, the term "lipid mixture" in this context applies to the lipid mixture components of both the aqueous dispersion and the nucleic acid-lipid particles.

[0345] Anionic amphiphiles In one embodiment, the composition of the present disclosure also includes a negatively charged amphiphile ("anionic amphiphile"). As used herein, the term "amphiphile" is generally defined as a molecule having both hydrophilic and hydrophobic portions (as defined above). The negative charge is located in the hydrophilic portion of the amphiphile. The negatively charged amphiphile can have one negatively charged group or multiple (e.g., 2, 3, 4, or 5) negatively charged groups. Anionic amphiphiles with a single negatively charged group are preferred.

[0346] In the present invention, anionic amphiphiles have a pH-sensitive charge. In this context, "pH-sensitive charge" means that the amphiphile may carry a negative charge at alkaline or neutral pH, but be neutral at acidic pH. In certain embodiments, such amphiphiles combine a pH-sensitive charge with a constitutive charge, such as in organic phosphates, which carry two negative charges at alkaline or neutral pH, but only one negative charge at acidic pH. Amphiphiles carrying a constitutively charged anionic moiety are typically salts of weak organic acids (i.e., organic acids that, when dissolved in a solvent, remain largely undissociated, with only a partial transfer of protons to solvent molecules).

[0347] In one embodiment, the anionic amphiphile has a charged polar moiety selected from the group consisting of carboxylate and phosphate.

[0348] In one embodiment, the negatively charged amphiphile is a carboxylic acid or carboxylate (as defined above in its broadest or preferred aspect).

[0349] In one embodiment, the negatively charged amphiphile has a pH-sensitive charge and the pH-sensitive anionic moiety is a carboxylic acid. One or more charged groups may be present in the amphiphile, and in a preferred embodiment, a single charged moiety is present in the amphiphile.

[0350] The polar region of the negatively charged amphiphile may further comprise an uncharged polar moiety. Preferred uncharged polar moieties are hydroxyl or amide groups, and one or more uncharged polar moieties may be present in the negatively charged amphiphile.

[0351] In one embodiment, the negatively charged amphiphile is a hemiester of a dicarboxylic acid and a diacylglycerol. The hydrocarbyl portion of the acyl portion of the diacylglycerol moiety is as defined above, but is preferably an alkyl group (as defined above) having 6 to 40, preferably 14 to 22, carbon atoms, or an alkenyl group (as defined above) having 6 to 40, preferably 14 to 22, carbon atoms. The acyl portions of the diacylglycerol moiety may be the same or different. In one embodiment, the acyl portion is a saturated fatty acid moiety, preferably selected from the group consisting of behenoyl, arachinoyl, stearoyl, palmitoyl, and myristoyl moieties. In one embodiment, the acyl portion is an unsaturated fatty acid moiety, preferably selected from the group consisting of oleoyl, linoyl, and lineoyl moieties. The dicarboxylic acid portion is as defined above, and preferably has 2 to 8 carbon atoms, more preferably 2 to 6, and even more preferably 2 to 4 carbon atoms. Examples of dicarboxylic acid moieties include oxalate, malonate, succinate, glutarate, adipate, pimelate, and suberate. Typical examples of such negatively charged amphiphiles include dimyristoyl glyceryl hemisuccinate (DMGS), dipalmitoyl glyceryl hemisuccinate (DPGS), palmitoyl stearoyl glyceryl hemisuccinate (PSGS), distearoyl glyceryl hemisuccinate (DSGS), dioleoyl glycerol hemisuccinate (DOGS), palmitoyl oleoyl glyceryl hemisuccinate (POGS), and homologs of any of the above in which the dicarboxylic acid moiety is oxalate, malonate, succinate, glutarate, adipate, pimelate, or suberate. Dimyristoyl glyceryl hemisuccinate (DMGS) or dioleoyl glyceryl hemisuccinate (DOGS) are preferred.

[0352] In one embodiment, the negatively charged amphiphile is a hemiester of a dicarboxylic acid and a steroid. The dicarboxylic acid moiety is as defined and exemplified above and typically contains a total of 2 to 6, preferably 3 to 5, and most preferably 4 carbon atoms (including the acyl carbon). The ester group may preferentially esterify the 3' hydroxyl group on the steroid molecule.

[0353] In one embodiment, the negatively charged amphiphile is a hemiester of a dicarboxylic acid and cholesterol. The dicarboxylic acid moiety is as defined above and preferably has 2 to 6 carbon atoms, more preferably 3 to 5 carbon atoms, and even more preferably 4 carbon atoms. Examples of the dicarboxylic acid moiety include oxalate, malonate, succinate, glutarate, and adipate, with succinate being preferred. Typical examples of such negatively charged amphiphiles include cholesteryl hemisuccinate and cholesteryl hemiadipate, with cholesterol hemisuccinate being preferred.

[0354] In one embodiment, the negatively charged amphiphile is a monoester or diester of phosphoric acid, with one of the hydroxyl groups of the phosphoric acid esterified with diacylglycerol. The hydrocarbyl moieties of the acyl moieties of the diacylglycerol moiety are as defined above, but are preferably alkyl groups (as defined above) having 6 to 40, preferably 14 to 22, carbon atoms, or alkenyl groups (as defined above) having 6 to 40, preferably 14 to 22, carbon atoms. The acyl moieties of the diacylglycerol moiety can be the same or different. In one embodiment, the acyl moieties are saturated fatty acid moieties, preferably selected from the group consisting of behenoyl, arachinoyl, stearoyl, palmitoyl, and myristoyl moieties. In one embodiment, the acyl moieties are unsaturated fatty acid moieties, preferably selected from the group consisting of oleoyl, linoyl, and lineoyl moieties.

[0355] In one embodiment, the anionic amphiphile is a carboxylic acid preferably selected from the group consisting of hexanoic acid, octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, eicosanoic acid, tricosanoic acid, 2-hydroxytetradecanoic acid, 2-methyloctadecanoic acid, 2-bromohexadecanoic acid, 2-propylpentanoic acid, 2-butyloctanoic acid, 2-hexyldecanoic acid, 9-hydroxystearic acid, trans-2-decenoic acid, (9Z)-9-hexadecenoic acid, linolic acid, linolenic acid, oleic acid, elaidic acid, arachidonic acid, cyclododecanoic acid, adamantylacetic acid, dicyclohexylacetic acid, trans-4-pentylcyclohexane-carboxylic acid, 4-(decyloxy)benzoic acid, 4-octylbenzoic acid, cholic acid, and lithocholic acid, and mixtures of any thereof.

[0356] In one embodiment, the anionic amphiphile is a hemiester of a dicarboxylic acid with a diacylglycerol, preferably selected from the group consisting of dimyristoyl hemisuccinate and dioleoyl hemisuccinate, and mixtures of any of these.

[0357] In one embodiment, the anionic amphiphile is a hemiester of a dicarboxylic acid with cholesterol, preferably selected from the group consisting of cholesterol hemisuccinate, cholesterol hemimalonate, and cholesterol hemiadipate, and mixtures of any of these.

[0358] In one embodiment, the anionic amphiphile is an organic sulfate or sulfonate, preferably selected from the group consisting of sodium lauryl sulfate, sodium hexadecanesulfonate, and sodium dodecylbenzenesulfonate, and mixtures of any of these.

[0359] In one embodiment, the anionic amphiphile is an organic phosphonate, preferably selected from the group consisting of octadecylphosphonic acid and dodecylphosphonic acid, and mixtures of any of these.

[0360] In one embodiment, the anionic amphiphile is an anionic phospholipid, preferably selected from the group consisting of phosphatidylserine, phosphatidylglycerol and phosphatidic acid, and mixtures of any of these.

[0361] In one embodiment, the anionic amphiphile is Carboxylic acids; Hemiesters of dicarboxylic acids with cholesterol; Hemiesters of dicarboxylic acids with diacylglycerol; phosphate esters with diacylglycerol; and any mixtures thereof is selected from the group consisting of:

[0362] In one embodiment, the anionic amphiphile is cholesterol hemisuccinate (CHEMS); Dimyristoyl hemisuccinate (DMGS); Dioleoylglycerol hemisuccinate (DOGS); and any mixtures thereof is selected from the group consisting of:

[0363] In one embodiment, the anionic amphiphile is CHEMS. In one embodiment, the anionic amphiphile is DMGS. In one embodiment, the anionic amphiphile is DOGS.

[0364] In one embodiment, the anionic amphiphile is present in an amount of 0-50 mol % of the total lipids present in the lipid mixture. In one embodiment, the anionic amphiphile is present in an amount of 5-45 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 and the nucleic acid-lipid particles.

[0365] 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).

[0366] 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.

[0367] 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.

[0368] 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.

[0369] In some embodiments, the PEG-conjugated lipid (pegylated lipid) is a lipid having a structure of the following general formula, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof: [ka] (In the formula, R 12 and R 13are each independently a linear or branched alkyl or alkenyl chain containing 10 to 30 carbon atoms, the alkyl / alkenyl chain optionally being interrupted by one or more ester bonds; and w has an average value in the range of 30 to 60.

[0370] In some embodiments of this formula, R 12 and R 13 are each independently a linear alkyl chain containing 10 to 18 carbon atoms, preferably 12 to 16 carbon atoms.

[0371] In some embodiments of this formula, R 12 and R 13 are identical. In some embodiments, R 12 and R 13 Each of R is a linear alkyl chain containing 12 carbon atoms. 12 and R 13 Each of R is a linear alkyl chain containing 14 carbon atoms. 12 and R 13 Each of the is a linear alkyl chain containing 16 carbon atoms.

[0372] In some embodiments of this formula, R 12 and R 13 In some embodiments, R 12 and R 13 is a linear alkyl chain containing 12 carbon atoms, and R 12 and R 13 The other is a linear alkyl chain containing 14 carbon atoms.

[0373] In some embodiments of this formula, w has an average value in the range of 40 to 50, for example, an average value of 45.

[0374] In some embodiments of this formula, w is in a range such that the PEG portion of the pegylated lipid has an average molecular weight of about 400 to about 6000 g / mol, e.g., about 1000 to about 5000 g / mol, about 1500 to about 4000 g / mol, or about 2000 to about 3000 g / mol. 12 and R 13 Each of is a linear alkyl chain containing 14 carbon atoms, and w has an average value of 45.

[0375] Various PEG-conjugated lipids are known in the art, including, but not limited to, pegylated diacylglycerols (PEG-DAGs), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), pegylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerols (PEG-S-DAGs), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), pegylated ceramides (PEG-cer), or PEG dialkoxypropylcarbamate. PEGylated lipids include PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8, PEG-9, PEG-10, PEG-11, PEG-12, PEG-13, PEG-14, PEG-15, PEG-16, PEG-17, PEG-18, PEG-19, PEG-20, PEG-21, PEG-22, PEG-23, PEG-24, PEG-25, PEG-26, PEG-27, PEG-28, PEG-29, PEG-30, PEG-31, PEG-32, PEG-33, PEG-34, PEG-35, PEG-36, PEG-37, PEG-38, PEG-39, PEG-40, PEG-41, PEG-42, PEG-43, PEG-44, PEG-45, PEG-46, PEG-47, PEG-48, PEG-49, PEG-49, PEG-49, PEG-49, PEG-49, PEG-41, PEG-42, PEG-44, PEG-45, PEG-46, PEG-47, PEG-48 ...

[0376] In some embodiments, the PEG-conjugated lipid (pegylated lipid) is or comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide. In some embodiments, the pegylated lipid has the following structure: [ka] It has.

[0377] 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.

[0378] 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, preferably 10 to 70 sarcosine units, preferably 15 to 50 sarcosine units, more preferably 20 to 30 sarcosine units, and even more preferably 21 to 25 sarcosine units.

[0379] In one embodiment, the grafted lipid has a carbonyl terminus (C 6-30 In one embodiment, the grafted lipid comprises a polysarcosine moiety (as defined and exemplified above) linked to a (C alkyl)amine (as defined and exemplified above), the amino terminus of which may be linked to an acetyl group.12-20 In one embodiment, the grafted lipid comprises a polysarcosine moiety (as defined and exemplified above) linked to a (C alkyl)amine (as defined and exemplified above), the amino terminus of which may be linked to an acetyl group. 14 The polysarcosine moiety (as defined and exemplified above) is linked to a (alkyl)amine (as defined and exemplified above), the amino terminus of which may be linked to an acetyl group.

[0380] In one particularly preferred embodiment, the grafted lipid has the following structure: [ka] (wherein n is 23) The compound is n-tetradecylpoly(sarcosine) 23 (C14-pSar23).

[0381] In one particularly preferred embodiment, the grafted lipid has the following structure: [ka] (wherein n is 23) The compound is n-tetradecylpoly(sarcosine)23 acetate (C14-pSar23Ac).

[0382] 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.

[0383] 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]

[0384] 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]

[0385] 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]

[0386] 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 repeating units in the polymer may include 1 to 100, 5 to 50, 5 to 25, 7 to 14, preferably 10 to 20, and more preferably 12 to 16.

[0387] The lipid moiety of the (pAEEA)-conjugated lipid can be any of the moieties defined above in connection with lipids in its broadest or preferred aspects. In one embodiment, the lipid moiety is a tocopherol or tocotrienol residue. In one embodiment, the lipid moiety is α-tocopherol. In one embodiment, the lipid moiety is β-tocopherol. In one embodiment, the lipid moiety is γ-tocopherol. In one embodiment, the lipid moiety is δ-tocopherol. In one embodiment, the lipid moiety is α-tocotrienol. In one embodiment, the lipid moiety is β-tocotrienol. In one embodiment, the lipid moiety is γ-tocotrienol. In one embodiment, the lipid moiety is δ-tocotrienol.

[0388] In one embodiment, the grafted lipid is α-tocopherol pAEEA14.

[0389] In one embodiment, the grafted lipid is a peptide-conjugated lipid. The compositions described herein may also contain a lipid conjugated to a binding moiety. In some embodiments, the lipid covalently bound to the binding moiety comprises the compound L-X1-P-X2-B, as described in detail herein. Preferably, the binding moiety is a peptide, and the compositions described herein may also contain a peptide-conjugated lipid. As used herein, the term "peptide-conjugated lipid" refers in its broadest sense to a lipid or lipid-like substance, as defined above (in its broadest or preferred embodiment), conjugated to a peptide. In this aspect, "peptide" is synonymous with "polypeptide" and "protein." In one embodiment, the peptide contains an ALFA-tag (i.e., the peptide-conjugated lipid may be an ALFA-conjugated lipid). Such peptide-conjugated lipids are described in more detail in U.S. Patent Application No. 63 / 305,905 (unpublished at the time of filing).

[0390] In some embodiments, the peptide-conjugated lipid has the formula (A): L-X1-P-X2-B(A) (In the formula, P comprises a polymer; L comprises a hydrophobic moiety attached to a first end of the polymer; B comprises a linking moiety attached to the second end of the polymer; X1 is absent or a first linking moiety; X2 is absent or a second linking moiety This includes compounds of the formula:

[0391] In some embodiments, X1 comprises a carbonyl group.

[0392] In some embodiments, X2 comprises the reaction product of a maleimide group with a thiol or cysteine ​​group of the compound comprising the binding moiety.

[0393] In some embodiments, the hydrophobic moiety is or is included in a lipid, hi some embodiments, the lipid comprises a phospholipid, such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0394] In some embodiments, the polymer provides stealth properties, increases circulation half-life, and / or reduces non-specific protein binding or cell adhesion.

[0395] In some embodiments, the polymer comprises polyethylene glycol (PEG), which may have an average molecular weight in the range of 200 to 10,000, preferably 500 to 5000, more preferably 1000 to 4000, and most preferably 2000.

[0396] In some embodiments, the hydrophobic moiety to which the binding moiety is covalently attached comprises a distearoyl-glycero-phosphoethanolamine-polyethylene glycol-conjugate (DSPE-PEG).

[0397] In some embodiments, the binding moiety covalently attached to the hydrophobic moiety comprises a peptide, preferably the binding moiety comprises an ALFA-tag.

[0398] In some embodiments, the ALFA-tag comprises the amino acid sequence -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-: (wherein the amino acids AAO, AAI, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13, and AA14 are as follows: AA0 is Pro or deleted; AA1 is Ser, Gly, Thr, or Pro; AA2 is Arg, Gly, Ala, Glu, or Pro; AA3 is Leu, Ile, or Val; AA4 is Glu or Gln; AA5 is Glu or Gln; AA6 is Glu or Gln; AA7 is Leu, Ile, or Val; AA8 is Arg, Ala, Gln, or Glu; AA9 is Arg, Ala, Gln, or Glu; AA10 is Arg; AA11 is Leu; AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or is deleted; AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or is deleted; AA14 is either Pro or deleted).

[0399] In some embodiments, the ALFA-tag comprises a sequence selected from the group consisting of SRLEEELRRRLTE, PSRLEEELRRRLTE, SRLEEELRRRLTEP, and PSRLEEELRRRLTEP.

[0400] In some embodiments, the ALFA-tag comprises the cyclized amino acid sequence -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, where any two side chains of the amino acids AAO, AAI, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AAI0, AA11, AA12, AA13, and AA14 (X1, X2) are connected by a covalent bond; The amino acids AAO, AAI, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13, and AA14 that are not X1 and X2 are as follows: AA0 is Pro or deleted; AA1 is Ser, Gly, Thr, or Pro; AA2 is Arg, Gly, Ala, Glu, or Pro; AA3 is Leu, Ile, or Val; AA4 is Glu or Gln; AA5 is Glu or Gln; AA6 is Glu or Gln; AA7 is Leu, Ile, or Val; AA8 is Arg, Ala, Gln, or Glu; AA9 is Arg, Ala, Gln, or Glu; AA10 is Arg; AA11 is Leu; AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or is deleted; AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or is deleted; AA14 is Pro or deleted) Includes.

[0401] In some embodiments, X1 and X2 are separated by 2 or 3 amino acids.

[0402] In some embodiments, AA5 is X1 and AA9 is X2, AA5 is X1 and AA8 is X2, AA9 is X1 and AA13 is X2, AA6 is X1 and AA9 is X2, AA9 is X1 and AA12 is X2, AAIO is X1 and AA13 is X2, AA6 is X1 and AAIO is X2, or AA4 is X1 and AA8 is X2.

[0403] In some embodiments, the ALFA-tag is a. -AA0-AA1-AA2-AA3-AA4-cyclo(X1-AA6-AA7-AA8-X2)-Arg-Leu-AA12-AA13-AA14-, b. -AA0-AA1-AA2-AA3-AA4-cyclo(X1-AA6-AA7-X2)-AA9-Arg-Leu-AA12-AA13-AA14-, c. -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-cyclo(X1-Arg-Leu-AA12-X2)-AA14-, d. -AA0-AA1-AA2-AA3-AA4-AA5-cyclo(X1-AA7-AA8-X2)-Arg-Leu-AA12-AA13-AA14-, e. -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-cyclo(X1-Arg-Leu-X2)-AA13-AA14-, f. -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-cyclo(X1-Leu-AA12-X2)-AA14-, g. -AA0-AA1-AA2-AA3-AA4-AA5-cyclo(X1-AA7-AA8-AA9-X2)-Leu-AA12-AA13-AA14-, and h. -AA0-AA1-AA2-AA3-cyclo(X1-AA5-AA6-AA7-X2)-AA9-Arg-Leu-AA12-AA13-AA14- and a cyclized amino acid sequence selected from the group consisting of: wherein the side chains of the X1 and X2 amino acid residues are covalently connected; AA0 is Pro or deleted; AA1 is Ser, Gly, Thr, or Pro; AA2 is Arg, Gly, Ala, Glu, or Pro; AA3 is Leu, Ile, or Val; AA4 is Glu or Gln; AA5 is Glu or Gln; AA6 is Glu or Gln; AA7 is Leu, Ile, or Val; AA8 is Arg, Ala, Gln, or Glu; AA9 is Arg, Ala, Gln, or Glu; AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or is deleted; AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or is deleted; AA14 is either Pro or deleted.

[0404] In some embodiments, X1 and X2 in the peptides disclosed herein are covalently linked via an amide, disulfide, thioether, ether, ester, thioester, thioamide, alkylene, alkenylene, alkynylene, and / or 1,2,3-triazole.

[0405] In some embodiments, the cyclized amino acid sequences described herein are produced by linking the amino group on the side chain of one of X1 and X2 to the carboxyl group on the side chain of the other of X1 and X2 via an amide bond. An amino group on the side chain of an amino acid having a pendant amine group, e.g., lysine or a lysine derivative, and a carboxyl group on the side chain of an acidic amino acid, e.g., aspartic acid, glutamic acid, or a derivative thereof, can be used to produce a cyclized amino acid sequence via an amide bond.

[0406] In some embodiments, the cyclized amino acid sequences described herein are generated by linking a sulfhydryl group on the side chain of one of X1 and X2 to a sulfhydryl group on the side chain of the other of X1 and X2 via a disulfide bond. Sulfhydryl-containing amino acids include cysteine ​​and other sulfhydryl-containing amino acids such as Pen.

[0407] In some embodiments, X1 and X2 are independently selected from the group consisting of Glu, DGlu, Asp, DAsp, Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, DDap, Cys, DCys, hCys, DhCys, Pen, and DPen, provided that when X1 is Glu, DGlu, Asp, or DAsp, X2 is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, or DDap; when X1 is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, or DDap, X2 is Glu, DGlu, Asp, or DAsp; when X1 is Cys, DCys, hCys, DhCys, Pen, or DPen, X2 is Cys, DCys, hCys, DhCys, Pen, or DPen.

[0408] In some embodiments, X1 is Glu and X2 is Lys. In some embodiments, -cyclo(Glu--------Lys)-, -c(Glu-------Lys)-, -cyclo(E-------K)-, -c(E---------K)-, -E------K-cyclo, or -cycloE----cycloK include the following structures: [ka]

[0409] In some embodiments, X1 is Lys and X2 is Glu. In some embodiments, -cyclo(Lys------Glu)-, -c(Lys------Glu)-, -cyclo(K------E)-, -c(K------E)-, -KE-cyclo, or cycloK------cycloE include the following structures: [ka]

[0410] In some embodiments, X1 is Cys and X2 is Cys. In some embodiments, -cyclo(Cys------Cys)-, c(Cys------Cys)-, -cyclo(C------C)-, -c(C--------C)-, -C----C-cyclo, or -cycloC----cycloC include the following structures:

[0411] In some embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In some other embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In still other embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-. In yet other embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)-.

[0412] Cyclic peptides can have different cyclic bridging moieties that form ring structures. Preferably, the ring structure contains a chemically stable bridging moiety, such as an amide group, a lactone group, an ether group, a thioether group, a disulfide group, an alkylene group, an alkenyl group, or a 1,2,3-triazole. The following are examples illustrating the diversity of bridging moieties in peptides: [ka]

[0413] The peptide-conjugated lipid may be incorporated into an aqueous dispersion and included in the lipid mixture described herein. The peptide-conjugated lipid may not be included in the lipid mixture, but instead may be added later to lipid particles contained in the dispersed phase of the aqueous dispersion. The peptide-conjugated lipid may not be included in the lipid mixture, but instead may be added later to nucleic acid-lipid particles. When the peptide-conjugated lipid is added to lipid particles or nucleic acid-lipid particles contained in the dispersed phase of an aqueous dispersion, the amount of peptide-conjugated lipid added may replace a corresponding amount of steroid (e.g., cholesterol) in the particles. The peptide-conjugated lipid is typically added to the particles at a final molar ratio of 0.1-0.3 mol% of the total lipid, optionally about 0.2 mol%.

[0414] When nucleic acid-lipid particles contain peptide-conjugated lipids, this allows the nucleic acid-lipid particles to be functionalized.For example, a binding moiety that specifically binds to the peptide of peptide-conjugated lipid can be attached to the nucleic acid-lipid particles, and the binding moiety can also bind to target cells (for example, by specifically binding to target cell surface antigens).This can provide targeted delivery of the nucleic acid contained in the functionalized nucleic acid-lipid particles.The binding moiety that specifically binds to the peptide of peptide-conjugated lipid can be an ALFA-tag binding moiety.

[0415] In some embodiments, the ALFA-tag binding moiety comprises an antibody or antibody fragment, such as a Camelidae VHH domain. In some embodiments, the ALFA-tag binding moiety comprises a single domain antibody (sdAb), an NbALFA-nanobody. In some embodiments, the ALFA-tag binding moiety comprises a single domain antibody, such as a Camelidae VHH domain comprising the CDR1 sequence VTX1SALNAMAMG (wherein X1 is I or V), the CDR2 sequence AVSX2RGNAM (wherein X2 is E, H, N, D, or S), and the CDR3 sequence LEDRVDSFHDY.

[0416] In some embodiments, the ALFA-tag binding moiety comprises a single domain antibody, e.g., a Camelidae VHH domain comprising the CDR1 sequence GVTX1SALNAMAMG (wherein X1 is I or V), the CDR2 sequence AVSX2RGNAM (wherein X2 is E, H, N, D, or S), and the CDR3 sequence LEDRVDSFHDY.

[0417] In some embodiments, the ALFA-tag binding moiety comprises a single domain antibody, e.g., a Camelidae VHH domain comprising the amino acid sequence EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity thereto, or a fragment of said amino acid sequence or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity thereto. In some embodiments, the amino acid sequence comprises the CDR1, CDR2, and CDR3 sequences defined above.

[0418] In some embodiments, the ALFA-tag binding moiety comprises a bispecific antibody that targets the ALFA-tag and a cell surface antigen, hi some embodiments, the ALFA-tag binding moiety comprises a moiety that binds to a peptide comprising an ALFA-tag and a moiety that targets a cell surface antigen.

[0419] In one embodiment, the grafted lipid 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 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 present in the lipid mixture in an amount of 1-2.5 mol% of the total lipids 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 and the nucleic acid-lipid particles.

[0420] In one embodiment, the grafted lipid is a PEG-conjugated 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-conjugated 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-conjugated 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. In one embodiment, the grafted lipid is a PEG-conjugated lipid and is present in the lipid mixture in an amount of about 1.8 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.

[0421] 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. In one embodiment, the grafted lipid is ALC-0159 and is present in the lipid mixture in an amount of about 1.8 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 (typically containing pre-LNPs) and the nucleic acid-lipid particles.

[0422] In one embodiment, the grafted lipid is a poly(sarcosine) (pSar)-conjugated lipid 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 a poly(sarcosine) (pSar)-conjugated lipid 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 a poly(sarcosine) (pSar)-conjugated lipid and is present in the lipid mixture in an amount of 1-2.5 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is a poly(sarcosine) (pSar)-conjugated lipid and is present in the lipid mixture in an amount of about 1.8 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is a poly(sarcosine) (pSar)-conjugated lipid and is present in the lipid mixture in an amount of 3-5 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is a poly(sarcosine) (pSar)-conjugated lipid and is present in the lipid mixture in an amount of about 4 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.

[0423] In one embodiment, the grafted lipid is n-tetradecylpoly(sarcosine)23 (C14-pSar23) or n-tetradecylpoly(sarcosine)23 (C14-pSar23) acetate 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 n-tetradecylpoly(sarcosine)23 (C14-pSar23) or n-tetradecylpoly(sarcosine)23 (C14-pSar23) acetate 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 n-tetradecylpoly(sarcosine)23 (C14-pSar23) or n-tetradecylpoly(sarcosine)23 (C14-pSar23) acetate and is present in the lipid mixture in an amount of 1-2.5 mol % of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is n-tetradecylpoly(sarcosine)23 (C14-pSar23) or n-tetradecylpoly(sarcosine)23 (C14-pSar23) acetate and is present in the lipid mixture in an amount of about 1.8 mol % of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is n-tetradecylpoly(sarcosine)23 (C14-pSar23) or n-tetradecylpoly(sarcosine)23 (C14-pSar23) acetate and is present in the lipid mixture in an amount of 3-5 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is n-tetradecylpoly(sarcosine)23 (C14-pSar23) or n-tetradecylpoly(sarcosine)23 (C14-pSar23) acetate and is present in the lipid mixture in an amount of about 4 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 (typically containing pre-LNPs) and the nucleic acid-lipid particles.

[0424] In one embodiment, the grafted lipid is a poly(aminoethyl-ethylene glycol acetyl) (pAEEA)-conjugated lipid 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 a poly(aminoethyl-ethylene glycol acetyl) (pAEEA)-conjugated lipid 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 a poly(aminoethyl-ethylene glycol acetyl) (pAEEA)-conjugated lipid and is present in the lipid mixture in an amount of 1-2.5 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is a poly(aminoethyl-ethylene glycol acetyl) (pAEEA)-conjugated lipid and is present in the lipid mixture in an amount of 1.8-2 mol% of the total lipids present in the lipid mixture.

[0425] In one embodiment, the grafted lipid is α-tocopherol-pAEEA14 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 α-tocopherol-pAEEA14 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 α-tocopherol-pAEEA14 and is present in the lipid mixture in an amount of 1-2.5 mol % of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is α-tocopherol-pAEEA14 and is present in the lipid mixture in an amount of 1.8-2 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 (typically containing pre-LNPs) and the nucleic acid-lipid particles.

[0426] 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.

[0427] The nucleic acid-lipid particle compositions described herein may be administered in the form of any suitable pharmaceutical composition.

[0428] 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.

[0429] 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%.

[0430] 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.

[0431] 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.

[0432] 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.

[0433] 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.

[0434] 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.

[0435] 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.

[0436] 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).

[0437] 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.

[0438] 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).

[0439] 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.

[0440] 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).

[0441] 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.

[0442] 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.

[0443] 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.

[0444] 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).

[0445] 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.

[0446] 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).

[0447] Pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and a "pharmaceutically acceptable preparation."

[0448] 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.

[0449] 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.

[0450] 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.

[0451] 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.

[0452] 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.

[0453] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0454] 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.

[0455] 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.

[0456] 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.

[0457] 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).

[0458] Pharmaceutical carriers, excipients, or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0459] 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.

[0460] 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.

[0461] Thus, in one aspect, there is disclosed a nucleic acid-lipid particle, or pharmaceutical composition, as defined herein, for use in medicine.

[0462] 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).

[0463] 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.

[0464] 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.

[0465] 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.

[0466] 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.

[0467] 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.

[0468] 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.

[0469] 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.

[0470] 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.

[0471] 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.

[0472] 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.

[0473] 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.

[0474] 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."

[0475] The term "patient" means an individual or subject for treatment, particularly an individual or subject with a disease.

[0476] In some embodiments of the present disclosure, the goal is to provide protection from infectious diseases by vaccination.

[0477] 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.

[0478] 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.

[0479] In some embodiments of the present disclosure, the goal is to modulate / reprogram immune cells in the blood.

[0480] 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.

[0481] 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.

[0482] 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."

[0483] 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.

[0484] 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.

[0485] 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.

[0486] 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.

[0487] 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]

[0488] The examples provide detailed information regarding the manufacturing process according to the present invention, highlighting the problems overcome by the present invention using a model formulation containing an ionizable lipid (HY501), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and cholesterol as the helper lipid, and C14 amine-terminated polysarcosine (NH-Psar)23 as the stealth moiety. The final drug product formulated in the present invention uses luciferase as the RNA payload. [Example]

[0489] Formulations containing grafted lipids The manufacturing process was carried out by a two-step process: (i) manufacturing preformed lipid nanoparticles (i.e., aqueous dispersion), and (ii) mixing the preformed lipid nanoparticles with mRNA. Details of each step are detailed below.

[0490] (i) Preparation of HY501 / C14PSar(23)-NH preformed lipid nanoparticles by solvent injection Preformed lipid nanoparticles 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 the cationically ionizable lipid HY-501, cholesterol, DSPC, and C14-Psar(23)-NH dissolved in isopropanol in a molar ratio of 47.5:38.5:10:4, respectively. The organic solvent in the resulting raw colloidal nanoparticles was removed by diafiltration against 5 mM AcOH using tangential flow filtration (TFF) with a hollow fiber (mPES / 100 kD, REPLIGEN) and concentrated as needed. After TFF, the nanoparticles were diluted with 40% sucrose in 5 mM AcOH to a 10% sucrose concentration, filtered through a 0.22 μm polyethersulfone (PES) filter, and stored at −20° C. until further use for the preparation of RNA-lipid particles. A schematic diagram for the preparation of preformed lipid nanoparticles is reproduced in Figure 1.

[0491] (ii) Mixing preformed lipid nanoparticles with mRNA RNA-lipid particles were prepared by complexing an aqueous dispersion of preformed lipid nanoparticles with N1-methylpseudouridine-modified mRNA (modRNA) encoding a model antigen by mixing equal volumes of RNA and preformed lipid nanoparticle phases in a T-shaped mixing channel (2.4 mm inner diameter) at a total flow rate of 360 mL / min using a semi-automated process.

[0492] The RNA phase was prepared by diluting modRNA to a 0.25 mg / mL RNA concentration in its native buffer, i.e., 10 mM HEPES / 0.1 mM EDTA at pH 7. The second phase contained an aqueous dispersion of preformed lipid nanoparticles containing HY501, cholesterol, DSPC, and NH-Psar in a molar ratio of 47.5:38.5:10:4, respectively. The preformed lipid nanoparticles were provided in 5 mM acetic acid and 10% (wt / vol) sucrose at pH 4.5. If necessary, the preformed lipid nanoparticles were further diluted to the target concentration with the native buffer matrix. The two phases were mixed at an N / P ratio of 6 and a total flow rate of 360 mL / min.

[0493] After mixing the two streams of RNA and preformed lipid particles, the raw RNA-lipid nanoparticles, with an RNA concentration of 0.125 mg / mL, were conditioned by diluting them with a storage matrix containing 60 mM HEPES and 30% (wt / vol) sucrose to a final RNA concentration of 0.1 mg / mL and a target pH of approximately 5.5. In the next step, the RNA-lipid particles were sterile filtered using a 0.22 μm polyethersulfone (PES) filter and filled into vials. The entire manufacturing process was carried out at room temperature. The formulation composition is shown in Table 1. A schematic diagram of the RNA-lipid particle manufacturing process is also shown in Figure 2. [Table 1] JPEG2026503621000038.jpg80170

[0494] Stability of preformed lipid nanoparticles The stability of the preformed lipid nanoparticles was investigated. The colloidal stability of the nanoparticles in both liquid (2–8°C and 25°C) and frozen (–20°C) conditions was maintained for the 3 months currently being evaluated, as shown in Figure 3.

[0495] Long-term stability of RNA-lipid particles The long-term stability of the RNA-lipid particles was investigated in a stability study. As seen in Figure 4, no changes in particle size, polydispersity, or RNA integrity were observed as a function of time when the drug product was stored frozen at -20°C or -80°C.

[0496] The biological functionality of the RNA-lipid particles (LNP2) produced by the method of the present invention was compared with that of the RNA-lipid particles (LNP1) produced by the conventional LNP manufacturing process. Herein, the two drug products were compared in vivo using a model antigen via intramuscular administration. The administration regime was a priming dose on day 1 followed by a booster dose 21 days later.

[0497] As shown in Figure 5, all tested groups exhibited higher CD8+ peptide pools than CD4+ peptide pools. The RNA-lipid particles (LNP2) produced by the two-step process of the present invention exhibited higher T cell responses compared to particles (LNP1) produced by the conventional LNP manufacturing process (one-step process). [Example]

[0498] Fabrication of DODMA / C14-Psar(23)-Ac preformed lipid nanoparticles by ethanol injection The stability of preformed lipid nanoparticles was investigated. Preformed lipid nanoparticles were prepared by flow-mixing an organic phase containing dissolved lipids with an aqueous phase (5 mM AcOH, pH 3.5). Mixing was performed using a syringe pump and a T-piece as the 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 consisted of a cationically ionizable lipid (DODMA), cholesterol, DSPC, and C14-PSar(23)-Ac 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 diafiltration against 5 mM AcOH using a hollow fiber (mPES / 100 kD, REPLIGEN) tangential flow filtration (TFF) and concentrated twice (2x). After TFF, the nanoparticles were diluted as needed to a 10% sucrose concentration, filtered through a 0.22 μm polyethersulfone (PES) filter, and stored at 4° C. until further use in the preparation of RNA-lipid particles.

[0499] The stability of the preformed lipid nanoparticles was investigated, and it was observed that the colloidal stability of the nanoparticles at 2–8°C and 25°C conditions was maintained for the 3 months currently being evaluated, as shown in Figure 6 . [Example]

[0500] Fabrication of DODMA / DMG-PEG2k preformed lipid nanoparticles by ethanol injection The effect of different buffer conditions on the stability of preformed lipid nanoparticles was investigated. To prepare preformed lipid nanoparticles, a lipid mix consisting of a cationically ionizable lipid (DODMA), cholesterol, DSPC, and DMG-PEG2k was dissolved in ethanol at a molar ratio of 47.5:40.7:10:1.8, respectively, and mixed with an aqueous phase (5 mM AcOH) at a total flow rate of 200 mL / min and a volume ratio of 1:4 (organic phase:aqueous phase) using a standard syringe pump-based setup and a T-piece as the mixing element. The organic solvent in the resulting raw colloidal nanoparticles was removed by dialysis against either (i) 5 mM AcOH, (ii) 40 mM acetate buffer, or (iii) 10 mM HEPES over different pH ranges (see Figure 7) in a 10K molecular weight cutoff (MWCO) Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific, Waltham, MA, USA). After dialysis, the nanoparticles were diluted to a concentration of 10% sucrose, filtered through a 0.22 μm polyethersulfone (PES) filter, and freeze-thaw and stability tests were performed under different conditions.

[0501] Freeze-thaw tests were performed by cycling the nanoparticles from -80°C (overnight) to room temperature (25°C) (2 hours) at least three times. Between thaw and freeze cycles, the nanoparticles were mixed by gently inverting before the next freeze cycle. The particle size and polydispersity index of the nanoparticles were measured after each freeze-thaw cycle.

[0502] Stability of DODMA / DMG-PEG2k preformed lipid nanoparticles The colloidal stability of the nanoparticles was maintained for all three freeze-thaw cycles at different pH levels with 5 mM acetic acid and 10 mM HEPES, whereas in the case of 40 mM acetate buffer, the particle size was observed to increase with all freeze-thaw cycles, as shown in Figure 7. [Example]

[0503] Preparation of HY501 / 10% DSPC (without grafted lipids) RNA-lipid particles Preformed lipid nanoparticles were prepared by flow-mixing an organic phase containing dissolved lipids with an aqueous phase (5 mM AcOH). Mixing was performed at a total flow rate of 200 mL / min using a syringe pump and a T-piece as the mixing element. The lipid mixture (total concentration 50.0 mM) consisted of the ionizable lipid HY-501, cholesterol, and DSPC dissolved in isopropanol in a molar ratio of 47.5:42.5:10. The organic solvent in the resulting raw colloidal nanoparticles was removed by diafiltration against 5 mM AcOH using tangential flow filtration with a hollow fiber (mPES / 100 kD, REPLIGEN) and concentrated twice. After TFF, the nanoparticles were diluted to a 10% sucrose concentration, filtered through a 0.22 μm polyethersulfone (PES) filter, and stored at -20 °C until further use in RNA-lipid particle preparation. The particle size of the preformed lipid nanoparticles was approximately 40 nm with a polydispersity index of approximately 0.2.

[0504] Preformed lipid...

Claims

1. 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 an anion of an aqueous acid; the aqueous dispersion is substantially free of inorganic cations, organic solvents, and RNA; The aqueous dispersion, wherein the aqueous mobile phase comprises a cryoprotectant.

2. 10. The aqueous dispersion of claim 1 having a pH of from 2.5 to 5.

5.

3. 3. The aqueous dispersion of claim 2 having a pH of from 2.5 to 4.

5.

4. 4. Aqueous dispersion according to any one of claims 1 to 3, wherein the molar ratio of cationic lipid or cationically ionizable lipid to anion of aqueous acid is from 20:1 to 1:

20.

5. 5. Aqueous dispersion according to any one of claims 1 to 4, wherein the molar ratio of cationic lipid or cationically ionizable lipid to anion of aqueous acid is from 5:1 to 1:

5.

6. 6. The aqueous dispersion according to claim 1, wherein the aqueous acid is an inorganic acid or a water-soluble organic acid.

7. 7. The aqueous dispersion according to claim 1, wherein the aqueous acid is acetic acid, malic acid, or succinic acid.

8. 8. The aqueous dispersion according to claim 1, wherein the concentration of the aqueous acid is in the range of 1 to 20 mM.

9. 9. The aqueous dispersion according to claim 1, wherein the concentration of the aqueous acid is in the range of 2.5 to 10 mM.

10. The cationic lipid or cationically ionizable lipid is [(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 (BHD-C2C2-PipZ); Bis(2-octyldodecyl)3,3′-((2-(1-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-1Me-Pyr); 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); structure 【Chemistry 1】 a compound having the formula: and any mixtures thereof The aqueous dispersion according to any one of claims 1 to 9, selected from the group consisting of:

11. 11. The aqueous dispersion of claim 1, wherein the lipid mixture further comprises one or more additional lipids.

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

13. 13. The aqueous dispersion of claim 12, wherein the one or more additional lipids comprise 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 mixture thereof.

14. The aqueous dispersion of claim 13, selected from the group consisting of:

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

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

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

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

19. 19. The aqueous dispersion of claim 18, wherein the grafted 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. 20. The aqueous dispersion of any one of claims 11 to 19, wherein the one or more additional lipids comprise a peptide-conjugated lipid.

21. 21. The aqueous dispersion of claim 20, wherein the peptide-conjugated lipid is an ALFA-tag conjugated lipid.

22. A freeze-dried composition comprising the aqueous dispersion of any one of claims 1 to 21.

23. A frozen composition comprising the aqueous dispersion of any one of claims 1 to 21 at a temperature of from -15°C to -90°C.

24. A method of forming the aqueous dispersion of any one of claims 1 to 21, comprising the steps of: (i) a lipid mixture comprising a cationically ionizable lipid; (ii) an aqueous phase comprising an aqueous acid and a cryoprotectant; to produce an aqueous dispersion comprising an anion of said aqueous acid.

25. A method of forming the aqueous dispersion of any one of claims 1 to 23, comprising the steps of: (a)(i) a lipid mixture comprising a cationically ionizable lipid; and (ii) an aqueous phase comprising an aqueous acid; to produce a first intermediate aqueous dispersion comprising an anion of said aqueous acid; and (b) adding a cryoprotectant to the first intermediate aqueous dispersion to produce an aqueous dispersion; The method comprising:

26. 22. A method for forming lipid particles containing RNA, comprising the step of mixing an aqueous dispersion of any one of claims 1 to 21 with an aqueous solution containing RNA to produce lipid particles containing RNA.

27. 27. The method of claim 26, wherein the cryoprotectant in the aqueous dispersion is selected from the group consisting of sucrose, trehalose, and glucose, and mixtures thereof.

28. 28. The method of claim 27, wherein the cryoprotectant is sucrose or trehalose and is present in the aqueous dispersion at a concentration of about 15% to about 25%.

29. 29. The method of any one of claims 26 to 28, which does not include any further dialysis, filtration, dilution, or addition of a cryoprotectant.

30. The method of any one of claims 26 to 29, wherein the RNA is mRNA.

31. 31. The method of claim 30, wherein the mRNA encodes one or more patient-specific antigens suitable for personalized cancer therapy.

32. Lipid particles comprising RNA obtained or obtainable by the method according to any one of claims 26 to 31.

33. 33. The lipid particle of claim 32, which is a lipid nanoparticle.

34. 34. A lipid particle according to claim 32 or 33 for use in medicine.

35. 34. The lipid particle of claim 32 or 33 for use in the treatment of cancer.

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