Stable lipid or lipid-like nanoparticle suspensions

Abstract

The present invention provides stable lipid nanoparticle (LNP) / lipid-like nanoparticle (LiNP) formulations and LNP / LiNP suspensions, their use and use in methods of treatment based on the discovery that addition of surfactants in the formulation or suspension avoids aggregation, making them amazing long shelf life and extended oscillation stability. According to the invention, the reduction of aggregation reduces the side effects of the formulations and suspensions of the invention, in particular the side effects caused by vaccine formulations and anti-cancer formulations including LNP and / or LiNP.

Description

Technical Field

[0001] The present invention relates to stabilizing suspension formulations comprising lipid nanoparticles or lipid-like nanoparticles for delivering nucleic acids. Background Art

[0002] Lipid or lipid-like nanoparticles (LNP or LiNP) are often used to deliver active pharmaceutical ingredients in patients. For example, lipid or lipid-like formulations of nucleic acids are very useful and effective for introducing nucleic acids into cells. For decades, this advantageous property of lipid or lipid-like formulations of nucleic acids has been used in biological and medical research and therapeutic methods to i) overexpress genes or supplement genetic defects in target cells, or ii) downregulate or upregulate endogenous gene expression in cells, or iii) repair genetic defects (mutations). Currently, nanoparticle-dependent mRNA formulations have also been established as COVID-19 vaccines.

[0003] However, it has been found that during handling or transportation, the physical stress conditions to which suspension formulations comprising lipid nanoparticles or lipid-like nanoparticles may be subjected can affect the formulation efficiency of the formulation for delivering nucleic acids to patients. In particular, exposing the formulation to vibrational stress or shaking the formulation may have destabilizing effects or reduce its potency (Kudsiova L, Lansley A, Scutt G et al., Stability testing of the Pfizer-BioNTech BNT162b2 COVID-19 vaccine: a translational study in UK vaccination centres, BMJ Open Science 2021;5:e100203.doi:10.1136 / bmjos-2021-100203; S. Grau et al., Clinical Microbiology and Infection 27(2021)1698.e1698.e4).

[0004] Therefore, there is a desire for reliable strategies to stabilize suspension formulations comprising lipid nanoparticles or lipid-like nanoparticles for delivering nucleic acids. Summary of the Invention

[0005] In the context of the present invention, it has been found that the presence of a surfactant stabilizes the suspension of lipid nanoparticles or lipid-like nanoparticles in an aqueous carrier solution, which prevents the particles from aggregating under physical stress conditions.

[0006] To this end, the following aspects provided by the present invention are shown in a non-exclusive manner.

[0007] In one aspect, the present invention provides a suspension of lipid nanoparticles or lipid-like nanoparticles in an aqueous carrier solution, wherein the aqueous carrier solution comprises a surfactant, and wherein the lipid nanoparticles or lipid-like nanoparticles comprise components (a) and (b) as follows:

[0008] (a) A therapeutic agent, which is preferably a nucleic acid, and

[0009] (b) At least one selected from the group consisting of permanent cationic lipids, ionizable lipids, and ionizable lipid-like compounds. A method for preparing the suspension is also provided.

[0010] According to another aspect, the present invention provides the use of a surfactant for stabilizing a suspension of lipid nanoparticles or lipid-like nanoparticles in an aqueous carrier solution to prevent particle aggregation under physical stress conditions, wherein the lipid nanoparticles or lipid-like nanoparticles comprise components (a) and (b) as follows:

[0011] (a) A therapeutic agent, which is preferably a nucleic acid, and

[0012] (b) At least one selected from the group consisting of permanent cationic lipids, ionizable lipids, and ionizable lipid-like compounds.

[0013] In a related aspect, the present invention provides a method for stabilizing a suspension of lipid nanoparticles or lipid-like nanoparticles in an aqueous carrier solution to prevent particle aggregation under physical stress conditions, wherein the lipid nanoparticles or lipid-like nanoparticles comprise components (a) and (b) as follows:

[0014] (a) A therapeutic agent, which is preferably a nucleic acid, and

[0015] (b) At least one selected from the group consisting of permanent cationic lipids, ionizable lipids, and ionizable lipid-like compounds,

[0016] and wherein the method comprises incorporating a surfactant into the suspension of lipid nanoparticles or lipid-like nanoparticles.

[0017] Furthermore, the present invention provides a suspension according to the present invention, including a suspension obtained by the method according to the present invention, which is used as a drug. In a related context, the formulation according to the present invention, including a suspension obtained by the method according to the present invention, is suitable for reducing side effects in lipid or lipid-like nanoparticle therapy.

[0018] Without being bound by theory, the present invention provides stable LNP / LiNP formulations and LNP / LiNP suspensions, their uses, and uses in methods of treatment, which are based on the discovery that the addition of a surfactant to the formulation or suspension avoids aggregation, thereby allowing, for example, an unexpectedly long shelf life and extended shaking stability. According to the present invention, the reduction of such aggregation reduces side effects of the formulations and suspensions of the present invention, such as reducing side effects caused by vaccine formulations or anti-cancer formulations comprising LNP or LiNP.

[0019] An overview of aspects of the present invention is provided in the following first set of entries.

[0020] 1. A suspension of lipid nanoparticles or lipid-like nanoparticles in an aqueous carrier solution, wherein the aqueous carrier solution comprises a surfactant, and wherein the lipid nanoparticles or lipid-like nanoparticles comprise components (a) and (b) below:

[0021] (a) A therapeutic agent, and

[0022] (b) At least one selected from the group consisting of permanent cationic lipids, ionizable lipids, and ionizable lipid-like substances.

[0023] 2. The suspension according to entry 1, wherein the therapeutic agent is a nucleic acid.

[0024] 3. The suspension according to entry 2, wherein the nucleic acid is selected from RNA and plasmid DNA.

[0025] 4. The suspension according to entry 2 or 3, wherein the nucleic acid is selected from mRNA, siRNA, miRNA, antisense RNA, tRNA, and non-coding RNA.

[0026] 5. The suspension according to entry 4, wherein the nucleic acid is mRNA.

[0027] 6. The suspension according to any one of entries 2 to 5, wherein, based on the total volume of the suspension, the concentration of the nucleic acid in the suspension ranges from 0.01 to 10 mg / mL, more preferably from 0.02 to 10 mg / mL, still more preferably from 0.05 to 5 mg / mL, and most preferably from 0.05 to 2.5 mg / ml.

[0028] 7. The suspension according to any one of entries 1 to 6, wherein the weight / volume ratio of the nanoparticles in the aqueous carrier solution (in g / L) is in the range of 0.1 g / L to 300 g / L, more preferably 0.2 g / L to 300 g / L, still more preferably 0.5 g / L to 250 g / L, and most preferably 0.5 g / L to 125 g / L.

[0029] 8. A suspension according to any one of items 1 to 7, wherein the suspended nanoparticles have a Z-average diameter measured by dynamic light scattering in the range of 10 to 500 nm, more preferably in the range of 10 to 250 nm, still more preferably in the range of 20 to 200 nm.

[0030] 9. A suspension according to any one of items 1 to 8, wherein the suspended nanoparticles have a polydispersity index measured by dynamic light scattering in the range of 0.02 to 0.4, more preferably in the range of 0.03 to 0.2.

[0031] 10. A suspension according to any one of items 1 to 9, wherein the nanoparticles further comprise one or more of the following components (c1) to (c6):

[0032] (c1) An ionizable lipid having a sterol structure;

[0033] (c2) A phosphoglyceride lipid;

[0034] (c3) A PEG-conjugated lipid;

[0035] (c4) A poly(sarcosine)-conjugated lipid;

[0036] (c5) A PASylated lipid; and

[0037] (c6) A cationic polymer.

[0038] 11. A suspension according to any one of items 1 to 10, wherein the nanoparticles comprise:

[0039] 30 to 65 mol% of at least one (b) selected from permanent cationic lipids, ionizable lipids, and ionizable lipidoids, preferably an ionizable lipid or an ionizable lipidoid (b), and one or more of the following components:

[0040] 10 to 50 mol% of a lipid (c1) having a sterol structure,

[0041] 4 to 50 mol% of a phosphoglyceride lipid (c2),

[0042] 0.5 to 10 mol% of one or any combination of a PEG-conjugated lipid (c3), a poly(sarcosine)-conjugated lipid (c4), and a PASylated lipid (c5),

[0043] 0.5 to 10 mol% of a cationic polymer (c6),

[0044] such that the sum of (b) and (c1) to (c6) is equal to 100 mol%.

[0045] 12. A suspension according to any one of items 1 to 11, wherein the nanoparticles further comprise the following components (c1) to (c3):

[0046] (c1) A non-ionizable lipid having a sterol structure;

[0047] (c2) A phosphoglyceride lipid; and

[0048] (c3) A PEG-conjugated lipid.

[0049] 13. A suspension according to item 12, wherein the nanoparticles comprise:

[0050] 30 to 65 mol% of at least one of a permanent cationic lipid, an ionizable lipid, and an ionizable lipidoid (b), preferably an ionizable lipid or an ionizable lipidoid (b),

[0051] 10 to 50 mol% of a lipid having a sterol structure (c1),

[0052] 4 to 50 mol% of a phosphoglyceride lipid (c2), and

[0053] 0.5 to 10 mol% of a PEG-conjugated lipid (c3),

[0054] such that the sum of (b) and (c1) to (c3) is equal to 100 mol%.

[0055] 14. A suspension according to any one of items 2 to 13, wherein the nanoparticles further comprise a polyanionic component different from nucleic acid.

[0056] 15. A suspension according to any one of items 2 to 14, wherein the composition of the nanoparticles is such that the weight ratio of the sum of the weights of the components other than nucleic acid in the nanoparticles to the weight of nucleic acid is in the range of 50:1 to 1:1, more preferably in the range of 40:1 to 2:1, and most preferably in the range of 30:1 to 3:.

[0057] 16. A suspension according to any one of items 1 to 15, wherein the nanoparticles comprise an ionizable lipidoid (b) of the following formula (b-1),

[0058]

[0059] wherein:

[0060] a is 1 and b is an integer from 2 to 4; or a is an integer from 2 to 4 and b is 1,

[0061] p is 1 or 2,

[0062] m is 1 or 2; n is 0 or 1, and m + n ≥ 2; and

[0063] R 1A to R 6A are each independently selected from: hydrogen; -CH 2 -CH(OH)-R 7A 、-CH(R 7A )-CH 2 -OH、-CH 2 -CH 2 -(C=O)-O-R 7A 、-CH 2 -CH 2 -(C=O)-NH-R 7A ;-CH 2 -R 7A ;-C(NH)-NH 2 ;a poly(ethylene glycol) chain; and a receptor ligand; wherein R 7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond;

[0064] provided that at least two residues of R 1A to R 6A are selected from -CH 2 -CH(OH)-R 7A 、-CH(R 7A )-CH 2 -OH、-CH 2 -CH 2 -(C=O)-O-R 7A 、-CH 2 -CH 2 -(C=O)-NH-R 7A and -CH 2 -R 7A ,wherein R 7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond;

[0065] or its protonated form, wherein one or more of the nitrogen atoms contained in the compound of formula (b-1) are protonated to provide a compound carrying a positive charge.

[0066] 17. The suspension according to any one of entries 1 to 16, wherein the nanoparticles comprise an ionizable lipid (b-1) of the following formula (b-1b),

[0067]

[0068] wherein R 1A to R 6A are as defined in entry 16,

[0069] or its protonated form, in which one or more of the nitrogen atoms contained in the compound of formula (b-1b) are protonated to provide a compound carrying a positive charge.

[0070] 18. The suspension according to entry 16 or 17, wherein R 1A to R 6A are independently selected from hydrogen and -CH 2 -CH(OH)-R 7A , wherein R 7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond, provided that at least two residues, preferably at least three residues and more preferably at least four residues of R 1A to R 6A are -CH 2 -CH(OH)-R 7A , wherein R 7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond.

[0071] 19. The suspension according to any one of entries 1 to 18, wherein the nanoparticles comprise an ionizable lipid dL_05(R) having the following formula:

[0072]

[0073] 20. The suspension according to any one of entries 1 to 15, wherein the nanoparticles comprise an ionizable lipid (b) of formula (a-III):

[0074]

[0075] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0076] One of L 1 or L 2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O-; and the other of L 1 or L 2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x-, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O- or a direct bond;

[0077] G 1 and G 2 each independently is C 1 -C 12 alkylene or C 1 -C 12 alkenylene;

[0078] G 3 is C 1 -C 24 alkylene, C 1 -C 24 alkenylene, C 3 -C 8 cycloalkylene, C 3 -C 8 cycloalkenylene, wherein the alkylene, alkenylene, cycloalkylene and cycloalkenylene are each optionally substituted;

[0079] R a is H or C 1 -C 12 alkyl, wherein the alkyl is optionally substituted;

[0080] R 1 and R 2 each independently is C 6 -C 24 alkyl or C 6 -C 24 alkenyl, wherein the alkyl and alkenyl are each optionally substituted;

[0081] R 3 is H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 is C 1 -C 12 alkyl, wherein the alkyl is optionally substituted;

[0082] R 5 is H or C 1 -C 6an alkyl group, wherein the alkyl group is optionally substituted; and

[0083] x is 0, 1 or 2.

[0084] 21. The suspension according to any one of entries 1 to 15, wherein the nanoparticles comprise (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butyrate or a protonated form thereof as the ionizable lipid (b), wherein the nitrogen atom of the compound is protonated to provide a positively charged compound.

[0085] 22. The suspension according to any one of entries 1 to 15, wherein the nanoparticles comprise ((4-hydroxybutyl)azanediyl)bis(hexan-6,1-diyl)bis(2-hexyldecanoate) or a protonated form thereof as the ionizable lipid (b), wherein the nitrogen atom of the compound is protonated to provide a positively charged compound.

[0086] 23. The suspension according to any one of entries 1 to 15 or 22, wherein the nanoparticles comprise heptadec-9-yl 8-[(2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino]octanoate (SM-102) or a protonated form thereof as the ionizable lipid (b), wherein the nitrogen atom of the compound is protonated to provide a positively charged compound.

[0087] 24. The suspension according to any one of entries 10 to 23, wherein the non-ionizable lipid (c1) having a sterol structure comprises a non-ionizable lipid of formula (c1-1):

[0088]

[0089] wherein R 1L is a C3-C12 alkyl group.

[0090] 25. The suspension according to any one of entries 10 to 24, wherein the non-ionizable lipid (c1) having a sterol structure comprises cholesterol.

[0091] 26. The suspension according to any one of entries 10 to 25, wherein the phosphoglyceride lipid (c2) comprises a phosphoglyceride lipid of formula (c2-1),

[0092]

[0093] wherein,

[0094] R 1Fand R 2F independently is a C8-C18 alkyl or a C8-C18 alkenyl, preferably a C12-C18 alkyl or a C12-C18 alkenyl,

[0095] or a pharmaceutically acceptable salt thereof;

[0096] or a phosphatidylglycerol lipid of formula (c2-2),

[0097]

[0098] wherein,

[0099] R 1G and R 2G independently is a C8-C18 alkyl or a C8-C18 alkenyl, preferably a C12-C18 alkyl or a C12-C18 alkenyl,

[0100] or a pharmaceutically acceptable salt thereof.

[0101] 27. The suspension according to any one of items 10 to 26, wherein the phosphatidylglycerol lipid (c2) comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or a pharmaceutically acceptable salt thereof or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a pharmaceutically acceptable salt thereof.

[0102] 28. The suspension according to any one of items 10 to 27, wherein the PEG-conjugated lipid (c3) comprises a PEG-conjugated lipid of formula (c3-1),

[0103]

[0104] wherein,

[0105] R 1H and R 2H independently is a C8-C18 alkyl or a C8-C18 alkenyl, preferably a C12-C18 alkyl or a C12-C18 alkenyl, and p is an integer from 5 to 200, preferably from 10 to 100, and more preferably from 20 to 60;

[0106] or a PEG-conjugated lipid of formula (c3-2),

[0107]

[0108] wherein,

[0109] R 1J and R 2JIndependently a C8-C18 alkyl or C8-C18 alkenyl group, preferably a C12-C18 alkyl or C12-C18 alkenyl group, and q is an integer from 5 to 200, preferably from 10 to 100, and more preferably from 20 to 60,

[0110] or a pharmaceutically acceptable salt thereof;

[0111] or a PEG-conjugated lipid of formula (c3-3),

[0112]

[0113] wherein,

[0114] R 1K and R 2K Independently a C8-C18 alkyl or C8-C18 alkenyl group, preferably a C12-C18 alkyl or C12-C18 alkenyl group, and q is an integer from 5 to 200, preferably from 10 to 100, and more preferably from 20 to 60.

[0115] 29. The suspension according to items 10 to 28, wherein the PEG-conjugated lipid (c3) comprises 1,2-dimyristoyl-sn-glycero methoxy(polyethylene glycol)-2000 (DMG-PEG2k) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylethanamide (ALC-0159).

[0116] 30. The suspension according to item 22, wherein the nanoparticles comprise ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) or its protonated form as the ionizable lipid (b), wherein the nitrogen atom of the compound is protonated to provide a positively charged compound, and further comprises one or more of the following components (d1) to (d8):

[0117] (d1) 2-[(polyethylene glycol)-2000]-N,N-ditetradecylethanamide (ALC-0159);

[0118] (d2) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);

[0119] (d3) cholesterol;

[0120] (d4) potassium chloride;

[0121] (d5) potassium dihydrogen phosphate;

[0122] (d6) sodium chloride;

[0123] (d7) disodium hydrogen phosphate dihydrate;

[0124] (d8) sucrose.

[0125] 31. A suspension according to item 23, wherein the nanoparticles comprise octadec-9-yl 8-[(2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino]octanoate (SM-102) or its protonated form as the ionizable lipid (b), wherein the nitrogen atom of the compound is protonated to provide a positively charged compound, and further comprises one or more of the following components (e1) to (e7):

[0126] (e1) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);

[0127] (e2) Cholesterol;

[0128] (e3) 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000 DMG);

[0129] (e4) Tromethamol hydrochloride;

[0130] (e5) Sodium acetate trihydrate;

[0131] (e6) Acetic acid;

[0132] (e7) Sucrose.

[0133] 32. A suspension according to any one of items 1 to 31, wherein the N / P ratio in the nanoparticles is in the range of 0.5 to 20, more preferably in the range of 0.5 to 10.

[0134] 33. A suspension according to any one of items 1 to 32, wherein the surfactant is substantially not attached to the nanoparticles.

[0135] 34. A suspension according to any one of items 1 to 33, wherein the surfactant is a non-ionic surfactant.

[0136] 35. A suspension according to item 34, wherein the non-ionic surfactant is at least one selected from the group consisting of fatty alcohol ethoxylates, fatty acid ethoxylates, block copolymers of ethylene oxide and propylene oxide, alkylphenol ethoxylates or oligomers of alkylphenol ethoxylates, sorbitan fatty acid esters, ethoxylated sorbitan fatty acid esters, glycerol fatty acid esters, ethoxylated castor oil, and ethoxylated vitamin E.

[0137] 36. A suspension according to item 35, wherein the block copolymer of ethylene oxide and propylene oxide is poloxamer.

[0138] 37. The suspension according to item 36, wherein the poloxamer comprises a poly(propylene oxide) block B of formula (p-1):

[0139]

[0140] where s is an integer from 15 to 60, and

[0141] two poly(ethylene oxide) blocks A of formula (p-2):

[0142]

[0143] where r is independently for each block an integer from 8 to 150, preferably an integer from 10 to 150.

[0144] 38. The suspension according to item 35, wherein the nonionic surfactant is at least one selected from the group consisting of fatty alcohol ethoxylates, fatty acid ethoxylates, alkylphenol ethoxylates or oligomers of alkylphenol ethoxylates, sorbitan fatty acid esters, ethoxylated sorbitan fatty acid esters, glycerol fatty acid esters, ethoxylated castor oil, and ethoxylated vitamin E.

[0145] 39. The suspension according to any one of items 35 to 37, wherein the nonionic surfactant is at least one selected from the group consisting of poloxamer 124, poloxamer 188, poloxamer 338, poloxamer 407, polysorbate 20, polysorbate 80, polyoxyethylene lauryl ether, polyoxyethylene-35 castor oil, D-α-tocopheryl polyethylene glycol 1000 succinate, and tyloxapol.

[0146] 40. The suspension according to item 39, wherein the nonionic surfactant is at least one selected from the group consisting of polysorbate 20, polysorbate 80, polyoxyethylene lauryl ether, polyoxyethylene-35 castor oil, D-α-tocopheryl polyethylene glycol 1000 succinate, and tyloxapol.

[0147] 41. The suspension according to any one of items 1 to 40, wherein the surfactant does not include poloxamer 188.

[0148] 42. The suspension according to any one of items 1 to 41, wherein the surfactant does not include poloxamer 407.

[0149] 43. A suspension according to any one of items 1 to 42, relative to the total volume of the suspension of the nanoparticles in the aqueous carrier solution, including a surfactant at a concentration of 0.01 to 10% (w / v), preferably 0.1 to 10% (w / v), more preferably 0.25 to 5% (w / v), still more preferably 0.33 to 2.5% (w / v), even more preferably 0.45 to 10% (w / v), and most preferably 0.5 to 1.5% (w / v).

[0150] 44. A suspension according to item 43, relative to the total volume of the suspension of the nanoparticles in the aqueous carrier solution, including a surfactant at a concentration of 0.5 to 1.5% (w / v).

[0151] 45. A suspension according to any one of items 1 to 44, wherein the nanoparticles have not been lyophilized.

[0152] 46. A method for preparing a suspension according to any one of items 1 to 45, comprising:

[0153] generating a formulation of lipid nanoparticles or lipid-like nanoparticles by mixing at least one selected from permanent cationic lipids, ionizable lipids, and ionizable lipid-like substances dissolved in an organic phase with a therapeutic agent dissolved in an aqueous solution; and

[0154] combining the nanoparticles with a surfactant to obtain a suspension of the nanoparticles in an aqueous carrier solution.

[0155] 47. The method according to item 46, wherein the method comprises the following steps:

[0156] i) generating a formulation of lipid nanoparticles or lipid-like nanoparticles by mixing at least one selected from permanent cationic lipids, ionizable lipids, and ionizable lipid-like substances dissolved in an organic phase with a therapeutic agent dissolved in an aqueous solution;

[0157] ii) diluting the lipid nanoparticle or lipid-like nanoparticle formulation by dilution with a first solution;

[0158] iii) concentrating the diluted lipid nanoparticle or lipid-like nanoparticle formulation by buffer exchange using TFF ultrafiltration / diafiltration, wherein the ultrafiltration / diafiltration uses a second solution;

[0159] iv) obtaining a suspension of lipid nanoparticles or lipid-like nanoparticles in an aqueous carrier solution comprising a surfactant;

[0160] Wherein the first solution comprises a surfactant in an amount between 0.01% w / v and 10% w / v, preferably between 0.1% w / v and 10% w / v, more preferably between 0.25% w / v and 5% w / v, still more preferably between 0.33% w / v and 2.5% w / v, even more preferably between 0.45% w / v and 1.5% w / v, and most preferably between 0.5% w / v and 1.5% w / v; and / or,

[0161] Wherein the second solution comprises a surfactant in an amount between 0.01% w / v and 10% w / v, preferably between 0.1% w / v and 10% w / v, more preferably between 0.25% w / v and 5% w / v, still more preferably between 0.33% w / v and 2.5% w / v, even more preferably between 0.45% w / v and 1.5% w / v, and most preferably between 0.5% w / v and 1.5% w / v;

[0162] And wherein, relative to the total volume of the suspension of the nanoparticles in the aqueous carrier solution, the final concentration of the surfactant in the combined first and second solutions is a surfactant in an amount between 0.01% w / v and 10% w / v, preferably between 0.1% w / v and 10% w / v, more preferably between 0.25% w / v and 5% w / v, still more preferably between 0.33% w / v and 2.5% w / v, even more preferably between 0.45% w / v and 1.5% w / v, and most preferably between 0.5% w / v and 1.5% w / v.

[0163] 48. The method according to entry 47, wherein the surfactant is not incorporated into the suspension before or during step i).

[0164] 49. The method according to entry 47 or 48, wherein the surfactant is added together with the first and second solutions.

[0165] 50. The method according to any one of items 47 to 49, based on the total weight of the surfactant in the suspension obtained in step iv), wherein 30 to 70 wt% of the surfactant, preferably 40 to 60 wt%, more preferably 45 to 55 wt% of the surfactant is added together with the first solution, and based on the total weight of the surfactant in the suspension obtained in step iv), 70 to 30 wt% of the surfactant, preferably 60 to 40 wt%, more preferably 55 to 45 wt% of the surfactant is added together with the second solution, such that the sum of the amounts of the surfactant added together with the first and second solutions is 100 wt%.

[0166] 51. The method according to any one of items 47 to 50, wherein approximately half of the surfactant is added together with the first solution, and approximately half of the surfactant is added together with the second solution.

[0167] 52. A lipid nanoparticle or lipid-like nanoparticle suspension (LNP or LiNP suspension), which is obtained by the method according to any one of items 46 to 51.

[0168] 53. A formulation of lipid nanoparticles or lipid-like nanoparticles, which comprises a suspension of lipid nanoparticles or lipid-like nanoparticles according to any one of items 1 to 45 or 52.

[0169] 58. A suspension or formulation of lipid nanoparticles or lipid-like nanoparticles according to any one of items 1 to 45, 52 or 53, which is used for treating or preventing a disease.

[0170] 59. A suspension or formulation of lipid nanoparticles or lipid-like nanoparticles according to any one of items 1 to 45, 52 or 53, which is used as a drug.

[0171] 60. A suspension or formulation of lipid nanoparticles or lipid-like nanoparticles according to any one of items 1 to 45, 52 or 53, which is used for vaccination or immunization.

[0172] 61. A method for inducing an immune response against a target pathogen in a subject in need thereof, the method comprising administering to the subject a formulation comprising a suspension of lipid nanoparticles or lipid-like nanoparticles according to any one of items 1 to 45 or 52.

[0173] 62. A method for reducing the occurrence or severity of one or more side effects associated with an LNP / LiNP-based vaccine in a subject, the method comprising administering to the subject a vaccine formulation or an anti-cancer formulation comprising a suspension of lipid nanoparticles or lipid-like nanoparticles according to any one of items 1 to 45 or 52.

[0174] 63. The method according to entry 62, wherein the occurrence or severity of one or more side effects is reduced by reducing LNP / LiNP aggregation.

[0175] 64. The method according to entry 63, wherein the reduction in aggregation is measured by determining the hydrodynamic diameter of the nanoparticles, for example by dynamic light scattering or photon correlation spectroscopy.

[0176] A further aspect of the invention is outlined in the following second set of entries.

[0177] 1b. Use of a surfactant for stabilizing a suspension of lipid nanoparticles or lipid-like nanoparticles in an aqueous carrier solution against aggregation of the particles under conditions of physical stress, wherein the lipid nanoparticles or lipid-like nanoparticles comprise components (a) and (b) below:

[0178] (a) A therapeutic agent, and

[0179] (b) At least one selected from the group consisting of permanent cationic lipids, ionizable lipids, and ionizable lipid-like compounds.

[0180] 2b. A method for stabilizing a suspension of lipid nanoparticles or lipid-like nanoparticles in an aqueous carrier solution against aggregation of the particles under conditions of physical stress, wherein the lipid nanoparticles or lipid-like nanoparticles comprise components (a) and (b) below:

[0181] (a) A therapeutic agent, and

[0182] (b) At least one selected from the group consisting of permanent cationic lipids, ionizable lipids, and ionizable lipid-like compounds,

[0183] and wherein the method comprises incorporating a surfactant into the suspension of the lipid nanoparticles or lipid-like nanoparticles.

[0184] 3b. The use or method according to entry 1b or 2b, wherein the therapeutic agent is a nucleic acid.

[0185] 4b. The use or method according to entry 3b, wherein the nucleic acid is selected from RNA and plasmid DNA.

[0186] 5b. The use or method according to entry 3b, wherein the nucleic acid is selected from mRNA, siRNA, miRNA, antisense RNA, tRNA, and non-coding RNA, and more preferably is mRNA.

[0187] 6b. For the use or method according to any one of items 3b to 5b, based on the total volume of the suspension, the concentration range of the nucleic acid in the suspension is from 0.01 to 10 mg / mL, more preferably from 0.02 to 10 mg / mL, still more preferably from 0.05 to 5 mg / mL, and most preferably from 0.05 to 2.5 mg / ml.

[0188] 7b. For the use or method according to any one of items 1b to 6b, wherein the weight - volume ratio (in g / L) of the nanoparticles in the aqueous carrier solution is in the range of 0.1 g / L to 300 g / L, more preferably 0.2 g / L to 300 g / L, still more preferably 0.5 g / L to 250 g / L, and most preferably 0.5 g / L to 125 g / L.

[0189] 8b. For the use or method according to any one of items 1b to 7b, wherein the suspended nanoparticles have a Z - average diameter in the range of 10 to 500 nm, more preferably 10 to 250 nm, still more preferably 20 to 200 nm, as measured by dynamic light scattering.

[0190] 9b. For the use or method according to any one of items 1b to 8b, wherein the suspended nanoparticles have a polydispersity index in the range of 0.02 to 0.4, more preferably in the range of 0.03 to 0.2, as measured by dynamic light scattering.

[0191] 10b. For the use or method according to any one of items 1b to 9b, wherein the nanoparticles further comprise one or more of the following components (c1) to (c6):

[0192] (c1) an ionizable lipid having a sterol structure;

[0193] (c2) a phosphoglyceride lipid;

[0194] (c3) a PEG - conjugated lipid;

[0195] (c4) a poly - sarcosine - conjugated lipid;

[0196] (c5) a PAS - modified lipid; and

[0197] (c6) a cationic polymer.

[0198] 11b. For the use or method according to any one of items 1b to 10b, wherein the nanoparticles comprise:

[0199] 30 to 65 mol% of at least one of (b) a permanent cationic lipid, an ionizable lipid, and an ionizable lipidoid, preferably an ionizable lipid or an ionizable lipidoid (b), and one or more of the following components:

[0200] 10 to 50 mol% of a lipid (c1) having a sterol structure,

[0201] 4 to 50 mol% of a phosphoglyceride lipid (c2),

[0202] 0.5 to 10 mol% of one or any combination of a PEG-conjugated lipid (c3), a poly(sarcosine)-conjugated lipid (c4), and a PASylated lipid (c5),

[0203] 0.5 to 10 mol% of a cationic polymer (c6),

[0204] such that the sum of (b) and (c1) to (c6) is equal to 100 mol%.

[0205] 12b. Use or method according to any one of entries 1b to 11b, wherein the nanoparticles further comprise the following components (c1) to (c3):

[0206] (c1) an ionizable lipid having a sterol structure;

[0207] (c2) a phosphoglyceride lipid; and

[0208] (c3) a PEG-conjugated lipid.

[0209] 13b. Use or method according to entry 12b, wherein the nanoparticles comprise:

[0210] 30 to 65 mol% of at least one of a permanently cationic lipid, an ionizable lipid, and an ionizable lipidoid (b), preferably an ionizable lipid or an ionizable lipidoid (b),

[0211] 10 to 50 mol% of a lipid (c1) having a sterol structure,

[0212] 4 to 50 mol% of a phosphoglyceride lipid (c2), and

[0213] 0.5 to 10 mol% of a PEG-conjugated lipid (c3),

[0214] such that the sum of (b) and (c1) to (c3) is equal to 100 mol%.

[0215] 14b. Use or method according to any one of entries 3b to 13b, wherein the nanoparticles further comprise a polyanionic component different from nucleic acid.

[0216] 15b. A use or method according to any one of clauses 3b to 14b, wherein the composition of the nanoparticles is such that the sum of the weights of the components other than the nucleic acid in the nanoparticles and the weight of the nucleic acid have a weight ratio in the range of 50:1 to 1:1, more preferably 40:1 to 2:1, and most preferably 30:1 to 3:1.

[0217] 16b. A use or method according to any one of clauses 1b to 15b, wherein the nanoparticles comprise an ionizable lipid (b) of the following formula (b-1),

[0218]

[0219] wherein:

[0220] a is 1 and b is an integer from 2 to 4; or a is an integer from 2 to 4 and b is 1,

[0221] p is 1 or 2,

[0222] m is 1 or 2; n is 0 or 1, and m + n ≥ 2; and

[0223] R 1A to R 6A are each independently selected from: hydrogen; -CH 2 -CH(OH)-R 7A 、-CH(R 7A )-CH 2 -OH、-CH 2 -CH 2 -(C=O)-O-R 7A 、-CH 2 -CH 2 -(C=O)-NH-R 7A ;-CH 2 -R 7A ;-C(NH)-NH 2 ;a poly(ethylene glycol) chain; and a receptor ligand; where R 7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond;

[0224] provided that at least two residues among R 1A to R 6A are selected from -CH 2 -CH(OH)-R 7A 、-CH(R 7A )-CH 2 -OH、-CH 2 -CH 2 -(C=O)-O-R 7A 、-CH 2 -CH 2-(C=O)-NH-R 7A and -CH 2 -R 7A , wherein R 7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond;

[0225] or its protonated form, wherein one or more of the nitrogen atoms contained in the compound of formula (b-1) are protonated to provide a compound carrying a positive charge.

[0226] 17b. The use or method according to any one of entries 1b to 16b, wherein the nanoparticles comprise an ionizable lipid (b-1) of the following formula (b-1b),

[0227]

[0228] wherein R 1A to R 6A are as defined in entry 16,

[0229] or its protonated form, wherein one or more of the nitrogen atoms contained in the compound of formula (b-1b) are protonated to provide a compound carrying a positive charge.

[0230] 18b. The use or method according to entry 16b or 17b, wherein R 1A to R 6A are independently selected from hydrogen and -CH 2 -CH(OH)-R 7A , wherein R 7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond, provided that at least two residues, preferably at least three residues and more preferably at least four residues among R 1A to R 6A are -CH 2 -CH(OH)-R 7A , wherein R 7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond.

[0231] 19b. The use or method according to any one of entries 1b to 18b, wherein the nanoparticles comprise an ionizable lipid dL_05(R) having the following formula:

[0232]

[0233] 20b. The use or method according to any one of entries 1b to 15b, wherein the nanoparticles comprise an ionizable lipid (b) of the following formula (a-III):

[0234]

[0235] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0236] L 1 or L 2 one of which is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O-; and L 1 or L 2 the other of which is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O- or a direct bond;

[0237] G 1 and G 2 each independently is C 1 -C 12 alkylene or C 1 -C 12 alkenylene;

[0238] G 3 is C 1 -C 24 alkylene, C 1 -C 24 alkenylene, C 3 -C 8 cycloalkylene, C 3 -C 8 cycloalkenylene, wherein the alkylene, alkenylene, cycloalkylene and cycloalkenylene are each optionally substituted;

[0239] R a is H or C 1 -C 12 alkyl, wherein the alkyl is optionally substituted;

[0240] R 1 and R 2 are each independently C 6 -C 24 -alkyl or C 6 -C 24 -alkenyl, wherein the alkyl and alkenyl are each optionally substituted;

[0241] R 3 is H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 is C 1 -C 12 -alkyl, wherein the alkyl is optionally substituted;

[0242] R 5 is H or C 1 -C 6 -alkyl, wherein the alkyl is optionally substituted; and

[0243] x is 0, 1 or 2.

[0244] 21b. Use or method according to any one of entries 1b to 15b, wherein the nanoparticles comprise (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butyrate or its protonated form as the ionizable lipid (b), wherein the nitrogen atom of the compound is protonated to provide a positively charged compound.

[0245] 22b. Use or method according to any one of entries 1b to 15b, wherein the nanoparticles comprise ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) or its protonated form as the ionizable lipid (b), wherein the nitrogen atom of the compound is protonated to provide a positively charged compound.

[0246] 23b. Use or method according to any one of entries 1b to 15b or 22b, wherein the nanoparticles comprise octadec-9-yl 8-[(2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino]octanoate (SM-102) or its protonated form as the ionizable lipid (b), wherein the nitrogen atom of the compound is protonated to provide a positively charged compound.

[0247] 24b. Use or method according to any one of entries 10b to 23b, wherein the non-ionizable lipid (c1) having a sterol structure comprises a non-ionizable lipid of formula (c1-1):

[0248]

[0249] wherein R 1L is a C3-C12 alkyl group.

[0250] 25b. A use or method according to any one of entries 10b to 24b, wherein the non-ionizable lipid (c1) having a sterol structure includes cholesterol.

[0251] 26b. A use or method according to any one of entries 10b to 25b, wherein the phosphatidylglycerol lipid (c2) includes a phosphatidylglycerol lipid of formula (c2-1),

[0252]

[0253] wherein,

[0254] R 1F and R 2F are independently a C8-C18 alkyl group or a C8-C18 alkenyl group, preferably a C12-C18 alkyl group or a C12-C18 alkenyl group,

[0255] or a pharmaceutically acceptable salt thereof;

[0256] or a phosphatidylglycerol lipid of formula (c2-2),

[0257]

[0258] wherein,

[0259] R 1G and R 2G are independently a C8-C18 alkyl group or a C8-C18 alkenyl group, preferably a C12-C18 alkyl group or a C12-C18 alkenyl group,

[0260] or a pharmaceutically acceptable salt thereof.

[0261] 27b. A use or method according to any one of entries 10b to 26b, wherein the phosphatidylglycerol lipid (c2) includes 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or a pharmaceutically acceptable salt thereof or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a pharmaceutically acceptable salt thereof.

[0262] 28b. A use or method according to any one of entries 10b to 27b, wherein the PEG-conjugated lipid (c3) includes a PEG-conjugated lipid of formula (c3-1),

[0263]

[0264] wherein,

[0265] R 1H and R 2H are independently C8-C18 alkyl or C8-C18 alkenyl, preferably C12-C18 alkyl or C12-C18 alkenyl, and p is an integer from 5 to 200, preferably from 10 to 100, and more preferably from 20 to 60;

[0266] or a PEG-conjugated lipid of formula (c3-2),

[0267]

[0268] wherein,

[0269] R 1J and R 2J are independently C8-C18 alkyl or C8-C18 alkenyl, preferably C12-C18 alkyl or C12-C18 alkenyl, and q is an integer from 5 to 200, preferably from 10 to 100, and more preferably from 20 to 60,

[0270] or a pharmaceutically acceptable salt thereof;

[0271] or a PEG-conjugated lipid of formula (c3-3),

[0272]

[0273] wherein,

[0274] R 1K and R 2K are independently C8-C18 alkyl or C8-C18 alkenyl, preferably C12-C18 alkyl or C12-C18 alkenyl, and q is an integer from 5 to 200, preferably from 10 to 100, and more preferably from 20 to 60.

[0275] 29b. Use or method according to items 10b to 28b, wherein the PEG-conjugated lipid (c3) comprises 1,2-dimyristoyl-sn-glycero methoxy(polyethylene glycol)-2000 (DMG-PEG2k) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylethanamide (ALC-0159).

[0276] 30b. Use or method according to item 22b, wherein the nanoparticles comprise ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) or its protonated form as the ionizable lipid (b), wherein the nitrogen atom of the compound is protonated to provide a positively charged compound, and further comprises one or more of the following components (d1) to (d8):

[0277] (d1) 2-[(Polyethylene glycol)-2000]-N,N-ditetradecylethanamide (ALC-0159);

[0278] (d2) 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC);

[0279] (d3) Cholesterol;

[0280] (d4) Potassium chloride;

[0281] (d5) Potassium dihydrogen phosphate;

[0282] (d6) Sodium chloride;

[0283] (d7) Disodium hydrogen phosphate dihydrate;

[0284] (d8) Sucrose.

[0285] 31b. The use or method according to item 23b, wherein the nanoparticles comprise 9-octadecanyl 8-[(2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino]octanoate (SM-102) or its protonated form as an ionizable lipid (b), wherein the nitrogen atom of the compound is protonated to provide a positively charged compound, and further comprises one or more of the following components (e1) to (e7):

[0286] (e1) 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC);

[0287] (e2) Cholesterol;

[0288] (e3) 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000 DMG);

[0289] (e4) Tromethamol hydrochloride;

[0290] (e5) Sodium acetate trihydrate;

[0291] (e6) Acetic acid;

[0292] (e7) Sucrose.

[0293] 32b. The use or method according to any one of items 1b to 31b, wherein the N / P ratio in the nanoparticles is in the range of 0.5 to 20, more preferably in the range of 0.5 to 10.

[0294] 33b. A use or method according to any one of items 1b to 32b, wherein the physical stress conditions are selected from shaking, stirring, vibrating, mixing, inverting, tapping or dripping the nanoparticle suspension, or any combination thereof, or physical stress conditions caused by pumping the nanoparticle suspension or drawing it into a syringe.

[0295] 34b. A use or method according to any one of items 1b to 33b, wherein a surfactant is incorporated into the aqueous carrier solution as an excipient.

[0296] 35b. A use or method according to any one of items 1b to 34b, wherein the surfactant is substantially not attached to the nanoparticles.

[0297] 36b. A use or method according to any one of items 1b to 35b, wherein the surfactant is a nonionic surfactant.

[0298] 37b. A use or method according to item 36b, wherein the nonionic surfactant is at least one selected from the group consisting of fatty alcohol ethoxylates, fatty acid ethoxylates, block copolymers of ethylene oxide and propylene oxide, alkylphenol ethoxylates or oligomers of alkylphenol ethoxylates, sorbitan fatty acid esters, ethoxylated sorbitan fatty acid esters, glycerol fatty acid esters, ethoxylated castor oil, and ethoxylated vitamin E.

[0299] 38b. A use or method according to item 37b, wherein the block copolymer of ethylene oxide and propylene oxide is poloxamer.

[0300] 39b. A use or method according to item 38b, wherein the poloxamer comprises a poly(propylene oxide) block B of formula (p-1):

[0301]

[0302] wherein s is an integer from 15 to 60, and

[0303] two poly(ethylene oxide) blocks A of formula (p-2):

[0304]

[0305] wherein r is independently for each block an integer from 8 to 150, preferably from 10 to 150.

[0306] 40b. A use or method according to item 36b, wherein the nonionic surfactant is at least one selected from the group consisting of fatty alcohol ethoxylates, fatty acid ethoxylates, alkylphenol ethoxylates or oligomers of alkylphenol ethoxylates, sorbitan fatty acid esters, ethoxylated sorbitan fatty acid esters, glycerol fatty acid esters, ethoxylated castor oil, and ethoxylated vitamin E.

[0307] 41b. A use or method according to entry 36b or 37b, wherein the nonionic surfactant is at least one selected from the group consisting of poloxamer 124, poloxamer 188, poloxamer 338, poloxamer 407, polysorbate 20, polysorbate 80, laureth, polyoxyethylene-35 castor oil, D-α-tocopheryl polyethylene glycol 1000 succinate, and tyloxapol.

[0308] 42b. A use or method according to entry 36b or 37b, wherein the nonionic surfactant is at least one selected from the group consisting of laureth, polyoxyethylene-35 castor oil, D-α-tocopheryl polyethylene glycol 1000 succinate, and tyloxapol.

[0309] 43b. A use or method according to any one of entries 1b to 41b, wherein the surfactant does not include poloxamer 188.

[0310] 44b. A use or method according to any one of entries 1b to 43b, wherein the surfactant does not include poloxamer 407.

[0311] 45b. A use according to any one of entries 1b to 44b, wherein, relative to the total volume of the suspension of the nanoparticles in the aqueous carrier solution, the suspension of the lipid nanoparticles or lipid-like nanoparticles in the aqueous carrier solution comprises a surfactant at a concentration of 0.01 to 10% (w / v), preferably 0.1 to 10% (w / v), more preferably 0.25 to 5% (w / v), still more preferably 0.33 to 2.5% (w / v), even more preferably 0.45 to 1.5% (w / v), and most preferably 0.5 to 1.5% (w / v).

[0312] 46b. A use according to entry 45b, wherein, relative to the total volume of the suspension of the nanoparticles in the aqueous carrier solution, the suspension of the nanoparticles comprises a surfactant at a concentration of 0.5 to 1.5% (w / v).

[0313] 47b. A use according to any one of entries 1b to 46b, wherein the nanoparticles have not been lyophilized.

[0314] 48b. A use according to any one of entries 1b to 46b, wherein the surfactant is not present in the carrier solution during the lyophilization process.

[0315] 49b. A use according to any one of entries 1b to 46b, wherein the surfactant is added before the lyophilization process.

[0316] 50b. A method according to any one of items 2b to 44b, wherein a surfactant is incorporated into a suspension of lipid nanoparticles or lipid-like nanoparticles to achieve a surfactant concentration of 0.01 to 10% (w / v), preferably 0.1 to 10% (w / v), more preferably 0.25 to 5% (w / v), still more preferably 0.33 to 2.5% (w / v), even more preferably 0.45 to 1.5% (w / v), and most preferably 0.5 to 1.5% (w / v) relative to the total volume of the suspension of the nanoparticles in the aqueous carrier solution.

[0317] 51b. A method according to item 50b, wherein a surfactant is incorporated into a suspension of the nanoparticles to achieve a surfactant concentration of 0.5 to 1.5% (w / v) relative to the total volume of the suspension of the nanoparticles in the aqueous carrier solution.

[0318] 52b. A method according to any one of items 2b to 44b, 50b or 51b, which does not include a lyophilization step.

[0319] 53b. A method according to any one of items 2b to 44b, 50b or 51b, wherein the surfactant is not present in the carrier solution during the lyophilization step.

[0320] 54b. A method according to any one of items 2b to 44b, 50b or 51b, wherein the surfactant is added before the lyophilization step.

[0321] 55b. A method according to any one of items 2b to 44b, 50b or 51b, wherein the method comprises the steps of:

[0322] i) generating a formulation of lipid nanoparticles or lipid-like nanoparticles by mixing at least one selected from the group consisting of permanent cationic lipids, ionizable lipids and ionizable lipid-like substances dissolved in an organic phase with a therapeutic agent dissolved in an aqueous solution;

[0323] ii) diluting the formulation of lipid nanoparticles or lipid-like nanoparticles by dilution with a first solution;

[0324] iii) concentrating the diluted formulation of lipid nanoparticles or lipid-like nanoparticles by buffer exchange using TFF ultrafiltration / diafiltration, wherein the ultrafiltration / diafiltration uses a second solution;

[0325] iv) obtaining a suspension of lipid nanoparticles or lipid-like nanoparticles in an aqueous carrier solution;

[0326] Wherein the first solution comprises a surfactant in an amount between 0.01% w / v and 10% w / v, preferably between 0.1% w / v and 10% w / v, more preferably between 0.25% w / v and 5% w / v, still more preferably between 0.33% w / v and 2.5% w / v, even more preferably between 0.45% w / v and 1.5% w / v, and most preferably between 0.5% w / v and 1.5% w / v; and / or

[0327] Wherein the second solution comprises a surfactant in an amount between 0.01% w / v and 10% w / v, preferably between 0.1% w / v and 10% w / v, more preferably between 0.25% w / v and 5% w / v, still more preferably between 0.33% w / v and 2.5% w / v, even more preferably between 0.45% w / v and 1.5% w / v, and most preferably between 0.5% w / v and 1.5% w / v;

[0328] And wherein, relative to the total volume of the suspension of the nanoparticles in the aqueous carrier solution, the final concentration of the surfactant in the combined first and second solutions is a surfactant in an amount between 0.01% w / v and 10% w / v, preferably between 0.1% w / v and 10% w / v, more preferably between 0.25% w / v and 5% w / v, still more preferably between 0.33% w / v and 2.5% w / v, even more preferably between 0.45% w / v and 1.5% w / v, and most preferably between 0.5% w / v and 1.5% w / v.

[0329] 56b. The method according to entry 55b, wherein the surfactant is not incorporated into the suspension before or during step i).

[0330] 57b. The method according to entry 55b or 56b, wherein the surfactant is added together with the first and second solutions.

[0331] 58b. A method according to any one of items 55b to 57b, wherein based on the total weight of the surfactant in the suspension obtained in step iv), 30 to 70 wt% of the surfactant, preferably 40 to 60 wt%, more preferably 45 to 55 wt% of the surfactant is added together with the first solution, and based on the total weight of the surfactant in the suspension obtained in step iv), 70 to 30 wt% of the surfactant, preferably 60 to 40 wt%, more preferably 55 to 45 wt% of the surfactant is added together with the second solution, such that the sum of the amounts of surfactant added together with the first and second solutions is 100 wt%.

[0332] 59b. A method according to any one of items 55b to 58b, wherein approximately half of the surfactant is added together with the first solution and approximately half of the surfactant is added together with the second solution.

[0333] 60b. A suspension of lipid nanoparticles or lipid-like nanoparticles (LNP or LiNP suspension) obtained by a method according to any one of items 55b to 59b.

[0334] 61b. A suspension of lipid nanoparticles or lipid-like nanoparticles according to item 60b for treating or preventing a disease.

[0335] 62b. A suspension of lipid nanoparticles or lipid-like nanoparticles according to item 60b for use as a medicament.

[0336] 63b. A suspension of lipid nanoparticles or lipid-like nanoparticles according to item 60b for vaccination or immunization.

[0337] 64b. A method for avoiding side effects in the treatment using lipid nanoparticles or lipid-like nanoparticles carrying at least one therapeutic agent, the method comprising the steps of:

[0338] i) determining whether the lipid nanoparticles or lipid-like nanoparticles in the pharmaceutical composition aggregate when subjected to the mechanical stress or temperature stress by determining their aggregation levels before and after the pharmaceutical composition comprising the lipid nanoparticles or lipid-like nanoparticles is subjected to the mechanical stress or temperature stress;

[0339] ii) If the lipid nanoparticles or lipid-like nanoparticles show aggregation after the test in step (i), a surfactant is added to the lipid nanoparticle or lipid-like nanoparticle formulation to obtain an LNP or LiNP suspension with a final surfactant concentration between 0.01% w / v and 10% w / v, preferably between 0.1% w / v and 10% w / v, more preferably between 0.25% w / v and 5% w / v, still more preferably between 0.33% w / v and 2.5% w / v, even more preferably between 0.45% w / v and 1.5% w / v, and most preferably between 0.5% w / v and 1.5% w / v;

[0340] iii) Mix and reconstitute to generate a stable lipid nanoparticle or lipid-like nanoparticle suspension.

[0341] 65b. A method for reducing one or more side effects associated with a vaccine formulation or an anti-cancer formulation comprising lipid nanoparticles or lipid-like nanoparticles carrying nucleic acids, the method comprising modifying the vaccine formulation or the anti-cancer formulation by adding a surfactant to the vaccine formulation or the anti-cancer formulation comprising a lipid nanoparticle or lipid-like nanoparticle suspension.

[0342] 66b. The method according to entry 65b, wherein the surfactant is between 0.01% w / v and 10% w / v, preferably between 0.1% w / v and 10% w / v, more preferably between 0.25% w / v and 5% w / v, still more preferably between 0.33% w / v and 2.5% w / v, even more preferably between 0.45% w / v and 1.5% w / v, and most preferably between 0.5% w / v and 1.5% w / v.

[0343] 67b. The method according to entry 66b, wherein the surfactant is at least one selected from the group consisting of fatty alcohol ethoxylates, fatty acid ethoxylates, block copolymers of ethylene oxide and propylene oxide, alkylphenol ethoxylates or oligomers of alkylphenol ethoxylates, sorbitan fatty acid esters, ethoxylated sorbitan fatty acid esters, glycerol fatty acid esters, ethoxylated castor oil, and ethoxylated vitamin E, preferably at least one selected from the group consisting of poloxamer 124, poloxamer 188, poloxamer 338, poloxamer 407, polysorbate 20, polysorbate 80, laureth, polyoxyethylene-35 castor oil, D-α-tocopheryl polyethylene glycol 1000 succinate, and tyloxapol.

[0344] 68b. The method according to entry 67b, wherein the surfactant is a block copolymer of ethylene oxide and propylene oxide, preferably a poloxamer selected from the group consisting of poloxamer 124, poloxamer 188, poloxamer 338, and poloxamer 407.

[0345] 69b. The method according to entry 67b, wherein the poloxamer is not poloxamer 188 or poloxamer 407.

[0346] 70b. A method of inducing an immune response against a target pathogen in a subject in need thereof, the method comprising administering to the subject a formulation comprising a suspension of the lipid nanoparticles or lipid-like nanoparticles as described in any one of entries 1b to 49b or 60b.

[0347] 71b. A method of reducing the occurrence or severity of one or more side effects associated with an LNP / LiNP-based vaccine in a subject, the method comprising administering to the subject a vaccine formulation or an anti-cancer formulation comprising a suspension of the lipid nanoparticles or lipid-like nanoparticles as described in any one of entries 1b to 49b or 60b.

[0348] 72b. The method according to entry 71b, wherein the reduction in the occurrence or severity of one or more side effects is caused by a reduction in LNP / LiNP aggregation.

[0349] 73b. The method according to entry 72b, wherein the reduction in aggregation is measured by determining the hydrodynamic diameter of the nanoparticles, for example by dynamic light scattering or photon correlation spectroscopy.

[0350] It should be understood that the above summaries in the entries form part of the general disclosure of the present invention, such that the information provided in the following detailed description, for example, information regarding further preferred embodiments or optional features, also applies to the above entries, and vice versa. Description of the Drawings

[0351] Figure 1 shows the particle size distribution (A) and polydispersity index (B) of LNP before (black bars) and after (white bars) shaking in the presence of different concentrations of excipients.

[0352] Figure 2 shows the particle size distribution (A) and polydispersity index (B) of LNP at different concentrations before (black bars) and after (white bars) shaking in the presence of 1% (w / v) poloxamer 188.

[0353] Figure 3 shows the particle size distribution (A) and polydispersity index (B) of MC3-LNP before (black bars) and after (white bars) shaking in the presence of different concentrations of excipients.

[0354] Figure 4 shows the particle size distribution (A) and polydispersity index (B) of ALC-0315 LNP before (black bars) and after (white bars) shaking in the presence of different concentrations of excipients.

[0355] Figure 5Shows the particle size distribution of LNP before (black bars) and after (white bars) shaking in the presence of excipients at different concentrations. The particle size distribution of LNP before (black bars) and after (white bars) shaking.

[0356] Figure 6 Shows the particle size distribution and polydispersity index of LNP before (black bars) and after (white bars) shaking in the presence of 1% (w / v) Kolliphor P188. The particle size distribution and polydispersity index of LNP before (black bars) and after (white bars) shaking.

[0357] Figure 7 Shows the size exclusion chromatography of LNP formulated with poloxamer P188 (lipids based on protocol 1). The X-axis corresponds to the elution volume in mL, and the Y-axis represents the UV 260 nm signal.

[0358] Figure 8 Shows the HPLC calibration curves for 0.2, 0.5, and 1 mg / mL poloxamer P188.

[0359] Figure 9 Shows the HPLC calibration curves for 3, 5, and 7 mg / mL poloxamer P188.

[0360] Figure 10 Shows the HPLC calibration curve for poloxamer P188.

[0361] Figure 11 Shows the signal comparison between a reference sample containing 0.5 mg / mL poloxamer and fraction 9 containing LiNP.

[0362] Figure 12 Shows the HPLC chromatograms of fraction 23 and fraction 24 compared with a 0.5 mg / mL poloxamer P188 reference sample.

[0363] Figure 13 Shows the HPLC chromatograms of fraction 25 and fraction 26 compared with a 0.5 mg / mL poloxamer P188 reference sample. Detailed Description

[0364] Unless otherwise indicated in any specific context, the following explanations, for example, regarding therapeutic agents, lipid nanoparticles or lipid-like nanoparticles or surfactants, apply to all aspects of the present invention.

[0365] For the sake of discussion, lipid nanoparticles ("LNPs") or lipid-like nanoparticles ("LiNPs") may be collectively referred to herein as "nanoparticles". Similarly, a suspension of lipid nanoparticles or lipid-like nanoparticles in an aqueous carrier solution, wherein the lipid nanoparticles or lipid-like nanoparticles comprise (a) a nucleic acid and (b) at least one of a permanent cationic lipid, an ionizable lipid, and an ionizable lipid-like, may be referred to herein simply as a "nanoparticle suspension". It should be understood that, unless otherwise stated, the "or" used when referring to lipid nanoparticles or lipid-like nanoparticles does not have an exclusive meaning. Thus, a suspension of lipid nanoparticles or lipid-like nanoparticles may comprise lipid nanoparticles but not lipid-like nanoparticles, comprise lipid-like nanoparticles but not lipid nanoparticles, or comprise both lipid nanoparticles and lipid-like nanoparticles. For example, they may be represented by the abbreviation LNP / LiNP.

[0366] The nanoparticles in the suspension and their components will be explained below. Unless otherwise specifically stated to the contrary, the term "nanoparticles" as used herein includes lipid nanoparticles (also referred to as LNPs) and lipid-like nanoparticles (also referred to as LiNPs). According to aspects of the present invention, the nanoparticles of the nanoparticle suspension comprise: (a) a therapeutic agent and (b) at least one selected from the group consisting of a permanent cationic lipid, an ionizable lipid, and an ionizable lipid-like. Thus, if the suspension in the context of the present invention comprises only LNPs and not LiNPs, both components (a) and (b) are included in the LNPs. If the suspension comprises only LiNPs and not LNPs, components (a) and (b) are included in the LiNPs. If the suspension in the context of the present invention comprises both LNPs and LiNPs, both the LNPs and the LiNPs generally include components (a) and (b).

[0367] As component (a), the nanoparticles comprise a therapeutic agent. The therapeutic agent is preferably a nucleic acid, which thus generally provides the pharmaceutically active ingredient of the nanoparticles.

[0368] The nature of the nucleic acid is not particularly limited. In principle, any type of nucleic acid may be used in the context of the present invention. Nucleic acids are known to those skilled in the art and refer to biopolymers or small biomolecules composed of nucleotides, which are monomers consisting of the following three components: a 5-carbon sugar, a phosphate group, and a nitrogenous base.

[0369] The term nucleic acid refers to the collective name of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), that is, members of the above-mentioned family of biopolymers. If the sugar is complex ribose, the polymer is RNA; if the sugar is derived from ribose in the form of deoxyribose, the polymer is DNA. The term "nucleic acid" includes oligonucleotides or polynucleotides. Since nucleic acids are biopolymers composed of nucleotides, the term "nucleic acid" is also often referred to as "nucleotide sequence". Therefore, as understood by those skilled in the art, the terms "nucleic acid" and "nucleic acid sequence" are often used interchangeably.

[0370] In a preferred embodiment, the nanoparticles comprise ribonucleic acid (RNA) as the nucleic acid, more preferably single-stranded RNA, and most preferably mRNA.

[0371] The term "nucleic acid" includes all forms of naturally occurring types of nucleic acids as well as chemically and / or enzymatically synthesized nucleic acids, and also includes nucleic acid analogs and nucleic acid derivatives. The term specifically includes any single-stranded or double-stranded nucleic acid with backbone modifications, sugar modifications or base modifications, such as, for example, locked nucleic acid (LNA), peptide nucleic acid (PNA), oligonucleoside phosphorothioates and phosphotriesters, morpholino oligonucleotides, cationic oligonucleotides (US6017700A, WO / 2007 / 09092), substituted ribooligonucleotides or phosphorothioates. In addition, the term "nucleic acid" also refers to any molecule containing nucleotides or nucleotide analogs. There is no limitation on the sequence or size of the nucleic acid contained in the nanoparticles of the present invention. The nucleic acid is mainly defined by the biological effect to be achieved at the biological target to which the nanoparticles of the present invention are delivered. For example, as will be outlined in more detail below, in the case of application to gene or nucleic acid therapy, the nucleic acid or nucleic acid sequence can be defined by the gene or gene fragment to be expressed or by the expected replacement or repair of a defective gene or any gene target sequence or by the target sequence of a gene to be inhibited, knocked down, down-regulated or up-regulated.

[0372] The nanoparticles in the suspension can comprise a nucleic acid that is a DNA molecule. A preferred embodiment of such a DNA molecule is a DNA molecule that can be transcribed into an mRNA molecule. Transcription is the first step of gene expression, in which a specific fragment of the DNA molecule is copied into an mRNA molecule by the enzyme RNA polymerase. During transcription, the DNA sequence is read by RNA polymerase, which produces a complementary, antiparallel RNA strand called the primary transcript.

[0373] DNA molecules can be introduced into a vector, preferably an expression vector, by standard molecular biological techniques (see, e.g., Sambrook et al., Molecular Cloning, A laboratory manual, 2nd Ed, 1989). The term "vector", such as an "expression vector" or "cloning vector" in the sense of the present invention, is understood as a circular double-stranded unit of DNA which is preferably capable of replicating independently of chromosomal DNA within a cell and serves as a vehicle for carrying genetic material into a cell, where it can be (replicated and / or) expressed (i.e., transcribed into RNA and translated into an amino acid sequence). A vector containing foreign DNA is called recombinant DNA. A vector is usually a DNA sequence which generally consists of an insert (e.g., a nucleic acid molecule / DNA molecule of the present invention) and a larger sequence serving as the "backbone" of the vector. Plasmids in the sense of the present invention are most commonly found in bacteria and are used in recombinant DNA research to transfer genes between cells and are a subgroup of the "vectors" used in the sense of the present invention.

[0374] It will be apparent to the person skilled in the art that further regulatory sequences can be added to the DNA molecules of the present invention. For example, transcriptional enhancers and / or sequences which allow inducible expression can be used. Suitable inducible systems are, for example, tetracycline-regulated gene expression, as described, e.g., by Gossen and Bujard, Proc. Natl. Acad. Sci. USA 89 (1992), 5547-5551) and Gossen, Trends Biotech. 12 (1994), 58-62, or the dexamethasone-inducible gene expression system, as described, e.g., by Crook, EMBO J. 8 (1989), 513-519. The present invention can also use vectors containing the DNA molecules, preferably expression vectors. The vector can be, for example, a plasmid, cosmid, virus, phage or another vector conventionally used in genetic engineering and can include further genes, such as marker genes, which allow selection of the vector in a suitable host cell and under suitable conditions.

[0375] If the nucleic acid used in the context of the present invention is a DNA molecule, it can be a plasmid DNA (pDNA) molecule.

[0376] As described above, the nanoparticles preferably comprise ribonucleic acid (RNA) as the nucleic acid, more preferably single-stranded RNA, and most preferably mRNA.

[0377] Regarding RNA, in principle any type of RNA can be employed in the context of the present invention. In a preferred embodiment, the RNA is single-stranded RNA. The term "single-stranded RNA" refers to a single continuous chain of ribonucleotides, as opposed to an RNA molecule in which two or more separate chains form a double-stranded molecule due to hybridization of the separate chains. The term "single-stranded RNA" does not exclude that the single-stranded molecule itself forms double-stranded structures, such as secondary structures (e.g., loops and stem-loops) or tertiary structures. Examples are tRNA and mRNA, but also include any other type of single-stranded RNA, such as antisense RNA, siRNA, miRNA, etc.

[0378] The term "RNA" includes RNA that encodes an amino acid sequence and RNA that does not encode an amino acid sequence. It has been proposed that more than 80% of the genome contains functional DNA elements that do not encode proteins. These non-coding sequences include regulatory DNA elements (binding sites for transcription factors, regulators, and co-regulators, etc.) and sequences that encode transcripts that are never translated into proteins. These transcripts encoded by the genome and transcribed into RNA but not translated into proteins are called non-coding RNAs (ncRNAs). Thus, in one embodiment, the RNA is a non-coding RNA. Preferably, the non-coding RNA is a single-stranded molecule. Studies have shown that ncRNAs play key roles in gene regulation, maintenance of genomic integrity, cell differentiation and development, and are misregulated in various human diseases. There are different types of ncRNAs: short (20-50 nt), medium (50-200 nt), and long (>200 nt) ncRNAs. Short ncRNAs include microRNAs (miRNAs), small interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs), and transcription initiation RNAs (tiRNAs). Examples of medium ncRNAs are small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), transfer RNAs (tRNAs), transcription start site-associated RNAs (TSSaRNAs), promoter-associated small RNAs (PASRs), and promoter upstream transcripts (PROMPTs). Long non-coding RNAs (lncRNAs) include long intergenic non-coding RNAs (lincRNAs), antisense IncRNAs, intronic IncRNAs, and transcribed ultra-conserved RNAs (T-UCRs), etc. (Bhan A, Mandal SS, ChemMedChem. 2014 Mar 26. doi:10.1002 / cmdc.201300534). Among the above non-coding RNAs, only siRNAs are double-stranded. Thus, since in a preferred embodiment, the non-coding RNA is single-stranded, it is preferred that the non-coding RNA is not an siRNA. In another embodiment, the RNA is coding RNA, i.e., RNA that encodes an amino acid sequence. Such RNA molecules are also called mRNAs (messenger RNAs) and are single-stranded RNA molecules. RNAs can be made by chemical synthesis and enzymatic methods known to those of ordinary skill in the art, or by using recombinant techniques, or can be isolated from natural sources, or made by a combination thereof.

[0379] Messenger RNA (mRNA) is a copolymer composed of phosphoronucleotide building blocks mainly of adenosine, cytidine, uridine, and guanosine as nucleosides, which serves as an intermediate carrier to bring the genetic information of DNA in the nucleus into the cytoplasm, where it is translated into proteins. Thus, they are suitable as substitutes for gene expression.

[0380] In the context of the present invention, mRNA shall be understood to mean any polynucleotide molecule which, if introduced into a cell, is suitable for the expression of a protein or a fragment thereof or is translatable into a protein or a fragment thereof. The term "protein" herein includes any kind of amino acid sequence, i.e., a chain of two or more amino acids, each amino acid being linked by a peptide bond and also includes peptides and fusion proteins.

[0381] The mRNA contains a ribonucleotide sequence which encodes a protein or a fragment thereof that is essential or beneficial for its function in or near a cell, e.g., a protein whose absence or defective form is the cause of a disease or disorder, the provision of which protein can mitigate or prevent the disease or disorder, or a protein that can promote a process beneficial to the body in or near a cell. The mRNA can contain the sequence of a complete protein or a functional variant thereof. Further, the ribonucleotide sequence can encode a protein which is a factor, inducer, regulator, stimulator or enzyme, or a functional fragment thereof, wherein the protein is such that its function is essential for treating a disorder, in particular a metabolic disorder, or initiating a process in the body, such as the formation of new blood vessels, tissues, etc. Examples of proteins that can be encoded by mRNA include antibodies, cytokines or chemokines. Herein, a functional variant is understood to mean a fragment that can assume the function of a protein in a cell, the function of which protein in the cell is essential, or whose absence or defective form is pathogenic. In addition, the mRNA can also have further functional regions and / or 3' or 5' untranslated regions, in particular 3' and / or 5' UTRs. The 3' and / or 5' untranslated regions can be regions that are naturally located on either side of a protein-coding sequence or an artificial sequence, e.g., sequences that contribute to RNA stability. A person skilled in the art can determine the sequences suitable therefor in each case by routine experiments.

[0382] In a preferred embodiment, the mRNA contains a 5'-cap (five-prime-cap; cap-0) which consists of m7GpppG linked to the mRNA by a 5' to 5' triphosphate bond, an additional methyl group on the second nucleotide from the 5'-end of the mRNA (cap-1, anti-reverse cap analogue (ARCA)) and / or an internal ribosome entry site (IRES) and / or a polyA tail at the 3'-end, in particular in order to improve translation. The mRNA can have further regions that promote translation, such as, for example, a cap-2 structure or a histone stem-loop structure.

[0383] RNA that can be present in nanoparticles can contain unmodified and modified nucleotides. As used herein, the term "unmodified nucleotide" refers to A, C, G, and U nucleotides. As used herein, the term "modified nucleotide" refers to any naturally occurring or non-naturally occurring isomer of A, C, G, and U nucleotides, as well as any naturally occurring or naturally occurring analog, alternative, or modified nucleotide or its isomer having, for example, a chemical modification or a substituted residue. Modified nucleotides can have base modifications and / or sugar modifications. Modified nucleotides can also have phosphate group modifications, e.g., relative to the 5'-primer cap of an mRNA molecule. Modified nucleotides also include nucleotides synthesized post-transcriptionally by covalently modifying nucleotides. Further, any suitable mixture of unmodified and modified nucleotides is possible. Non-limiting examples of the number of modified nucleotides can be found in the literature (e.g., US2013 / 0123481A1; Cantara et al., Nucleic Acids Res, 2011, 39(Issue suppl_1):D195-D201; Helm and Alfonzo, Chem Biol, 2014, 21(2):174-185; or Carell et al., Angew Chem Int Ed Engl, 2012, 51(29):7110-31), and some preferred modified nucleotides are exemplified below based on their respective nucleoside residues: 1-methyladenosine, 2-methylthio-N6-hydroxy-n-valylcarbamoyladenosine, 2-methyladenosine, 2'-O-ribosylphosphoadenosine, N6-methyl-N6-threonylcarbamoyladenosine, N6-acetyladenosine, N6-glycidylcarbamoyladenosine, N6-isopentenyladenosine, N6-methyladenosine, N6-threonyladenosine, N6,N6-dimethyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, N6-hydroxy-n-valylcarbamoyladenosine, 1,2'-O-dimethyladenosine, N6,2'-O-dimethyladenosine, 2'-O-methyladenosine, N6,N6,2'-O-trimethyladenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-methyladenosine, 2-methylthio-N6-isopentenyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6-2-methylthio-N6-threonylcarbamoyladenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 7-methyladenosine, 2-methylthio-adenosine, 2-methoxyadenosine, 2'-amino-2'-deoxyadenosine, 2'-azido-2'-deoxyadenosine, 2”-fluoro-2'-deoxyadenosine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenosine, 7-deaza-8-aza-adenosine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine; 2-thiocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-hydroxycytidine, lysidine, N4-acetyl-2'-O-methylcytidine, 5-formyl-2'-O-methylcytidine, 5,2'-O-dimethylcytidine, 2-O-methylcytidine, N4,2'-O-dimethylcytidine, N4,N4,2'-O-trimethylcytidine, isocytidine, pseudocytidine, pseudoisocytidine, 2-thio-cytidine, 2'-methyl-2'-deoxycytidine, 2'-amino-2'-deoxycytidine, 2'-fluoro-2'-deoxycytidine, 5-iodocytidine, 5-bromocytidine, 2'-azido-2'-deoxycytidine, 2'-amino-2'-deoxycytidine, 2'-fluoro-2'-deoxycytidine, 5-azacytidine, 3-methyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-l-methyl-1-deaza-pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-l-methyl-pseudoisocytidine, zebularine, 5-azazebularine, 5-methylzebularine, 5-aza-2-thiozebularine, 2-thiozebularine; 1-methylguanosine, N2,7-dimethylguanosine, N2-methylguanosine, 2'-O-ribosylphosphoguanosine, 7-methylguanosine, hydroxywybutosine, 7-aminomethyl-7-deazaguanosine, 7-cyano-7-deazaguanosine, N2,N2-dimethylguanosine, N2,7,2'-O-trimethylguanosine, N2,2'-O-dimethylguanosine, 1,2'-O-dimethylguanosine, 2'-O-methylguanosine, N2,N2,2'-O-trimethylguanosine, N2,N2J-trimethylguanosine, queuosine, 4-demethylwyosine, epoxyqueuosine, undermodified hydroxywybutosine, methylated undermodified hydroxywybutosine, queuoisine, peroxowybosine, galactosyl-queuosine, mannosyl-queuosine, queuosine, archaeosine, wybutosine, methylwyosine, wyosine, 7-aminocarboxypropyl-demethylwyosine, 7-aminocarboxypropylwyosine, 7-aminocarboxypropylwyosine methyl ester, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-azaguanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, N1-methylguanosine, 2'-amino-3'-deoxyguanosine, 2'-azido-2'-deoxyguanosine, 2'-fluoro-2'-deoxyguanosine, 2-thiouridine, 3-(3-amino-3-carboxypropyl)uridine, 3-methyluridine, 4-thiouridine, 5-methyl-2-thiouridine, 5-methylaminomethyluridine, 5-carboxymethyluridine, 5-carboxymethylaminomethyluridine, 5-hydroxyuridine, 5-methyluridine, 5-tauromethyluridine, 5-carbamoylmethyluridine, 5-(carboxyhydroxymethyl)uridine methyl ester, dihydrouridine, 5-methyldihydrouridine, 5-methylaminomethyl-2-thiouridine, 5-(carboxyhydroxymethyl)uridine, 5-(carboxyhydroxymethyl)-2'-O-methyluridine methyl ester, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thiouridine, 3,2'-O-dimethyluridine, 5-carboxymethylaminomethyl-2'-O-methyluridine, 5-carbamoylhydroxymethyluridine, 5-carbamoylmethyl-2'-O-methyluridine, 5-carbamoylmethyl-2-thiouridine, 5-methoxycarbonylmethyl-2'-O-methyluridine, 5-(isopentenylaminomethyl)-2'-O-methyluridine, 5,2'-O-dimethyluridine, 2'-O-methyluridine, 2'-O-methyl-2-thiouridine, 2-thio-2'-O-methyluridine, uridine 5-oxyacetic acid, 5-methoxycarbonylmethyluridine, methyl uridine 5-oxyacetate, 5-methoxyuridine, 5-aminomethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-tauromethyl-2-thiouridine, pseudouridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 1-methylpseudouridine, 3-methylpseudouridine, 2'-O-methylpseudouridine, 5-formyluridine, 5-aminomethyl-2-geranyluridine, 5-tauromethyluridine, 5-iodouridine, 5-bromouridine, 2'-methyl-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine, 2'-fluoro-2'-deoxyuridine, inosine, 1-methylinosine, 1,2'-O-dimethylinosine, 2'-O-methylinosine, 5-aza-uridine, 2-thio-5-aza-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 1-tauromethyl-pseudouridine, 5-tauromethyl-2-thio-uridine, 1-tauromethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 1,2'-O-dimethyladenosine, 1,2'-O-dimethylguanosine, 1,2'-O-dimethylinosine, 2,8-dimethyladenosine, 2-methylthiomethylthio-N6-isopentenyl-adenosine, 2-geranylthiouridine, 2-lyxidine, 2-methylthiocyclo-N6-threonylcarbamoyladenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-hydroxy-norvalylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, 2-selenouridine, 2-thio-2′-O-methyluridine, 2′-O-methyladenosine, 2′-O-methylcytidine, 2′-O-methylguanosine, 2′-O-methylinosine, 2′-O-methylpseudouridine, 2′-O-methyluridine, methyl 2′-O-ribosyladenosine phosphate, 2′-O-ribosylguanosine phosphate, 3,2′-O-dimethyluridine, 3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine, 3(3-amino3-carboxypropyl)pseudouridine, 5,2′-O-dimethylcytidine, 5,2'-O-dimethyluridine, methyl 5-(carboxymethyl)-2'-O-methyluridine, 5-(isopentenylaminomethyl)-2'-O-methyluridine, 5-aminomethyl-2-geranylthiouridine, 5-aminomethyl-2-selenouridine, 5-aminomethyluridine, 5-carbamoylmethyl-2'-O-methyluridine, 5-carboxymethyluridine, 5-carboxymethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-geranylthiouridine, 5-carboxymethylaminomethyl-2-selenouridine, 5-carboxymethylaminomethyl-2'-O-methyluridine, 5-cyanomethyluridine, 5-formyl-2'-O-methylcytidine, methyl 5-methoxycarbonylmethyl-2'-O-methyluridine, 5-methylaminomethyl-2-geranylthiouridine, 7-aminocarboxypropyl-demethylwybutosine, 7-methylguanosine, 8-methyladenosine, N2,2'-O-dimethylguanosine, N2,7,2'-O-trimethylguanosine, N2,7-dimethylguanosine, N2,N2,2'-O-trimethylguanosine, N2,N2,7-trimethylguanosine, N2,N2,7-trimethylguanosine, N4,2'-O-dimethylcytidine, N4,N4,2'-O-trimethylcytidine, N4,N4-dimethylcytidine, N4-acetyl-2'-O-methylcytidine, N6,2'-O-dimethyladenosine, N6,N6,2'-O-trimethyladenosine, N6-formyladenosine, N6-hydroxymethyladenosine, agmatidine, 2-methylthio-cyclo-N6-threonylcarbamoyladenosine, glutamyl-queuosine, guanosine added to any nucleotide, guanosylated 5'-end, hydroxy-N6-threonylcarbamoyladenosine; most preferably pseudouridine, N1-methylpseudouridine, 2'-fluoro-2'-deoxycytidine, 5-iodocytidine, 5-methylcytidine, 2-thiouridine, 5-iodouridine and / or 5-methyl-uridine.,

[0384] In addition, the term "modified nucleotide" includes nucleotides containing isotopes such as deuterium. The term "isotope" refers to elements having the same number of protons but different numbers of neutrons, resulting in different mass numbers. Thus, for example, the isotopes of hydrogen are not limited to deuterium, but also include tritium. In addition, polynucleotides can also contain isotopes of other elements, which include, for example, carbon, oxygen, nitrogen, and phosphorus. It is also possible for modified nucleotides to be deuterated or to contain another isotope of hydrogen, oxygen, carbon, nitrogen, or phosphorus.

[0385] Among the U, C, A, and G nucleotides, none, one, two, three, or all of them can be modified. Thus, in some embodiments, at least one nucleotide of a nucleotide type, e.g., at least one U nucleotide, can be a modified nucleotide. In some embodiments, at least one nucleotide of a total of two nucleotide types, e.g., at least one U nucleotide and at least one C nucleotide, can be a modified nucleotide. In some embodiments, at least one nucleotide of a total of three nucleotide types, e.g., at least one G nucleotide, at least one U nucleotide, and at least one C nucleotide, can be a modified nucleotide. In some embodiments, at least one nucleotide of all four nucleotide types can be a modified nucleotide. In all these embodiments, one or more nucleotides of each nucleotide type can be modified, and the percentage of the modified nucleotides of each nucleotide type is 0%, 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100%.

[0386] In some embodiments, the total percentage of modified nucleotides contained in the mRNA molecule is 0%, 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100%.

[0387] In a preferred embodiment, the mRNA is an mRNA containing a combination of modified and unmodified nucleotides. Preferably, it is an mRNA containing a combination of modified and unmodified nucleotides as described in WO2011 / 012316. It has been reported that the mRNA described therein exhibits increased stability and reduced immunogenicity. In a preferred embodiment, in such a modified mRNA, 5 - 50% of the cytidine nucleotides and 5 - 50% of the uridine nucleotides are modified. In another preferred embodiment, 5 - 50% of the uridine nucleotides are replaced with N1 - methyl - pseudouridine. The nucleotides containing adenosine and guanosine can be unmodified. The adenosine and guanosine nucleotides can be unmodified or partially modified, and they preferably exist in an unmodified form.

[0388] In certain embodiments of any of the foregoing, the percentage of an analogue of a given nucleotide refers to the input percentage (e.g., the percentage of the analogue in the starting reaction, e.g., the starting in vitro transcription reaction). In certain embodiments of any of the foregoing, the percentage of an analogue of a given nucleotide refers to the output (e.g., the percentage in the synthesized or transcribed compound). These two options are considered equally.

[0389] RNA, preferably an mRNA molecule, can be recombinantly produced in an in vivo system by methods known to those skilled in the art.

[0390] Optionally, a modified RNA, preferably an mRNA molecule, can be produced in an in vitro system using, for example, an in vitro transcription system known to those skilled in the art. An in vitro transcription system capable of producing RNA (preferably mRNA) requires an input mixture of modified and unmodified nucleoside triphosphates to produce unmodified RNA. In certain embodiments, in such an input mixture, 5 to 50% of the cytidine is an analogue of cytidine, and in such an input mixture, 5 to 50% of the uridine is an analogue of uridine. In certain embodiments, in such an input mixture, 5 to 40% of the cytidine is an analogue of cytidine, and in such an input mixture, 5 to 40% of the uridine is an analogue of uridine. In certain embodiments, in such a mixture, 5 to 30% of the cytidine is an analogue of cytidine, and in such an input mixture, 5 to 30% of the uridine is an analogue of uridine. In certain embodiments, in such a mixture, 5 to 30% of the cytidine is an analogue of cytidine, and in such a mixture, 10 to 30% of the uridine is an analogue of uridine. In certain embodiments, in such an input mixture, 5 to 20% of the cytidine is an analogue of cytidine, and in such an input mixture, 5 to 20% of the uridine is an analogue of uridine. In certain embodiments, in such an input mixture, 5 to 10% of the cytidine is an analogue of cytidine, and in such an input mixture, 5 to 10% of the uridine is an analogue of uridine. In certain embodiments, in such an input mixture, 25% of the cytidine is an analogue of cytidine, and in such an input mixture, 25% of the uridine is an analogue of uridine. In certain embodiments, the input mixture does not contain analogues of adenosine and / or guanosine. In other embodiments, optionally, the input mixture contains one or more analogues of adenosine and / or guanosine (or neither or both).

[0391] In certain embodiments, the percentage of cytidine in the input mixture that is an analogue of cytidine is different from the percentage of uridine in the input mixture that is an analogue of uridine. In certain embodiments, the percentage of cytidine analogue in the input mixture is lower than the percentage of uridine analogue in the input mixture. As described above, this can be the case in the presence or absence of analogues of adenosine and guanosine in the input mixture, but in certain embodiments, it is in the case where there are no analogues of adenosine and guanosine in the input mixture.

[0392] In certain embodiments, the input mixture of nucleotides for an in vitro transcription system for generating RNA (preferably, the mRNA of the present invention) includes analogs of cytidine and analogs of uridine, and 5% to 20% of the cytidine in the input mixture is an analog of cytidine, and 25% to 45% of the uridine in the input mixture is an analog of uridine. In other words, the input mixture includes modified and unmodified cytidine as well as modified and unmodified uridine, and 5 to 20% of the cytidine in the input mixture includes an analog of cytidine, while 25 to 45% of the uridine in the input mixture includes an analog of uridine. In other embodiments, the input mixture contains 5 to 10% of an analog of cytidine and 30 to 40% of an analog of uridine, such as 7 to 9% of an analog of cytidine, such as 7%, 7.5% or 8%, and such as 32 to 38% of an analog of uridine, such as 33%, 34%, 35%, 36%.

[0393] In certain embodiments, any one of the analogs of uridine and analogs of cytidine described herein can be used, optionally excluding pseudouridine. In certain embodiments, the analog of cytidine comprises 5-iodocytidine or consists of 5-iodocytidine (e.g., it is the single type of C analog used), and the analog of uridine comprises 5-iodouridine or consists of 5-iodouridine (e.g., it is the single type of U analog used).

[0394] Exemplary analogs are described above. It should be understood that for a modified polynucleotide encoding a desired polypeptide, unless otherwise stated, analogs and modification levels are considered throughout the entire polynucleotide encoding the desired polypeptide - including the 5' and 3' untranslated regions (e.g., the modification level is based on the input ratio of the analog in the in vitro transcription reaction such that the analog can be incorporated at the site of transcription).

[0395] In addition, the modified RNA, preferably the mRNA molecule, can be chemically synthesized, for example, by conventional chemical synthesis on an automated nucleotide sequence synthesizer using a solid support and standard techniques, or by chemically synthesizing the corresponding DNA sequence and subsequently transcribing it in vitro or in vivo.

[0396] In another preferred embodiment, the mRNA can be bound to a target binding site, a targeting sequence, and / or a microRNA binding site in order to allow the activity of the desired mRNA only in relevant cells. In a further preferred embodiment, the RNA can be bound to a microRNA or shRNA in the untranslated region.

[0397] Generally, the therapeutic effect can be achieved through the interaction of ribonucleic acid with cellular molecules and organelles. For example, such interaction alone can activate the innate immune system, as is the case with certain CpG oligonucleotides and sequences designed to specifically interact with toll-like receptors and other extracellular or intracellular receptors. In addition, the uptake or introduction of nucleic acids (preferably, ribonucleic acid, more preferably mRNA) into cells can be aimed at causing the expression of nucleotide sequences - such as genes contained in nucleic acids (preferably ribonucleotides, more preferably mRNA), can be aimed at downregulating, silencing or knocking down endogenous gene expression due to the presence of the introduced exogenous nucleic acid in the cell, or can be aimed at modifying endogenous nucleic acid sequences, such as repairing, excising, inserting or exchanging selected bases or entire fragments of the endogenous nucleic acid sequence, or can be aimed at interfering with almost any cellular process due to the presence and interaction of the introduced exogenous ribonucleic acid (preferably, mRNA) in the cell. Overexpression of the introduced exogenous nucleic acid (preferably ribonucleic acid, more preferably mRNA) can be aimed at compensating for or supplementing endogenous gene expression, especially in cases where the endogenous gene is defective or silenced, resulting in no gene expression, insufficient gene expression or defective or dysfunctional products of gene expression, such as, in the case of many metabolic and genetic diseases, such as cystic fibrosis, hemophilia or muscular dystrophy. Overexpression of the introduced exogenous nucleic acid (preferably ribonucleic acid, more preferably mRNA) can also be aimed at interacting with the expression product with any endogenous cellular process or interfering with any endogenous cellular process, such as the regulation of gene expression, signal transduction and other cellular processes. Overexpression of the introduced exogenous nucleic acid (preferably ribonucleic acid, more preferably mRNA) can also be aimed at eliciting an immune response in the context of an organism in which the transfected or transduced cells are present or made to be present. Examples are the genetic modification of antigen-presenting cells such as dendritic cells to make them present antigens for vaccination purposes. Other examples are the overexpression of cytokines in tumors in order to elicit a tumor-specific immune response. In addition, overexpression of the introduced exogenous ribonucleic acid (preferably mRNA) can also be aimed at generating transiently genetically modified cells in vivo or ex vivo for cell therapy, such as modified T cells, NK cells and other lymphocytes or progenitor cells or stem cells or other cells for regenerative medicine.

[0398] For example, downregulation, silencing or knockdown of endogenous gene expression for therapeutic purposes can be achieved by using ribozymes, antisense oligonucleotides, tRNA, RNA interference (RNAi) of long double-stranded RNA, wherein such downregulation can be sequence-specific or non-specific and can also result in cell death, as is the case when long double-stranded RNA is introduced into cells. Downregulation, silencing or knockdown of endogenous or pre-existing gene expression is useful in the treatment of acquired, inherited or spontaneously occurring diseases, including viral infections and cancer. It is also contemplated that introduction of nucleic acids into cells can be practiced as a prophylactic measure to prevent, for example, viral infections or tumors. Downregulation, silencing or knockdown of endogenous gene expression can act at the transcriptional and translational levels. A variety of mechanisms are known to those skilled in the art and include, for example, epigenetic modifications, changes in chromatin structure, selective binding of introduced nucleic acids to transcription factors, hybridization of introduced nucleic acids to complementary sequences in genomic DNA, mRNA or other RNA by base pairing, which includes non-conventional base pairing mechanisms such as triple helix formation. Similarly, gene repair, base or sequence alterations can be achieved at the genomic level and at the mRNA level (including exon skipping). For example, base or sequence alterations can be achieved by RNA-guided site-specific DNA cleavage, by using the splicing and ligation mechanisms of trans-splicing, trans-splicing ribozymes, chimeras, spliceosome-mediated RNA trans-splicing, or by using group II or redirected introns, or by using virus-mediated insertional mutagenesis or genomic insertion targeted by prokaryotic, eukaryotic or viral integrase systems. Since nucleic acids are the carriers of the construction plan of the living system and since they are involved in many cellular processes in direct and indirect ways, any cellular process can theoretically be affected by introducing nucleic acids into cells from the outside. It is noted that such introduction can be carried out directly in in vivo and in vitro cell or organ cultures, and then the thus modified organs or cells can be transplanted into a recipient. Particles used as therapeutic active agents together with nucleic acids in the context of the present invention can be used for all of the above purposes.

[0399] As described above, RNA, preferably mRNA, can comprise a ribonucleotide sequence that encodes a protein or a fragment thereof whose function is desired or beneficial in or near a cell, for example, a protein whose absence or defective form is an inducer of a disease or disorder, and providing such protein can alleviate or prevent the disease or disorder, or a protein that can promote a process beneficial to the body in or near the cell.

[0400] In fact, in recent years, RNA (especially mRNA) has become increasingly important as a new drug entity. In contrast to DNA-based gene therapies, mRNA does not need to be transported into the nucleus but is directly translated into proteins in the cytoplasm (J Control Release, 2011, 150:238-247, and Eur J Pharm Biopharm, 2009, 71:484-489).

[0401] In addition, many genetic diseases caused by single gene mutations are known and are candidates for RNA (preferably, mRNA) therapeutic approaches. Diseases caused by single gene mutations, such as cystic fibrosis, hemophilia, and many other diseases, can be dominant or recessive in terms of the likelihood of a certain trait appearing in offspring. While dominant alleles exhibit a phenotype in individuals with only one copy of the allele, for recessive alleles, an individual must have two copies, one from each parent, to exhibit the phenotype. In contrast, polygenic diseases are caused by two or more genes, and the manifestation of each disease is often smooth and related to environmental factors. Examples of polygenic diseases are hypertension, elevated cholesterol levels, cancer, neurodegenerative diseases, mental diseases, etc. Also in these cases, therapeutic RNA, preferably mRNA, representing one or more of these genes may be beneficial to these subjects. In addition, genetic disorders are not necessarily inherited from the genes of parents and may also be caused by new mutations. Also in these cases, therapeutic RNA, preferably mRNA, representing the correct gene sequence may be beneficial to the subject.

[0402] Currently, an online catalog of 22,993 human gene and genetic disorder entries with their respective gene and phenotype descriptions is available on the OMIM (Online Mendelian Inheritance in Man) web page (http: / / omim.org); each sequence can be obtained from the Uniprot database (http: / / www.uniprot.org). As a non-limiting example, Table A below lists some congenital diseases and disorders and the corresponding gene(s). Due to the highly interactive nature of cellular signaling pathways, mutations in a certain gene can lead to multiple pathogenic symptoms, and only the characteristic symptoms are listed in Table A.

[0403] In some embodiments of the present invention, the RNA, preferably mRNA-encoded therapeutic proteins that can be present in the suspension formulations and aerosols of the present invention are selected from the cellular proteins listed in Table A. Thus, the RNA, preferably the mRNA molecule, can encode a therapeutic cellular protein, wherein the encoded therapeutic protein is the therapeutic protein listed in Table A or its homolog.

[0404] In another embodiment of the present invention, the therapeutic protein encoded by RNA, preferably mRNA, is selected from the secreted proteins listed in Table A. Thus, RNA, preferably mRNA, can encode a therapeutic fusion protein, wherein the encoded therapeutic protein or its homolog is one of those listed in Table A, and the second protein is a signal peptide that allows secretion of the therapeutic protein. The signal peptide is a short sequence, typically 5 - 30 amino acids long, present at the N-terminus of the therapeutic protein and directs the fusion protein to the secretory pathway of the cell via certain organelles (i.e., endoplasmic reticulum, Golgi apparatus, or endosome). Thus, such a fusion protein is secreted from the cell or organelle, or inserted into the cell membrane at the cell compartment or cell surface (e.g., multi-transmembrane transmembrane protein).

[0405] Thus, in a preferred embodiment of the present invention, RNA, preferably mRNA, can encode one or more (but not limited to) proteins of the genes that cause, predispose to, or prevent diseases. Non-limiting examples of such diseases or disorders that can be treated (or prevented) include those in which the polypeptide, protein, or peptide is selected from those outlined in Table A below.

[0406] In some embodiments, the coding sequence of RNA (preferably mRNA) can be transcribed and translated into a partial or full-length protein that contains cellular activity equal to or greater than the native protein level. In some embodiments, RNA (preferably mRNA) encodes a therapeutic or pharmaceutically active polypeptide, protein, or peptide having a therapeutic or prophylactic effect, wherein the polypeptide, protein, or peptide is selected from those outlined in Table A below. RNA, preferably mRNA, more specifically its coding sequence can be used to express a partial or full-length protein having cellular activity equal to or less than the native protein level. This can allow treatment of diseases that can indicate administration of the RNA molecule.

[0407] Table A: Non-limiting examples of human genes and genetic diseases or disorders

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414] Table A above shows examples of genes in which a defect causes a disease that can be treated with the RNA, preferably mRNA, that may be present in the suspension formulations and aerosols of the present invention, wherein the RNA, preferably mRNA, comprises a ribonucleotide sequence that encodes a full version of the protein of the defective gene disclosed above or a functional fragment thereof. In a particularly preferred embodiment, a genetic disease can be addressed, such as a genetic disease that affects the lungs, such as SPB (surfactant protein B) deficiency, ABCA3 deficiency, cystic fibrosis, and alpha-1 antitrypsin deficiency, or a genetic disease that affects plasma proteins (e.g., congenital hemochromatosis (hepcidin deficiency), thrombotic thrombocytopenic purpura (TPP, ADAMTS13 deficiency)) and causes coagulation defects (e.g., hemophilia A and B) and complement defects (e.g., protein C deficiency), immunodeficiencies such as, for example, SCID (caused by mutations in different genes, such as: RAG1, RAG2, JAK3, IL7R, CD45, CD3δ, CD3ε) or defects caused by a lack of adenosine deaminase, for example (ADA-SCID), chronic granulomatous disease (e.g., caused by mutations in the gp-91-phox gene, p47-phox gene, p67phox gene, or p33-phox gene) and storage diseases such as Gaucher disease, Fabry disease, Krabbe disease, MPS I, MPS II (Hunter syndrome), MPS VI, glycogen storage disease type II, or mucopolysaccharidosis.

[0415] Other disorders for which the RNA of the present invention, preferably mRNA, can be used include the following disorders such as SMN1-related spinal muscular atrophy (SMA); amyotrophic lateral sclerosis (ALS); GALT-related galactosemia; cystic fibrosis (CF); SLC3A1-related disorders including cystinuria; COL4A5-related disorders including Alport syndrome; galactocerebrosidase deficiency; X-linked adrenoleukodystrophy and adrenomyeloneuropathy; Friedrich ataxia; Pelizaeus-Merzbacher disease; TSC1 and TSC2-related tuberous sclerosis; Sanfilippo B syndrome (MPS IIIB); CTNS-related cystinosis; FMR1-related diseases which include fragile X syndrome, fragile X-related tremor / ataxia syndrome and fragile X premature ovarian failure syndrome; Prader-Willi syndrome; hereditary hemorrhagic telangiectasia; Niemann-Pick disease type C1; neuronal ceroid lipofuscinosis-related diseases including juvenile neuronal ceroid lipofuscinosis (JNCL), juvenile Batten disease, Santavuori Haltia disease, Jansky-Bielschowsky disease and PTT-1 and TPP1 deficiencies; EIF2B1, EIF2B2, EIF2B3, EIF2B4 and EIF2B5-related childhood ataxia with central nervous system hypomyelination / leukodystrophy; CACNA1A and CACNB4-related episodic ataxia type 2; MECP2-related disorders including classic Rett syndrome, MECP2-related severe neonatal encephalopathy and PPM-X syndrome; CDKL5-related atypical Rett syndrome; Kennedy disease (SBMA); Notch-3-related cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL); SCN1A and SCN1B-related epileptic seizure disorders; polymerase G-related diseases including Alpers-Huttenlocher Sydrome, POLG-related sensory ataxic neuropathy, dysarthria and ophthalmoparesis, and autosomal dominant and recessive progressive external ophthalmoplegia with mitochondrial DNA deletions; X-linked adrenal hypoplasia; X-linked agammaglobulinemia; Fabry disease; and Wilson's disease.

[0416] In all of these diseases, proteins, such as, for example, enzymes, are defective and can be treated with RNA encoding any of the above proteins, preferably mRNA, which makes available the protein encoded by the defective gene or a functional fragment thereof. Transcript replacement therapy / protein replacement therapy does not affect the underlying genetic defect but increases the concentration of the protein that the subject lacks. For example, in Pompe disease, transcript replacement therapy / enzyme replacement therapy replaces the defective lysosomal enzyme acid alpha-glucosidase (GAA).

[0417] Thus, non-limiting examples of proteins that can be encoded by mRNA are erythropoietin (EPO), growth hormone (somatotropin, hGH), cystic fibrosis transmembrane conductance regulator (CFTR), growth factors such as GM-SCF, G-CSF, MPS, protein C, hepcidin, ABCA3, and surfactant protein B. Further examples of diseases that can be treated with RNA according to the invention are hemophilia A / B, Fabry disease, CGD, ADAMTS13, Heller disease, X-chromosome-mediated agammaglobulinemia, adenosine deaminase-related immunodeficiency, and neonatal respiratory distress syndrome, which is associated with SP-B. Particularly preferably, the RNA according to the invention, preferably mRNA, comprises the coding sequence of surfactant protein B (SP-B) or erythropoietin. Further examples of proteins that can be encoded by the RNA according to the invention, preferably mRNA, of the invention are growth factors such as human growth hormone hGH, BMP-2, or angiogenic factors.

[0418] Although the above embodiments have been described in the context of RNA, preferably mRNA molecules, that may be present in the nanoparticles used in the present invention, as described above, the present invention is not limited to the use of RNA, preferably mRNA, but other nucleic acid molecules, such as DNA molecules, can be employed.

[0419] The DNA molecules can encode the above RNA, preferably the above mRNA, and accordingly, contain the genetic information of the correspondingly transcribed RNA molecules.

[0420] Thus, with respect to the preferred embodiments, as described above and below in the context of RNA molecules, preferably mRNA molecules, that may be present in the nanoparticles used in the present invention, with the necessary modifications, the same applies to the DNA molecules of the present invention.

[0421] Optionally, the RNA, preferably mRNA, can comprise ribonucleotide sequences encoding full-length antibodies or smaller antibodies (e.g., heavy and light chains), which can be used in a therapeutic setting, for example, to confer immunity to a subject. The corresponding antibodies and their therapeutic applications (s) are known in the art. The antibody can be encoded by a single mRNA strand or can be encoded by more than one mRNA strand.

[0422] In another embodiment, RNA, preferably mRNA, can encode a functional monoclonal or polyclonal antibody that can be used to target and / or inactivate a biological target (e.g., a stimulatory cytokine such as tumor necrosis factor). Similarly, an RNA, preferably an mRNA sequence, can encode, for example, a functional anti-nephritic factor antibody that can be used to treat membranoproliferative glomerulonephritis type II or acute hemolytic uremic syndrome, or alternatively can encode an anti-vascular endothelial growth factor (VEGF) antibody that can be used to treat VEGF-mediated diseases such as cancer.

[0423] In another embodiment, RNA, preferably mRNA, can encode a functional monoclonal or polyclonal antibody that can be used to neutralize or otherwise inhibit a virus or viral replication.

[0424] Optionally, RNA, preferably mRNA, can comprise a ribonucleotide sequence encoding an antigen that is preferably useful for preventing or treating in an environment.

[0425] In another embodiment, mRNA can encode one or more proteins that can induce immunomodulation, such as cytokines, including chemokines, interferons (such as interferon lambda), interleukins, lymphokines, and tumor necrosis factor.

[0426] In another embodiment, RNA, preferably mRNA, can comprise a ribonucleotide sequence encoding a polypeptide or protein that can be used in genome editing techniques. Genome editing is a type of genetic engineering that uses nucleases to insert, delete, or replace DNA in an organism's genome. These nucleases create site-specific breaks at desired locations in the genome. The induced breaks are repaired by non-homologous end joining or homologous recombination, resulting in targeted mutations in the genome, thereby "editing" the genome. The breaks can be single-strand breaks or double-strand breaks (DSBs), and double-strand breaks (DSBs) are preferred. Many genome editing systems that utilize different polypeptides or proteins are known in the art, i.e., for example, the CRISPR-Cas system, meganucleases, zinc finger nucleases (ZFNs), and transcription activator-like effector-based nucleases (TALENs). Genome engineering methods are reviewed in Trends in Biotechnology, 2013, 31(7), 397-405.

[0427] Thus, in a preferred embodiment, the RNA, preferably mRNA, may comprise a ribonucleotide sequence that encodes a polypeptide or protein of the Cas (CRISPR-associated protein) protein family - preferably Cas9 (CRISPR-associated protein 9). The proteins of the Cas protein family, preferably Cas9, can be used in CRISPR / Cas9-based methods and / or CRISPR / Cas9 genome editing techniques. The CRISPR-Cas systems for genome editing, regulation, and targeting are reviewed in Nat. Biotechnol., 2014, 32(4):347-355.

[0428] In another preferred embodiment, the RNA, preferably mRNA, may comprise a ribonucleotide sequence that encodes a meganuclease. Meganucleases are endodeoxyribonucleases that recognize large recognition sites (e.g., double-stranded DNA sequences of 12 - 40 base pairs) compared to "conventional" endodeoxyribonucleases. As a result, in any given genome, the corresponding sites occur only a few times, preferably only once. Thus, meganucleases are considered the most specific naturally occurring restriction enzymes and are therefore suitable tools in genome editing techniques.

[0429] In another preferred embodiment, the RNA, preferably mRNA, comprises a ribonucleotide sequence that encodes a zinc finger nuclease (ZFN). ZFNs are artificial restriction enzymes produced by fusing a zinc finger DNA-binding domain to a DNA cleavage domain. The zinc finger domain can be engineered to target specific desired DNA sequences, and this enables zinc finger nucleases to target unique sequences in complex genomes. By utilizing the endogenous DNA repair machinery, ZFNs can be used to precisely alter the genomes of higher organisms and are therefore suitable tools in genome editing techniques.

[0430] In another preferred embodiment, the RNA, preferably mRNA, may comprise a ribonucleotide sequence that encodes a transcription activator-like effector nuclease (TALEN). TALENs are restriction enzymes that can be engineered to cut DNA at specific sequences. TALENs are fusion proteins in which the TAL effector DNA-binding domain is fused to the cleavage domain of a DNA nuclease. The transcription activator-like effectors (TALEs) can be engineered to actually bind to any desired DNA sequence. Thus, when combined with a nuclease, the DNA can be cut at specific desired locations.

[0431] Although the above embodiments are described in the context of RNA, preferably mRNA molecules, as described above, the present invention is not limited to the use of RNA, preferably mRNA, and any nucleic acid molecule, such as a DNA molecule, can be used.

[0432] The DNA molecule can encode the above RNA, preferably the above mRNA, and correspondingly, contains the genetic information of the correspondingly transcribed RNA molecule.

[0433] Thus, in terms of preferred embodiments, the RNA molecules that may be present in the nanoparticles as used in the present invention above and below, preferably in the context of mRNA molecules, are, with the necessary modifications, equally applicable to DNA molecules.

[0434] In addition, RNA contains ribonucleotide sequences that are not expressed as proteins or polypeptides. Therefore, the term RNA should not be understood only to refer to any polynucleotide molecule that, if introduced into a cell, can be translated into a polypeptide / protein or a fragment thereof. On the contrary, it is also contemplated that RNA contains ribonucleotide sequences that are not translated into proteins. In this context, it is contemplated that RNA contains ribonucleotide sequences that preferably provide the genetic information for antisense RNA, siRNA or miRNA sequences or another desired non-coding ribonucleotide sequence.

[0435] Thus, RNA can also be an antisense RNA, siRNA or miRNA sequence. Antisense RNA, siRNA or miRNA sequences can be used to silence the action of certain RNA molecules at some stage. This may be desirable and useful in certain medical settings and in the treatment of certain diseases, particularly in the RNA-based therapies described above and below herein.

[0436] The action of the silencing RNA molecule can be utilized by using nucleic acid strands complementary to certain RNA sequences to exploit the RNAi (RNA interference) mechanism. The term "RNA interference" or "inhibitory RNA" (RNAi / iRNA) describes the use of double-stranded RNA to target specific mRNA for degradation, thereby silencing its translation. Preferred inhibitory RNA molecules can be selected from double-stranded RNA (dsRNA), siRNA, shRNA, and stRNA. dsRNA matching the gene sequence can be synthesized in vitro and introduced into cells. dsRNA can also be introduced into cells in the form of a vector expressing the target gene sequence in sense and antisense orientations, for example, in the form of a hairpin mRNA. The sense and antisense sequences can also be expressed from separate vectors, whereby the individual antisense and sense molecules form double-stranded RNA after their expression. It is known in the art that in some cases, due to the internal amplification mechanism in cells, the expression of the sense-oriented sequence or even the promoter sequence is sufficient to produce dsRNA and subsequently siRNA. Thus, according to the present invention, all means and methods that result in a decrease in the activity of the polypeptide or protein encoded by the coding region will be used. For example, sense constructs, antisense constructs, hairpin constructs, sense and antisense molecules, and combinations thereof can be used to generate / introduce these siRNAs. dsRNA enters a natural process that includes the highly conserved endonuclease (dicer), which cleaves the dsRNA precursor molecule into short interfering RNA (siRNA). The production and preparation of siRNA(s) and the method of inhibiting target gene expression are particularly described in WO02 / 055693, Wei (2000) Dev. Biol. 15:239-255; La Count (2000) Biochem. Paras. 111:67-76; Baker (2000) Curr. Biol. 10:1071-1074; Svoboda (2000) Development 127:4147-4156 or Marie (2000) Curr. Biol. 10:289-292. These siRNAs then construct the sequence-specific part of the RNA-induced silencing complex (RISC), which is a multi-complex nuclease that destroys messenger RNA homologous to the silencing trigger. Elbashir (2001) EMBO J. 20:6877-6888 showed that duplexes of 21-nucleotide RNAs can be used in cell culture to interfere with gene expression in mammalian cells.

[0437] Methods for the derivation and construction of siRNAs are known in the art and are described in Elbashir (2002) Methods 26:199-213, on the internet websites of commercial suppliers of siRNAs such as Qiagen GmbH (https: / / www1.qiagen.com / GeneGlobe / Default.aspx); Dharmacon (www.Dharmacon.com); Xeragon Inc. (http: / / www.dharmacon.com / Default.aspx), and Ambion (www.Ambion.com), or on the website of the Tom Tuschl research group ( http: / / www.rockefeller.edu / labheads / tuschl / sirna.html ). In addition, programs are available online for inferring siRNAs from a given mRNA sequence (e.g., http: / / www.ambion.com / techlib / misc / siRNA_finder.html or http: / / katahdin.cshl.org:9331 / RNAi / html / rnai.html ). The uridine residue in the 2-nt 3' overhang can be replaced by 2'-deoxythymidine without loss of activity, which significantly reduces the cost of RNA synthesis and, when applied to mammalian cells, can also enhance the resistance of the siRNA duplex (Elbashir (2001) loc. cit). siRNAs can also be synthesized enzymatically using T7 or other RNA polymerases (Donze (2002) Nucleic Acids Res 30:e46). Short RNA duplexes that mediate efficient RNA interference (esiRNAs) can also be generated by hydrolysis with Escherichia coli RNase III (Yang (2002) PNAS 99:9942-9947). In addition, expression vectors have been developed to express double-stranded siRNAs linked by small hairpin RNA loops in eukaryotic cells (e.g., (Brummelkamp (2002) Science 296:550-553). All of these constructs can be developed with the help of the above programs. In addition, commercially available sequence prediction tools incorporated into sequence analysis programs or sold separately, such as the siRNA design tool provided by www.oligoEngine.com (Seattle, WA), can be used for siRNA sequence prediction.

[0438] MicroRNA (miRNA) is similar to the above-described small interfering RNA (siRNA). MicroRNA (miRNA) is a small non-coding RNA molecule (containing approximately 22 nucleotides) found in plants, animals, and some viruses, which plays a role in RNA silencing and post-transcriptional regulation of gene expression. miRNA acts by base pairing with complementary sequences within the mRNA molecule. Thus, these mRNA molecules are silenced through one or more of the following processes: (1) cleavage of the mRNA strand into two segments, (2) destabilization of the mRNA by shortening its poly(A) tail, and (3) less efficient translation of the mRNA into protein by ribosomes. As described above, miRNA is similar to small interfering RNA (siRNA) in the RNA interference (RNAi) pathway, except that miRNA is derived from regions of an RNA transcript that fold back on themselves to form short hairpins, while siRNA is derived from longer regions of double-stranded RNA.

[0439] The DNA molecules used in the suspension formulations and aerosols of the present invention can also be DNA molecules encoding the above-described RNAs, such as the above siRNA or miRNA, and accordingly, contain the genetic information of the corresponding transcribed RNA molecules. Thus, with respect to the preferred embodiments, as described above for the RNA molecules that may be present in the nanoparticles used in the present invention, preferably in the context of mRNA molecules, with the necessary modifications, the same applies to DNA molecules.

[0440] It should be understood that in the context of the present invention, the nanoparticles can include a single type of nucleic acid, preferably RNA, such as mRNA, but can optionally include a combination of two or more types of nucleic acids, preferably RNA in the form of particles containing two or more types of nucleic acids, preferably RNA in a single particle, or in the form of a mixture of particles in which the types of nucleic acids contained therein (preferably RNA, such as mRNA) are different.

[0441] Together with the therapeutic agent (a), the nanoparticles further comprise a permanent cationic lipid, an ionizable lipid or an ionizable lipidoid as component (b), i.e., component (b) is at least one selected from permanent cationic lipids, ionizable lipids and ionizable lipidoids. It should be understood that this encompasses the following possibilities: the nanoparticles comprise a combination of different permanent cationic lipids, a combination of different ionizable lipids, a combination of different ionizable lipidoids, or a combination of one or more permanent cationic lipids, one or more ionizable lipids and / or one or more ionizable lipidoids. Preferred component (b) is an ionizable lipid and an ionizable lipidoid, i.e., preferably the nanoparticles comprise at least one selected from ionizable lipids and ionizable lipidoids as component (b). The nanoparticles used in the present invention generally comprise a nucleic acid (a) and a permanent cationic lipid, an ionizable lipid or an ionizable lipidoid (b) in the form of a mixture of components (a) and (b).

[0442] The term "permanent cationic lipid" is used in the field of lipid nanoparticles to refer to a lipid containing a permanent cationic charge, for example, in the form of a quaternary nitrogen atom.

[0443] The terms "ionizable lipid" and "ionizable lipidoid" are used in the fields of lipid nanoparticles and lipidoid nanoparticles to refer to a lipid or lipidoid that is protonated to carry a cationic charge, or can be protonated to carry a cationic charge. Thus, ionizable lipids and lipidoids are also referred to as "protonable lipids" and "protonable lipidoids", as "ionizable cationic lipids" and "ionizable cationic lipidoids" or are also referred to as "titratable lipids" or "titratable lipidoids", respectively. As will be understood by the reader of the prior art, reference to an "ionizable lipid" or "ionizable lipidoid" includes the ionizable lipid or lipidoid in protonated or non-protonated form. As will be further understood, the protonated or non-protonated state of a lipid or lipidoid is generally determined by the pH value of the medium surrounding the lipid or lipidoid, for example, by the pH value of the aqueous carrier solution in which the nanoparticles are suspended. Thus, the terms "ionizable lipid" and "ionizable lipidoid" also include lipids or lipidoids that are positively charged at neutral pH.

[0444] In the context of the present invention, the counterions (anions) of the positive charges of permanently positively charged cationic lipids, ionizable lipids or ionizable lipidoids are typically provided by the anionic moieties contained in the nucleic acid. If the positively charged groups are present in excess compared to the anionic moieties in the nucleic acid, the positive charges can be balanced by other pharmaceutically acceptable anions such as chloride, bromide or iodide, sulfate, nitrate, phosphate, hydrogen phosphate, dihydrogen phosphate, carbonate or bicarbonate, or by a polyanionic component different from the nucleic acid, which can be present as an optional component in the nanoparticles.

[0445] It is well known that permanently positively charged cationic lipids, ionizable lipids and ionizable lipidoids are components of lipid nanoparticles or lipidoid nanoparticles. In the context of the present invention, there is no particular limitation on the type of permanently positively charged cationic lipid, ionizable lipid or ionizable lipidoid contained in the nanoparticles.

[0446] Typically, ionizable lipids or lipidoids respectively include primary, secondary or tertiary amino groups, which can act as proton acceptors and which can therefore be protonated or non-protonated. Ionizable lipidoids typically include a plurality of such amino groups, such as two or more, preferably three or more.

[0447] Preferably, the ionizable lipid that the nanoparticles can contain is a lipid that comprises a protonatable head group that comprises one or more, preferably one primary, secondary or tertiary amino group as the protonatable or protonated group, and one or more, preferably one or two hydrophobic moieties attached to the head group.

[0448] Examples of these preferred ionizable lipids are

[0449] i) lipids that comprise a protonatable head group and one hydrophobic moiety attached to the head group, the protonatable head group comprising one or more, preferably one primary, secondary or tertiary amino group as the protonatable or protonated group;

[0450] ii) lipids that comprise a secondary or tertiary amino group as the protonatable or protonated head group and two hydrophobic moieties attached to the head group.

[0451] The hydrophobic moiety included in these preferred lipids preferably comprises one or more straight-chain aliphatic residues, e.g., straight-chain residues containing 8 to 18 carbon atoms, branched-chain aliphatic residues, e.g., branched-chain residues containing 8-18 carbon atoms, or may be an alicyclic structure which can be a fused-ring structure, e.g., an alicyclic structure containing 10 to 18 carbon atoms. In addition, the hydrophobic moiety may include one or more linking groups which facilitate the attachment of the moiety to the head group or allow two or more of the above aliphatic residues to bind to each other. Further, it may contain one or more substituents, provided that the hydrophobic nature of the moiety is maintained.

[0452] Preferably, the ionizable lipid that can be included in the nanoparticles is an oligoamine, more preferably an oligoalkylamine, which contains at least two, preferably at least three amino groups selected from protonatable or protonated secondary and tertiary amino groups, each of which may carry a hydrophobic moiety attached thereto. In addition to the amino groups carrying hydrophobic residues, the lipid may further include protonatable or protonated amino groups selected from primary, secondary and tertiary amino groups. Preferably, the total number of amino groups is from 2 to 10, more preferably from 3 to 6. Preferably, the total number of hydrophobic moieties attached to the amino groups is from 2 to 6, more preferably from 3 to 6. Preferably, the ratio of the total number of hydrophobic moieties attached to the amino groups to the total number of amino groups in the oligoalkylamine is from 0.5 to 2, more preferably from 0.75 to 1.5.

[0453] The hydrophobic moiety included in such a preferred lipid preferably comprises one or more straight-chain aliphatic residues, e.g., straight-chain residues containing 8 to 18 carbon atoms, and branched-chain aliphatic residues, e.g., branched-chain residues containing 8 to 18 carbon atoms. In addition, the hydrophobic moiety may include one or more linking groups which facilitate the attachment of the moiety to the amino group or allow two or more of the above aliphatic residues to bind to each other. Further, it may contain one or more substituents, provided that the hydrophobic nature of the moiety is maintained.

[0454] For example, in WO2006 / 138380A2, EP2476756A1, US2016 / 0114042A1, US 8,058,069 B2, US 8,492,359B2, US 8,822,668B2, US 8,969,535, US 9,006,417B2, US 9,018,187B2, US9,345,780B2, US 9,352,042B2, US 9,364,435B2, US 9,394,234B2, US 9,492,386B2, US 9,504,651 B2, US 9,518,272 B2, DE 19834683 A1, WO 2010 / 053572 A2, US 9,227,917B2, US9,556,110 B2, US 8,969,353 B2, US 10,189,802B2, WO 2012 / 000104A1, WO 2010 / 053572, WO 2014 / 028487, WO 2015 / 095351, US 2013 / 0156849 A1 (e.g., claims 13, 33, 34), US9254311 B2 (e.g., claim 14), US 10501512 B2 (e.g., claims 1, 6, 9), US 2014 / 0010861 A1 (e.g., claims 44 and 78 - 82), US 2013 / 0115272A1 (e.g., claim 12), or in Akinc, A., et al., Nature Biotechnology, 26(5), 2008, 561 - 569; Sabnis, S. et al., Molecular Therapy, 26(6), 2018, Vol.26 No 6 June 2018, 1509 - 1519; Kowalski, P.S., et al., Molecular Therapy, 27(4), 2019, 710 - 728; Kulkarni, J.A. et al, Nucleic Acid Therapeutics, 28(3), 2018, 146 - 157; and Li, B. et al., Nano Letters, 15, 2015, 8099 - 8107, suitable exemplary ionizable lipids or ionizable lipidoids are disclosed that can be included as component (b) in nanoparticles used in the context of the present invention.

[0455] Preferably, the permanent cationic lipid that can be included in the nanoparticles is a lipid that includes a head group containing a quaternary nitrogen atom and one or more (preferably one or two) hydrophobic moieties attached to the head group. Preferably, the quaternary nitrogen atom is provided by a group of the formula -N(Me) 3 + wherein Me is a methyl group.

[0456] The hydrophobic moieties included in these preferred lipids preferably include one or more of the following: linear-chain aliphatic residues, for example, linear-chain residues including 8 to 18 carbon atoms, or branched-chain aliphatic residues, for example, branched-chain residues including 8 to 18 carbon atoms. In addition, the hydrophobic moiety may include one or more linking groups that facilitate the attachment of the moiety to the head group or bind two or more of the above aliphatic residues to each other. In addition, one or more substituents may be included provided that the hydrophobic nature of the moiety is maintained. As examples of permanent cationic lipids, reference may be made to DOTMA (dioleoyl-3-trimethylammonium propane) and DOTAP (dioleoyl-3-trimethylammonium propane).

[0457] In a preferred embodiment, component (b) of the nanoparticles comprises or more preferably consists of an ionizable lipid or lipidoid of formula a-I:

[0458]

[0459] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0460] L 1 or L 2 one of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O-; and L 1 or L 2 the other of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR aC(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O- or a direct bond;

[0461] R a is H or C 1 -C 12 alkyl;

[0462] R 1a and R 1b each occurrence is independently (a) H or C 1 -C 12 alkyl, or (b) R 1a is H or C 1 -C 12 alkyl, and R 1b together with the carbon atom to which it is attached and the adjacent R 1b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0463] R 2a and R 2b each occurrence is independently (a) H or C 1 -C 12 alkyl, or (b) R 2a is H or C 1 -C 12 alkyl, and R 2b together with the carbon atom to which it is attached and the adjacent R 2b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0464] R 3a and R 3b each occurrence is independently (a) H or C 1 -C 12 alkyl, or (b) R 3a is H or C 1 -C 12 alkyl, and R 3b together with the carbon atom to which it is attached and the adjacent R 3b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0465] R 4a and R 4b each occurrence is independently (a) H or C 1 -C 12 alkyl, or (b) R 4a is H or C 1 -C 12 alkyl, and R 4b together with the carbon atom to which it is attached and the adjacent R 4bforms a carbon-carbon double bond together with the carbon atom to which it is attached;

[0466] R 5 and R 6 are each independently methyl or cycloalkyl;

[0467] R 7 is independently H or C 1 -C 12 alkyl each time it appears;

[0468] R 8 and R 9 are each independently unsubstituted C 1 -C 12 alkyl; or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocycle containing one nitrogen atom; a and d are each independently an integer from 0 to 24;

[0469] b and c are each independently an integer from 1 to 24;

[0470] e is 1 or 2; and

[0471] x is 0, 1 or 2.

[0472] In some embodiments, the ionizable lipid has a structure of formula a-II:

[0473]

[0474] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0475] L 1 or L 2 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O-; and L 1 or L 2 The other one is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR aC(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O- or a direct bond;

[0476] G 1 is C 1 -C 2 alkylene, -C(=O)-, -O(C=O)-, -SC(=O)-, -NR a C(=O)- or a direct bond;

[0477] G 2 is -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O)NR a - or a direct bond;

[0478] G 3 is C 1 -C 6 alkylene;

[0479] R a is H or C 1 -C 12 alkyl;

[0480] R 1a and R 1b each occurrence is independently (a) H or C 1 -C 12 alkyl, or (b) R 1a is H or C 1 -C 12 alkyl, and R 1b together with the carbon atom to which it is attached and the adjacent R 1b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0481] R 2a and R 2b each occurrence is independently (a) H or C 1 -C 12 alkyl, or (b) R 2a is H or C 1 -C 12 alkyl, and R 2b together with the carbon atom to which it is attached and the adjacent R 2b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0482] R 3a and R 3b each occurrence is independently (a) H or C1 -C 12 alkyl, or (b) R 3a is H or C 1 -C 12 alkyl, and R 3b together with the carbon atom to which it is attached and the adjacent R 3b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0483] R 4a and R 4b each independently, upon each occurrence, is (a) H or C 1 -C 12 alkyl, or (b) R 4a is H or C 1 -C 12 alkyl, and R 4b together with the carbon atom to which it is attached and the adjacent R 4b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0484] R 5 and R 6 are each independently H or methyl;

[0485] R 7 is C 4 -C 20 alkyl;

[0486] R 8 and R 9 are each independently C 1 -C 12 alkyl; or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocycle;

[0487] a, b, c and d are each independently an integer from 1 to 24; and

[0488] x is 0, 1 or 2.

[0489] In some embodiments, the ionizable lipid has the structure of formula a-III:

[0490]

[0491] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0492] L 1 or L 2 one of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x-, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O-; and L 1 or L 2 in which the other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O- or a direct bond;

[0493] G 1 and G 2 each independently is an unsubstituted C 1 -C 12 alkylene or C 1 -C 12 alkenylene;

[0494] G 3 is C 1 -C 24 alkylene, C 1 -C 24 alkenylene, C 3 -C 8 cycloalkylene, C 3 -C 8 cycloalkenylene;

[0495] R a is H or C 1 -C 12 alkyl;

[0496] R 1 and R 2 each independently is C 6 -C 24 alkyl or C 6 -C 24 alkenyl;

[0497] R 3 is H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R4 or -NR 5 C(=O)R 4 ; R 4 is C 1 -C 12 alkyl;

[0498] R 5 is H or C 1 -C 6 alkyl; and

[0499] x is 0, 1 or 2.

[0500] In some embodiments, the ionizable lipid has a structure of Formula a-IV:

[0501]

[0502] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0503] G 1 or G 2 in each occurrence is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y -, -S-S-, -C(=O)S-, SC(=O)-, -N(R a C(=O)-, -C(=O)N(R a )-, N(R a )C(=O)N(R a )-, -OC(=O)N(R a )- or -N(R a )C(=O)O-; and G 1 or G 2 in each occurrence is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y -, -S-S-, -C(=O)S-, SC(=O)-, -N(R a )C(=O)-, -C(=O)N(R a )-, N(R a )C(=O)N(R a )-, -OC(=O)N(R a )- or -N(R a )C(=O)O- or a direct bond;

[0504] L in each occurrence is ~O(C=O)-, where ~ represents a covalent bond with X;

[0505] X is CR a ;

[0506] When n is 1, Z is an alkyl group, a cycloalkyl group or a monovalent moiety comprising at least one polar functional group; or when n is greater than 1, Z is an alkylene group, a cycloalkylene group or a polyvalent moiety comprising at least one polar functional group;

[0507] R a is independently, in each occurrence, H, C 1 -C 12 alkyl, C 1 -C 12 hydroxyalkyl, C 1 -C 12 aminoalkyl, C 1 -C 12 alkylaminoalkyl, C 1 -C 12 alkoxyalkyl, C 1 -C 12 alkoxycarbonyl, C 1 -C 12 alkylcarbonyloxy, C 1 -C 12 alkylcarbonyloxyalkyl or C 1 -C 12 alkylcarbonyl;

[0508] R is independently, in each occurrence, (a) H or C 1 -C 12 alkyl; or (b) R and the carbon atom to which it is attached, together with the adjacent R and the carbon atom to which it is attached, form a carbon-carbon double bond;

[0509] R 1 and R 2 each have the following structure, respectively, in each occurrence:

[0510]

[0511] a 1 and a 2 are independently, in each occurrence, an integer from 3 to 12; b 1 and b 2 are independently, in each occurrence, 0 or 1;

[0512] c 1 and c 2 are independently, in each occurrence, an integer from 5 to 10; d 1 and d 2 are independently, in each occurrence, an integer from 5 to 10; y is independently, in each occurrence, an integer from 0 to 2; and n is an integer from 1 to 6,

[0513] Each of the alkyl, alkylene, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituents.

[0514] In some embodiments, the ionizable lipid has the following formula (a-V):

[0515]

[0516] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0517] G 1 or G 2 One of them is, each time it appears, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y -, -S-S-, -C(=O)S-, SC(=O)-, -N(R a )C(=O)-, -C(=O)N(R a )-, N(R a )C(=O)N(R a )-, -OC(=O)N(R a )- or -N(R a )C(=O)O-; and G 1 or G 2 The other one is, each time it appears, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y -, -S-S-, -C(=O)S-, SC(=O)-, -N(R a )C(=O)-, -C(=O)N(R a )-, N(R a )C(=O)N(R a )-, -OC(=O)N(R a )- or -N(R a )C(=O)O- or a direct bond;

[0518] L is, each time it appears, -O(C=O)-, where ~ represents a covalent bond with X;

[0519] X is CR a ;

[0520] When n is 1, Z is an alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group; or when n is greater than 1, Z is an alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group;

[0521] R aIndependently at each occurrence, H, C 1 -C 12 alkyl, C 1 -C 12 hydroxyalkyl, C 1 -C 12 aminoalkyl, C 1 -C 12 alkylaminoalkyl, C 1 -C 12 alkoxyalkyl, C 1 -C 12 alkoxycarbonyl, C 1 -C 12 alkylcarbonyloxy, C 1 -C 12 alkylcarbonyloxyalkyl or C 1 -C 12 alkylcarbonyl;

[0522] R independently at each occurrence is (a) H or C 1 -C 12 alkyl; or (b) R and the carbon atom to which it is attached, together with the adjacent R and the carbon atom to which it is attached, form a carbon-carbon double bond;

[0523] R 1 and R 2 each have the following structure at each occurrence:

[0524]

[0525] R ’ independently at each occurrence is H or C 1 -C 12 alkyl; a 1 and a 2 independently at each occurrence is an integer from 3 to 12; b 1 and b 2 independently at each occurrence is 0 or 1;

[0526] c 1 and c 2 independently at each occurrence is an integer from 2 to 12; d 1 and d 2 independently at each occurrence is an integer from 2 to 12; y independently at each occurrence is an integer from 0 to 2; and n is an integer from 1 to 6,

[0527] where a 1 、a 2 、c 1 、c 2 、d 1 and d 2 are selected such that a 1 +c1 +d 1 The sum of which is an integer from 18 to 30, a 2 +c 2 +d 2 The sum of which is an integer from 18 to 30, and each alkyl, alkylene, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl, and alkylcarbonyl is optionally substituted with one or more substituents.

[0528] In some embodiments, the ionizable lipid is selected from the lipids in Table 1, Table 2, Table 3, or Table 4.

[0529] Table 1:

[0530]

[0531]

[0532]

[0533]

[0534]

[0535]

[0536]

[0537] Table 2:

[0538]

[0539]

[0540]

[0541]

[0542]

[0543]

[0544]

[0545]

[0546] Table 3:

[0547]

[0548]

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555] Table 4:

[0556]

[0557] In some embodiments, the ionizable lipid has one of the following structures:

[0558]

[0559] In some embodiments, the ionizable lipid has the following structure:

[0560]

[0561] or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein: R 1 and R 2 each independently at each occurrence is optionally substituted C 10 -C 30 alkyl, optionally substituted C 10 -C 30 alkenyl, optionally substituted C 10 -C 30 alkynyl, or optionally substituted C 10 -C 30 acyl;

[0562] R 3 is H, optionally substituted C 10 -C 10 alkyl, optionally substituted C 2 -C 10 alkenyl, optionally substituted C 2 -C 10Alkynyl, alkyl heterocycle, alkyl phosphate, alkyl thiophosphate, alkyl dithiophosphate, alkyl phosphonate, alkylamine, hydroxyalkyl, ω-aminoalkyl, ω-(substituted) aminoalkyl, ω-phosphinoalkyl, ω-thiophosphinoalkyl, optionally substituted polyethylene glycol (PEG, mw 100 - 40K), optionally substituted mPEG (mw 120 - 40K), heteroaryl or heterocycle or linking ligand; and

[0563] E is O, S, N(Q), C(O), N(Q)C(O), C(O)N(Q), (Q)N(CO)O, O(CO)N(Q), S(O), NS(O) 2 N(Q), S(O) 2 、N(Q)S(O) 2 、SS, O=N, aryl, heteroaryl, cyclic or heterocyclic; and

[0564] Q is H, alkyl, ω-aminoalkyl, ω-(substituted) aminoalkyl, ω-phosphinoalkyl or ω-thiophosphinoalkyl.

[0565] In some embodiments, the ionizable lipid has one of the following structures:

[0566]

[0567] In a preferred embodiment, the ionizable lipid is ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), also known as ALC-0315, and is shown as follows:

[0568]

[0569] In some embodiments, the molar ratio of the ionizable lipid to the neutral lipid ranges from about 4.1:1.0 to about 4.9:1.0, 4.5:1.0 to about 4.8:1.0, or 4.7:1.0 to about 4.8:1.0. In some embodiments, the molar ratio of the cationic lipid to the neutral lipid ranges from about 2:1 to about 8:1, preferably 5:1 to 1:1.

[0570] In some embodiments, the molar ratio of the ionizable lipid to the polymer-conjugated lipid ranges from about 35:1 to about 25:1, or 100:1 to about 20:1.

[0571] In some embodiments, the ionizable lipid has the following structure:

[0572]

[0573] A further aspect of the invention relates to a lipid nanoparticle comprising:

[0574] i) a first cationic lipid as the ionizable lipid (a), which has a first effective pKa;

[0575] ii) a second cationic lipid as the ionizable lipid (a), which has a second effective pKa, and the second effective pKa is greater than the first effective pKa;

[0576] iii) a neutral lipid;

[0577] iv) a steroid;

[0578] v) a polymer-conjugated lipid;

[0579] vi) a therapeutic agent encapsulated in or associated with the lipid nanoparticle or a pharmaceutically acceptable salt or prodrug thereof; and

[0580] vii) a surfactant,

[0581] wherein the effective pKa of the lipid nanoparticle is between the first and second effective pKas.

[0582] In some embodiments, the first effective pKa is less than 5.75. In some embodiments, the second effective pKa is greater than 6.25. In some embodiments, the lipid nanoparticle of any one of claims 48 - 50, wherein the effective pKa of the lipid nanoparticle ranges from 5.90 to 6.35. In some embodiments, the molar ratio of the first cationic lipid to the second cationic lipid ranges from 1:20 to 1:2.

[0583] In some embodiments, the LNP or LiNP of the present invention comprises a first cationic lipid as the ionizable lipid (a), or a second cationic lipid as the ionizable lipid (a), and one or both of them have a structure of formula a-I:

[0584]

[0585] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0586] L 1 or L 2 one of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NRa C(=O)O-; and L 1 or L 2 and the other of L is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -, or -NR a C(=O)O- or a direct bond;

[0587] R a is H or C 1 -C 12 alkyl;

[0588] R 1a and R 1b each occurrence is independently (a) H or C 1 -C 12 alkyl, or (b) R 1a is H or C 1 -C 12 alkyl, and R 1b together with the carbon atom to which it is attached and the adjacent R 1b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0589] R 2a and R 2b each occurrence is independently (a) H or C 1 -C 12 alkyl, or (b) R 2a is H or C 1 -C 12 alkyl, and R 2b together with the carbon atom to which it is attached and the adjacent R 2b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0590] R 3a and R 3b each occurrence is independently (a) H or C 1 -C 12 alkyl, or (b) R 3a is H or C 1 -C 12 alkyl, and R 3b together with the carbon atom to which it is attached and the adjacent R 3b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0591] R 4a and R 4b each independently at each occurrence is (a) H or C 1 -C 12 -alkyl, or (b) R 4a is H or C 1 -C 12 -alkyl, and R 4b together with the carbon atom to which it is attached and the adjacent R 4b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0592] R 5 and R 6 are each independently methyl or cycloalkyl;

[0593] R 7 each independently at each occurrence is H or C 1 -C 12 -alkyl;

[0594] R 8 and R 9 are each independently unsubstituted C 1 -C 12 -alkyl; or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocycle including one nitrogen atom;

[0595] a and d are each independently an integer from 0 to 24;

[0596] b and c are each independently an integer from 1 to 24;

[0597] e is 1 or 2; and

[0598] x is 0, 1 or 2.

[0599] In some embodiments, the first and second cationic lipids as the ionizable lipid (a) are each independently selected from lipids of formula a-I. In some embodiments, the first cationic lipid or the second cationic lipid or both have the structure of formula a-II:

[0600]

[0601] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0602] L 1 or L 2 one of is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x-, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O-; and L 1 or L 2 in which the other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O- or a direct bond;

[0603] G 1 is C 1 -C 2 alkylene, -C(=O)-, -O(C=O)-, -SC(=O)-, -NR a C(=O)- or a direct bond;

[0604] G 2 is -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O)NR a - or a direct bond; G 3 is C 1 -C 6 alkylene;

[0605] R a is H or C 1 -C 12 alkyl;

[0606] R 1a and R 1b each occurrence is independently (a) H or C 1 -C 12 alkyl, or (b) R 1a is H or C 1 -C 12 alkyl, and R 1b together with the carbon atom to which it is attached and the adjacent R 1b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0607] R 2aand R 2b is independently, at each occurrence, (a) H or C 1 -C 12 alkyl, or (b) R 2a is H or C 1 -C 12 alkyl, and R 2b together with the carbon atom to which it is attached and the adjacent R 2b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0608] R 3a and R 3b is independently, at each occurrence, (a) H or C 1 -C 12 alkyl, or (b) R 3a is H or C 1 -C 12 alkyl, and R 3b together with the carbon atom to which it is attached and the adjacent R 3b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0609] R 4a and R 4b is independently, at each occurrence, (a) H or C 1 -C 12 alkyl, or (b) R 4a is H or C 1 -C 12 alkyl, and R 4b together with the carbon atom to which it is attached and the adjacent R 4b and the carbon atom to which it is attached form a carbon-carbon double bond;

[0610] R 5 and R 6 are each independently H or methyl;

[0611] R 7 is C 4 -C 20 alkyl;

[0612] R 8 and R 9 are each independently C 1 -C 12 alkyl; or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocycle;

[0613] a, b, c and d are each independently an integer from 1 to 24; and

[0614] x is 0, 1 or 2.

[0615] In some embodiments, the first and second cationic lipids as the ionizable lipid (a) are each independently selected from the lipids of formula a-II.

[0616] In some embodiments, the first cationic lipid as the ionizable lipid (a) or the second cationic lipid as the ionizable lipid (a) or both have the structure of formula a-III:

[0617]

[0618] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0619] L 1 or L 2 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O-; and L 1 or L 2 The other of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O- or a direct bond;

[0620] G 1 and G 2 are each independently unsubstituted C 1 -C 12 alkylene or C 1 -C 12 alkenylene;

[0621] G 3 is C 1 -C 24 alkylene, C 1 -C 24 alkenylene, C 3 -C 8Cycloalkylene, C 3 -C 8 Cycloalkenylene;

[0622] R a is H or C 1 -C 12 alkyl;

[0623] R 1 and R 2 are each independently C 6 -C 24 alkyl or C 6 -C 24 alkenyl;

[0624] R 3 is H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 is C 1 -C 12 alkyl;

[0625] R 5 is H or C 1 -C 6 alkyl; and

[0626] x is 0, 1 or 2.

[0627] In some embodiments, the first and second cationic lipids as the ionizable lipid (a) are each independently selected from the lipids of formula a-III.

[0628] In some embodiments, the first cationic lipid as the ionizable lipid (a) or the second cationic lipid as the ionizable lipid (a) or both have the structure of formula a-IV:

[0629]

[0630] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0631] G 1 or G 2 One of them is, each time it appears, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y -, -S-S-, -C(=O)S-, SC(=O)-, -N(R a )C(=O)-, -C(=O)N(R a )-, N(R a)C(=O)N(R a )-, -OC(=O)N(R a )- or -N(R a )C(=O)O-; and G 1 or G 2 in each occurrence is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y -, -S-S-, -C(=O)S-, SC(=O)-, -N(R a )C(=O)-, -C(=O)N(R a )-, N(R a )C(=O)N(R a )-, -OC(=O)N(R a )- or -N(R a )C(=O)O- or a direct bond; L in each occurrence is ~O(C=O)-, where ~ represents a covalent bond with X;

[0632] X is CR a ;

[0633] When n is 1, Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group; or when n is greater than 1, Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group;

[0634] R a in each occurrence is independently H, C 1 -C 12 alkyl, C 1 -C 12 hydroxyalkyl, C 1 -C 12 aminoalkyl, C 1 -C 12 alkylaminoalkyl, C 1 -C 12 alkoxyalkyl, C 1 -C 12 alkoxycarbonyl, C 1 -C 12 alkylcarbonyloxy, C 1 -C 12 alkylcarbonyloxyalkyl or C 1 -C 12 alkylcarbonyl;

[0635] R in each occurrence is independently (a) H or C 1 -C 12 alkyl; or (b) R and the carbon atom to which it is attached together with the adjacent R and the carbon atom to which it is attached form a carbon-carbon double bond;

[0636] R 1 and R 2 each has the following structure respectively at each occurrence:

[0637]

[0638] a 1 and a 2 is independently an integer from 3 to 12 at each occurrence; b 1 and b 2 is independently 0 or 1 at each occurrence;

[0639] c 1 and c 2 is independently an integer from 5 to 10 at each occurrence; d 1 and d 2 is independently an integer from 5 to 10 at each occurrence; y is independently an integer from 0 to 2 at each occurrence; and n is an integer from 1 to 6,

[0640] wherein each alkyl, alkylene, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted by one or more substituents.

[0641] In some embodiments, the first and second cationic lipids as the ionizable lipid (a) are each independently selected from lipids of formula a-IV.

[0642] In some embodiments, the first cationic lipid as the ionizable lipid (a) or the second cationic lipid as the ionizable lipid (a) or both have the structure of formula a-V:

[0643]

[0644] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0645] G 1 or G 2 one of which is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y -, -S-S-, -C(=O)S-, SC(=O)-, -N(R a )C(=O)-, -C(=O)N(R a )-, N(R a )C(=O)N(R a )-, -OC(=O)N(R a )- or -N(R a )C(=O)O- at each occurrence; and G1 or G 2 in which another, at each occurrence, is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y -, -S-S-, -C(=O)S-, SC(=O)-, -N(R a )C(=O)-, -C(=O)N(R a )-, N(R a )C(=O)N(R a )-, -OC(=O)N(R a )- or -N(R a )C(=O)O- or a direct bond;

[0646] L, at each occurrence, is ~O(C=O)-, where ~ represents a covalent bond with X;

[0647] X is CR a ;

[0648] When n is 1, Z is an alkyl group, a cycloalkyl group or a monovalent moiety comprising at least one polar functional group; or when n is greater than 1, Z is an alkylene group, a cycloalkylene group or a polyvalent moiety comprising at least one polar functional group;

[0649] R a , at each occurrence, is independently H, C 1 -C 12 alkyl, C 1 -C 12 hydroxyalkyl, C 1 -C 12 aminoalkyl, C 1 -C 12 alkylaminoalkyl, C 1 -C 12 alkoxyalkyl, C 1 -C 12 alkoxycarbonyl, C 1 -C 12 alkylcarbonyloxy, C 1 -C 12 alkylcarbonyloxyalkyl or C 1 -C 12 alkylcarbonyl;

[0650] R, at each occurrence, is independently (a) H or C 1 -C 12 alkyl; or (b) R and the carbon atom to which it is attached, together with the adjacent R and the carbon atom to which it is attached, form a carbon-carbon double bond;

[0651] R 1 and R 2 , at each occurrence, respectively have the following structures:

[0652]

[0653] R ’ independently H or C at each occurrence 1 -C 12 alkyl; a 1 and a 2 independently an integer from 3 to 12 at each occurrence; b 1 and b 2 independently 0 or 1 at each occurrence;

[0654] c 1 and c 2 independently an integer from 2 to 12 at each occurrence; d 1 and d 2 independently an integer from 2 to 12 at each occurrence; y is independently an integer from 0 to 2 at each occurrence; and n is an integer from 1 to 6,

[0655] wherein a 1 、a 2 、c 1 、c 2 、d 1 and d 2 are selected such that the sum of a 1 +c 1 +d 1 is an integer from 18 to 30, a 2 +c 2 +d 2 is an integer from 18 to 30, and wherein each alkyl, alkylene, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituents.

[0656] In some embodiments, the first and second cationic lipids as the ionizable lipid (a) are each independently selected from lipids of formula a-V.

[0657] In some embodiments, the first and second cationic lipids as the ionizable lipid (a) have the following structures, respectively:

[0658]

[0659] In some embodiments, the first cationic lipid as the ionizable lipid (a), the second cationic lipid as the ionizable lipid (a), or both have one of the following structures:

[0660]

[0661] In some embodiments, when using a compound of formula a-II, based on the total lipids present in the lipid nanoparticle, the total molar percentage of the cationic lipid as the ionizable lipid (a) in the lipid nanoparticle ranges from 40 to 55 mole percent. In some embodiments, the molar ratio of the total amount of cationic lipid to neutral lipid ranges from about 2:1 to about 8:1. In some embodiments, the molar ratio of the total amount of cationic lipid to steroid is from 5:1 to 1:1. In some embodiments, the molar ratio of the total cationic lipid to the polymer-conjugated lipid ranges from about 100:1 to about 20:1.

[0662] In some embodiments, the neutral lipid is distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOGG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyloleoyl phosphatidylcholine (POPC), palmitoyloleoyl phosphatidylethanolamine (POPE), and dioleoyl phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE), or 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE). The neutral lipid is preferably DSPC, DPPC, DMPC, DOPC, POPC, DOPE, or SM. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol.

[0663] In some embodiments, the concentration of the polymer-conjugated lipid is from 1.0 to 2.5 mole percent, preferably about 1.7 mole percent, wherein the concentration of the polymer-conjugated lipid is about 1.5 mole percent.

[0664] In some embodiments, the polymer-conjugated lipid is a polyethylene glycolated lipid. In some embodiments, the polyethylene glycolated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG dialkoxypropyl carbamate. In some embodiments, the polyethylene glycolated lipid has the following formula (a-VI):

[0665]

[0666] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

[0667] R12 and R 13 each independently is a straight-chain or branched-chain, saturated or unsaturated alkyl chain having 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and

[0668] The average value of w ranges from 30 to 60.

[0669] Optionally, R 12 and R 13 each independently is a straight-chain saturated alkyl chain having 12 to 16 carbon atoms. Optionally, the average value of w ranges from 42 to 55, and preferably the average value of w is about 49. In some embodiments, the polyethylene glycolylated lipid has the following formula (VIa):

[0670]

[0671] wherein the average value of w is about 49.

[0672] In some embodiments, the lipid nanoparticles form a plurality of nanoparticles having a polydispersity of less than 0.12. Preferably, the polydispersity is less than 0.08.

[0673] In some embodiments, the average diameter ranges from 50 nm to 100 nm, and preferably the diameter ranges from 60 nm to 85 nm.

[0674] Aspects of the present invention relate to a method of administering a therapeutic agent to a patient in need thereof, the method comprising administering to the patient the lipid nanoparticles of the present invention or the pharmaceutical composition of the present invention.

[0675] A further aspect of the present invention relates to a method of treating a disease in a patient in need thereof, the method comprising administering to the patient the lipid nanoparticles comprising a surfactant of the present invention, or a pharmaceutical composition comprising a surfactant, wherein the therapeutic agent is effective for treating the disease.

[0676] In a preferred embodiment, component (b) of the nanoparticles for the various aspects of the present invention comprises or more preferably consists of an ionizable lipid of formula (b-1) or its protonated form. The ionizable lipid of formula (b-1) or its protonated form that can be used as the preferred component (b) in the context of the present invention is described in detail in PCT application WO 2014 / 207231A1.

[0677] Accordingly, component (b) preferably comprises a lipid of formula (b-1) or consists of a lipid of formula (b-1)

[0678]

[0679] wherein the variables a, b, p, m, n and R 1A to R6A is defined as follows:

[0680] a is 1 and b is an integer from 2 to 4; or a is an integer from 2 to 4 and b is 1,

[0681] p is 1 or 2,

[0682] m is 1 or 2; n is 0 or 1, and m + n ≥ 2; and

[0683] R 1A to R 6A are independently selected from hydrogen; -CH 2 -CH(OH)-R 7A 、-CH(R 7A )-CH 2 -OH、-CH 2 -CH 2 -(C=O)-O-R 7A 、-CH 2 -CH 2 (C=O)-NH-R 7A ;-CH 2 -R 7A ;-C(NH)-NH 2 ;a poly(ethylene glycol) chain; and a receptor ligand; wherein R 7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond;

[0684] provided that at least two residues in R 1A -R 6A are selected from -CH 2 -CH(OH)-R 7A 、-CH(R 7A )CH 2 -OH、-CH 2 -CH 2 (C=O)-O-R 7A 、-CH 2 -CH 2 -(C=O)-NH-R 7A and -CH 2 -R 7A ,wherein R 7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond;

[0685] or its protonated form, wherein one or more of the nitrogen atoms contained in the compound of formula (I) are protonated to provide a compound carrying a positive charge.

[0686] Preferably, R 1A to R 6A are independently selected from hydrogen; the group -CH2 -CH(OH)-R 7A ,-CH(R 7A )-CH 2 -OH,-CH 2 -CH 2 -(C=O)-O-R 7A ,-CH 2 -CH 2 -(C=O)-NH-R 7A ; and -CH 2 -R 7A , wherein R 7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond; provided that at least two residues in R 1A -R 6A , more preferably at least three residues in R 1A -R 6A and still more preferably at least four residues in R 1A to R 6A are selected from -CH 2 -CH(OH)-R 7A , -CH(R 7A )-CH 2 -OH, -CH 2 -CH 2 -(C=O)-O-R 7A , -CH 2 -CH 2 -(C=O)-NH-R 7A and -CH 2 -R 7A , wherein R 7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond. More preferably, R 1A -R 6A are independently selected from hydrogen and the group -CH 2 -CH(OH)-R 7A , wherein R 7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond; provided that at least two residues in R 1A to R 6A , more preferably at least three residues in R 1A to R 6A and still more preferably at least four residues in R 1A to R 6A are the group -CH 2 -CH(OH)-R 7A , wherein R 7ASelected from C3-C18 alkyl groups and C3-C18 alkenyl groups having one C-C double bond.

[0687] Preferably, R 7A is selected from C8-C18 alkyl groups and C8-C18 alkenyl groups having one C-C double bond, and more preferably is selected from C8-C12 alkyl groups and C8-C12 alkenyl groups having 1 C-C double bond. Generally, as R 7A , alkyl groups are preferred over alkenyl groups.

[0688] Regarding any one of the groups R 1A to R 6A being a protecting group for an amino group, such as those described in WO2006 / 138380, its preferred embodiments are tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), or carbobenzyloxy (Cbz).

[0689] Regarding any one of the groups R 1A to R 6A being a receptor ligand, useful examples are given by Philipp and Wagner in Chapter 15 of "Gene and Cell Therapy - Therapeutic Mechanisms and Strategy", 3rd Edition, CRC Press, Taylor & Francis Group LLC, Boca Raton 2009. Preferred receptor ligands for lung tissue are described in Pfeifer et al. 2010, Ther Deliv. 1(1):133-48. Preferred receptor ligands include synthetic cyclic or linear peptides, such as those derived from screening peptide libraries for binding to specific cell surface structures or specific cell types, cyclic or linear RGD peptides, synthetic or natural carbohydrates, such as sialic acid, galactose, or mannose, or synthetic ligands derived from carbohydrates, such as those reacting with peptides, antibodies specifically recognizing cell surface structures, folic acid, epidermal growth factor and its derived peptides, transferrin, anti-transferrin receptor antibodies, nanobodies, and antibody fragments, or approved drugs that bind to known cell surface molecules.

[0690] Regarding any one of the groups R 1A to R 6A being a poly(ethylene glycol) chain, the preferred molecular weight of the poly(ethylene glycol) chain is 100 - 20000 g / mol, more preferably 1000 - 10000 g / mol, and most preferably 1000 - 5000 g / mol.

[0691] The variable p in formula (b-1) is preferably 1.

[0692] In formula (b-1), m is 1 or 2; n is 0 or 1, and m + n ≥ 2. In other words, if m is 1, then n must also be 1, and if m is 2, then n can be 0 or 1. If n is 0, then m must be 2. If n is 1, then m can be 1 or 2.

[0693] The variable n in formula (b-1) is preferably 1. More preferably, m is 1 and n is 1.

[0694] Therefore, the combination of p = 1, m = 1, and n = 1 is equally preferred.

[0695] For the variables a and b in formula (b-1), preferably, one of a and b is 1 and the other is 2 or 3. More preferably, a is 1 and b is 2, or a is 2 and b is 1. Most preferably, a is 1 and b is 2.

[0696] In view of the above, it is further preferred that the compound of formula (b-1) is a compound of formula (b-1a), and component (b) comprises a lipid of the following formula (b-1a) or consists of a lipid of the following formula (b-1a):

[0697] R 1A -NR 2A -CH 2 -(CH 2 ) a -NR 3A -CH 2 -(CH 2 ) b -NR 4A -CH 2 -(CH 2 ) a -NR 5A -R 6A (b-1a),

[0698] wherein a, b, and R 1A to R 6A are as defined in formula (b-1), including its preferred embodiments;

[0699] or its protonated form, wherein one or more of the nitrogen atoms shown in formula (b-1a) are protonated to provide a compound carrying a positive charge.

[0700] According to a still further preferred embodiment, the compound of formula (b-1) is a compound of formula (b-1b), and component (b) comprises a lipid compound of the following formula (b-1b) or consists of a lipid compound of the following formula (b-1b),

[0701]

[0702] wherein R 1A to R 6A is as defined in formula (Ia), including its preferred embodiments;

[0703] or a protonated form thereof, wherein one or more of the nitrogen atoms shown in formula (b-1b) are protonated to provide a compound carrying a positive charge.

[0704] Thus, according to a particularly preferred embodiment, component (b) comprises a lipid compound of the above formula (b-1b) or a protonated form thereof or consists of a lipid compound of the above formula (b-1b) or a protonated form thereof, and R 1A to R 6A are independently selected from hydrogen and -CH 2 -CH-(OH)R 7A wherein R 7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one -C-C double bond, provided that at least two of the residues R 1A to R 6A are -CH 2 -CH(OH)-R 7A , more preferably at least three of the residues R 1A to R 16A , and still more preferably at least four of the residues R 1A to R 6A are -CH 2 -CH(OH)-R 7A wherein R 7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond.

[0705] As an example of a suitable lipid compound that can be used as an ionizable lipid in the context of the present invention, reference can be made to the cationic lipid dL_05(R), the structure of which is as follows:

[0706]

[0707] According to a further exemplary embodiment, component (b) comprises an ionizable lipid of formula (b-2) or consists of an ionizable lipid of formula (b-2)

[0708]

[0709] wherein R 1B is an organic group containing one or more primary, secondary or tertiary amino groups,

[0710] or a protonated form thereof, wherein one or more of the nitrogen atoms contained in the primary, secondary or tertiary amino groups contained in R 1B are protonated to provide a compound carrying a positive charge.

[0711] Preferably, the compound of formula (b-2) has the following structure:

[0712]

[0713] According to another exemplary embodiment, component (b) comprises an ionizable lipid of formula (b-3) or consists of an ionizable lipid of formula (b-3)

[0714]

[0715] wherein

[0716] R 1C and R 2C are independently selected from C8-C18 alkyl and C8-C18 alkenyl, preferably selected from C12-C18 alkyl and C12-C18 alkenyl,

[0717] R 3C is a C1-C6 alkanediyl group, preferably a C2 or C3 alkanediyl group, and

[0718] R 4C and R 5C are independently hydrogen or C1-C3 alkyl, and preferably methyl;

[0719] or its protonated form, wherein one or more of the nitrogen atoms contained in the compound of formula (b-3) are protonated to provide a compound carrying a positive charge. As an example of the ionizable lipid of formula (b-3), reference may be made to DLin-MC3-DMA ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butyrate).

[0720] According to still another exemplary embodiment, component (b) comprises an ionizable lipid of formula (b-4) or consists of an ionizable lipid of formula (b-4)

[0721]

[0722] wherein

[0723] R 1D and R 2D are independently selected from C8-C18 alkyl and C8-C18 alkenyl, preferably selected from C12-C18 alkyl and C12-C18 alkenyl,

[0724] R 3D is a C1-C6 alkanediyl group, preferably a C2 alkanediyl group, and

[0725] R 4Dand R 5D are each independently hydrogen or a C1-C3 alkyl group, and preferably a methyl group;

[0726] or a protonated form thereof, wherein one or more of the nitrogen atoms contained in the compound of formula (b-4) are protonated to provide a compound carrying a positive charge.

[0727] According to yet another exemplary embodiment, component (b) comprises an ionizable lipid of formula (b-5) or consists of an ionizable lipid of formula (b-5)

[0728]

[0729] wherein R 1E to R 5E are each independently selected from hydrogen, -CH 2 -CH(OH)-R 7E 、-CH(R 7E )-CH 2 -OH、-CH 2 -CH 2 -(C=O)-O-R 7E 、-CH 2 -CH 2 (C=O)-NH-R 7E and -CH 2 -R 7E ,wherein R 7E is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond, provided that at least two of the residues from R 1E to R 5E are selected from -CH 2 -CH(OH-)-R 7E 、-CH(R 7E )-CH 2 -OH、-CH 2 -CH 2 -(C=O)-O-R 7E 、-CH 2 -CH 2 -(C=O)-NH-R 7E and -CH 2 -R 7E ,wherein R 7E is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond;

[0730] or a protonated form thereof, wherein one or more of the nitrogen atoms contained in the compound of formula (b-5) are protonated to provide a compound carrying a positive charge.

[0731] In formula (b-5), R1E to R 5E is preferably independently -CH 2 -CH(OH)-R 7E , wherein R 7E is selected from C8-C18 alkyl or C8-C18 alkenyl having one C-C double bond.

[0732] Still another exemplary ionizable lipid suitable for use in the present invention and which may be included in component (b) or of which component (b) may consist is the "cationic lipid of formula I" disclosed in PCT application WO2012 / 001004A1, starting on page 104 of that document and including all of its specific embodiments also discussed in that document.

[0733] A further exemplary ionizable lipid suitable for use in the present invention and which may be included in component (b) or of which component (b) may consist is the ionizable lipid disclosed in PCT application WO 2010 / 053572A2 and claimed as "amino alcohol lipids", which includes compounds of all general formulas shown in the summary of the invention on page 4 of that document and further defined in the remainder of the application.

[0734] Still further exemplary ionizable lipids suitable for use in the present invention and which may be included in component (b) or of which component (b) may consist are the ionizable lipids disclosed in PCT application WO 2014 / 028487A1 as amine-containing lipids of formulas I to V, including their specific embodiments.

[0735] A further preferred example of an ionizable lipid suitable for use in the present invention and which may be included in component (b) or of which component (b) may consist is the ionizable lipid ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) or its protonated form, wherein the nitrogen atom of the compound is protonated to provide a positively charged compound.

[0736] Still further preferred examples of an ionizable lipid suitable for use in the present invention and which may be included in component (b) or of which component (b) may consist are the ionizable lipid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate) or its protonated form, wherein the nitrogen atom of the compound is protonated to provide a positively charged compound.

[0737] Still further preferred examples of ionizable lipids that can be included in component (b) or of which component (b) can consist for use in the present invention are ionizable lipid 8-[(2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino]octanoic acid heptadec-9-yl ester (SM-102) or its protonated form, wherein the nitrogen atom of the compound is protonated to provide a positively charged compound.

[0738] As a preferred optional component in addition to (a) a therapeutic agent (preferably a nucleic acid) and (b) at least one selected from the group consisting of permanent cationic lipids, ionizable lipids, and ionizable lipidoids (preferably ionizable lipids and ionizable lipidoids), the nanoparticles may include one or more of the following components (c1) to (c6):

[0739] (c1) a non-ionizable lipid having a sterol structure;

[0740] (c2) a phosphoglyceride lipid;

[0741] (c3) a PEG-conjugated lipid;

[0742] (c4) a poly(sarcosine)-conjugated lipid;

[0743] (c5) a PASylated lipid; and

[0744] (c6) a cationic polymer.

[0745] Those skilled in the art will understand that the possibility of the nanoparticles including one or more of components (c1) to (c6) includes not only combinations between (c1) to (c6), but also combinations of different components of one type, for example, two components (c2), or combinations of different components of one type with other components of (c1) to (c6).

[0746] Component (c1) is a lipid having a sterol structure. Accordingly, suitable lipids are compounds having a steroid core structure and having a hydroxyl group at the 3-position of the A ring.

[0747] Exemplary non-ionizable lipids having a sterol structure included by or of which component (c1) can consist have the structure of formula (c1-1)

[0748]

[0749] wherein R 1L is a C3-C12 alkyl group.

[0750] Further exemplary non-ionizable lipids having a sterol structure that are included by or from which component (c1) can be composed include those disclosed in S. Patel et al., Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA, Nature Communications, 2020, 11:983, particularly those shown in Figure 2 of that publication.

[0751] Preferably, component (c1) comprises cholesterol or consists of cholesterol.

[0752] Component (c2) is a phosphoglyceride.

[0753] Preferably, component (c2) comprises or consists of a phospholipid selected from

[0754] compounds of formula (c2-1)

[0755]

[0756] wherein

[0757] R 1F and R 2F are independently selected from C8-C18 alkyl and C8-C18 alkenyl, preferably selected from C12-C18 alkyl and C12-C18 alkenyl,

[0758] or a pharmaceutically acceptable salt thereof;

[0759] and phospholipids of formula (c2-2)

[0760]

[0761] wherein

[0762] R 1G and R 2G are independently selected from C8-C18 alkyl and C8-C18 alkenyl, preferably selected from C12-C18 alkyl and C12-C18 alkenyl,

[0763] or a pharmaceutically acceptable salt thereof.

[0764] More preferably, component (c2) comprises or consists of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or a pharmaceutically acceptable salt thereof or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a pharmaceutically acceptable salt thereof.

[0765] Exemplary salt forms of the compound of formula (c2-1) include salts formed from acidic -OH groups with bases, or salts formed from amino groups with acids. As salts formed with bases, mention may be made of alkali metal salts such as sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts and ammonium salts. As exemplary salts formed with acids, mention may be made of salts formed with the acidic groups of nucleic acids, but other salts are not excluded, and mineral acid salts such as chlorides, bromides or iodides, sulfates, nitrates, phosphates, hydrogen phosphates or dihydrogen phosphates, carbonates and bicarbonates may be mentioned as examples.

[0766] Exemplary salt forms of the compound of formula (c2-2) include salts formed from acidic -OH groups attached to the P atom with bases, or salts formed from quaternary amino groups with anions. As salts formed with bases, mention may be made of alkali metal salts such as sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts and ammonium salts. As exemplary salts formed with anions, mention may be made of salts formed with the acidic groups of nucleic acids, but other salts are not excluded, and mineral acid salts such as chlorides, bromides or iodides, sulfates, nitrates, phosphates, hydrogen phosphates or dihydrogen phosphates, carbonates and bicarbonates may be mentioned as examples.

[0767] Component (c3) is a PEG-conjugated lipid, i.e., a lipid covalently linked to a polyethylene glycol chain.

[0768] Preferably, component (c3) comprises or consists of a PEG-conjugated lipid selected from the following

[0769] Compound of formula (c3-1)

[0770]

[0771] wherein

[0772] R 1H and R 2H are independently selected from C8-C18 alkyl and C8-C18 alkenyl, preferably selected from C12-C18 alkyl or C12-C18 alkenyl, and p is an integer from 5 to 200, preferably 10 to 100, more preferably 20 to 60;

[0773] Compound of formula (c3-2)

[0774]

[0775] wherein

[0776] R 1J and R 2JIndependently selected from C8-C18 alkyl and C8-C18 alkenyl, preferably selected from C12-C18 alkyl or C12-C18 alkenyl, and q is an integer from 5 to 200, preferably from 10 to 100, and more preferably from 20 to 60

[0777] or a pharmaceutically acceptable salt thereof,

[0778] or a compound of formula (c3-3)

[0779]

[0780] wherein

[0781] R 1K and R 2K Independently selected from C8-C18 alkyl and C8-C18 alkenyl, preferably selected from C12-C18 alkyl or C12-C18 alkenyl, and q is an integer from 5 to 200, preferably from 10 to 100, and more preferably from 20 to 60.

[0782] Exemplary salt forms of the compound of formula (c3-2) include salts formed with a base by an acidic -OH group attached to the P atom. As salts formed with a base, mention may be made of alkali metal salts such as sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts and ammonium salts.

[0783] More preferably, component (c3) comprises or consists of 1,2-dimyristoyl-sn-glycero methoxy(polyethylene glycol) (DMG-PEG), and still more preferably component (d) comprises or consists of 1,2-dimyristoyl-sn-glycero methoxy(polyethylene glycol)-2000 (DMG-PEG2k) or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylethanamide (ALC-0159).

[0784] Component (c4) is a poly(sarcosine)-conjugated lipid, i.e., a lipid covalently linked to a polymeric moiety of formula (c4-1):

[0785] -[C(O)-CH 2 -N(CH 3 )] r -(c4-1)

[0786] where r represents the number of repeating units and is preferably from 10 to 100.

[0787] Component (c5) is a PASylated lipid, for example, a lipid covalently linked to a polymeric moiety formed by repeating residues of proline (pro) / alanine (ala) / serine (ser).

[0788] Regarding the PAS-acylated lipids used herein, the contents of WO 2017 / 109087 A1 and EP 3394266 B1 are incorporated herein by reference. In particular, the definitions and embodiments incorporated herein by reference as described below, specifically, embodiments of nucleic acids encoding PAS polypeptides are enumerated. The PASylated lipids can include, for example, polypeptides consisting of at least 100 amino acid residues of proline, alanine, and optionally serine, wherein the polypeptides form random coils.

[0789] Component (c6) is a cationic polymer. Such polymers suitable for forming nanoparticles containing nucleic acids are known in the art. Exemplary suitable cationic polymers are discussed in A.C. Silva et al., Current Drug Metabolism, 16, 2015, 3-16 and the references cited therein, in J.C. Kasper et al., J. Contr. Rel. 151 (2011), 246-255, in WO 2014 / 207231 and the references cited therein, and in WO 2016 / 097377 and the references cited therein.

[0790] Suitable cationic oligomers or polymers specifically include cationic polymers containing multiple units in which amino groups are included. The amino groups can be protonated to provide the cationic charge of the polymer.

[0791] The polymer is preferably one that contains multiple units independently selected from the following (1), (2), (3), and (4):

[0792] -CH 2 -CH 2 -NH-(1)

[0793]

[0794] -CH 2 -CH 2 -CH 2 -NH-(3)

[0795]

[0796] wherein one or more of the nitrogen atoms in the repeating units (1), (2), (3), and / or (4) can be protonated to provide the cationic charge of the polymer.

[0797] As the cationic polymer, four types of polymers containing multiple units in which amino groups are included are particularly preferred.

[0798] As a first preferred class, poly(ethyleneimine) (“PEI”) is mentioned, which includes branched poly(ethyleneimine) (“brPEI”).

[0799] A second preferred class of cationic polymers are polymers that contain multiple groups of the following formula (c6-1) as side chains and / or as end groups, as they are disclosed as formula (II) groups in WO 2014 / 207231 (applicant ethris GmbH):

[0800]

[0801] where for each group of formula (c6-1) among multiple such groups, the variables a, b, p, m, n, and R 2 to R 6 are independently defined as follows:

[0802] a is 1 and b is an integer from 2 to 4; or a is an integer from 2 to 4 and b is 1,

[0803] p is 1 or 2,

[0804] m is 1 or 2; n is 0 or 1, and m + n ≥ 2; and

[0805] R 2 to R 5 are independently selected from hydrogen; the group -CH 2 -CH(OH)-R 7 、-CH(R 7 )-CH 2 -OH、-CH 2 -CH 2 -(C=O)-O-R 7 、-CH 2 -CH 2 -(C=O)-NH-R 7 or CH 2 -R 7 where R 7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; protecting groups for amino groups; and poly(ethylene glycol) chains;

[0806] R 6 is selected from hydrogen; the group -CH 2 -CH(OH)-R 7 、-CH(R 7 )-CH-OH、-CH 2 -CH 2 -(C=O)-O-R 7 、-CH 2 -CH 2-(C=O)-NH-R 7 or -CH 2 -R 7 , where R 7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; a protecting group for an amino group; -C(NH)-NH 2 ; a poly(ethylene glycol) chain; and a receptor ligand,

[0807] and one or more of the nitrogen atoms indicated in formula (c6-1) may be protonated to provide a cationic group of formula (c6-1).

[0808] For further preferred definitions of these polymers and the variables included in the above formula (c6-1), the corresponding disclosure in WO2014 / 207231 regarding its group of formula (II) also applies to the invention described herein.

[0809] A third preferred class of cationic polymers is a polymer comprising a plurality of groups of formula (c6-2) as repeating units, as disclosed by them in WO 2014 / 207231 (applicant ethris GmbH) as groups of formula (III):

[0810]

[0811] where for each group of formula (c6-2) in a plurality of such groups, the variables a, b, p, m, n, and R 2 to R 5 are independently defined as follows:

[0812] a is 1 and b is an integer from 2 to 4; or a is an integer from 2 to 4 and b is 1,

[0813] p is 1 or 2,

[0814] m is 1 or 2; n is 0 or 1, and m + n ≥ 2; and

[0815] R 2 to R 5 are independently selected from hydrogen; the group -CH 2 -CH(OH)-R 7 , -CH(R 7 )-CH 2 -OH, -CH 2 -CH 2 -(C=O)-O-R 7 , CH 2 -CH 2 -(C=O)-NH-R 7 or -CH 2 -R 7 , where R7 Selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; a protecting group for an amino group; C(NH)-NH 2 ; and a poly(ethylene glycol) chain;

[0816] And one or more of the nitrogen atoms indicated in formula (c6-2) may be protonated to provide a cationic group of formula (c6-2).

[0817] For further preferred definitions of these polymers and the variables included in the above formula (c6-2), the corresponding disclosure in WO2014 / 207231 regarding the repeating units of its formula (III) also applies to the invention described herein.

[0818] The fourth preferred class of cationic polymers is provided by statistical copolymers, as disclosed in WO2016 / 07377 (applicant ethris GmbH). It comprises a plurality of repeating units (a) independently selected from repeating units of the following formula (a1) and (a2):

[0819] -CH 2 -CH 2 -NH- (a1)

[0820]

[0821] and a plurality of repeating units (b) independently selected from repeating units of the following formula (b1) to (b4):

[0822] -CH 2 -CH 2 -CH 2 -NH-(b1)

[0823]

[0824] -CH 2 -CH 2 -CH 2 -CH 2 -NH-(b3)

[0825]

[0826] And the molar ratio of the sum of the repeating units (a) to the sum of the repeating units (b) is in the range of 0.7 / 1.0 to 1.0 / 0.7, and one or more of the nitrogen atoms of the repeating units (a) and / or (b) comprised in the copolymer may be protonated to provide a cationic copolymer.

[0827] For a further preferred definition of the copolymer, the corresponding disclosure in WO2016 / 07377 also applies to the invention described herein. As described therein, a particularly preferred copolymer is a linear copolymer comprising or consisting of repeating units (a1) and (b1).

[0828] As an optional component of the nanoparticles, a polyanionic component different from nucleic acid may also be included, especially if the nanoparticles include nucleic acid as a preferred therapeutic agent, and a polyanionic component different from nucleic acid may be included in addition to nucleic acid. Examples of such polyanions are polyglutamic acid and chondroitin sulfate. If such a polyanionic component different from nucleic acid is used in the nanoparticles, it is preferred to limit its amount such that the amount of anionic charge provided by the polyanionic component is not higher than the amount of anionic charge provided by the nucleic acid.

[0829] As described above, lipid or lipid-like nanoparticles suspended in an aqueous carrier solution comprise (a) a therapeutic agent, which is preferably nucleic acid, and (b) at least one selected from the group consisting of permanent cationic lipids, ionizable lipids and ionizable lipid-like substances, and is preferably an ionizable lipid or ionizable lipid-like substance. If a lipid-like substance is included, the nanoparticles shall be referred to as lipid-like nanoparticles herein.

[0830] Preferably, the nanoparticles preferably comprise the following components, more preferably consist of,

[0831] Nucleic acid (a) as a therapeutic agent,

[0832] At least one selected from the group consisting of permanent cationic lipids, ionizable lipids and ionizable lipid-like substances, preferably an ionizable lipid or ionizable lipid-like substance (b),

[0833] And optionally one or more of the following:

[0834] Non-ionizable lipid (c1) having a sterol structure;

[0835] Phosphoglyceride lipid (c2);

[0836] PEG-conjugated lipid (c3);

[0837] Poly(sarcosine)-conjugated lipid (c4);

[0838] PASylated lipid (c5);

[0839] Cationic polymer (c6).

[0840] Exemplary suspensions comprising nanoparticles formed from the components listed above, which are also applicable in the context of the present invention, include those disclosed in S. Patel et al., Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA, Nature Communications, 2020, 11:983.

[0841] It should be understood that the components of the nanoparticles, in particular components (a) and (b), and optionally one or more of (c1) to (c6), are generally included in the nanoparticles as a mixture.

[0842] In terms of the amounts of these components, it is further preferred that the nanoparticles comprise the following components, more preferably consist of the following components:

[0843] A therapeutic agent, which is preferably a nucleic acid, and

[0844] 30 to 65 mol% of at least one selected from permanent cationic lipids, ionizable lipids, and ionizable lipidoids, preferably an ionizable lipid or ionizable lipidoid (b),

[0845] And one or more of the following components:

[0846] 10 to 50 mol% of a lipid (c1) having a sterol structure,

[0847] 4 to 50 mol% of a phosphoglyceride lipid (c2),

[0848] 0.5 to 10 mol% of one or any combination of a PEG-conjugated lipid (c3), a poly(sarcosine)-conjugated lipid (c4), and a PASylated lipid (c5),

[0849] 0.5 to 10 mol% of a cationic polymer (c6),

[0850] Such that the sum of (b) and (c1) to (c6) is equal to 100 mol%.

[0851] For 30 to 65 mol% of at least one selected from permanent cationic lipids, ionizable lipids, and ionizable lipidoids as component (b), it is understood that if two or more of the cationic lipid, ionizable lipid, and ionizable lipidoid are present as component (b), the indicated mole percentage refers to the total amount of these components in the nanoparticles. Similarly, it should be understood that the mole percentages of components (c1) to (c6) are indicated, provided that not all of these components need to be present in the nanoparticles. Thus, for example, in the context of this preferred embodiment, the cationic polymer may be present or absent, but if present, it is used in an amount of 0.5 - 10 mol%. As further indicated above, in the context of the preferred embodiment, the amounts of components (c1), (c2), (c3), (c4), (c5), and / or (c6) are such that the sum of (b) and (c1) to (c6) equals 100 mol%.

[0852] Still further preferably, the nanoparticles comprise or consist of the following components:

[0853] Nucleic acid (a),

[0854] At least one selected from permanent cationic lipids, ionizable lipids, and ionizable lipidoids, preferably an ionizable lipid or ionizable lipidoid (b),

[0855] An non-ionizable lipid (c1) having a sterol structure,

[0856] Phosphoglyceride lipid (c2), and

[0857] PEG-conjugated lipid (c3).

[0858] Regarding the amounts of these components, still further preferably, the nanoparticles comprise, more preferably consist of, the following components:

[0859] Nucleic acid (a),

[0860] 30 to 65 mol% of at least one selected from permanent cationic lipids, ionizable lipids, and ionizable lipidoids, preferably an ionizable lipid or ionizable lipidoid (b),

[0861] 10 to 50 mol% of a lipid (c1) having a sterol structure,

[0862] 4 to 50 mol% of phosphoglyceride lipid (c2), and

[0863] 0.5 to 10 mol% of PEG-conjugated lipid (c3),

[0864] such that the sum of (b) and (c1) to (c3) equals 100 mol%.

[0865] Based on the above information related to preferred therapeutic agents (especially nucleic acids) and preferred components of lipid compositions other than therapeutic agents, the lipid nanoparticles according to the context of the present invention preferably comprise

[0866] (a) mRNA as a nucleic acid;

[0867] (b) an ionizable lipid of formula (b-1b)

[0868]

[0869] wherein R 1A to R 6A are independently selected from hydrogen and -CH 2 -CH(OH)-R 7A wherein R 7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond, provided that at least two residues among R 1A to R 1A are -CH 2 -CH(OH)-R 7A more preferably at least four residues among R 1A to R 6A are -CH 2 -CH(OH)-R 7A wherein R 7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond;

[0870] or its protonated form, wherein one or more of the nitrogen atoms shown in formula (b-1b) are protonated to provide a cationic lipid;

[0871] (c1) a non-ionizable lipid having a sterol structure of formula (c1-1)

[0872]

[0873] wherein R 1L is C3-C12 alkyl;

[0874] (c2) a phosphoglyceride of formula (c2-2)

[0875]

[0876] wherein R 1G and R 2G are independently selected from C8-C18 alkyl and C8-C18 alkenyl, preferably selected from C12-C18 alkyl and C12-C18 alkenyl,

[0877] or a pharmaceutically acceptable salt thereof; and

[0878] (c3) a PEG-conjugated lipid of formula (c3-1)

[0879]

[0880] wherein R 1H and R 2H are independently selected from C8-C18 alkyl and C8-C18 alkenyl, preferably selected from C12-C18 alkyl or C12-C18 alkenyl, and p is an integer from 5 to 200, preferably from 10 to 100, and more preferably from 20 to 60. In such a lipid particle composition, the lipid dL_05(R) of the formula shown above will be a particularly preferred variant of the ionizable lipid.

[0881] Another preferred exemplary composition of lipid nanoparticles suitable for use in the context of the present invention comprises a nucleic acid, more preferably mRNA, as a therapeutic agent, ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) or its protonated form as an ionizable lipid (b), wherein the nitrogen atom of the compound is protonated to provide a positively charged compound, and optionally further comprises one or more of the following components (d1) to (d8):

[0882] (d1) 2-[(Polyethylene glycol)-2000]-N,N-ditetradecylethanamide (ALC-0159);

[0883] (d2) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);

[0884] (d3) cholesterol;

[0885] (d4) potassium chloride;

[0886] (d5) potassium dihydrogen phosphate;

[0887] (d6) sodium chloride;

[0888] (d7) disodium hydrogen phosphate dihydrate;

[0889] (d8) sucrose.

[0890] More preferably, they further comprise at least (d1), (d2) and (d3), and more preferably, they comprise all of (d1) to (d8).

[0891] Yet another preferred exemplary composition of lipid nanoparticles suitable for the context of the present invention includes a nucleic acid as a therapeutic agent, more preferably mRNA, octadec-9-yl 8-[(2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino]octanoate (SM-102) or its protonated form as the ionizable lipid (b), wherein the nitrogen atom of the compound is protonated to provide a positively charged compound, and optionally includes one or more of the following components (e1) to (e7):

[0892] (e1) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);

[0893] (e2) cholesterol;

[0894] (e3) 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000 DMG);

[0895] (e4) tris(hydroxymethyl)aminomethane hydrochloride;

[0896] (e5) sodium acetate trihydrate;

[0897] (e6) acetic acid;

[0898] (e7) sucrose.

[0899] More preferably, they further include at least (e1), (e2) and (e3), and still more preferably, they include all of (e1) to (e7).

[0900] Yet another preferred exemplary composition of lipid nanoparticles suitable for the context of the present invention includes a nucleic acid as a therapeutic agent, more preferably mRNA, DLin-MC3-DMA ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate) or its protonated form, wherein the nitrogen atom of the compound is protonated, and optionally one or more of the following components (e1) to (e7):

[0901] (e1) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);

[0902] (e2) cholesterol;

[0903] (e3) PEG2000-C-DMG (α-(3'-{[1,2-bis(myristyloxy)propoxy]carbonylamino}propyl)-ω-methoxy, polyoxyethylene);

[0904] (e4) 2-amino-2-(hydroxymethyl)propane-1,3-diol (tris(hydroxymethyl)aminomethane) hydrochloride;

[0905] (e5) Disodium hydrogen phosphate heptahydrate;

[0906] (e6) Potassium dihydrogen phosphate anhydrous;

[0907] (e7) Sodium chloride.

[0908] More preferably, components (e1), (e2) and (e3) are present, and still more preferably, they include all of (e1) to (e6).

[0909] The composition of the nanoparticles is preferably such that the weight ratio of the total weight of the components other than nucleic acid in the nanoparticles to the weight of the nucleic acid is in the range of 50:1 to 1:1, more preferably in the range of 40:1 to 2:1, and most preferably in the range of 30:1 to 3:1.

[0910] The N / P ratio, i.e., the ratio of the number of amine nitrogen atoms provided by ionizable lipids or ionizable lipidoids to the number of phosphate groups provided by the nucleic acid of the nanoparticles (if the nucleic acid is included therein as a therapeutic agent) is preferably in the range of 0.5 to 20, more preferably in the range of 0.5 to 10.

[0911] The Z-average diameter of the suspended lipid or lipidoid nanoparticles is preferably in the range of 10 to 500 nm, more preferably in the range of 10 to 250 nm, and still more preferably in the range of 20 to 200 nm. The indicated particle diameter is the hydrodynamic diameter of the particles determined by dynamic light scattering (DLS). The measurement is usually carried out at 25 °C. Due to the stabilizing effect of the non-ionic surfactant, this average diameter can be maintained even when the suspension is exposed to physical stress conditions.

[0912] The polydispersity index of the suspended nanoparticles is preferably in the range of 0.02 to 0.4, more preferably in the range of 0.03 to 0.2. The polydispersity index can be determined by dynamic light scattering (DLS). The measurement is usually carried out at 25 °C. Due to the stabilizing effect of the non-ionic surfactant, this polydispersity index can be maintained even when the suspension is exposed to physical stress conditions.

[0913] A suspension containing different lipid or lipidoid nanoparticles as defined above - i.e., particles different in their components - can be provided. However, preferably, the nanoparticles contained in the suspension are composed of the same components.

[0914] Nanoparticles can be conveniently prepared by mixing a solution containing nucleic acid in an aqueous solvent, such as a citrate buffer with a pH of 4.5 and optionally containing a salt such as sodium chloride, with a solution containing an ionizable lipid or ionizable lipidoid in an organic solvent, such as ethanol. Further optional components can be incorporated, for example, by adding them to one of the two solutions. The nanoparticles produced in this way can be further processed by chromatography and / or dialysis and / or tangential flow filtration (TFF) in order to obtain the nanoparticles in the desired liquid composition. TFF is preferably used for further processing.

[0915] To provide a nanoparticle suspension, it is also possible to rely on lyophilized nanoparticles prepared according to the above procedure and then lyophilized, which are subsequently resuspended in an aqueous carrier solution.

[0916] In the stable suspensions provided in aspects of the present invention, the nanoparticles are suspended in an aqueous carrier solution.

[0917] The carrier solution is an aqueous solution, that is, a solution in which the main solvent is water in terms of the total volume of the solvent(s), preferably containing more than 70% water, more preferably containing more than 90% water as the solvent, expressed as the volume percentage of water in the total volume of the solvent(s) contained in the carrier solution (at a temperature of 25 °C). Most preferably, water is the only solvent in the carrier solution. Thus, the carrier solution is liquid at room temperature (e.g., 25 °C).

[0918] The weight / volume ratio of the nanoparticles in the carrier solution is preferably in the range of 0.1 g / L to 300 g / L, more preferably in the range of 0.2 g / L to 300 g / L, still more preferably in the range of 0.5 g / L to 250 g / L, and most preferably in the range of 0.5 g to 125 g / L (measured at 25 °C).

[0919] If the nanoparticles comprise nucleic acid as a therapeutic agent, the concentration of nucleic acid provided by the lipid or lipidoid nanoparticles in the suspension is preferably in the range of 0.01 to 10 mg / ml, more preferably in the range of 0.02 to 10 mg / ml, still more preferably in the range of 0.05 to 5 mg / ml, and most preferably in the range of 0.05 to 2.5 mg / ml (measured at 25 °C), based on the total volume of the suspension.

[0920] As described above, the Z-average diameter of the lipid or lipidoid nanoparticles contained in the suspension is preferably in the range of 10 to 500 nm, more preferably in the range of 10 to 250 nm, still more preferably in the range of 20 to 200 nm. The particle diameter shown is the hydrodynamic diameter of the particles determined by dynamic light scattering (DLS). The measurement is usually carried out at 25 °C.

[0921] The polydispersity index of the nanoparticles contained in the suspension is preferably in the range of 0.02 to 0.4, more preferably in the range of 0.03 to 0.2. The polydispersity index can be determined by dynamic light scattering (DLS). The measurement is typically carried out at 25 °C.

[0922] By using a surfactant, preferably a non-ionic surfactant, the nanoparticle suspension is stabilized in the context of the present invention, preventing the particles from aggregating under conditions of physical stress. To achieve this effect, the surfactant is incorporated into the suspension, preferably as an excipient into an aqueous carrier solution.

[0923] In some embodiments, the LNP and / or LiNP are not lyophilized. In some embodiments, the surfactant is added before the lyophilization process. In some embodiments, the surfactant is not present in the carrier solution during lyophilization.

[0924] Generally, the presence of the surfactant does not cause a change in the bioactivity of the nanoparticles. Bioactivity refers to the expression level of the therapeutic nucleic acid in target cells. Bioactivity can be quantified, for example, by transfecting a cell line (e.g., HEK-293) in vitro with the nanoparticles and then quantifying the resulting nucleic acid by Southern / northern blotting, or by quantifying the protein by ELISA. The difference in the detected protein levels calculated as the average of three measurements at each concentration should not exceed 10%, preferably not exceed 5%, and more preferably there is no statistical difference when performing the same detection with the same LNP or LiNP without surfactant.

[0925] Generally, the presence of the surfactant does not cause a change in the physical properties of the nanoparticles, as measured by the hydrodynamic diameter of the nanoparticles and the proportion of encapsulated nucleic acid.

[0926] The hydrodynamic diameter of the nanoparticles can be measured, for example, by methods such as dynamic light scattering (which can also be by photon correlation spectroscopy). Optionally, in the presence of a surfactant, the average of three measurements of the hydrodynamic diameter of the nanoparticles should not differ by more than 5%, preferably not more than 1%, more preferably there should be no statistical difference, compared to the same nanoparticles in the absence of a surfactant. The viscosity change of the surfactant must be taken into account during the measurement. The percentage of encapsulated nucleic acid can be determined, for example, by measuring the fluorescence intensity in a RiboGreen assay. The nanoparticles are analyzed under two different conditions, with the untreated sample for external nucleic acid and the sample treated with Triton X-100 for total mRNA. The percentage content of the encapsulated nucleic acid is calculated. Optionally, in the absence of a surfactant, the value calculated as the average of three measurements of the nanoparticles should not differ by more than 5%, optionally not more than 3%, preferably there should be no statistical difference, compared to the same nanoparticles in the presence of a surfactant.

[0927] Those of ordinary skill in the art will understand that the stabilizing measures taken for the nanoparticle suspension to prevent particle aggregation can prevent the aggregation of nanoparticles or, compared to the situation where no relevant measures are taken, can reduce the degree of nanoparticle aggregation. Preferably, the stability of the nanoparticle suspension is manifested as the Z-average particle size of the suspended particles increasing by less than 50% under physical stress conditions, more preferably less than 20%, still more preferably less than 10%, and most preferably not increasing.

[0928] Similarly, it should be understood that the stability of the nanoparticle suspension in preventing particle aggregation under physical stress conditions means that when the nanoparticle suspension is exposed to physical stress conditions, it can prevent or reduce the aggregation of nanoparticles, and if there is no such stability, the aggregation of nanoparticles will be observed.

[0929] The physical stress conditions to which the nanoparticle suspension may be exposed are generally those encountered during the handling or transportation of the suspension. They include, for example, the rapid movement of the suspension volume, which can cause collisions of the nanoparticles in an unstable suspension. As examples of physical stress conditions, reference can be made to the shaking, stirring, vibration, mixing, inversion, tapping or dropping of the nanoparticle suspension, or, for example, the physical stress conditions caused by pumping the nanoparticle suspension or drawing it into a syringe. Those of ordinary skill in the art will understand that the physical stress conditions include not only the conditions to which the nanoparticle suspension is exposed during routine handling but also the conditions to which the suspension may be exposed under special circumstances (such as transportation under difficult conditions) or inadvertently (such as the suspension sample falling).

[0930] In the context of the present invention, various types of surfactants can be used, but nonionic surfactants are most preferably used to stabilize the suspension. Thus, it is preferred that the surfactant comprises, most preferably, a nonionic surfactant. Examples of suitable nonionic surfactants include fatty alcohol ethoxylates, fatty acid ethoxylates, block copolymers of ethylene oxide and propylene oxide, alkylphenol ethoxylates or oligomers of alkylphenol ethoxylates, sorbitan fatty acid esters, ethoxylated sorbitan fatty acid esters, glycerol fatty acid esters, ethoxylated castor oil, and ethoxylated vitamin E.

[0931] Thus, in the context of the present invention, the surfactant used in the nanoparticle suspension preferably comprises, more preferably, at least one selected from the group consisting of fatty alcohol ethoxylates, fatty acid ethoxylates, block copolymers of ethylene oxide and propylene oxide, alkylphenol ethoxylates or oligomers of alkylphenol ethoxylates, sorbitan fatty acid esters, ethoxylated sorbitan fatty acid esters, glycerol fatty acid esters, ethoxylated castor oil, and ethoxylated vitamin E.

[0932] According to another embodiment, the surfactant used in the nanoparticle suspension in the context of the present invention preferably comprises, more preferably, at least one selected from the group consisting of fatty alcohol ethoxylates, fatty acid ethoxylates, alkylphenol ethoxylates or oligomers of alkylphenol ethoxylates, sorbitan fatty acid esters, ethoxylated sorbitan fatty acid esters, glycerol fatty acid esters, ethoxylated castor oil, and ethoxylated vitamin E.

[0933] The block copolymer of ethylene oxide and propylene oxide is preferably a poloxamer. The poloxamer preferably contains a poly(propylene oxide) block B of formula (p-1):

[0934]

[0935] where s is an integer from 15 to 60, and

[0936] two poly(ethylene oxide) blocks A of formula (p-2):

[0937]

[0938] where r is independently for each block an integer from 8 to 150, preferably an integer from 10 to 150.

[0939] Thus, it is preferred that the surfactant comprises, more preferably, a poloxamer, for example, the preferred poloxamer discussed above. However, the present invention also provides and relates in its various aspects to suspensions that do not comprise poloxamer.

[0940] Particularly preferably, the nonionic surfactants for stabilization according to the present invention include or are preferably selected from at least one of the group consisting of poloxamer 124, poloxamer 188, poloxamer 338, poloxamer 407, polysorbate 20, polysorbate 80, laureth, polyoxyethylene-35 castor oil, D-α-tocopheryl polyethylene glycol 1000 succinate, and tyloxapol. According to another embodiment, the nonionic surfactant for stabilization according to the present invention includes or is selected from at least one of the group consisting of polysorbate 20, polysorbate 80, laureth, polyoxyethylene-35 castor oil, D-α-tocopheryl polyethylene glycol 1000 succinate, and tyloxapol.

[0941] The carrier solution for suspending the nanoparticles generally includes a surfactant dissolved therein. As will be understood by those of ordinary skill in the art, this does not exclude the possibility that a certain amount of surfactant molecules adsorb onto the lipid or lipid-like nanoparticles contained in the suspension.

[0942] In the context of the present invention, it has been found that when the concentration of the surfactant is relatively low, for example, when the surfactant concentration in the suspension is 0.01% (w / v), the surfactant can produce beneficial effects. Therefore, generally, in terms of the total volume of the suspension of nanoparticles in the aqueous carrier solution (usually measured at 25 °C), the content of the surfactant in the suspension is 0.01% (w / v) or more.

[0943] For example, the use according to the present invention includes incorporating the surfactant into the nanoparticle suspension, preferably adding it to the aqueous carrier solution, and the addition amount is 0.01 to 10% (w / v), preferably 0.1 to 10% (w / v), more preferably 0.25 to 5% (w / v), still more preferably 0.33 to 2.5% (w / v), even more preferably 0.45 to 1.5% (w / v), and most preferably 0.5 to 1.5% (w / v) relative to the total volume of the nanoparticle suspension in the aqueous carrier solution. It can be understood that the concentration of a substance expressed in % (w / v) or (weight / volume) is equivalent to the content of the substance in grams in a volume of 100 mL, usually measured at 25 °C. Therefore, 1% (w / v) is equivalent to containing 1 g of surfactant in every 100 mL of the total volume of the suspension.

[0944] Similarly, the method according to the present invention may include incorporating a surfactant into the nanoparticle suspension, and the amount added, relative to the total volume of the nanoparticle suspension in the aqueous carrier solution, is, for example, 0.01 to 10% (w / v), preferably 0.1 to 10% (w / v), more preferably 0.25 to 5% (w / v), still more preferably 0.33 to 2.5% (w / v), even more preferably 0.45 to 1.5% (w / v), and most preferably 0.5 to 1.5% (w / v) (usually measured at 25 °C).

[0945] Although, as described above, a concentration of 0.5 - 1.5% (w / v) is particularly preferred, the present invention also provides and relates to suspensions with lower surfactant concentrations in its various aspects, for example, in the range of 0.01 to 0.45% (w / v), or 0.1 to 0.40% (w / v).

[0946] In the context of the suspensions of lipid nanoparticles or lipid-like nanoparticles in an aqueous carrier solution in various aspects of the present invention, it is generally preferred that the surfactant is substantially not attached to the nanoparticles, for example, is substantially not contained in the nanoparticles and is substantially not attached to the nanoparticles. For example, in the context of the suspensions of lipid nanoparticles or lipid-like nanoparticles in an aqueous carrier solution in various aspects of the present invention, more than 90 wt%, preferably more than 95 wt% of the total amount of surfactant contained or incorporated is present in the aqueous carrier solution without being attached to the nanoparticles.

[0947] In addition to the surfactant, other excipients may also be present in the carrier solution. Preferably, the carrier solution further includes at least one of sugar and salt, more preferably at least one of sucrose and NaCl.

[0948] The surfactant can be conveniently incorporated into the nanoparticle suspension. For example, the addition method includes adding the surfactant to a suspension including an aqueous carrier solution and lipid or lipid-like nanoparticles, or including adding lipid or lipid-like nanoparticles to an aqueous carrier solution including the surfactant. For example, as described above, if the nanoparticles are provided in a lyophilized form, they can be resuspended in an aqueous carrier solution containing the surfactant.

[0949] Within this range, the present invention also provides a method for preparing a suspension of lipid nanoparticles or lipid-like nanoparticles as defined herein, the method comprising generating a formulation of lipid nanoparticles or lipid-like nanoparticles by mixing at least one selected from permanent cationic lipids, ionizable lipids, and ionizable lipid-like lipids dissolved in an organic phase with a therapeutic agent dissolved in an aqueous solution, and

[0950] combining the nanoparticles with a surfactant to obtain a suspension of the nanoparticles in an aqueous carrier solution.

[0951] Preferably, the method comprises the following steps:

[0952] i) Generating a formulation of lipid nanoparticles or lipidoid nanoparticles by mixing at least one selected from permanent cationic lipids, ionizable lipids, and ionizable lipidoids dissolved in an organic phase with a therapeutic agent dissolved in an aqueous solution;

[0953] ii) Diluting the lipid nanoparticle or lipidoid nanoparticle formulation by dilution with a first solution;

[0954] iii) Concentrating the diluted lipid nanoparticle or lipidoid nanoparticle formulation by buffer exchange using a TFF ultrafiltration / diafiltration method, wherein the ultrafiltration / diafiltration uses a second solution;

[0955] iv) Obtaining a suspension of lipid nanoparticles or lipidoid nanoparticles in an aqueous carrier solution comprising a surfactant;

[0956] wherein the first solution comprises a surfactant between 0.01% w / v and 10% w / v, preferably between 0.1% w / v and 10% w / v, more preferably between 0.25% w / v and 5% w / v, still more preferably between 0.33% w / v and 2.5% w / v, even more preferably between 0.45% w / v and 1.5% w / v, and most preferably between 0.5% w / v and 1.5% w / v; and / or

[0957] wherein the second solution comprises a surfactant between 0.01% w / v and 10% w / v, preferably between 0.1% w / v and 10% w / v, more preferably between 0.25% w / v and 5% w / v, still more preferably between 0.33% w / v and 2.5% w / v, even more preferably between 0.45% w / v and 1.5% w / v, and most preferably between 0.5% w / v and 1.5% w / v;

[0958] And wherein the final concentration of surfactant in the combined first and second solutions is surfactant in the range of from 0.01% w / v to 10% w / v, preferably from 0.1% w / v to 10% w / v, more preferably from 0.25% w / v to 5% w / v, still more preferably from 0.33% w / v to 2.5% w / v, even more preferably from 0.45% w / v to 1.5% w / v, and most preferably from 0.5% w / v to 1.5% w / v, relative to the total volume of the nanoparticle suspension in the aqueous carrier solution.

[0959] In the above method, it is preferred not to add the surfactant to the suspension before or during step i).

[0960] In addition, it is preferred that the surfactant is added together with the first and second solutions. For example, based on the total weight of surfactant in the suspension obtained in step iv), 30 to 70 wt% of the surfactant, preferably 40 to 60 wt%, and more preferably 45 to 55 wt% of the surfactant can be added with the first solution, and based on the total weight of surfactant in the suspension obtained in step iv), 70 to 30 wt% of the surfactant, preferably 60 to 40 wt%, and more preferably 55 to 45 wt% of the surfactant is added with the second solution, such that the sum of the amounts of surfactant added with the first and second solutions is 100 wt%. Generally, it is preferred that approximately half of the surfactant is added with the first solution and approximately half of the surfactant is added with the second solution.

[0961] Therapeutic agents (preferably nucleic acids such as RNA, preferably mRNA) present in lipid or lipid-like nanoparticles used in the context of the present invention are particularly useful in a medical setting and for the treatment or prevention of diseases and disorders, preferably for the treatment or prevention of diseases or disorders that rely on nucleic acids as active agents. Thus, the suspension is generally provided or used as a medicament or pharmaceutical composition. The present invention also provides a formulation of a lipid nanoparticle or lipid-like nanoparticle comprising a suspension of a lipid nanoparticle or lipid-like nanoparticle according to the present invention as described herein.

[0962] In particular, the nanoparticle suspension or formulation is suitable for administration to a subject. In this way, the therapeutic agent, preferably a nucleic acid (such as RNA, preferably mRNA), contained in the nanoparticles of the suspension can also be administered to the subject.

[0963] By administering to a subject, a therapeutic agent, preferably a nucleic acid contained in a lipid or lipid-like nanoparticle, can be delivered to a target cell. The term "delivered to a target cell" preferably refers to the transfer of the nucleic acid into the cell. Administration can be accomplished by various means known to the skilled practitioner, including administration to or via the respiratory tract, for example, by aerosol administration of a suspension, or by intramuscular or intravenous administration.

[0964] By administering a suspension to a subject, a disease or disorder can be treated or prevented. The term "disease" refers to any conceivable pathological condition that can be treated, prevented or vaccinated against by using the suspension. The disease can be, for example, genetic, acquired, infectious or non-infectious, age-related, cardiovascular, metabolic, intestinal, neoplastic (especially cancer) or genetic. For example, a disease can be based on irregularities in physiological processes, molecular processes, biochemical reactions in an organism, which in turn can be based on, for example, the genetic endowment of the organism, behavior, social or environmental factors such as exposure to chemicals or radiation.

[0965] In this context, the invention also provides a suspension of lipid nanoparticles or lipid-like nanoparticles or a formulation of lipid nanoparticles or lipid-like nanoparticles according to the invention as described herein for the treatment or prevention of a disease. Similarly, a suspension of lipid nanoparticles or lipid-like nanoparticles or a formulation of lipid nanoparticles or lipid-like nanoparticles according to the invention as described herein can be used in a method for the treatment or prevention of a disease, the method comprising administering the suspension or formulation to a subject in need thereof.

[0966] In a related aspect, the invention also provides a suspension of lipid nanoparticles or lipid-like nanoparticles or a formulation of lipid nanoparticles or lipid-like nanoparticles according to the invention as described herein for use as a medicament.

[0967] For example, the invention provides a suspension of lipid nanoparticles or lipid-like nanoparticles or a formulation of lipid nanoparticles or lipid-like nanoparticles according to the invention as described herein for vaccination or immunization. Similarly, a suspension of lipid nanoparticles or lipid-like nanoparticles or a formulation of lipid nanoparticles or lipid-like nanoparticles according to the invention as described herein can be used in a method for vaccination or immunization, the method comprising administering the suspension or formulation to a subject in need thereof.

[0968] According to a further aspect, the invention provides a method of inducing an immune response against a target pathogen in a subject in need thereof, the method comprising administering to the subject a suspension of lipid nanoparticles or lipid-like nanoparticles or a formulation of lipid nanoparticles or lipid-like nanoparticles according to the invention as described herein.

[0969] In another instance, the present invention provides a lipid nanoparticle or lipidoid nanoparticle suspension or lipid nanoparticle or lipidoid nanoparticle formulation according to the present invention as described herein for treating cancer. Similarly, a lipid nanoparticle or lipidoid nanoparticle suspension or lipid nanoparticle or lipidoid nanoparticle formulation according to the present invention as described herein can be used in a method for treating cancer, the method comprising administering the suspension or formulation to a subject in need thereof.

[0970] In a further aspect, the present invention provides a method for avoiding or reducing side effects during treatment with a lipid nanoparticle or lipidoid nanoparticle comprising at least one therapeutic agent as described herein, wherein the method comprises the steps of:

[0971] i) determining whether the lipid nanoparticles or lipidoid nanoparticles in a pharmaceutical composition comprising the lipid nanoparticles or lipidoid nanoparticles aggregate when subjected to mechanical stress or temperature stress by determining their aggregation levels before and after the pharmaceutical composition comprising the lipid nanoparticles or lipidoid nanoparticles is subjected to mechanical stress or temperature stress;

[0972] ii) if the lipid nanoparticles or lipidoid nanoparticles exhibit aggregation after the test in step (i), adding a surfactant as defined herein to the lipid nanoparticle or lipidoid nanoparticle formulation to obtain an LNP or LiNP suspension having a final surfactant concentration between 0.01% w / v and 10% w / v, preferably between 0.1% w / v and 10% w / v, more preferably between 0.25% w / v and 5% w / v, still more preferably between 0.33% w / v and 2.5% w / v, even more preferably between 0.45% w / v and 1.5% w / v, and most preferably between 0.5% w / v and 1.5% w / v;

[0973] iii) mixing and reconstituting to produce a stable lipid nanoparticle or lipidoid nanoparticle suspension.

[0974] In related aspects, the present invention further provides a method for reducing one or more side effects associated with a vaccine formulation or an anti-cancer formulation comprising lipid nanoparticles or lipid-like nanoparticles carrying nucleic acids as described herein, the method comprising modifying the vaccine formulation or anti-cancer formulation by adding a surfactant as described herein to the vaccine formulation or anti-cancer formulation comprising a lipid nanoparticle or lipid-like nanoparticle suspension. Preferably, the surfactant is between 0.01% w / v and 10% w / v surfactant, preferably between 0.1% w / v and 10% w / v surfactant, more preferably between 0.25% w / v surfactant and 5% w / v surfactant, still more preferably between 0.33% w / v surfactant and 2.5% w / v surfactant, even more preferably between 0.45% w / v surfactant and 1.5% w / v surfactant, and most preferably between 0.5% w / v and 1.5% w / v surfactant.

[0975] As described above, the surfactant is preferably at least one non-ionic surfactant selected from the group consisting of fatty alcohol ethoxylates, fatty acid ethoxylates, block copolymers of ethylene oxide and propylene oxide, alkylphenol ethoxylates or oligomers of alkylphenol ethoxylates, sorbitan fatty acid esters, ethoxylated sorbitan fatty acid esters, glycerol fatty acid esters, ethoxylated castor oil, and ethoxylated vitamin E, more preferably at least one selected from the group consisting of poloxamer 124, poloxamer 188, poloxamer 338, poloxamer 407, polysorbate 20, polysorbate 80, laureth, polyoxyethylene-35 castor oil, D-α-tocopheryl polyethylene glycol 1000 succinate, and tyloxapol, or even more preferably one selected from this group. According to the above and preferred embodiments, the surfactant is a block copolymer of ethylene oxide and propylene oxide, preferably a poloxamer selected from the group consisting of poloxamer 124, poloxamer 188, poloxamer 338, and poloxamer 407. According to an alternative embodiment, the poloxamer is a poloxamer other than poloxamer 188 or poloxamer 407.

[0976] In related aspects, the present invention provides a method for reducing the occurrence or severity of one or more side effects associated with an LNP / LiNP-based vaccine in a subject, the method comprising administering to the subject a vaccine formulation or anti-cancer formulation comprising a lipid nanoparticle or lipid-like nanoparticle suspension as described herein.

[0977] The reduction in the occurrence or severity of one or more side effects may be caused by a reduction in LNP / LiNP aggregation, as can be measured, for example, by determining the hydrodynamic diameter of the nanoparticles by dynamic light scattering or photon correlation spectroscopy.

[0978] Generally, a therapeutic agent (preferably a nucleic acid) is included in the nanoparticles in an effective amount. The term "effective amount" refers to an amount sufficient to induce a detectable therapeutic response or prophylactic effect in a subject to whom the pharmaceutical composition is administered. According to the above, as long as the nucleic acid is useful for treatment or prophylaxis as described above, its content is not limited. As described above, the composition containing particles including a nucleic acid preferably includes an amount of particles such that the concentration of the nucleic acid contained in the particles is 0.01 to 10 mg / ml, more preferably 0.02 to 10 mg / ml, still more preferably 0.05 to 5 mg / ml, and most preferably 0.05 to 2.5 mg / ml based on the total volume of the composition. Similarly, it is understood that in the case of administering the lipid nanoparticles or lipid-like nanoparticles suspension or lipid nanoparticles or lipid-like nanoparticles formulation according to the present invention as described herein to a subject, it will be administered in an effective amount.

[0979] Exemplary subjects include mammals such as dogs, cats, pigs, cows, sheep, horses, rodents, e.g., rats, mice, and guinea pigs, or primates, e.g., gorillas, chimpanzees, and humans. In the most preferred embodiment, the subject is a human.

[0980] In this specification, many documents including patent applications and manufacturer manuals are cited. The disclosures of these documents, while not considered relevant to the patentability of the present invention, are hereby incorporated by reference in their entirety. More specifically, all cited documents are incorporated by reference as if each individual document was specifically and individually indicated to be incorporated by reference.

[0981] Examples

[0982] 1 Example 1 - Anti-oscillation of Nanoparticles in the Presence of Different Excipients at Different Excipient Concentrations

[0983] 1.1 Method

[0984] 1.1.1 Preparation of Nanoparticles

[0985] The lipid nanoparticles were formulated from ionizable lipid (dL_05(R), Scheme 1), co-lipid DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, Avanti Polar Lipids), cholesterol (Avanti Polar Lipids), and PEG lipid DMG-PEG2k (1,2-dimyristoyl-sn-glycero-methoxypoly(ethylene glycol)-2000, Avanti Polar Lipids) at a molar ratio of 8.00 / 5.29 / 4.41 / 0.88, respectively. Appropriate volumes of lipid stock solutions in HPLC-grade ethanol at concentrations of 50, 20, 20, and 20 mg / mL were combined. The formulation process was carried out by rapid solvent exchange. The lipid mixture in ethanol was mixed with mRNA in citrate buffer (10 mM citric acid, 150 mM NaCl, pH 4.5) at a volume ratio of 1:4 using a NanoAssembler benchtop (Precision NanoSystems). The resulting formulation had an mRNA concentration of 0.2 mg / mL and an N / P ratio of 8. After incubation at room temperature for 30 minutes, the formulation was dialyzed and purified using a Slide-A-Lyzer MINI dialysis device (20k, 2 mL, Thermo Scientific). If the suspension needed to be concentrated, it was done at 45 °C using a speed vac (concentrator plus, Eppendorf) in V-AQ mode.

[0986]

[0987] Scheme 1: Chemical structure of dL_05(R)

[0988] 1.1.2 Mixing of nanoparticles with excipients

[0989] Table 5 lists the excipients used in this experiment. Excipient dilutions were prepared at the stock solution concentrations shown in Table 5 according to the solubility of the excipients in water. The LNP was mixed with the excipients such that the excipient concentrations were 0% (w / v), 0.01% (w / v), 0.1% (w / v), 1% (w / v), and 10% (w / v) (as otherwise specified in the figure, if possible), and the LNP concentration was 0.2 mg mRNA / mL.

[0990] Table 5: Excipients used in this experiment

[0991] Materials Supplier Stock solution concentration [% w / v] Kolliphor P188 Sigma 20 Kolliphor P338Geismar BASF 20 Kolliphor P407Geismar BASF 2 Tween 20 Sigma 20 Tween 80 Roth 20 BRIJ35 Merck 10 Tyloxapol Sigma 2 VitE-PEG1000 Sigma 20 Kolliphor EL Sigma 20

[0992] 1.1.3 Oscillating the nanoparticle suspension

[0993] For stress testing, 100 μL of the LNP / excipient mixture was oscillated for 1 min at maximum speed on a vortex mixer (Vortex Genie 2, Scientific Industries).

[0994] 1.1.4 Measurement of complex size and PdI

[0995] The hydrodynamic diameter (Z-average, size) and polydispersity index (PdI) of the nanoparticles were measured by dynamic light scattering (DLS) using a Zetasizer Nano-ZS (Malvern Instruments) with an automatic attenuator and reported as an intensity particle size distribution. Samples were measured undiluted at 25 °C

[0996] 1.2 Results

[0997] This set of experiments aimed to test the particle stability of the LNP suspension under physical stress conditions. The results are shown in Figure 1. In the absence of excipient (0% (w / v)), the LNPs aggregated after oscillation (both size and PdI increased compared to before oscillation), indicating that the suspension lost its integrity. In the presence of excipient, when the excipient concentration was greater than 0.01% (w / v), the integrity of the nanoparticles remained stable. Generally, the overall increase in size at 10% (w / v) can be explained by the increased viscosity of the suspension contributed by the high excipient concentration. This led to a reduced Brownian motion of the nanoparticles, which the DLS software interpreted as larger particles. Since there was no difference in the particle size before and after oscillation, the stability of the nanoparticles could still be demonstrated.

[0998] 2. Example 2 - Oscillation resistance of nanoparticles at different nanoparticle concentrations

[0999] 2.1 Method

[1000] 2.1.1 Preparation of nanoparticles

[1001] See Section 1.1.1

[1002] 2.1.2 Mixing of nanoparticles with excipient

[1003] See Section 1.1.2. In this set of experiments, only poloxamer 188 (Kolliphor P188) was used with an excipient concentration of 1% (w / v). As a control, a sample without excipient was used. The mRNA concentrations of the LNPs were 0.01 mg / mL, 0.1 mg / mL, 1 mg / mL, and 2.5 mg / mL, respectively.

[1004] 2.1.3 Oscillation of nanoparticle suspension

[1005] See Section 1.1.3

[1006] 2.1.4 Measuring the size and PdI of the complex

[1007] See Section 1.1.4

[1008] 2.2 Results

[1009] This experiment aimed to determine the concentration limit of LNPs in suspension. The results are shown in Figure 2. The results indicate that oscillation leads to a decrease in nanoparticle mass at all tested concentrations. In the absence of excipients, the formation of aggregates (increase in size and / or PdI) could be detected for all concentrations of LNPs. In contrast, the presence of excipients stabilizes the nanoparticle suspension at all tested LNP concentrations, indicating that this beneficial effect applies to a wide range of LNP concentrations.

[1010] 3 Example 3 - Anti-oscillation properties of MC3-LNPs in the presence of different excipients at different excipient concentrations

[1011] 3.1 Method:

[1012] 3.1.1 Preparation of nanoparticles

[1013] To formulate MC3 LNPs, lipid stock solutions of DLin-MC3-DMA ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate), DSPC, cholesterol, and DMPE-PEG2k were prepared in ethanol at concentrations of 10 mg / mL, 20 mg / mL, 20 mg / mL, and 20 mg / mL, respectively. Ethanol stock solutions (557 μL DLin-MC3-DMA, 69 μL DSPC, 129 μL cholesterol, 35 μL DMPE-PEG2k, 461 μL ethanol) were mixed and fused with an aqueous mRNA solution (0.2667 mg / mL citrate buffer) using a NanoAssemblr device at a volume ratio of 3:1 (mRNA:lipid) and a total flow rate of 12 mL / min. After incubation at RT for 30 min, the formulation was purified by water dialysis using a Slide-A-Lyzer MINI dialysis device (20k, 2 mL, Thermo Scientific). If the suspension needed to be concentrated, rapid vacuum (Concentrator plus, Eppendorf) was used in V-AQ mode at 45 °C.

[1014] 3.1.2 Mixing of nanoparticles with excipients

[1015] See Section 1.1.2. In addition to the excipients already listed in Table 5, Kolliphor P124, Geismar (BASF) was also tested as an excipient. The stock solution of the substance was prepared at a concentration of 20% (w / v), and the treatment method was the same as that of other excipients.

[1016] 3.1.3 Oscillating nanoparticle suspension

[1017] See Section 1.1.3

[1018] 3.1.4 Measurement of complex size and PdI

[1019] See Section 1.1.4

[1020] 3.2 Results

[1021] This experiment aimed to determine whether the observed excipient protection effect was a unique property of the tested LNP (containing dL_05(R)) or could be considered a general property. For this purpose, the LNP used in Example 1 was replaced with MC3-LNP, a known LNP formulation for delivering various nucleic acids. Figure 3 summarizes the relevant data. The results showed a high similarity to the data generated in Example 1. In the absence of excipients, oscillation led to a decrease in particle quality (increase in size and PdI). When the concentration exceeded 0.01% (w / v), the presence of different excipients stabilized the suspension and prevented aggregation under physical stress. These data indicate that the protection effect is independent of the LNP components used.

[1022] 4 Example 4 - Anti-oscillation property of ALC-0315 LNP in the presence of different excipients at different excipient concentrations 4.1 Method

[1023] 4.1.1 Preparation of nanoparticles

[1024] To prepare ALC-0315 LNP, lipid stock solutions of ALC-0315 ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), DSPC, cholesterol, and ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylethanamide) were prepared at 25 mg / mL, 20 mg / mL, 20 mg / mL, and 25 mg / mL, respectively, in ethanol. The ethanol stock solutions were heated to 50 °C for 30 min and mixed. For an example amount of 1 mg mRNA, the following volumes were combined: 574 μL ALC-0315, 156 μL DSPC, 311 μL cholesterol, 71 μL ALC-0519, 138 μL ethanol. The mixture was fused with an mRNA aqueous solution (3750 μL, 0.2667 mg / mL citrate buffer) using a NanoAssemblr device at a volume ratio of 3:1 (mRNA:lipid) with a total flow rate of 12 mL / min. After incubation at room temperature for 30 minutes, the formulation was purified by aqueous dialysis using a Slide-A-Lyzer MINI dialysis device (20k, 2 mL, Thermo Scientific). If concentration of the suspension was required, it was done using a rapid vacuum (concentrator plus, Eppendorf) in V-AQ mode at 45 °C.

[1025] 4.1.2 Mixing of nanoparticles and excipients

[1026] See Section 1.1.2. In addition to the excipients already listed in Table 5, Kolliphor P124, Geismar (BASF) was also tested as an excipient. The stock solution of the substance was prepared at a concentration of 20% (w / v), and the treatment was the same as that of other excipients. The concentration range of the tested excipient was 0.1% (w / v) to 10% (w / v).

[1027] 4.1.3 Oscillating nanoparticle suspension

[1028] See Section 1.1.3

[1029] 4.1.4 Measurement of complex size and PdI

[1030] See Section 1.1.4

[1031] 4.2 Results

[1032] This experiment aims to determine whether the observed protective effect of the excipient is a unique property of the tested LNP (containing dL_05(R)) or can be regarded as a general property. To this end, the LNP used in Example 1 was replaced with an LNP containing ALC-0315, which contains the lipid components of the Covid vaccine Comirnaty. The results are summarized in Figure 4 and show a high degree of similarity to the data generated in Examples 1 and 3. Oscillation in the absence of excipient results in a decrease in particle quality (increase in size and PdI). The presence of different excipients at concentrations above 0.01% (w / v) stabilizes the suspension and prevents aggregation under physical stress. These data further demonstrate that the protective effect is independent of the LNP components used.

[1033] 5 Example 5 - At different excipient concentrations Anti-oscillation property in the presence of different excipients

[1034] 5.1 Method

[1035] 5.1.1 Mixing of nanoparticles and excipients

[1036] Commercially available. The excipients used in this example are listed in the table. According to the solubility of the excipient in water, the excipient diluent was prepared at the stock solution concentration shown in the table. The LNP was mixed with the excipient so that the excipient concentrations were 0% (w / v), 0.01% (w / v), 0.1% (w / v), 0.5% (w / v), 1% (w / v), 2.5% (w / v), and 5% (w / v) (if possible), and the LNP concentration was 0.075 mg mRNA / mL.

[1037] Table 6: Excipients used in Example 5

[1038] Materials Supplier Stock solution concentration [% w / v] Kolliphor P188 Sigma 20 Kolliphor P338Geismar BASF 20 Kolliphor P407Geismar BASF 2 Tween 20 Sigma 20 Tween 80 Roth 20 BRIJ35 Merck 10 Tyloxapol Sigma 2 VitE-PEG1000 Sigma 20 Kolliphor EL Sigma 20 Kollisolv P124Geismar BASF 50

[1039] 5.1.2 Oscillating the nanoparticle suspension

[1040] See Section 1.1.3

[1041] 5.1.3 Measurement of complex size and PdI

[1042] See Section 1.1.4

[1043] 5.2 Results

[1044] This experiment aims to determine whether the observed protective effect of the existing excipient is a unique property of the tested LNP (containing dL_05) or can be regarded as a general property. To this end, the LNP used in Example 1 was replaced with a commercially available mRNA / LNP preparation for vaccination: (BioNTech / Pfizer). Figure 5 Summarized the relevant data. The results showed a high similarity to the data generated in Example 1. Oscillation without excipients led to a decrease in particle quality (hydrodynamic diameter increased by >20%). The presence of different excipients at concentrations above 0.01% (w / v) stabilized the suspension and prevented aggregation under physical stress.

[1045] 6 Example 6 - Anti-oscillation property in the presence of representative excipients

[1046] 6.1 Method

[1047] 6.1.1 Mixing of nanoparticles and excipients

[1048] (Moderna) was obtained commercially. The LNP was mixed with Kolliphor P188 (poloxamer 188) to give an excipient concentration of 1% (w / v) and an LNP concentration of 0.15 mg mRNA / mL, as shown in Table 7.

[1049] Table 7: Sample preparation for Example 6

[1050]

[1051] 6.1.2 Oscillation of the nanoparticle suspension

[1052] For the stress test, 100 μL of the LNP / excipient mixture was oscillated at maximum speed for 1, 5, 30, 60, and 90 minutes on a vortex mixer (Vortex Genie 2, Scientific Industries).

[1053] 6.1.3 Measurement of complex size and PdI

[1054] See Section 1.1.4

[1055] 6.2 Results

[1056] This experiment aimed to determine whether the protective effect of the observed excipient was a unique property of the tested LNP (containing dL_05) or could be considered a general property. For this purpose, a commercially available mRNA / LNP formulation for vaccination was used: (Moderna) was used instead of the LNP used in Example 1. Figure 6The relevant data was summarized. The results showed a high similarity to the data generated in Example 1. Oscillation in the absence of excipients led to a decrease in particle quality (increase in size and PdI). The presence of Kolliphor P188 at a concentration of 1% (w / v) stabilized the suspension and prevented aggregation under physical stress.

[1057] 7 Example 7 - Analysis of the attachment of poloxamer to LiNP using preparative size exclusion chromatography

[1058] This experiment aimed to determine whether the poloxamer (such as p188) added to LNP after nanoprecipitation was attached (especially adhered or integrated) to LNP or LiNP, or whether it remained in the solution.

[1059] Preparations containing LiNP and P188 were separated on a size exclusion chromatography (SEC) column to determine whether P188 was attached / bound to the particles under these conditions.

[1060] Materials and methods

[1061] 7.1.1 Materials

[1062] Table 8: Materials

[1063]

[1064] 7.1.2 Methods

[1065] LiNP preparations containing LiNP and P188 were separated by size exclusion chromatography. The poloxamer content of individual fractions was analyzed by HPLC, its particle size distribution was analyzed by DLS, and its integrity was analyzed by Ribogreeen.

[1066] 7.1.2.1 Preparative SEC

[1067] A HiPrep 16 / 60 Sephacryl S-500HR (cv 120 mL) chromatography column was used for preparative size exclusion chromatography analysis on a Purifier system. The running buffer was 25 mM MOPS, 150 mM NaCl, pH 6.8. The loading and elution flow rates were set at 1 mL / min. During the preparative run, 2.5 mL fractions were collected during elution. The ultraviolet signal and conductivity at a wavelength of 260 nm were monitored during the run.

[1068] 7.1.2.2 HPLC analysis

[1069] Table 1 summarizes the parameter settings for HPLC. The solvent gradient curve is shown in Table 9.

[1070] Table 9: Equipment Parameters for HPLC Method

[1071]

[1072] Table 10: Gradient Curve for Analyzing LNP and Carrier Samples by HPLC

[1073]

[1074]

[1075] Results

[1076] An HPLC calibration curve was prepared using P188 reference solutions at 0.2 to 7 mg / mL ( Figure 8 , 9 and 10).

[1077] Fractions after size exclusion chromatography were analyzed by HPLC to determine the presence of poloxamer. Absorbance of the elution curve at 260 nm showed that LiNP eluted within an elution volume of 50 to 70 mL (fractions 3 - 13, Figure 7 ). The integrity of LiNP was confirmed by DLS and ribogreen assays.

[1078] In the fractions of the LiNP main peak, we could not detect any P188 by HPLC (see Figure 11 , example of fraction 9).

[1079] P188 at a concentration of 0.2 to 0.5 mg / mL was only detected in the later eluting fractions 23, 24, 25, and 26 ( Figure 12 and 13 ).

[1080] Summary

[1081] Using preparative size exclusion chromatography, poloxamer and intact LNP can be separately detected based on their different molecular weights. Intact LiNP was detected according to the elution curve measured at 260 nm for fractions 3 - 13. We can verify the integrity of the particles by DLS measurements and ribogreen assays. By HPLC, we detected poloxamer in the later eluting fractions 23 - 26. Based on these data, we conclude that the poloxamer added to LNP did not attach to the particles.

[1082] Table 11: Summary of Results from Preparative Size Exclusion Chromatography Analysis

[1083]

Claims

1. Use of a surfactant for stabilizing a suspension of lipid nanoparticles or lipid-like nanoparticles in an aqueous carrier solution and preventing the particles from aggregating under physical stress conditions, wherein the lipid nanoparticles or lipid-like nanoparticles comprise the following components (a) and (b): (a) a therapeutic agent; and (b) at least one selected from the group consisting of permanent cationic lipids, ionizable lipids, and ionizable lipid-like substances.

2. Use of the surfactant according to claim 1, wherein the physical stress conditions are selected from shaking, stirring, vibrating, mixing, inverting, tapping, or dropping into the suspension, or a combination thereof, or wherein the physical stress conditions are caused by pumping the suspension or drawing it into a syringe.

3. Use of the surfactant according to claim 1 or 2, wherein the surfactant is incorporated into the aqueous carrier solution as an excipient.

4. Use of the surfactant according to claim 3, wherein the surfactant is substantially not attached to the nanoparticles.

5. Use of the surfactant according to any one of claims 1 to 4, wherein the surfactant is not present in the carrier solution during the freeze-drying process.

6. Use of the surfactant according to any one of claims 1 to 5, wherein the surfactant is a non-ionic surfactant.

7. Use of the surfactant according to claim 6, wherein the surfactant is at least one non-ionic surfactant selected from the group consisting of fatty alcohol ethoxylates, fatty acid ethoxylates, block copolymers of ethylene oxide and propylene oxide, alkylphenol ethoxylates or oligomers of alkylphenol ethoxylates, sorbitan fatty acid esters, ethoxylated sorbitan fatty acid esters, glycerol fatty acid esters, ethoxylated castor oil, and ethoxylated vitamin E.

8. Use of the surfactant according to any one of claims 1 to 7, wherein the suspension of lipid nanoparticles or lipid-like nanoparticles in the aqueous carrier solution comprises the surfactant at a concentration of 0.01 to 10% (w / v).

9. Use of the surfactant according to claim 8, wherein the suspension of the nanoparticles comprises the surfactant at a concentration of 0.5 to 1.5% (w / v).

10. Use of the surfactant according to any one of claims 1 to 9, wherein the therapeutic agent is mRNA.

11. Use of the surfactant according to any one of claims 1 to 10, wherein the nanoparticles do not comprise poloxamer 188 and / or do not comprise poloxamer 407.

12. Use of the surfactant according to any one of claims 1 to 11, wherein the nanoparticles comprise an ionizable lipid-like substance (b) of the following formula (b-1), wherein: a is 1 and b is an integer from 2 to 4; or a is an integer from 2 to 4 and b is 1, p is 1 or 2, m is 1 or 2; n is 0 or 1, and m + n ≥ 2; and R 1A to R 6A each independently selected from: hydrogen; -CH 2 -CH(OH)-R 7A ,-CH(R 7A )-CH 2 -OH, -CH 2 -CH 2 -(C=O)-O-R 7A 、-CH 2 -CH 2 -(C=O)-NH-R 7A ;-CH 2 -R 7A ;-C(NH)-NH 2 ;a poly(ethylene glycol) chain; and a receptor ligand; wherein R 7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond; The condition is R 1A to R 6A at least two residues selected from -CH 2 -CH(OH)-R 7A , -CH(R 7A )-CH 2 -OH, -CH 2 -CH 2 -(C=O)-O-R 7A , -CH 2 -CH 2 -(C=O)-NH-R 7A and -CH 2 -R 7A , where R 7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; or its protonated form, wherein one or more of the nitrogen atoms contained in the compound of formula (b-1) are protonated to provide a compound carrying a positive charge.

13. Use of a surfactant according to any one of claims 1 to 13, wherein as the ionizable lipid (b), the nanoparticles comprise: (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate or its protonated form, wherein the nitrogen atom of the compound is protonated, ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate) or its protonated form, wherein the nitrogen atom of the compound is protonated, and / or octadec-9-yl 8-[(2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino]octanoate or its protonated form, wherein the nitrogen atom of the compound is protonated.

14. A suspension of lipid nanoparticles or lipid-like nanoparticles in an aqueous carrier solution, wherein the aqueous carrier solution comprises a surfactant, and wherein the lipid nanoparticles or lipid-like nanoparticles comprise the following components (a) and (b): (a) A therapeutic agent, which is preferably a nucleic acid, and (b) At least one selected from the group consisting of permanent cationic lipids, ionizable lipids, and ionizable lipid-like compounds, and wherein the suspension of the nanoparticles comprises the surfactant at a concentration of 0.5 to 1.5% (w / v).

15. A suspension of lipid nanoparticles or lipid-like nanoparticles in an aqueous carrier solution, wherein the aqueous carrier solution comprises a surfactant, and wherein the lipid nanoparticles or lipid-like nanoparticles comprise the following components (a) and (b): (a) A therapeutic agent, which is preferably a nucleic acid, and (b) At least one selected from the group consisting of permanent cationic lipids, ionizable lipids, and ionizable lipid-like compounds, and wherein the surfactant is substantially not attached to the nanoparticles.

16. A method for stabilizing a suspension of lipid nanoparticles or lipid-like nanoparticles in an aqueous carrier solution to prevent particle aggregation under physical stress conditions, wherein the lipid nanoparticles (LNP) or lipid-like nanoparticles (LiNP) comprise the following components (a) and (b): (a) A therapeutic agent, which is preferably a nucleic acid, and (b) At least one selected from the group consisting of permanent cationic lipids, ionizable lipids, and ionizable lipid-like compounds, and wherein the method comprises incorporating a surfactant into the suspension of the lipid nanoparticles or lipid-like nanoparticles.

17. The method according to claim 16, wherein the method comprises the following steps: i) Generating a formulation of lipid nanoparticles (LNP) or lipid-like nanoparticles (LiNP) by mixing at least one selected from the group consisting of permanent cationic lipids, ionizable lipids, and ionizable lipid-like compounds dissolved in an organic phase with a therapeutic agent dissolved in an aqueous solution; ii) Diluting the formulation of the lipid nanoparticles or lipid-like nanoparticles by dilution with a first solution; iii) Concentrating the diluted formulation of the lipid nanoparticles or lipid-like nanoparticles by buffer exchange through TFF ultrafiltration / diafiltration, wherein the ultrafiltration / diafiltration uses a second solution; iv) Obtain a suspension of lipid nanoparticles or lipid-like nanoparticles; wherein the first solution comprises a surfactant between 0.01% w / v and 10% w / v, preferably between 0.25% w / v and 5% w / v, more preferably between 0.33% w / v and 2.5% w / v, more preferably between 0.45% w / v and 1.5% w / v, and most preferably between 0.5% w / v and 1.5% w / v; and / or wherein the second solution comprises a surfactant between 0.01% w / v and 10% w / v, preferably between 0.25% w / v and 5% w / v, more preferably between 0.33% w / v and 2.5% w / v, more preferably between 0.45% w / v and 1.5% w / v, and most preferably between 0.5% w / v and 1.5% w / v; and wherein, relative to the total volume of the suspension of the nanoparticles in the aqueous carrier solution, the final concentration of the surfactant in the combined first and second solutions is a surfactant between 0.01% w / v and 10% w / v, preferably between 0.25% w / v and 5% w / v, more preferably between 0.33% w / v and 2.5% w / v, more preferably between 0.45% w / v and 1.5% w / v, and most preferably between 0.5% w / v and 1.5% w / v.

18. The method according to claim 17, wherein the surfactant is added together with the first solution and the second solution.

19. The method according to claim 17 or 18, wherein approximately half of the surfactant is added to the first solution and approximately half of the surfactant is added to the second solution.

20. An LNP or LiNP suspension obtained by the method according to any one of claims 17 to 19.

21. The LNP or LiNP suspension according to claim 20, which is used for vaccination or immunization.

22. A method for avoiding side effects in the treatment with an LNP or LiNP carrying at least one therapeutic agent, wherein the method comprises the following steps: i) Determining whether the LNP or LiNP in the pharmaceutical composition comprising the LNP or LiNP aggregates when subjected to the mechanical stress or temperature stress by determining their aggregation levels before and after the pharmaceutical composition of the LNP or LiNP is subjected to the mechanical stress or temperature stress; ii) If the LNP or LiNP exhibits aggregation after the test in step (i), a surfactant is added to the LNP or LiNP formulation to obtain an LNP or LiNP suspension having a final surfactant concentration between 0.01% w / v and 10% w / v, preferably between 0.1% w / v and 10% w / v, more preferably between 0.25% w / v and 5% w / v, still more preferably between 0.33% w / v and 2.5% w / v, even more preferably between 0.45% w / v and 1.5% w / v, and most preferably between 0.5% w / v and 1.5% w / v; iii) Mix and reconstitute to generate a stable LNP or LiNP suspension.

23. A method of reducing one or more side effects associated with a vaccine formulation or an anti-cancer formulation comprising an LNP or LiNP carrying nucleic acid, the method comprising modifying the vaccine formulation or the anti-cancer formulation by adding a surfactant to the vaccine formulation comprising an LNP or LiNP suspension.

24. The method according to claim 23, wherein the surfactant is between 0.01% w / v and 10% w / v, preferably between 0.1% w / v and 10% w / v, more preferably between 0.25% w / w and 5%, still more preferably between 0.33% and 2.5%, even more preferably between 0.45% and 1.5%, and most preferably between 0.5% and 1.5%.

25. A method of reducing the incidence or severity of one or more side effects associated with an LNP / LiNP-based vaccine or an LNP / LiNP cancer therapy in a subject, the method comprising administering to the subject a vaccine formulation or an anti-cancer formulation comprising the LNP or LiNP suspension according to any one of claims 1 to 15 or 21.

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