Formulations for forming aerosols and aerosols for delivering nucleic acids

Abstract

The present invention relates to an aqueous suspension formulation for forming an aerosol, comprising lipid or lipidoid nanoparticles suspended in an aqueous vehicle solution, the lipid or lipidoid nanoparticles comprising the following components (a) and (b): (a) a nucleic acid, and (b) an ionizable lipid or an ionizable lipidoid, and the aqueous vehicle solution comprises a triblock copolymer containing one poly(propylene oxide) block and two poly(ethylene oxide) blocks. Additionally, the present invention relates to an aerosol obtained from the formulation for forming an aerosol.

Description

[Technical field]

[0001] The present invention relates to aqueous suspension formulations for forming aerosols, and to aerosols that can be advantageously used to administer nucleic acids to a subject. [Background technology]

[0002] Lipid formulations, such as lipid vesicles, including liposomes, and lipid or lipidoid nanoparticles (LNPs), are often used to deliver active pharmaceutical ingredients in patients.In particular, lipid or lipidoid formulations of nucleic acid are very useful and effective for the introduction of nucleic acid into cells.These advantageous properties of lipid or lipidoid formulations of nucleic acid have been used for decades in biological and medical research, and in therapeutic approaches for i) overexpressing genes or complementing genetic defects in target cells, or ii) downregulating or upregulating the endogenous gene expression in cells, or iii) repairing genetic defects (mutations).

[0003] In gene overexpression and complementation of genetic defects, nucleic acid containing a protein-coding sequence is introduced into cells. These are any DNA constructs containing coding regions under the control of suitable promoters, and this construct is transcribed into mRNA in the nucleus of the cell. The mRNA is translocated to the cytoplasm, where it is translated into protein. Alternatively, in vitro transcribed mRNA can be introduced into the cytoplasm using lipid formulations to achieve the same effect. In gene therapy and mRNA transcription therapy, the concept of introducing exogenous genetic information into patient cells is utilized to induce the production of therapeutic proteins in patient cells.

[0004] Downregulation of endogenous gene expression can use synthetic (antisense) oligonucleotides or completely synthetic nucleic acids such as siRNAs or ribozymes, among others, or (plasmid) DNA constructs are transcribed into RNA suitable for downregulating endogenous gene expression in cells. Knockdown of endogenous gene expression can use nucleic acids encoding nucleases such as zinc finger nucleases, TALE nucleases or CRISPR-Cas systems. Similarly, upregulation of endogenous gene expression can be achieved with certain oligonucleotides by various mechanisms (Khorkova O, Hsiao J, Wahlestedt C. Oligonucleotides for upregulating gene expression. Pharm Pat Anal. 2013;2(2):215-29; Sargent RG, Kim S, Gruenert DC. Oligo / polynucleotide-based gene modification: strategies and therapeutic potential. Oligonucleotides. 2011;21(2):55-75), also known as therapeutic gene regulation. A special case of therapeutic gene regulation is immune stimulation with CpG motif-containing oligonucleotides (Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol. 2002;20:709-60).

[0005] Repair of genetic defects at the mRNA level can use nucleic acid constructs that affect splicing reactions, such as, but not limited to, oligonucleotides for exon skipping.Genome-level gene repair can use nucleic acids that code for nucleases that can change the nucleic acid sequence in chromosomes, such as zinc finger nucleases, TALE nucleases, or CRISPR-Cas systems.

[0006] In any of the three concepts of nucleic acid therapy (overexpression-gene complementation / down- or up-regulation of endogenous gene / gene repair), the lipid formulation of nucleic acid must be introduced into the patient's body in a manner that is tolerable to the patient and suitable for nucleic acid to exert its desired effect in target cell or target organ, or throughout the patient's body.The frequently used administration routes include local injection, such as intradermal, subcutaneous, intraocular, intramuscular, intramyocardial, intratumoral, or direct administration to other target tissue or organ, and also systemic administration (usually intravenous).

[0007] The aerosol administration of lipid or lipidoid formulations of active pharmaceutical ingredients, particularly nucleic acid, by inhalation is not used very frequently.This administration route seems to be the most convenient and useful for delivering active pharmaceutical ingredients, nucleic acid, to target cells in the airway, but this administration route has many challenges, especially for lipid or lipidoid formulations of nucleic acid, as outlined below.

[0008] These difficulties are due, on the one hand, to the requirements of medical use. When administered by inhalation, it must be ensured that therapeutically effective dose is deposited in such an area of ​​the patient's respiratory tract within a reasonable time, where it can produce the desired therapeutic effect. This can be, for example, either the upper respiratory tract or alveolar area, depending on the medical use. Furthermore, as far as the time required for depositing the required dose is concerned, the compliance of the patient must be taken into account. An inhalation time of 1 hour or longer is a high burden on the patient, while 30 minutes or less is obviously more convenient.

[0009] Further challenges stem from the regulatory requirements outlined in the FDA Guidance for Industry entitled “Liposome Drug Products Chemistry, Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability; and Labeling Documentation” (Liposome Drug Products Chemistry, Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability; and Labeling Documentation - Guidance for Industry, (2018)) and the EMA guideline EMEA / CHMP / QWP / 49313 / 2005 Corr, “Guideline on the Pharmaceutical Quality of Inhalation and Nasal Products”, 2006. Both guidelines emphasize the importance of drug product integrity. Physicochemical properties such as vesicle / particle size and size distribution, as well as geometry, are listed as critical quality attributes. “Development studies should include the physical characteristics of drug substances and excipients that are relevant to their effect on product function” (Guideline on the Pharmaceutical Quality of Inhalation and Nasal Products, (2006)). EMEA / CHMP / QWP / 49313 / 2005 Corr states that "physical characteristics such as solubility, size, shape, density, roughness, charge and crystallinity of drug substances and / or excipients can affect the uniformity and reproducibility of the final product." The guideline requires that the homogeneity of the delivered dose and particulate weight be guaranteed over the range of patient flow rates.The susceptibility to fusion (i.e., irreversible association of smaller liposomes to form larger liposomes), aggregation (i.e., reversible aggregation or pooling of two or more liposomes without fusion), and leakage of contained drug substance during storage can affect the stability of the drug product (Liposome Drug Products Chemistry, Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability; and Labeling Documentation - Guidance for Industry, (2018)). In other words, the drug product present in the liquid to be nebulized should not change during nebulization, i.e., its composition, particle size and encapsulation efficiency of the drug substance (in the case of a particulate drug product), as well as its performance, should remain unchanged.

[0010] For administration by inhalation, lipid formulations of active pharmaceutical ingredients need to be nebulized.Various types of nebulizers known to those skilled in the art are available for medical use.Regardless of the design of the nebulizer, nebulization of a liquid requires the introduction of significant energy into the liquid. It has been observed that lipid or lipidoid formulations of pharma- ceutical active ingredients, when nebulized, change the size, geometry and encapsulation of the drug substance (Elhissi AM, Faizi M, Naji WF, Gill HS, Taylor KM. Physical stability and aerosol properties of liposomes delivered using an air-jet nebulizer and a novel micropump device with large mesh apertures. Int J Pharm. 2007;334(1-2):62-70; Li Z, Zhang Y, Wurtz W, Lee JK, Malinin VS, Durwas-Krishnan S, et al. Characterization of nebulized liposomal amikacin (Arikace) as a function of droplet size. J Aerosol Med Pulm Drug Deliv. 2008;21(3):245-54). In addition, it has been observed that lipid formulations of mRNA can clog nebulizers during nebulization and that the formulation loses potency when nebulized.

[0011] Therefore, the problem underlying the present invention is to provide a composition comprising nucleic acid, which is suitable for delivering nucleic acid to cells in airways when said composition is nebulized and inhaled in a subject, so that the nucleic acid can effectively perform its intended function, such as producing protein in lung cells, down-regulating or up-regulating the expression of endogenous genes, or causing gene repair after inhalation.Furthermore, it should be possible to nebulize an effective dose of the composition within a reasonable time, and the components of the composition should remain intact during nebulization. Summary of the Invention

[0012] In the context of the present invention, it has been found that a lipid or lipidoid nanoparticle (LNP) suspension formulation, which comprises nucleic acid and ionizable lipid or ionizable lipidoid, and is suspended in an aqueous vehicle solution comprising poly(ethylene oxide)-poly(propylene oxide) block copolymer, can be efficiently nebulized while preventing the negative effect of the nebulization procedure on the integrity of the nanoparticles and nucleic acid contained therein.In particular, the use of such a formulation for the preparation of aerosols makes it possible to significantly improve the resistance of particles to aggregation during the nebulization process and to realize beneficial retention of transfection efficiency of nucleic acid after the nebulization process.

[0013] A summary of aspects of the present invention is presented in the following items.

[0014] 1. An aqueous suspension formulation for forming an aerosol comprising lipid or lipidoid nanoparticles suspended in an aqueous vehicle solution, The lipid or lipidoid nanoparticles comprise the following components (a) and (b): (a) a nucleic acid, and (b) an ionizable lipid or an ionizable lipidoid wherein the aqueous vehicle solution comprises a triblock copolymer containing one poly(propylene oxide) block and two poly(ethylene oxide) blocks.

[0015] 2. The suspension formulation according to item 1, wherein the nucleic acid is selected from RNA and plasmid DNA.

[0016] 3. The suspension formulation according to item 1 or 2, wherein the nucleic acid is selected from mRNA, siRNA, miRNA, antisense RNA, tRNA and non-coding RNA, more preferably, mRNA.

[0017] 4. The suspension formulation according to any of items 1 to 3, wherein the concentration of the nucleic acid in the suspension formulation is in the range of 0.01 to 10 mg / mL, more preferably 0.02 to 10 mg / mL, and most preferably 0.05 to 5 mg / mL, relative to the total volume of the suspension formulation.

[0018] 5. The suspension formulation according to any of items 1 to 4, wherein the weight to volume ratio (grams / liter) of the nanoparticles in the aqueous suspension is in the range of 0.5 g / L to 100 g / L, preferably 10 g / L to 100 g / L, more preferably 10 g / L to 50 g / L, and most preferably 10 g / L to 75 g / L.

[0019] 6. The suspension formulation according to any of items 1 to 5, wherein the nanoparticles have a Z-average diameter in the range of 10 to 500 nm, more preferably in the range of 10 to 250 nm, and even more preferably in the range of 20 to 200 nm, as measured by dynamic light scattering.

[0020] 7. A suspension formulation according to any of items 1 to 6, wherein the nanoparticles have a polydispersity index, measured by dynamic light scattering, in the range of 0.05 to 0.4, more preferably in the range of 0.05 to 0.2.

[0021] 8. The nanoparticles are composed of the following components (c1) to (c6): (c1) Non-ionizable lipids having a sterol structure (c2) Phosphoglyceride lipids (c3) PEG-conjugated lipid (c4) Polysarcosine-conjugated lipid (c5) PASylated lipids, and (c6) Cationic polymer 8. The suspension formulation according to any of items 1 to 7, further comprising one or more of:

[0022] 9. Nanoparticles 30-65 mol % of an ionizable lipid or ionizable lipidoid (b), and the following components: 10 to 50 mol% of a lipid having a sterol structure (c1), 4-50 mol% phosphoglyceride lipid (c2), 0.5 to 10 mol % of one of PEG-conjugated lipid (c3), polysarcosine-conjugated lipid (c4) and PAS-modified lipid (c5), or any combination thereof; 0.5 to 10 mol% of a cationic polymer (c6), (c1) to (c6) in an amount such that the sum of (b) and (c1) to (c6) is 100 mol %.

[0023] 10. Nanoparticles comprising the following components (c1) to (c3): (c1) a non-ionizable lipid having a sterol structure; (c2) phosphoglyceride lipids, and (c3) PEG-conjugated lipid 10. The suspension formulation according to any of items 1 to 9, further comprising:

[0024] 11. Nanoparticles are 30-65 mol % of an ionizable lipid or ionizable lipidoid (b); 10 to 50 mol% of a lipid having a sterol structure (c1), 4 to 50 mol% of a phosphoglyceride lipid (c2), and 0.5-10 mol% PEG-conjugated lipid (c3) The suspension formulation according to item 10, comprising (b) and (c1) to (c3) in an amount such that the sum of these is 100 mol %.

[0025] 12. The suspension formulation according to any of items 1 to 11, wherein the nanoparticles further comprise a polyanionic component different from the nucleic acid.

[0026] 13. The suspension formulation according to any of items 1 to 12, wherein the composition of the nanoparticles has a weight ratio of the sum of the weights of components other than the nucleic acid in the nanoparticles to the weight of the nucleic acid in the nanoparticles in the range of 30:1 to 1:1, more preferably 20:1 to 2:1 and most preferably 15:1 to 3:1.

[0027] 14. The nanoparticles are formed from an ionizable lipidoid (b) of the following formula (b-1):

[0028] [ka] (In the formula, 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 is ≧2; R 1A ~R 6A are, independently of each other, hydrogen; -CH 2 -CH(OH)-R 7A , -CH(R 7A )-CH 2 -OH, -CH 2 -CH 2 -(C=O)-OR 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, R 7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond; However, R 1A ~R 6A At least two of the residues are -CH 2 -CH(OH)-R 7A , -CH(R 7A )-CH 2 -OH, -CH 2 -CH 2 -(C=O)-OR 7A , -CH 2 -CH 2 -(C=O)-NH-R 7A and -CH 2 -R 7A Selected from R 7Ais selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond. or a protonated form thereof, in which one or more of the nitrogen atoms contained in the compound of formula (b-1) are protonated to provide a compound having a positive charge. 14. A suspension formulation according to any of items 1 to 13, comprising

[0029] 15. The nanoparticles are prepared by dissolving an ionizable lipidoid (b-1) of the following formula (b-1b):

[0030] [ka] (In the formula, R 1A ~R 6A is as defined in item 14) or a protonated form thereof, in which one or more of the nitrogen atoms contained in the compound of formula (b-1b) are protonated to provide a compound having a positive charge. 15. A suspension formulation according to any of items 1 to 14, comprising

[0031] 16. R 1A ~R 6A is hydrogen and -CH 2 -CH(OH)-R 7A are independently selected from R 7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond, with the proviso that R 1A ~R 6A At least two residues, preferably at least three residues and more preferably at least four residues of -CH 2 -CH(OH)-R 7A and R 7A 16. The suspension formulation according to item 14 or 15, provided that is selected from C8-C18 alkyl, and C8-C18 alkenyl having one CC double bond.

[0032] 17. The nanoparticles are formed by dissolving a non-ionizable lipid (c1) having a sterol structure of formula (c1-1):

[0033] [ka] (In the formula, R 1K is a C3 to C12 alkyl group. 17. A suspension formulation according to any of items 8 to 16, comprising

[0034] 18. A suspension formulation according to any of items 8 to 17, wherein the non-ionizable lipid having a sterol structure (c1-1) contains cholesterol.

[0035] 19. The nanoparticles are prepared by dissolving a phosphoglyceride lipid (c2) of formula (c2-1)

[0036] [ka] (In the formula, R 1F and R 2F are independently a C8 to C18 alkyl group or a C8 to C18 alkenyl group, preferably a C12 to C18 alkyl group or a C12 to C18 alkenyl group. or a pharma- ceutically acceptable salt thereof; or a phosphoglyceride lipid (c2) of the formula (c2-2):

[0037] [ka] (In the formula, R 1G and R 2G are independently a C8 to C18 alkyl group or a C8 to C18 alkenyl group, preferably a C12 to C18 alkyl group or a C12 to C18 alkenyl group. or a pharma- ceutically acceptable salt thereof 19. A suspension formulation according to any of items 8 to 18, comprising

[0038] 20. A suspension formulation according to any of items 8 to 19, wherein the phosphoglyceride lipid (c2) comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or a pharma- ceutically acceptable salt thereof.

[0039] 21. The nanoparticles are prepared by dissolving a PEG-conjugated lipid (c3) of formula (c3-1)

[0040] [ka] (In the formula, R 1H and R 2H are independently a C8 to C18 alkyl group or a C8 to C18 alkenyl group, preferably a C12 to C18 alkyl group or a C12 to C18 alkenyl group, and p is an integer of 5 to 200, preferably 10 to 100, more preferably 20 to 60. or a PEG-conjugated lipid (c3) of formula (c3-2):

[0041] [ka] (In the formula, R 1J and R 2J are independently a C8 to C18 alkyl group or a C8 to C18 alkenyl group, preferably a C12 to C18 alkyl group or a C12 to C18 alkenyl group, and q is an integer of 5 to 200, preferably 10 to 100, more preferably 20 to 60. or a pharma- ceutically acceptable salt thereof 21. A suspension formulation according to any of items 8 to 20, comprising

[0042] 22. The suspension formulation according to item 21, wherein the PEG-conjugated lipid (c3) comprises 1,2-dimyristoyl-sn-glycerol methoxy(polyethylene glycol)-2000 (DMG-PEG2k).

[0043] 23. A suspension formulation according to any of items 1 to 22, 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.

[0044] 24. A triblock copolymer comprising one poly(propylene oxide) block B of formula (p-1):

[0045] [ka] (wherein s is an integer of 15 to 67, preferably 20 to 40), and Two poly(ethylene oxide) blocks A of formula (p-2)

[0046] [ka] (In the formula, r is independently an integer of 2 to 130, preferably 50 to 100, and more preferably 60 to 90 for each block.) 24. The suspension formulation according to any of items 1 to 23, wherein the ABA triblock copolymer contains

[0047] 25. A triblock copolymer having the following structure:

[0048] [ka] (In the formula, r and t are each independently an integer of 2 to 130, preferably an integer of 50 to 100, and more preferably an integer of 60 to 90, and s is an integer of 15 to 67, preferably an integer of 20 to 40.) 25. The suspension formulation according to item 24, having

[0049] 26. The suspension formulation according to item 24 or 25, wherein the triblock copolymer is poloxamer P188.

[0050] 27. A suspension formulation according to any of items 1 to 26, comprising the triblock copolymer in a concentration of 0.05 to 5% (w / v, at a temperature of 25°C), preferably 0.1 to 2%, relative to the total volume of the suspension formulation.

[0051] 28. The suspension formulation according to any of items 1 to 27, wherein the vehicle solution further comprises at least one of sucrose or NaCl, more preferably sucrose and NaCl.

[0052] 29. A method for preparing an aqueous suspension formulation for forming an aerosol comprising lipid or lipidoid nanoparticles suspended in an aqueous vehicle solution according to any one of items 1 to 28, comprising the steps of mixing a solution containing a nucleic acid (a) and a solution containing an ionizable lipid or ionizable lipidoid (b) to form a suspension comprising lipid or lipidoid nanoparticles; adding to the above suspension a triblock copolymer containing one poly(propylene oxide) block and two poly(ethylene oxide) blocks as defined in the preceding paragraph; and subjecting the suspension to tangential flow filtration. A method comprising:

[0053] 30. An aqueous suspension formulation for forming an aerosol according to any one of items 1 to 28, obtained by the method of item 29.

[0054] 31. A nebulizer comprising a compartment containing an aqueous suspension formulation for forming an aerosol according to any of items 1 to 28 or 30.

[0055] 32. A nebulizer according to item 31, selected from a jet nebulizer, a soft mist inhaler and a mesh nebulizer, more preferably a soft mist inhaler or a vibrating mesh nebulizer.

[0056] 33. An aerosol comprising aerosol droplets dispersed in a gas phase, the aerosol droplets comprising lipid or lipidoid nanoparticles and an aqueous vehicle solution for the nanoparticles; The lipid or lipidoid nanoparticles comprise the following components (a) and (b): (a) a nucleic acid, and (b) an ionizable lipid or an ionizable lipidoid Including, An aerosol in which the aqueous vehicle solution comprises a triblock copolymer containing one poly(propylene oxide) block and two poly(ethylene oxide) blocks.

[0057] 34. An aerosol according to item 33, the gas phase of which is air.

[0058] 35. An aerosol according to item 33 or 34, wherein the mass median aerodynamic diameter (MMAD) of the aerosol droplets is in the range of 2 to 10 μm, preferably 3 to 8 μm.

[0059] 36. The aerosol according to any of items 33 to 35, wherein the nucleic acid is selected from RNA and plasmid DNA.

[0060] 37. The aerosol according to any of items 33 to 36, wherein the nucleic acid is selected from mRNA, siRNA, miRNA, antisense RNA, tRNA and non-coding RNA, more preferably mRNA.

[0061] 38. An aerosol according to any of items 33 to 37, wherein the nanoparticles have a Z-average diameter, as measured by dynamic light scattering, in the range of 10 to 500 nm, more preferably 10 to 250 nm, and even more preferably 20 to 200 nm.

[0062] 39. An aerosol according to any of items 33 to 38, in which the nanoparticles have a polydispersity index, measured by dynamic light scattering, in the range of 0.05 to 0.4, more preferably in the range of 0.05 to 0.2.

[0063] 40. A nanoparticle having the following components (c1) to (c6): (c1) Non-ionizable lipids having a sterol structure (c2) Phosphoglyceride lipids (c3) PEG-conjugated lipid (c4) Polysarcosine-conjugated lipid (c5) PAS-modified lipid, and (c6) Cationic polymer 40. The aerosol according to any of items 33 to 39, further comprising one or more of:

[0064] 41. Nanoparticles are 30-65 mol % of an ionizable lipid or ionizable lipidoid (b) and the following components: 10 to 50 mol% of lipids having a sterol structure (c1) 4-50 mol% phosphoglyceride lipids (c2) 0.5 to 10 mol % of one of PEG-conjugated lipid (c3), polysarcosine-conjugated lipid (c4) and PAS-modified lipid (c5), or any combination thereof; 0.5 to 10 mol% cationic polymer (c6) The aerosol according to any of items 33 to 40, comprising one or more of the following:

[0065] 42. A nanoparticle comprising the following components (c1) to (c3): (c1) Non-ionizable lipids having a sterol structure (c2) phosphoglyceride lipids, and (c3) PEG-conjugated lipid 40. The aerosol according to any of items 33 to 39, further comprising:

[0066] 43. Nanoparticles are 30-65 mol % of an ionizable lipid or an ionizable lipidoid (b) 10 to 50 mol% of lipids having a sterol structure (c1) 4 to 50 mol% of a phosphoglyceride lipid (c2), and 0.5-10 mol% PEG-conjugated lipid (c3) An aerosol according to item 42, comprising (b) and (c1) to (c3) in an amount such that the sum of these is 100 mol %.

[0067] 44. The aerosol according to any of items 33 to 43, wherein the nanoparticles further comprise a polyanionic component different from the nucleic acid.

[0068] 45. The aerosol according to any of items 33 to 44, wherein the composition of the nanoparticles has a weight ratio of the sum of the weights of components other than the nucleic acid in the nanoparticles to the weight of the nucleic acid in the nanoparticles in the range of 30:1 to 1:1, more preferably 20:1 to 2:1 and most preferably 15:1 to 3:1.

[0069] 46. ​​The nanoparticles are formed from an ionizable lipidoid (b) having the following formula (b-1):

[0070] [ka] (In the formula, 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 is ≧2; R 1A ~R 6A are, independently of each other, hydrogen; -CH 2 -CH(OH)-R 7A , -CH(R 7A )-CH 2 -OH, -CH 2 -CH 2 -(C=O)-OR 7A , -CH 2 -CH 2 -(C=O)-NH-R 7A ;-CH 2 -R7A ;-C(NH)-NH 2 a poly(ethylene glycol) chain; and a receptor ligand, R 7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond; However, R 1A ~R 6A At least two of the residues are -CH 2 -CH(OH)-R 7A , -CH(R 7A )-CH 2 -OH, -CH 2 -CH 2 -(C=O)-OR 7A , -CH 2 -CH 2 -(C=O)-NH-R 7A and -CH 2 -R 7A Selected from R 7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond. or a protonated form thereof, in which one or more of the nitrogen atoms contained in the compound of formula (b-1) are protonated to provide a compound having a positive charge. An aerosol according to any of items 33 to 45, including

[0071] 47. The nanoparticles are formed from an ionizable lipidoid (b) of the following formula (b-1b):

[0072] [ka] (In the formula, R 1A ~R 6A is as defined in item 46) or the compound of formula (Ia) in its protonated form, in which one or more of the nitrogen atoms contained in the compound are protonated to result in a compound having a positive charge.

[0073] 48. R 1A ~R 6Ais hydrogen and -CH 2 -CH(OH)-R 7A are independently selected from R 7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond, with the proviso that R 1A ~R 6A At least two residues, preferably at least three residues and more preferably at least four residues of -CH 2 -CH(OH)-R 7A and R 7A 48. The aerosol according to item 46 or 47, with the proviso that is selected from C8-C18 alkyl, and C8-C18 alkenyl having one CC double bond.

[0074] 49. The nanoparticles are formed by dissolving non-ionizable lipids (c1) having a sterol structure of formula (c1-1):

[0075] [ka] (In the formula, R 1K The aerosol according to any of items 40 to 48, comprising a C3 to C12 alkyl group.

[0076] 50. An aerosol according to any of items 40 to 49, in which the non-ionizable lipid having a sterol structure (c1) contains cholesterol.

[0077] 51. The nanoparticles are formed of a phosphoglyceride lipid (c2) of formula (c2-2):

[0078] [ka] (In the formula, R 1F and R 2F are independently a C8 to C18 alkyl group or a C8 to C18 alkenyl group, preferably a C12 to C18 alkyl group or a C12 to C18 alkenyl group. or a pharma- ceutically acceptable salt thereof; or a phosphoglyceride lipid (c2) of the formula (c2-2):

[0079] [ka] (In the formula, R 1G and R 2G are independently a C8 to C18 alkyl group or a C8 to C18 alkenyl group, preferably a C12 to C18 alkyl group or a C12 to C18 alkenyl group. or a pharma- ceutically acceptable salt thereof An aerosol according to any of items 40 to 50, including:

[0080] 52. An aerosol according to any of items 40 to 51, wherein the phosphoglyceride lipid (c2) comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or a pharma- ceutically acceptable salt thereof.

[0081] 53. The nanoparticles are prepared by dissolving a PEG-conjugated lipid (c3) of formula (c3-1)

[0082] [ka] (In the formula, R 1H and R 2H are independently a C8 to C18 alkyl group or a C8 to C18 alkenyl group, preferably a C12 to C18 alkyl group or a C12 to C18 alkenyl group, and p is an integer of 5 to 200, preferably 10 to 100, more preferably 20 to 60. or a PEG-conjugated lipid (c3) of formula (c3-2):

[0083] [ka] (In the formula, R 1J and R 2Jare independently a C8 to C18 alkyl group or a C8 to C18 alkenyl group, preferably a C12 to C18 alkyl group or a C12 to C18 alkenyl group, and q is an integer of 5 to 200, preferably 10 to 100, more preferably 20 to 60. or a pharma- ceutically acceptable salt thereof An aerosol according to any of items 40 to 52, including

[0084] 54. The aerosol according to item 53, wherein the PEG-conjugated lipid (c3) comprises 1,2-dimyristoyl-sn-glycerol methoxy(polyethylene glycol)-2000 (DMG-PEG2k).

[0085] 55. An aerosol according to any of items 33 to 54, in which the N / P ratio in nanoparticles is in the range of 0.5 to 20.

[0086] 56. A triblock copolymer comprising one poly(propylene oxide) block B of formula (p-1):

[0087] [ka] (wherein s is an integer of 15 to 67, preferably 20 to 40), and Two poly(ethylene oxide) blocks A of formula (p-2)

[0088] [ka] (In the formula, r is independently an integer of 2 to 130, preferably 50 to 100, and more preferably 60 to 90 for each block.) 56. The aerosol according to any of items 33 to 55, wherein the ABA triblock copolymer contains

[0089] 57. A triblock copolymer having the following structure:

[0090] [ka] (In the formula, r and t are each independently an integer of 2 to 130, preferably an integer of 50 to 100, and more preferably an integer of 60 to 90, s is an integer of 15 to 67, preferably 20 to 40. 57. The aerosol according to item 56, having

[0091] 58. The aerosol according to item 56 or 57, wherein the triblock copolymer is poloxamer P188.

[0092] 59. The aerosol according to any of items 33 to 58, wherein the vehicle solution further comprises at least one of sucrose or NaCl, more preferably sucrose and NaCl.

[0093] 60. An aerosol according to any of items 33 to 59, obtainable by atomization of an aqueous suspension formulation according to any of items 1 to 28 and 30.

[0094] 61. A method for preparing an aerosol, comprising the step of spraying an aqueous suspension formulation for forming an aerosol according to any one of items 1 to 28 and 30.

[0095] 62. The method of item 61, wherein the aerosol is an aerosol according to any one of items 33 to 60.

[0096] 63. The method of item 61 or 62, wherein nebulization is performed by an inhaler selected from a jet nebulizer, a soft mist inhaler and a mesh nebulizer, more preferably by a soft mist inhaler or a vibrating mesh nebulizer.

[0097] 64. An aqueous suspension formulation according to any of items 1 to 28 and 30 for use as a medicament, in which the suspension formulation is to be nebulized and the aerosol provided by the nebulization is to be administered to a subject.

[0098] 65. An aerosol according to any of items 33 to 60 for use as a medicine.

[0099] 66. An aqueous suspension formulation according to any of items 1 to 28 and 30 for use in the treatment or prevention of a disease or disorder by nucleic acid-based therapy, wherein the treatment or prevention comprises nebulization of the aqueous suspension formulation, and administration of an aerosol provided by nebulization to or through the respiratory tract of a subject, preferably by pulmonary or nasal administration.

[0100] 67. An aerosol according to any of items 33 to 60, for use in the treatment or prevention of a disease or disorder by nucleic acid-based therapy, wherein the treatment or prevention comprises administration of the aerosol to or via the respiratory tract of a subject, preferably by pulmonary or nasal administration.

[0101] 68. An aqueous suspension formulation for use according to item 66, or an aerosol for use according to item 67, wherein the disease or disorder to be treated or prevented is a pulmonary disease.

[0102] 69. A method of treatment comprising spraying an aqueous suspension formulation according to any of items 1 to 28 and 30, and administering the aerosol provided by spraying to or through the respiratory tract of a subject, preferably by pulmonary or nasal administration.

[0103] 70. A method of treatment comprising administration of an aerosol according to any of items 33 to 60 to or via the respiratory tract of a subject, preferably by pulmonary or nasal administration.

[0104] 71. The method according to item 69 or 70 for the treatment of a pulmonary disease.

[0105] It will be understood that the summary of the above items forms part of the general disclosure of the present invention, and thus information presented in the following detailed description, e.g., information regarding further preferred embodiments or optional features, also applies to the above items, and vice versa. [Brief description of the drawings]

[0106] [Figure 1] FIG. 1 shows the size and PdI of nanoparticle formulations before and after nebulization of 1 mL at an mRNA concentration of 0.5 mg / mL in 1% vehicle, 10% (w / v) sucrose, 50 mM NaCl. [Diagram 2] Figure 2 shows the encapsulation efficiency of nanoparticle formulations at an mRNA concentration of 2.5 mg / mL in x% (w / v) excipient, 10% (w / v) sucrose, 50 mM NaCl. Data were recorded 6 hours after mixing. [Diagram 3] Figure 3 shows the biophysical characteristics of 1 mL of 2.5 mg / mL nanoparticle suspension before (untreated) and after nebulization at various poloxamer concentrations: (a) size, (b) encapsulation efficiency, and (c) relative mRNA integrity. [Figure 4] FIG. 4 shows the results of biophysical characterization of fractionated aerosols of 10 mL of a formulation with an mRNA concentration of 2.5 mg / mL in the presence of 5% poloxamer (buffer: 5% (w / v) P188, 10% (w / v) sucrose, 50 mM NaCl): (a) size, (b) PdI, (c) encapsulation efficiency, and (d) relative mRNA integrity over nebulization time. [Diagram 5] Figure 5 shows eGFP levels in cell lysates 24 hours after transfection of 16HBEo-ALI with nanoparticles encapsulating eGFP mRNA before (untreated) and after nebulization in the presence of 5% (w / v) excipient (poloxamer or Tween-80). Dotted line: baseline eGFP levels after transfection with nanoparticles without excipient. [Figure 6] FIG. 6 shows eGFP levels upon intratracheal instillation of nanoparticles in 10% (w / v) sucrose, 50 mM NaCl with and without excipient (w / o). [Figure 7] FIG. 7 shows the size and PdI of nanoparticle formulations before and after nebulization in the presence or absence of poloxamer. [Figure 8]FIG. 8 shows the size of nanoparticle formulations (containing ICE as the cationic lipid) before and after nebulization of 1 mL at an mRNA concentration of 0.5 mg / mL in the presence or absence of 5% (w / v) poloxamer. [Figure 9] FIG. 9 shows the size and PdI of nanoparticle formulations (containing DLin-MC3-DMA as the cationic lipid) before and after nebulization of 1 mL at an mRNA concentration of 0.5 mg / mL in the presence or absence of 5% (w / v) poloxamer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0107] In the following, the present invention is described in detail.As understood by those skilled in the art, and unless otherwise indicated in a particular context, the information provided in this context applies to all aspects of the present invention, including the aqueous suspension formulation for forming aerosol according to the present invention (also referred to herein as "aqueous suspension formulation" or simply "suspension formulation"), the aerosol according to the present invention, and the method and use comprising the suspension formulation or aerosol.

[0108] First, nanoparticles and their components are described. The aqueous suspension formulation for forming aerosol according to the present invention and the aerosol comprise lipid nanoparticles or lipidoid nanoparticles. Therefore, unless otherwise specifically indicated, in this specification, when "nanoparticles" or "LNP" is mentioned, lipid nanoparticles and lipidoid nanoparticles are included. Furthermore, since the aerosol according to the present invention can be conveniently prepared using an aqueous suspension formulation, it should be understood that the information provided in this specification about the components of nanoparticles applies to the nanoparticles contained in the formulation for forming aerosol according to the present invention and the nanoparticles contained in the aerosol according to the present invention.

[0109] As component (a), the nanoparticles contained in the formulation for forming an aerosol according to the invention, and the nanoparticles contained in the aerosol according to the invention, comprise a nucleic acid, which generally provides the pharmacologically active component of the nanoparticle.

[0110] The nature of nucleic acid is not particularly limited.In principle, any type of nucleic acid can be used in the context of the present invention.Nucleic acid is known to those skilled in the art and refers to biopolymers or small biomolecules that are composed of nucleotides, which are monomers made of three components: 5-carbon sugar, phosphate group and nitrogenous base.

[0111] The term nucleic acid is a collective name for DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), the above family members of biopolymers. When the sugar is the compound ribose, the polymer is RNA, and when the sugar is derived from ribose as deoxyribose, the polymer is DNA. The term "nucleic acid" encompasses oligonucleotides or polynucleotides. Because nucleic acids are biopolymers made of nucleotides, the term "nucleic acid" is also often referred to as "sequence of nucleotides," and therefore, as will be understood by those skilled in the art, the terms "nucleic acid" and "nucleic acid sequence" are often used interchangeably.

[0112] In a preferred embodiment, the nanoparticles of the aqueous suspension formulation according to the invention and the nanoparticles of the aerosol according to the invention comprise ribonucleic acid (RNA) as nucleic acid, more preferably single-stranded RNA, most preferably mRNA.

[0113] The term "nucleic acid" includes all forms of naturally occurring types of nucleic acid, as well as chemically and / or enzymatically synthesized nucleic acids, and also includes nucleic acid analogs and nucleic acid derivatives. This term includes any single-stranded or double-stranded nucleic acid, with modified backbone, sugar-modified, or base-modified, such as, for example, locked nucleic acid (LNA), peptide nucleic acid (PNA), oligonucleoside thiophosphates and phosphotriesters, morpholino oligonucleotides, cationic oligonucleotides (US Pat. No. 6,017,700, WO 2007 / 069092), substituted ribo oligonucleotides or phosphorothioates. Furthermore, the term "nucleic acid" also refers to any molecule that contains nucleotides or nucleotide analogs. There is no restriction on the sequence or size of the nucleic acid contained in the nanoparticles of the present invention. Nucleic acid is primarily defined by the biological action that will be achieved in the biological target to which the nanoparticles of the present invention are delivered. For example, as outlined in further detail below, for applications in gene or nucleic acid therapy, the nucleic acid or nucleic acid sequence may be defined by the gene or gene fragment to be expressed, or by the intended replacement or repair of a defective gene or any gene target sequence, or by the target sequence of the gene to be inhibited, knocked down, down-regulated or up-regulated.

[0114] The nanoparticles of the suspension and aerosol according to the present invention can contain nucleic acid, which 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 segment of a DNA molecule is copied into an mRNA molecule by the enzyme RNA polymerase. During transcription, the DNA sequence is read by the RNA polymerase, which generates a complementary antiparallel RNA strand, called the primary transcript.

[0115] The DNA molecule according to the invention can be introduced into a vector, preferably an expression vector, by standard molecular biology techniques (see, for example, Sambrook et al., Molecular Cloning, A laboratory manual, 2nd Ed, 1989). The term "vector" such as "expression vector" or "cloning vector" in the sense of the present invention is understood as a circular double-stranded unit of DNA that can preferably replicate in a cell independently of chromosomal DNA and is used as a vehicle to bring genetic material into a cell, where it can be (replicated and / or) expressed (i.e., transcribed into RNA and translated into amino acid sequence). A vector containing foreign DNA is called recombinant DNA. A vector is itself a DNA sequence that generally consists of an insert sequence (e.g., the nucleic acid molecule / DNA molecule of the present invention) and a larger sequence that serves as the "backbone" of the vector. Plasmids in the sense of the present invention are most often found in bacteria and are used in recombinant DNA research to transfer genes between cells, and therefore constitute a subset of the "vectors" used in the sense of the present invention.

[0116] It is clear to those skilled in the art that further regulatory sequences may be added to the DNA molecule of the present invention. For example, transcription enhancers and / or sequences that allow induction of expression may be used. Suitable inducible systems are, for example, tetracycline-regulated gene expression, as described, for example, 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, for example, by Crook, EMBO J. 8 (1989), 513-519. The present invention may also use a vector, preferably an expression vector, comprising the DNA molecule. The vector may be, for example, a plasmid, a cosmid, a virus, a bacteriophage, or another vector, for example, conventionally used in genetic engineering, and may comprise further genes, such as marker genes, that allow the selection of said vector under suitable conditions and in suitable host cells.

[0117] When a nucleic acid used in the context of the present invention is a DNA molecule, this nucleic acid may be a plasmid DNA (pDNA) molecule.

[0118] As specified above, the nanoparticles of the aqueous suspension formulation according to the invention and the nanoparticles of the aerosol according to the invention preferably contain ribonucleic acid (RNA) as nucleic acid, more preferably single-stranded RNA, most preferably mRNA.

[0119] With regard to RNA, in principle, any type of RNA can be used 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 strand of ribonucleotides, as opposed to an RNA molecule, where two or more separate strands form a double-stranded molecule by hybridization of the separate strands. The term "single-stranded RNA" does not exclude that the single-stranded molecule forms a double-stranded structure itself, such as a secondary structure (e.g., loop and stem-loop) or a tertiary structure. Examples are tRNA and mRNA, but also any other type of single-stranded RNA, such as antisense-RNA, siRNA, etc.

[0120] The term "RNA" encompasses RNA that codes for amino acid sequences and RNA that does not code for amino acid sequences. It has been suggested that over 80% of genomes contain functional DNA elements that do not code for proteins. These non-coding sequences include regulatory DNA elements (binding sites for transcription factors, regulators and co-regulators, etc.) and sequences that code for transcripts that are never translated into proteins. Such transcripts that are 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 demonstrated that ncRNAs are important players in gene regulation, maintenance of genome integrity, cell differentiation and development, and that they are misregulated in various human diseases. There are various 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 initiator 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-lncRNAs, intronic lncRNAs, transcribed ultraconserved region RNAs (T-UCRs), etc. (Bhan A, Mandal SS, ChemMedChem. 2014 Mar 26. doi: 10.1002 / cmdc.201300534). Of the above non-coding RNAs, only siRNAs are double-stranded. Thus, in a preferred embodiment, the non-coding RNA is single stranded, and therefore the non-coding RNA is preferably not an siRNA.In another embodiment, RNA is coding RNA, i.e., RNA that codes amino acid sequence.Such RNA molecule is also called mRNA (messenger RNA), and is a single-stranded RNA molecule.RNA can be produced by synthetic chemical and enzymatic methods known to those skilled in the art, or by using recombinant technology, or can be isolated from natural sources, or a combination thereof.

[0121] Messenger RNA (mRNA) is a copolymer constructed mainly from nucleoside phosphate building blocks with adenosine, cytidine, uridine and guanosine as nucleosides, which carries genetic information from DNA in the cell nucleus as an intermediate to the cytoplasm, where it is translated into proteins. Therefore, they are suitable as an alternative for gene expression.

[0122] In the context of the present invention, mRNA should be understood to mean any polyribonucleotide molecule, which, once in the cell, is suitable for the expression of a protein or a fragment thereof or can be translated into a protein or a fragment thereof. The term "protein" here encompasses any kind of amino acid sequence, i.e. a chain of two or more amino acids linked together via peptide bonds, and also encompasses peptides and fusion proteins.

[0123] mRNA encompasses ribonucleotide sequences that code for proteins or fragments thereof whose function in or near a cell is necessary or beneficial, for example, proteins whose missing or defective form triggers a disease or illness and whose provision can alleviate or prevent the disease or disorder, or proteins whose function in or near a cell can promote a process beneficial to the body. mRNA may contain the sequence of a complete protein or a functional variant thereof. Furthermore, the ribonucleotide sequence may code for a protein that acts as a factor, i.e., an inducer, regulator, stimulator or enzyme, or a functional fragment thereof, where such a protein is a protein whose function is required to treat a disorder, particularly a metabolic disorder, or to initiate a process in vivo, such as the formation of new blood vessels, tissues, etc. Examples of proteins that may be encoded by mRNA include antibodies, cytokines, or chemokines. Here, functional variants are understood to mean fragments that can assume the function of a protein whose function in the cell is necessary or whose missing or defective form is pathogenic. Furthermore, mRNA may also have additional functional regions and / or 3' or 5' non-coding regions, in particular 3' and / or 5' UTR. 3' and / or 5' non-coding regions may be regions that naturally flank the protein-encoding sequence or artificial sequences, for example sequences that contribute to the stabilization of RNA. Those skilled in the art can determine suitable sequences for this in each case by routine experimentation.

[0124] In preferred embodiments, the mRNA comprises a 5' cap consisting of m7GpppG linked to the mRNA via a 5'-to-5' triphosphate linkage (five prime cap; cap 0), an additional methyl group on the penultimate nucleotide from the 5' end of the mRNA (cap-1, anti-reverse cap analog (ARCA)), and / or an internal ribosome entry site (IRES) and / or a polyA tail at the 3' end, specifically to improve translation. The mRNA can have additional regions that facilitate translation, such as, for example, a cap 2 structure or a histone stem loop structure.

[0125] The RNA that may be present in the suspension formulation and aerosol according to the present invention may 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 isomers of A, C, G and U nucleotides, and any naturally occurring or naturally occurring analogs, alternative or modified nucleotides, or isomers thereof, for example, with chemically modified or substituted residues. Modified nucleotides can have base modifications and / or sugar modifications. Modified nucleotides can also have phosphate group modifications, for example, with respect to the 5' prime cap of an mRNA molecule. Modified nucleotides also include nucleotides that are post-transcriptionally synthesized by covalent modification of nucleotides. Furthermore, any suitable mixture of unmodified and modified nucleotides is also contemplated. A non-limiting number of examples of modified nucleotides can be found in the literature (e.g., U.S. Patent Application Publication No. 2013 / 0123481; 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), some preferred modified nucleotides are exemplarily specified below based on their individual nucleoside residues: 1-Methyladenosine, 2-Methylthio-N6-hydroxynorvalylcarbamoyl adenosine, 2-Methyladenosine, 2'-O-ribosylphosphate adenosine, N6-Methyl-N6-threonylcarbamoyl adenosine, N6-Acetyladenosine, N6-Glycinylcarbamoyl adenosine, N6-Isopentenyladenosine, N6-Methyladenosine, N6-Threonylcarbamoyl adenosine adenosine, N6,N6-dimethyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, N6-hydroxynorvalylcarbamoyl adenosine, 1,2'-O-dimethyladenosine, N6,2'-O-dimethyladenosine, 2'-O-methyladenosine, N6,N6,2'-O-trimethyladenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 2-methyl 2-methylthio-N6-isopentenyl adenosine, 2-methylthio-N6-threonylcarbamoyl adenosine, N6-2-methylthio-N6-threonylcarbamoyl adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, 7-methyl adenosine, 2-methylthio-adenosine, 2-methoxy-adenosine, 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'-fluor-2'-deoxycytidine, 5-aza-cytidine, 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-1-methyl-pseudoisocytidine, le-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine; 1-methylguanosine, N2,7-dimethylguanosine, N2-methylguanosine, 2'-O-ribosylphosphateguanosine, 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, isoguanosine, 4-demethylwybutosine, epoxyqueosine, undermodified hydroxywybutosine, Hypomethylated modifications Hydroxywybutosine, Isowybutosine, Peroxywybutosine, Galactosyl-queosine, Mannosyl-queosine, Queosine, Archaeosine, Wybutosine, Methylwybutosine, Wybutosine, 7-aminocarboxypropyl demethylwybutosine, 7-aminocarboxypropyl wybutosine, 7-aminocarboxypropyl wybutosine methyl ester, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 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-Taurinomethyluridine, 5-Carbamoylmethyluridine, 5-(Carboxyhydroxymethyl)uridine methyl ester, Dihydrouridine, 5-Methyldihydrouridine, 5-Methylaminomethyl-2-thiouridine, 5-(Carboxyhydroxymethyl)uridine methyl ester Lysine, 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, uridine 5-oxyacetic acid methyl ester, 5-methoxyuridine, 5-aminomethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-taurinomethyl-2-thiouridine Auridine, pseudouridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 1-methylpseudouridine, 3-methylpseudouridine, 2'-O-methylpseudouridine, 5-formyluridine, 5-aminomethyl-2-geranyluridine, 5-taurinomethyluridine, 5-iodouridine, 5-bromouridine, 2'-methyl-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine, 2'-fluoro-2'-deoxyuridine, i. 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-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-propynyl-pseudouridine 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-dimethyl adenosine, 1,2'-O-dimethyl guanosine, 1,2'-O-dimethylinosine, 2,8-dimethyladenosine, 2-methylthiomethylenethio-N6-isopentenyl-adenosine, 2-geranylthiouridine, 2-lysidine, 2-methylthiocyclic N6-threonylcarbamoyl adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyl adenosine, 2-methylthio-N6-threonylcarbamoyl adenosine, 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, 2'-O-methyluridine 5-oxyacetic acid methyl ester, 2'-O-ribosyladenosine phosphate, 2'-O-ribosylagnosine phosphate, 3,2'-O-dimethyluridine, 3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine, 3-(3-amino-3-carboxypropyl)pseudouridine, 5,2'-O-dimethylcytidine, 5,2'-O-dimethyluridine, 5-(carbo 5-(hydroxyhydroxymethyl)-2'-O-methyluridine methyl ester, 55-(isopentenylaminomethyl)-2'-O-methyluridine, 5-aminomethyl-2-geranylthiouridine, 5-aminomethyl-2-selenouridine, 5-aminomethyluridine, 5-carbamoylmethyl-2'-O-methyluridine, 5-carboxyhydroxymethyluridine, 5-carboxymethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-geranylthiouridine, 5-carboxymethylaminomethyl-2-selenouridine, 5-carboxymethyl Aminomethyl-2'-O-methyluridine, 5-cyanomethyluridine, 5-formyl-2'-O-methylcytidine, 5-methoxycarbonylmethyl-2'-O-methyluridine, 5-methylaminomethyl-2-geranylthiouridine, 7-aminocarboxypropyl-demethyl-2'-O-uridine, 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-formyl adenosine, N6-hydroxymethyl adenosine, agamatidine, 2-methylthiocyclic N6-threonylcarbamoyl adenosine, glutamyl-queosine, guanosine attached to any nucleotide, guanylylated 5' terminus, hydroxy-N6-threonylcarbamoyl adenosine; Most preferred are pseudo-uridine, N1-methyl-pseudo-uridine, 2'-fluoro-2'-deoxycytidine, 5-iodocytidine, 5-methylcytidine, 2-thiouridine, 5-iodouridine and / or 5-methyl-uridine.

[0126] Furthermore, the term "modified nucleotide" includes nucleotides that contain isotopes such as deuterium. The term "isotope" refers to an element that has the same number of protons but different numbers of neutrons, resulting in different mass numbers. Thus, for example, isotopes of hydrogen include but are not limited to deuterium, but also tritium. Furthermore, polyribonucleotides can also contain isotopes of other elements, including, for example, carbon, oxygen, nitrogen and phosphorus. Modified nucleotides can be deuterated or contain other isotopes of hydrogen, or isotopes of oxygen, carbon, nitrogen or phosphorus.

[0127] Of U, C, A and G nucleotides, none of them can be modified, one, two, three or all of them can be modified.Thus, in some embodiments, at least one nucleotide of one nucleotide type, for example, at least one U nucleotide, can be modified nucleotide.In some embodiments, at least one nucleotide of two nucleotide types in total, for example, at least one U nucleotide and at least one C nucleotide, can be modified nucleotide.In some embodiments, at least one nucleotide of three nucleotide types in total, for example, at least one G nucleotide, at least one U nucleotide and at least one C nucleotide, can be modified nucleotide.In some embodiments, at least one nucleotide of all four nucleotide types can be modified nucleotide. In all these embodiments, one or more nucleotides per nucleotide type may be modified, and the percentage of said modified nucleotides per 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%.

[0128] In some embodiments, the total percentage of modified nucleotides in an 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%.

[0129] In a preferred embodiment, the mRNA is an mRNA comprising a combination of modified and unmodified nucleotides. Preferably, it is an mRNA containing a combination of modified and unmodified nucleotides as described in WO 2011 / 012316. The mRNA described therein is reported to exhibit improved stability and reduced immunogenicity. In a preferred embodiment, in such modified mRNA, 5-50% of the cytidine nucleotides and 5-50% of the uridine nucleotides are modified. The adenosine and guanosine containing nucleotides may be unmodified. The adenosine and guanosine nucleotides may be unmodified or partially modified, and they are preferably present in unmodified form.

[0130] In certain embodiments of any of the above, the percentage of analogs of a given nucleotide refers to the input percentage (e.g., the percentage of analogs in an initial reaction, such as an initial in vitro transcription reaction). In certain embodiments of any of the above, the percentage of analogs of a given nucleotide refers to the output (e.g., the percentage in a synthesized or transcribed compound). Both options are equally contemplated.

[0131] The RNA, preferably mRNA, molecules of the present invention can be produced recombinantly in vivo by methods known to those of skill in the art.

[0132] Alternatively, the modified RNA, preferably mRNA molecules of the invention may be produced in an in vitro system, for example using 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 modified RNA, preferably mRNA molecules with the desired properties of the invention. In certain embodiments, 5-50% of the cytidines are cytidine analogs in such input mixtures and 5-50% of the uridines are uridine analogs in such input mixtures. In certain embodiments, 5-40% of the cytidines are cytidine analogs in such input mixtures and 5-40% of the uridines are uridine analogs in such input mixtures. In certain embodiments, 5-30% of the cytidines are cytidine analogs in such mixtures and 5-30% of the uridines are uridine analogs in such input mixtures. In certain embodiments, 5-30% of the cytidine is an analog of cytidine in such a mixture, and 10-30% of the uridine is an analog of uridine in such a mixture. In certain embodiments, 5-20% of the cytidine is an analog of cytidine in such an input mixture, and 5-20% of the uridine is an analog of uridine in such an input mixture. In certain embodiments, 5-10% of the cytidine is an analog of cytidine in such an input mixture, and 5-10% of the uridine is an analog of uridine in such an input mixture. In certain embodiments, 25% of the cytidine is an analog of cytidine in such an input mixture, and 25% of the uridine is an analog of uridine in such an input mixture. In certain embodiments, the input mixture does not include an analog of adenosine and / or guanosine. In other embodiments, the input mixture optionally includes one or more analogs of adenosine and / or guanosine (or includes neither, or both).

[0133] In certain embodiments, the proportion of cytidine analogs in the input mixture is not the same as the proportion of uridine analogs in the input mixture.In certain embodiments, the proportion of cytidine analogs in the input mixture is lower than the proportion of uridine analogs in the input mixture.As stated above, this means that adenosine and guanosine analogs may or may not be present in the input mixture, but in certain embodiments, adenosine and guanosine analogs are not present in the input mixture.

[0134] In certain embodiments, an input mixture of nucleotides for an in vitro transcription system producing an RNA, preferably an mRNA, of the invention comprises a cytidine analog and a uridine analog, wherein 5-20% of the cytidines in the input mixture are cytidine analogs and 25-45% of the uridines in the input mixture are uridine analogs. In other words, the input mixture comprises modified and unmodified cytidines, and modified and unmodified uridines, wherein 5-20% of the cytidines in the input mixture comprise cytidine analogs, while 25-45% of the uridines in the input mixture comprise uridine analogs. In other embodiments, the input mixture comprises 5-10% cytidine analog, such as 7-9% cytidine analog, such as 7%, 7.5% or 8%, and 32-38% uridine analog, such as 33%, 34%, 35%, 36%, etc.

[0135] In certain embodiments, any of the uridine analogs and cytidine analogs described herein may be used, and in some cases, excluding pseudouridine.In certain embodiments, the cytidine analog comprises or consists of 5-iodocytidine (e.g., it is the type of single C analog used), and the uridine analog comprises or consists of 5-iodouridine (e.g., it is the type of single U analog used).

[0136] Exemplary analogs are described above. In the case of modified polyribonucleotides encoding a desired polypeptide, it should be understood that the level of analogs and modifications is considered throughout the entire polyribonucleotide encoding the desired polypeptide, including the 5' and 3' untranslated regions, unless otherwise indicated (e.g., the level of modification is based on the input ratio of the analog in an in vitro transcription reaction such that the analog can be incorporated into the transcribed position).

[0137] Furthermore, modified RNA, preferably mRNA molecules, may be chemically synthesized, for example by conventional chemical synthesis on an automated nucleotide sequence synthesizer using a solid phase support and standard techniques, or by chemical synthesis of the respective DNA sequence, followed by in vitro or in vivo transcription of the DNA sequence.

[0138] In another preferred embodiment, the mRNA may be combined with a target binding site, a target sequence, and / or a microRNA binding site to allow the desired mRNA activity only in relevant cells. In a further preferred embodiment, the RNA may be combined with a microRNA or shRNA in the untranslated region.

[0139] In general, therapeutic action can be achieved by the interaction of ribonucleic acid with cellular molecules and organelles.Such interaction can activate the innate immune system by itself, as in the case of certain CpG oligonucleotides and sequences designed to specifically interact with toll-like receptors and other extracellular or intracellular receptors.Furthermore, the uptake or introduction of nucleic acid (preferably ribonucleic acid, more preferably mRNA) into cells can be intended to cause the expression of nucleotide sequences, such as genes contained in nucleic acid (preferably ribonucleic acid, more preferably mRNA), can be intended to downregulate, silence or knock down endogenous gene expression as a result of the presence of introduced exogenous nucleic acid in cells, or can be intended to modify endogenous nucleic acid sequences, such as repair, removal, insertion or replacement of selected bases or entire stretches of endogenous nucleic acid sequences, or can be intended to interfere with virtually any cellular process as a result of the presence and interaction of introduced exogenous ribonucleic acid (preferably mRNA) in cells. The overexpression of the introduced exogenous nucleic acid (preferably ribonucleic acid, more preferably mRNA) is intended to counterbalance or complement endogenous gene expression, especially when the endogenous gene is defective or silent, and does not result in insufficient, defective or dysfunctional products of gene expression, as in the case of many metabolic and genetic diseases, such as cystic fibrosis, hemophilia or muscular dystrophies, to name a few. The overexpression of the introduced exogenous nucleic acid (preferably ribonucleic acid, more preferably mRNA) may also be intended to cause the product of expression to interact or interfere with any endogenous cellular process, such as gene expression, signal transduction and the control of other cellular processes. The overexpression of the introduced exogenous nucleic acid (preferably ribonucleic acid, more preferably mRNA) may also be intended to cause an immune response in the context of an organism in which the transfected or transduced cells are present or in which these cells are present. An example is the genetic modification of antigen-presenting cells, such as dendritic cells, to make them present antigens for vaccination purposes.Another example is the overexpression of cytokines in tumors to induce tumor-specific immune responses. Moreover, overexpression of introduced exogenous ribonucleic acids (preferably mRNA) may also be intended to generate in vivo or ex vivo transiently genetically modified T cells, NK cells and other lymphocytes for cell therapy, or precursor or stem cells or other cells for regenerative medicine.

[0140] Downregulation, silencing or knockdown of endogenous gene expression for therapeutic purposes can be achieved by RNA interference (RNAi), for example, using ribozymes, antisense oligonucleotides, tRNA, long double-stranded RNA, where such downregulation can be sequence-specific or non-specific, and can also result in cell death, as when long double-stranded RNA is introduced into cells. Downregulation, silencing or knockdown of endogenous or existing gene expression can be useful for treating acquired, hereditary or spontaneously developing diseases, including viral infection and cancer. It can also be envisioned that the introduction of nucleic acid into cells can be practiced as a preventive measure, for example, to prevent viral infection or neoplasia. Downregulation, silencing or knockdown of endogenous gene expression can be exerted at the transcriptional and translational levels. Several mechanisms are known to those skilled in the art, including, for example, epigenetic modification, changes in chromatin structure, selective binding of transcription factors by the introduced nucleic acid, hybridization of the introduced nucleic acid with complementary sequences in genomic DNA, mRNA or other RNA species by base pairing, including non-conventional base pairing mechanisms such as triple helix formation.Similarly, gene repair, base or sequence change can be achieved at genome level and mRNA level, including exon skipping.Base or sequence change can be achieved, for example, by RNA-induced site-specific DNA cleavage, by trans-splicing, trans-splicing ribozyme, chimeraplast, splicosome-mediated RNA trans-splicing by cut-and-paste mechanism, by using group II or retargeting intron, by using virus-mediated insertion mutagenesis, or by using targeted genome insertion using prokaryotic, eukaryotic or viral integrase system. Since nucleic acids are carriers of the architectural plans of living systems and they are directly and indirectly involved in numerous cellular processes, theoretically any cellular process can be affected by the introduction of nucleic acids into cells from outside.In particular, such introduction can be performed directly in vivo, in cell or organ cultures, and ex vivo, followed by transplantation of such modified organs or cells into a recipient. Particles for use in the context of the present invention having nucleic acids as therapeutically active agents can be useful for all of the above purposes.

[0141] As described above, the RNA, preferably the mRNA, may comprise a ribonucleotide sequence that encodes a protein or a fragment thereof, the function of which is necessary or beneficial in or near a cell, such as a protein, the supply of which, in its defective or defective form, triggers a disease or illness and the supply of which can alleviate or prevent the disease or illness, or a protein, the supply of which can promote a process in or near a cell that is beneficial to the body.

[0142] Indeed, in recent years, RNA (especially mRNA) has become increasingly relevant as a novel drug entity. In contrast to DNA-based gene therapy agents, mRNA does not need to be transported into the nucleus, but is directly translated into protein in the cytoplasm (J Control Release, 2011, 150:238-247, and Eur J Pharm Biopharm, 2009, 71:484-489).

[0143] Furthermore, numerous genetic disorders caused by mutations in single genes are known and are candidates for therapeutic approaches with RNA, preferably mRNA. Disorders caused by single gene mutations, such as cystic fibrosis, hemophilia, and many others, can be dominant or recessive in terms of the likelihood that a particular trait will appear in offspring. A dominant allele will manifest a phenotype in an individual who has only one copy of the allele, while a recessive allele requires that the individual has two copies, one from each parent, to be manifested. In contrast, polygenic disorders are caused by two or more genes, and the symptoms of the individual diseases are often mild and related to environmental factors. Examples of polygenic disorders are hypertension, elevated cholesterol levels, cancer, neurodegenerative disorders, psychiatric disorders, etc. Similarly, in these cases, therapeutic RNA, preferably mRNA, that represents one or more of these genes may be beneficial for such subjects. Furthermore, genetic disorders are not necessarily inherited from parental genes, and may also be caused by new mutations. Similarly, in these cases, therapeutic RNA, preferably mRNA, expressing the correct gene sequence may be beneficial to the subject.

[0144] An online catalog of 22,993 current entries of human genes and genetic disorders, together with their individual genes and their phenotypic descriptions, is available at the ONIM (Online Mendelian Inheritance in Man) webpage (http: / / onim.org). The respective sequences are available from the Uniprot database (http: / / www.uniprot.org). As a non-limiting example, the following Table A lists some congenital diseases and disorders and the corresponding genes. Due to the high degree of interaction of cell signaling pathways, mutations in certain genes cause multiple pathogenic symptoms, and only characteristic ones of these symptoms are listed in Table A.

[0145] In some embodiments of the invention, the therapeutic proteins encoded by RNA, preferably mRNA, and which may be present in the suspension formulations and aerosols of the invention are selected from the cellular proteins listed in Table A. Thus, the RNA molecule, preferably the mRNA molecule, may encode a therapeutic cellular protein, where the encoded therapeutic proteins are those listed in Table A or homologs thereof.

[0146] In another embodiment of the invention, the therapeutic protein encoded by the RNA, preferably the mRNA, is selected from the secreted proteins listed in Table A. Thus, the RNA, preferably the mRNA, can encode a therapeutic fusion protein, the encoded therapeutic protein or its homologue being one listed in Table A, and the second protein is a signal peptide allowing the secretion of the therapeutic protein. A signal peptide is a short, usually 5-30 amino acid long sequence present at the N-terminus of said therapeutic protein, which directs the fusion protein to the secretory pathway of the cell, via certain organelles (i.e. the endoplasmic reticulum, the Golgi apparatus or endosomes). Thus, such fusion proteins are either secreted from the cell or from organelles of the cell, or inserted into the cell membrane (e.g. multi-spanning membrane proteins), in an intracellular compartment or at the cell surface.

[0147] Thus, in a preferred embodiment of the invention, the RNA, preferably the mRNA, can encode one or more of the following proteins (but not limited to): of genes that cause, predispose to, or protect against a disease: Non-limiting examples of such diseases or disorders that may be treated (or prevented) include those in which said polypeptides, proteins or peptides are selected from the group consisting of those outlined in Table A below:

[0148] In some embodiments, the encoding sequence of the RNA, preferably the mRNA, can be transcribed and translated into a partial or full-length protein that has cellular activity at a level equal to or higher than the level of the natural protein. In some embodiments, the RNA, preferably the mRNA, encodes a therapeutically or pharma- ceutically active polypeptide, protein or peptide that has a therapeutic or prophylactic effect, said polypeptide, protein or peptide being selected from the group consisting of those outlined in Table A below. The RNA, preferably the mRNA, more particularly its encoding sequence, can be used to express a partial or full-length protein that has cellular activity at a level equal to or lower than the level of the natural protein. This can allow for the treatment of diseases for which administration of the RNA molecule is indicated.

[0149] [Table 1-1]

[0150] [Table 1-2]

[0151] [Table 1-3]

[0152] [Table 1-4]

[0153] [Table 1-5]

[0154] [Table 1-6]

[0155] [Table 1-7]

[0156] [Table 1-8]

[0157] [Table 1-9]

[0158] Table A above shows examples of genes whose defects result in diseases that can be treated by RNA, preferably mRNA, that can be present in the suspension formulations and aerosols of the invention, the RNA, preferably mRNA, comprising a ribonucleotide sequence that codes for an intact form of the protein of the defective gene disclosed above, or a functional fragment thereof. In particularly preferred embodiments, the RNA, preferably mRNA, is used to treat diseases affecting the lungs, such as SPB (surfactant protein B) deficiency, ABCA3 deficiency, cystic fibrosis and α1-antitrypsin deficiency, or affecting plasma proteins (e.g. congenital hemochromatosis (hepcidin deficiency), thrombotic thrombocytopenic purpura (TPP, ADAMTS13 deficiency), as well as genetic disorders of coagulation disorders (e.g. hemophilia a and b) and complement disorders (e.g. protein C deficiency), immune disorders such as SCID. Inherited diseases causing deficiency (caused by mutations in various genes such as RAG1, RAG2, JAK3, IL7R, CD45, CD3δ, CD3ε, etc.) or deficiency due to adenosine deaminase deficiency, e.g. (ADA-SCID), septic granulomatosis (caused by mutations in the gp-91-phox, p47-phox, p67-phox or p33-phox genes, for example), 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 mucopolysaccharidoses may be addressed.

[0159] Other disorders for which the RNA, preferably mRNA, of the invention may be useful include SMN1-associated spinal muscular atrophy (SMA); amyotrophic lateral sclerosis (ALS); GALT-associated galactosemia; cystic fibrosis (CF); SLC3A1-associated disorders, including cystinuria; COL4A5-associated disorders, including Alport syndrome; galactocerebrosidase deficiency; X-linked adrenoleukodystrophy and adrenomeloneuropathy; Friedreich's ataxia; Pelizaeus-Merzbacher disease; TSC1- and TSC2-associated tuberous sclerosis; Sanfilippo B syndrome (MPS). IIIB);CTNS-associated cystinosis;FMR1-associated disorders including fragile X syndrome, fragile X associated tremor / ataxia syndrome, and fragile X premature ovarian failure syndrome;Prader-Willi syndrome;Hereditary hemorrhagic telangiectasia (AT);Niemann-Pick disease type C1;neuronal ceroid lipofuscinosis-associated disorders including juvenile neuronal ceroid lipofuscinosis (JNCL), juvenile Batten disease, Santavuori-Haltia disease, Jansky-Bielschowsky disease, and PTT-1 and TPP1 deficiency;EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5-associated childhood ataxia with central nervous system white matter hypoplasia / vanishing white matter;CACNA1A- and CACNB4-associated recurrent seizures 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 autosomal dominant cerebral arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL); SCN1A and SCN1B-related seizure disorders; Alpers-Huttenlocher syndrome, POLG-related sensory ataxic neuropathy, dysarthria, and ophthalmoplegia, and polymerase G-related disorders including autosomal dominant, and recessive progressive external ophthalmoplegia with mitochondrial DNA deletions; X-linked adrenal hypoplasia; X-linked agammaglobulinemia; Fabry disease; and Wilson's disease.

[0160] In all of these diseases, proteins, such as enzymes, are defective and can be treated with RNA, preferably mRNA, encoding any of the above proteins of the present invention, thereby making the protein encoded by the defective gene or its functional fragment available.Transcript replacement therapy / protein replacement therapy does not affect the underlying genetic defect, but improves the concentration of the protein that the subject is deficient in.As an example, in Pompe disease, transcript replacement therapy / enzyme replacement therapy replaces the defect of the lysosomal enzyme acid alpha-glucosidase (GAA).

[0161] Thus, non-limiting examples of proteins that can be encoded by the mRNA of the present invention are erythropoietin (EPO), growth hormone (somatotropin, hGH), cystic fibrosis transmembrane conductance regulator (CFTR), growth factors (GM-SCF, G-CSF, etc.), MPS, protein C, hepcidin, ABCA3 and surfactant protein B. Further examples of diseases that can be treated by the RNA of the present invention are hemophilia A / B, Fabry disease, CGD, ADAMTS13, Hurler disease, X-chromosome-mediated A-gamma-globulinemia, adenosine deaminase-associated immunodeficiency and respiratory distress syndrome in newborns linked with SP-B. Particularly preferably, the RNA, preferably the mRNA, of the present invention contains coding sequences for surfactant protein B (SP-B) or erythropoietin. Further examples of proteins that can be encoded by the RNA, preferably the mRNA of the present invention are growth factors such as human growth hormone hGH, BMP-2 or angiogenic factors.

[0162] Although the above embodiments are described in the context of RNA, preferably mRNA, molecules that may be present in the nanoparticles used in the present invention, the above invention is not limited to the use of RNA, preferably mRNA, and other nucleic acid molecules, such as DNA molecules, may be used.

[0163] Said DNA molecule is capable of encoding said RNA, preferably said mRNA, and thus contains the genetic information for the RNA molecule which is transcribed accordingly.

[0164] Thus, with regard to preferred embodiments, the same applies mutatis mutandis to the DNA molecules of the invention as described above and below in the context of RNA molecules, preferably mRNA molecules, which may be present in the nanoparticles used in the present invention.

[0165] Alternatively, the RNA, preferably the mRNA, may contain ribonucleotide sequences encoding full-length or smaller antibodies (e.g., both heavy and light chains), which may be used in therapeutic situations, for example, to immunize a subject. The corresponding antibodies and their therapeutic uses are known in the art. The antibody may be encoded by a single mRNA strand or by two or more mRNA strands.

[0166] In another embodiment, the RNA, preferably the mRNA, may encode a functional monoclonal or polyclonal antibody that may be useful for targeting and / or inactivating a biological target (e.g., a stimulatory cytokine such as tumor necrosis factor). Similarly, the RNA, preferably the mRNA sequence may encode a functional anti-nephrotic factor antibody, useful for treating, for example, membranoproliferative glomerulonephritis type II or acute hemolytic uremic syndrome, or alternatively, an anti-VEGF antibody, useful for treating vascular endothelial growth factor (VEGF)-mediated diseases, such as cancer.

[0167] In another embodiment, the RNA, preferably mRNA, may encode a functional monoclonal or polyclonal antibody that may be useful for neutralizing or otherwise inhibiting the virus or viral replication.

[0168] Alternatively, the RNA, preferably the mRNA, may comprise a ribonucleotide sequence encoding an antigen which may preferably be used in a prophylactic or therapeutic context.

[0169] In another embodiment, the mRNA can encode a protein capable of inducing immune modulation, such as a cytokine, including chemokines, interferons (such as interferon lambda), interleukins, lymphokines, and tumor necrosis factors.

[0170] In another embodiment, the RNA, preferably the mRNA, may comprise a ribonucleotide sequence that codes for a polypeptide or protein that can be used in genome editing technology. Genome editing is a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using nucleases. These nucleases generate site-specific breaks at desired positions 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 may be either single-strand breaks or double-strand breaks (DSBs), although double-strand breaks (DSBs) are preferred. Numerous genome editing systems utilizing various polypeptides or proteins are known in the art, i.e., for example, CRISPR-Cas systems, 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.

[0171] Therefore, in a preferred embodiment, RNA, preferably mRNA, can contain ribonucleotide sequence coding for Cas (CRISPR-associated protein) protein family polypeptide or protein, preferably Cas9 (CRISPR-associated protein 9).Cas protein family protein, preferably Cas9, can be used in CRISPR / Cas9-based method and / or CRISPR / Cas9 genome editing technology.The CRISPR-Cas system for genome editing, control and targeting is reviewed in Nat. Biotechnol., 2014, 32(4):347-355.

[0172] In another preferred embodiment, the RNA, preferably the mRNA, can comprise a ribonucleotide sequence encoding a meganuclease. Meganucleases are endonucleases that, in contrast to "conventional" endonucleases, recognize large recognition sites (e.g., double-stranded DNA sequences consisting of 12-40 base pairs). As a consequence, each site occurs only a few times, preferably only once, in any given genome. Meganucleases are therefore considered to be the most specific naturally occurring restriction enzymes and are therefore suitable tools in genome editing technology.

[0173] In another preferred embodiment, the RNA, preferably the mRNA, contains a ribonucleotide sequence that codes for zinc finger nuclease (ZFN). ZFN is an artificial restriction enzyme that is generated by fusing zinc finger DNA binding domain with DNA cleavage domain. Zinc finger domain can be engineered to target specific desired DNA sequence, which allows zinc finger nuclease to target unique sequence in complex genome. By utilizing endogenous DNA repair mechanism, ZFN can be used to precisely modify genome of higher organism, therefore ZFN is a suitable tool for genome editing technology.

[0174] In another preferred embodiment, the RNA, preferably the mRNA, can contain a ribonucleotide sequence that codes for a transcription activator-like effector nuclease (TALEN). A TALEN is a restriction enzyme that can be engineered to cleave a specific sequence of DNA. A TALEN is a fusion protein in which a TAL effector DNA binding domain is fused to a DNA cleavage domain of a nuclease. A transcription activator-like effector (TALE) can be engineered to bind to practically any desired DNA sequence. Thus, when combined with a nuclease, it can cleave DNA at a specific desired position.

[0175] Although the above embodiments are described in the context of RNA, preferably mRNA molecules, as noted above the invention is not limited to the use of RNA, preferably mRNA, but any nucleic acid molecule, such as a DNA molecule, may be used.

[0176] Said DNA molecule is capable of encoding said RNA, preferably said mRNA, and thus contains the genetic information for the correspondingly transcribed RNA molecule.

[0177] Thus, with regard to preferred embodiments, the same applies mutatis mutandis to DNA molecules as described above and below in the context of RNA molecules, preferably mRNA molecules, which may be present in the nanoparticles used in the present invention.

[0178] As an alternative to the above, RNA contains ribonucleotide sequence that is not expressed as protein or polypeptide.Therefore, the term RNA should not be understood to mean only any polynucleotide molecule that can be translated into polypeptide / protein or their fragments when introduced into cell.Rather, it is also intended that RNA contains ribonucleotide sequence that is not translated into protein.In this context, it is assumed that RNA contains ribonucleotide sequence that preferably provides genetic information for antisense RNA, siRNA or miRNA sequence, or another desired non-coding ribonucleotide sequence.

[0179] Therefore, RNA can also be antisense RNA, siRNA or miRNA sequence.Antisense RNA, siRNA or miRNA sequence can be used to silence the action of certain RNA molecules at a certain stage.This can be desirable and useful in particular in the treatment of certain medical conditions and certain diseases, and in particular in the RNA-based therapeutic methods described above and below in this specification.

[0180] Silencing the action of RNA molecules can be achieved by utilizing the RNAi (RNA interference) mechanism by using a nucleic acid strand complementary to a certain RNA sequence. The term "RNA interference" or "inhibiting RNA" (RNAi / iRNA) describes the use of double-stranded RNA to target specific mRNAs for degradation, thereby silencing their translation. Preferred inhibiting RNA molecules can be selected from the group consisting of double-stranded RNA (dsRNA), siRNA, shRNA and stRNA. dsRNA that matches the gene sequence can be synthesized in vitro and introduced into the cell. dsRNA can also be introduced into the cell in the form of a vector that expresses the target gene sequence in a sense orientation and an antisense orientation, for example in the form of a hairpin mRNA. The sense and antisense sequences can be expressed from separate vectors, whereby the individual antisense and sense molecules form double-stranded RNA upon their expression. It is known in the art that in some cases, the expression of sequence in sense orientation, or even the expression of promoter sequence, is sufficient to produce dsRNA and then siRNA due to the internal amplification mechanism in cells.Therefore, all of the means and methods that lead to the reduction of the activity of the polypeptide or protein encoded by the coding region will be used by the present invention.For example, these siRNAs can be produced / introduced using sense constructs, antisense constructs, hairpin constructs, sense and antisense molecules, and their combinations.dsRNA is fed into the natural process, including the highly conserved nuclease Dicer, which cleaves dsRNA precursor molecules into short interfering RNA (siRNA).The generation and preparation of siRNA and the method of inhibiting the expression of target genes are described, inter alia, in WO 02 / 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 assemble the sequence-specific part of the RNA-induced silencing complex (RISC), which is a multicomplex nuclease that destroys messenger RNA that is homologous to the silencing trigger. Elbashir (2001) EMBO J. 20:6877-6888 showed that 21 nucleotide RNA duplexes may be used in cell culture to interfere with gene expression in mammalian cells.

[0181] The method of predicting and constructing siRNA is known in the art and described in Elbashir (2002) Methods 26:199-213, on the internet websites of commercial siRNA suppliers, 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 Tom Tuschl's research group (http: / / www.rockefeller.edu / labheads / tuschl / sirna.html).In addition, programs that predict siRNA from a given mRNA sequence are available online (e.g., http: / / www.ambion.com / techlib / misc / siRNA_finder.html or http: / / katahdin.cshl.org:9331 / RNAi / html / rnai.html). The uridine residues in the 2-nt 3' overhangs can be replaced by 2' deoxythymidine without loss of activity, which significantly reduces the cost of RNA synthesis and may also enhance the tolerance of siRNA duplexes when applied to mammalian cells (Elbashir (2001), in place of display). siRNAs can also be enzymatically synthesized using T7 or other RNA polymerases (Donze (2002) Nucleic Acids Res 30:e46). Short RNA duplexes that mediate effective RNA interference (esiRNA) can also be generated by hydrolysis with Escherichia coli RNase III (Yang (2002) PNAS 99:9942-9947). Furthermore, 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 aid of the programs named above.Additionally, commercially available sequence prediction tools, either integrated into sequence analysis programs or sold separately, such as the siRNA design tool provided by www.oligoEngine.com (Seattle, WA), can be used to predict siRNA sequences.

[0182] MicroRNA (miRNA) is similar to the short interfering RNA (siRNA) described above. MicroRNA (miRNA) is a short non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals and some viruses that functions in RNA silencing and post-transcriptional regulation of gene expression. miRNA functions by base pairing with complementary sequences in mRNA molecules. As a result, these mRNA molecules are silenced by one or more of the following processes: (1) cleavage of the mRNA strand into two small pieces, (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 the siRNA of the RNA interference (RNAi) pathway, except that miRNA is derived from a region of the RNA transcript that folds back on itself to form a short hairpin, whereas short interfering RNA (siRNA) is derived from a longer region of double-stranded RNA.

[0183] The DNA molecule used in the suspension formulation and aerosol of the present invention can also code for the above-mentioned RNA, for example the above-mentioned siRNA or miRNA, and thus contains the genetic information for the RNA molecule transcribed accordingly.Therefore, in the preferred embodiment, the same applies mutatis mutandis to the DNA molecule described above in the context of the RNA molecule, preferably the mRNA molecule, that can be present in the nanoparticle used in the present invention.

[0184] It will be understood that nanoparticles in the context of the present invention may contain a single type of nucleic acid, preferably RNA such as mRNA, but may alternatively contain a combination of two or more types of nucleic acid, preferably a combination of RNA, contained therein, for example in the form of a particle containing two or more types of nucleic acid, preferably RNA, in a single particle or in the form of a blend of particles with different types of nucleic acid, preferably RNA, such as mRNA.

[0185] As explained above, the nanoparticles of the aqueous suspension formulation according to the present invention and the nanoparticles of the aerosol according to the present invention further comprise ionizable lipid or ionizable lipidoid as component (b).It is understood that this includes the possibility that the nanoparticles comprise a combination of different ionizable lipids, a combination of different ionizable lipidoids, or a combination of one or more ionizable lipids and one or more ionizable lipidoids.The nanoparticles used in the context of the present invention usually comprise nucleic acid (a) and ionizable lipid or ionizable lipidoid (b) in the form of a mixture of these components.

[0186] The term "ionizable lipid" and "ionizable lipidoid" are used in the field of lipid nanoparticles and lipidoid nanoparticles to refer to lipid or lipidoid that is protonated and has a positive charge or can be protonated and has a positive charge.Thus, ionizable lipid and lipidoid are also referred to as "protonizable lipid" and "protonizable lipidoid", respectively, or as titratable lipid or titratable lipidoid, respectively.As will be understood by those skilled in the art, when referring to "ionizable lipid" or "ionizable lipidoid", it includes the ionizable lipid or lipidoid in its protonated or non-protonated form.As will be further understood, the protonated or non-protonated state of lipid or lipidoid is generally determined by the pH of the medium surrounding the lipid or lipidoid, for example, by the pH value of the aqueous vehicle solution contained in the aqueous suspension formulation, and by the aerosol according to the present invention.

[0187] In the context of the present invention, the counterion (anion) of the positive charge of the positively charged ionizable lipid or ionizable lipidoid is usually provided by the anionic moiety contained in nucleic acid.When the positively charged group is present in excess compared to the anionic moiety in nucleic acid, the positive charge can be balanced by other pharma-ceutically acceptable anions such as chloride, bromide or iodide, sulfate, nitrate, phosphate, hydrogen phosphate, dihydrogen phosphate, carbonate or bicarbonate, or by polyanion components other than nucleic acid, which can be present as optional components in nanoparticles.

[0188] Ionizable lipids and ionizable lipidoids are well known as components of lipid or lipidoid nanoparticles. In the context of the present invention, no particular restrictions are imposed on the type of ionizable lipid or ionizable lipidoid contained in the nanoparticles.

[0189] Generally, ionizable lipids or lipidoids contain primary, secondary or tertiary amino groups, respectively, that can act as proton acceptors, and thus may be protonated or unprotonated. Ionizable lipidoids generally contain a plurality of such amino groups, such as two or more, preferably three or more.

[0190] Preferably, the ionizable lipids that may be comprised by the nanoparticles used in the suspension formulations and aerosols according to the invention are lipids that comprise a protonizable head group that contains one or more, preferably one, primary, secondary or tertiary amino groups as the protonizable or protonated group, and one or more, preferably one or two, hydrophobic moieties linked to the head group.

[0191] Examples of these preferred ionizable lipids are: i) a lipid comprising a protonatable head group containing one or more, preferably one, primary, secondary or tertiary amino groups as the protonatable or protonated group, and one hydrophobic moiety linked to the head group; ii) lipids that contain a secondary or tertiary amino group as a protonatable or protonated head group, and two hydrophobic moieties linked to the head group. It is.

[0192] The hydrophobic moieties contained in these preferred lipids preferably contain one or more of a linear aliphatic residue, e.g., a linear residue containing 8-18 carbon atoms, a branched aliphatic residue, e.g., a branched residue containing 8-18 carbon atoms, or an alicyclic ring structure, which may be a fused ring structure, e.g., an alicyclic ring structure containing 10-18 carbon atoms. Additionally, the hydrophobic moiety may contain one or more linking groups that facilitate linking the moiety to a head group or that allow two or more of the above-mentioned aliphatic residues to be joined together. Additionally, the hydrophobic moiety may contain one or more substituents to the extent that the hydrophobic character of the moiety is maintained.

[0193] Preferably, the ionizable lipidoids that may be included in the nanoparticles used in the suspension formulations and aerosols according to the invention are oligoamines, more preferably oligoalkylamines, which contain at least two, preferably at least three, amino groups selected from secondary and tertiary amino groups that can be protonated or are protonated, each of which may have a hydrophobic moiety attached to the amino group. In addition to the amino group with the hydrophobic residue, the lipidoids may contain further protonable or protonated amino groups selected from primary, secondary and tertiary amino groups. Preferably, the total number of amino groups is between 3 and 10, more preferably between 3 and 6. Preferably, the total number of hydrophobic moieties attached to the amino groups is between 3 and 6. Preferably, the ratio of the number of hydrophobic moieties attached to the amino groups to the total number of amino groups in the oligoalkylamine is between 0.75 and 1.5.

[0194] The hydrophobic moiety contained in such preferred lipidoids preferably contains one or more of a linear aliphatic residue, e.g., a linear residue containing 8 to 18 carbon atoms, and a branched aliphatic residue, e.g., a branched residue containing 8 to 18 carbon atoms. Furthermore, the hydrophobic moiety may contain one or more linking groups that facilitate linking the moiety to an amino group or that allow two or more of the above aliphatic residues to be joined together. Furthermore, the hydrophobic moiety may contain one or more substituents to the extent that the hydrophobic character of the moiety is maintained.

[0195] Suitable exemplary ionizable lipids or ionizable lipidoids that may be included as component (b1) in the nanoparticles used in the context of the present invention are described, for example, in WO 2006 / 138380, EP 2476756, US 2016 / 0114042, U.S. Pat. No. 8,058,069, U.S. Pat. No. 8,492,359, U.S. Pat. No. 8,822,668, U.S. Pat. No. 8,969,535, U.S. Pat. No. 9,006,417, U.S. Pat. No. 9,018,187, U.S. Pat. No. 9,345,780, U.S. Pat. No. 9,352,042, U.S. Pat. No. 9,364,435, and the like. No. 9,394,234, U.S. Pat. No. 9,492,386, U.S. Pat. No. 9,504,651, U.S. Pat. No. 9,518,272, DE 19834683 A1, WO 2010 / 053572, U.S. Pat. No. 9,227,917, U.S. Pat. No. 9,556,110, U.S. Pat. No. 8,969,353, U.S. Pat. No. 10,189,802, WO 2012 / 000104, WO 2010 / 053572, WO 2014 / 028487 or WO 2015 / 095351 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, PS, et al., Molecular Therapy, 27(4), 2019, 710-728; Kulkarni, JA et al, Nucleic Acid Therapeutics, 28(3), 2018, 146-157; and Li, B. et al., Nano Letters, 15, 2015, 8099-8107.

[0196] Preferably, component (b) of the nanoparticles comprises or, more preferably, consists of an ionizable lipidoid of formula (Ia) below, or a protonated form thereof. Ionizable lipidoids of formula (Ia) below, or a protonated form thereof, which can be used as preferred component (b) in the context of the present invention, are described in detail in PCT application WO 2014 / 207231.

[0197] Thus, component (b) is a lipidoid of the following formula (b-1):

[0198] [ka] where the variables a, b, p, m, n and R 1A ~R 6A is defined as follows: 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 is ≧2; R 1A ~R 6A are, independently of each other, hydrogen; -CH 2 -CH(OH)-R 7A , -CH(R 7A )-CH 2 -OH, -CH 2 -CH 2 -(C=O)-OR 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, R 7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond; However, R 1A ~R 6AAt least two of the residues are -CH 2 -CH(OH)-R 7A , -CH(R 7A )-CH 2 -OH, -CH 2 -CH 2 -(C=O)-OR 7A , -CH 2 -CH 2 -(C=O)-NH-R 7A and -CH 2 -R 7A Selected from R 7A is selected from C3-C18 alkyl, or C3-C18 alkenyl having one C-C double bond. or preferably comprises or consists of a protonated form thereof, in which one or more of the nitrogen atoms contained in the compound of formula (I) are protonated to give a compound bearing a positive charge.

[0199] Preferably, R 1A ~R 6A is hydrogen; the group -CH 2 -CH(OH)-R 7A , -CH(R 7A )-CH 2 -OH, -CH 2 -CH 2 -(C=O)-OR 7A , -CH 2 -CH 2 -(C=O)-NH-R 7A ; and -CH 2 -R 7A are independently selected from R 7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond, provided that R 1A ~R 6A and more preferably at least two residues of R 1A ~R 6A and even more preferably at least three residues of R 1A ~R 6A At least four of the residues are -CH 2 -CH(OH)-R 7A , -CH(R7A )-CH 2 -OH, -CH 2 -CH 2 -(C=O)-OR 7A , -CH 2 -CH 2 -(C=O)-NH-R 7A and -CH 2 -R 7A R 7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond. More preferably, R 1A ~R 6A is hydrogen and the group -CH 2 -CH(OH)-R 7A are independently selected from R 7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond, provided that R 1A ~R 6A and more preferably at least two residues of R 1A ~R 6A and even more preferably at least three residues of R 1A ~R 6A At least four of the residues are -CH groups. 2 -CH(OH)-R 7A and R 7A is selected from C3-C18 alkyl, and C3-C18 alkenyl having one CC double bond.

[0200] Preferably, R 7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond, more preferably from C8-C12 alkyl and C8-C12 alkenyl having one C-C double bond. Generally, the alkyl group is R 7A As such, it is more preferred than an alkenyl group.

[0201] base R 1A ~R 6Ais a protecting group for an amino group, such as, for example, those described in WO 2006 / 138380, the preferred embodiments of which are t-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc) or carbobenzyloxy (Cbz).

[0202] base R 1A ~R 6A As long as any of the above is a receptor ligand, a useful example is the work of Philipp and Wagner in “Gene and Cell Therapy - Therapeutic Mechanisms and Strategy”, 3 rd Edition, Chapter 15. 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 peptides, antibodies specifically recognizing cell surface structures, folic acid, epidermal growth factor and peptides derived therefrom, transferrin, anti-transferrin receptor antibodies, nanobodies and antibody fragments, or approved drugs that bind to known cell surface molecules.

[0203] base R 1A ~R 6A As long as any one of the above is a poly(ethylene glycol) chain, the molecular weight of the poly(ethylene glycol) chain is preferably 100 to 20,000 g / mol, more preferably 1,000 to 10,000 g / mol, and most preferably 1,000 to 5,000 g / mol.

[0204] The variable p in formula (b-1) is preferably 1.

[0205] In formula (b-1), m is 1 or 2, n is 0 or 1, and m+n is ≧2. In other words, when m is 1, n must also be 1, and when m is 2, n can be 0 or 1. When n is 0, m must be 2. When n is 1, m can be 1 or 2.

[0206] The variable n in formula (b-1) is preferably 1. It is more preferable that m is 1 and n is 1.

[0207] Thus, the combination p=1, m=1 and n=1 is also preferred.

[0208] With respect to the variables a and b in formula (Ia), it is preferred that one of a and b is 1 and the other is 2 or 3. It is more preferred that a is 1 and b is 2, or a is 2 and b is 1. Most preferably, a is 1 and b is 2.

[0209] In view of the above, it is understood that the compound of formula (b-1) is a compound of formula (b-1a) and that the component (b) is a lipidoid of the following formula (b-1a):

[0210] [ka] (Wherein, a, b and R 1A ~R 6A is as defined in formula (b-1), including preferred embodiments thereof. or a protonated form thereof, in which one or more of the nitrogen atoms shown in formula (b-1a) are protonated to provide a compound having a positive charge. It is even more preferred that the compound comprises or consists of:

[0211] According to a further preferred embodiment, the compound of formula (b-1) is a compound of formula (b-1b), and component (b) is a lipidoid compound of formula (b-1b)

[0212] [ka] (In the formula, R 1A ~R 6A is defined as in formula (Ia), including preferred embodiments thereof. or a protonated form thereof, in which one or more nitrogen atoms shown in formula (b-1b) are protonated to provide a compound having a positive charge. It comprises or consists of:

[0213] Thus, according to a particularly preferred embodiment, component (b) comprises or consists of a lipidoid compound of formula (b-1b) above or a protonated form thereof, R 1A ~R 6A is hydrogen and -CH 2 -CH(OH)-R 7A are independently selected from R 7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond, provided that R 1A ~R 6A At least two of the residues are -CH 2 -CH(OH)-R 7A and more preferably R 1A ~R 6A and even more preferably at least three residues of R 1A ~R 6A At least four of the residues are -CH 2 -CH(OH)-R 7A and R 7A is selected from C8-C18 alkyl, and C8-C18 alkenyl having one CC double bond.

[0214] According to a further exemplary embodiment, component (b) is an ionizable lipid of formula (b-2):

[0215] [ka] (In the formula, R 1B is an organic group containing one or more primary, secondary or tertiary amino groups. or R 1B In the protonated form, one or more nitrogen atoms contained in a primary, secondary or tertiary amino group are protonated to provide a compound having a positive charge. It comprises or consists of:

[0216] Preferably, the compound of formula (b-2) has the following structure:

[0217] [ka] has.

[0218] According to another exemplary embodiment, component (b) is an ionizable lipid of formula (b-3):

[0219] [ka] (In the formula, R 1C and R 2C are independently selected from C8 to C18 alkyl groups and C8 to C18 alkenyl groups, preferably C12 to C18 alkyl groups and C12 to C18 alkenyl groups; R 3C is a C1 to C6 alkanediyl group, preferably a C2 or C3 alkanediyl group, R 4C and R 5C are independently hydrogen or C1-C3 alkyl, preferably methyl. or a protonated form thereof, in which one or more of the nitrogen atoms contained in the compound of formula (b-3) are protonated to provide a compound having a positive charge. An example of an ionizable lipid of formula (b-3) is DLin-MC3-DMA (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate).

[0220] According to yet another exemplary embodiment, component (b) is an ionizable lipid of formula (b-4):

[0221] [ka] (In the formula, R 1D and R 2D are independently selected from C8 to C18 alkyl groups and C8 to C18 alkenyl groups, preferably C12 to C18 alkyl groups and C12 to C18 alkenyl groups; R 3D is a C1 to C6 alkanediyl group, preferably a C2 alkanediyl group, R 4D and R 5D are independently hydrogen or C1-C3 alkyl, preferably methyl. or a protonated form thereof, in which one or more of the nitrogen atoms contained in the compound of formula (b-4) are protonated to provide a compound having a positive charge. It comprises or consists of:

[0222] According to yet another exemplary embodiment, component (b) is an ionizable lipidoid of formula (b-5):

[0223] [ka] (In the formula, R 1E ~R 5E are each independently hydrogen, -CH 2 -CH(OH)-R 7E , -CH(R 7E )-CH 2 -OH, -CH 2 -CH2 -(C=O)-OR 7E , -CH 2 -CH 2 -(C=O)-NH-R 7E and -CH 2 -R 7E Selected from R 7E is selected from C3-C18 alkyl, or C3-C18 alkenyl having one C-C double bond, provided that R 1E ~R 5E At least two of the residues are -CH 2 -CH(OH)-R 7E , -CH(R 7E )-CH 2 -OH, -CH 2 -CH 2 -(C=O)-OR 7E , -CH 2 -CH 2 -(C=O)-NH-R 7E and -CH 2 -R 7E Selected from R 7E is selected from C3-C18 alkyl, or C3-C18 alkenyl having one C-C double bond. or a protonated form thereof, in which one or more of the nitrogen atoms contained in the compound of formula (b-5) are protonated to provide a compound having a positive charge. It comprises or consists of:

[0224] In formula (b-5), R 1E ~R 5E is preferably independently -CH 2 -CH(OH)-R 7E and R 7E is selected from C8-C18 alkyl or C8-C18 alkenyl having one CC double bond.

[0225] Yet another exemplary ionizable lipid suitable for use in the present invention, which may be included in or consist of component (b), is the ionizable lipid disclosed in PCT Application WO 2012 / 000104, beginning on page 104 of said document, as "cationic lipids of formula I", including all of the specific embodiments thereof also discussed therein.

[0226] Further exemplary ionizable lipidoids suitable for use in the present invention, which may be comprised in or consist of component (b), are those disclosed and claimed in PCT Application WO 2010 / 053572 on page 4 as "amino alcohol lipidoids", including all compounds of the general formula set out in the Summary of the Invention, and further defined in the remainder of the application.

[0227] Further exemplary ionizable lipidoids suitable for use in the present invention, which may be comprised in or consist of component (b), are the ionizable lipidoids disclosed in PCT Application WO 2014 / 028487, including specific embodiments thereof, as amine containing lipidoids of formulae I-V.

[0228] In addition to the nucleic acid and the ionizable lipid or ionizable lipidoid, as preferred optional components, the nanoparticles in the aqueous suspension formulations and aerosols of the invention can include one or more of the following components (c1) to (c6): (c1) a non-ionizable lipid having a sterol structure; (c2) phosphoglyceride lipids, (c3) PEG-conjugated lipids, (c4) polysarcosine-conjugated lipids, (c5) PAS-modified lipid, and (c6) Cationic polymers.

[0229] Component (c1) is a lipid having a sterol structure. Thus, suitable lipids are compounds having a steroid core structure with a hydroxyl group at the 3-position of the A ring.

[0230] Exemplary non-ionizable lipids having a sterol structure that may be comprised by or that component (c1) may consist of are those having the structure of formula (c1-1):

[0231] [ka] (In the formula, R 1K is a C3 to C12 alkyl group.

[0232] Further exemplary non-ionizable lipids having a sterol structure that may be comprised by or of which component (c1) can consist include those disclosed by 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, in particular those illustrated in Figure 2 of this publication.

[0233] Preferably, component (c1) comprises or consists of cholesterol.

[0234] Component (c2) is a phosphoglyceride.

[0235] Preferably, component (c2) is a phospholipid selected from the compounds of formula (c2-1)

[0236] [ka] (In the formula, R 1F and R 2F are independently selected from C8 to C18 alkyl groups and C8 to C18 alkenyl groups, preferably C12 to C18 alkyl groups and C12 to C18 alkenyl groups. or a pharma- ceutically acceptable salt thereof, and a phospholipid of formula (c2-2)

[0237] [ka] (In the formula, R 1G and R 2G are independently selected from C8 to C18 alkyl groups and C8 to C18 alkenyl groups, preferably C12 to C18 alkyl groups and C12 to C18 alkenyl groups. or a pharma- ceutically acceptable salt thereof It comprises or consists of:

[0238] More preferably, component (c2) comprises or consists of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or a pharma- ceutically acceptable salt thereof.

[0239] Exemplary salt forms of the compound of formula (c2-1) include the salt formed by acidic -OH group and base, or the salt formed by amino group and acid.As the salt formed with base, can mention alkali metal salt such as sodium or potassium salt; alkaline earth metal salt such as calcium or magnesium salt and ammonium salt.As the exemplary salt formed with acid, can mention salt form formed with acidic group of nucleic acid, but other salts are not excluded, and can mention salts of inorganic acids such as chloride, bromide or iodide, sulfate, nitrate, phosphate, hydrogen phosphate or dihydrogen phosphate, carbonate and hydrogen carbonate as examples.

[0240] Exemplary salt forms of the compound of formula (c2-2) include the salt formed by the acidic -OH group bonded to the P atom and a base, or the salt formed by the quaternary amino group and an anion.As the salt formed with a base, mention can 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 the exemplary salt formed with an anion, mention can be made of the salt formed with the acidic group of nucleic acid, but other salts are not excluded, and mention can be made of the salts of inorganic acids such as chloride, bromide or iodide, sulfate, nitrate, phosphate, hydrogen phosphate or dihydrogen phosphate, carbonate and hydrogen carbonate as examples.

[0241] Component (c3) is a PEG-conjugated lipid, ie a lipid that is covalently linked to a polyethylene glycol chain.

[0242] Preferably, component (c3) is a compound of formula (c3-1)

[0243] [ka] (In the formula, R 1H and R 2H are independently selected from C8 to C18 alkyl groups and C8 to C18 alkenyl groups, preferably C12 to C18 alkyl groups and C12 to C18 alkenyl groups, and p is an integer of 5 to 200, preferably 10 to 100, more preferably 20 to 60. and a compound of formula (c3-2)

[0244] [ka] (In the formula, R 1J and R 2Jare independently selected from C8 to C18 alkyl groups and C8 to C18 alkenyl groups, preferably C12 to C18 alkyl groups and C12 to C18 alkenyl groups, and q is an integer of 5 to 200, preferably 10 to 100, more preferably 20 to 60. or a pharma- ceutically acceptable salt thereof The PEG-conjugated lipid may comprise or consist of a PEG-conjugated lipid selected from:

[0245] Exemplary salt forms of the compound of formula (c3-2) include salts formed by the acidic -OH group bonded to the P atom and a base. 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.

[0246] More preferably, component (c3) comprises or consists of 1,2-dimyristoyl-sn-glycerol methoxy(polyethylene glycol) (DMG-PEG), and even more preferably, component d) comprises or consists of 1,2-dimyristoyl-sn-glycerol methoxy(polyethylene glycol)-2000 (DMG-PEG2k).

[0247] Component (c4) is a polysarcosine-conjugated lipid, i.e., the formula (c4-1):

[0248] [ka] (wherein r represents the number of repeating units, preferably 10 to 100) is a lipid covalently linked to a polymer portion.

[0249] Component (c5) is a PAS-linked lipid, ie, a lipid covalently linked to a polymer moiety formed by repeating proline (pro) / alanine (ala) / serine (ser) residues.

[0250] Component (c6) is a cationic polymer.Suitable for use in forming nanoparticles containing nucleic acid, such polymers are known in the art.Exemplary suitable cationic polymers are discussed in AC Silva et al., Current Drug Metabolism, 16, 2015, 3-16 and references therein, JC Kasper et al., J. Contr. Rel. 151 (2011), 246-255, WO 2014 / 207231 and references therein, and WO 2016 / 097377 and references therein.

[0251] Suitable cationic oligomers or polymers include, in particular, cationic polymers that include units that include amino groups, which can be protonated to provide a positive charge on the polymer.

[0252] Items (1), (2), (3) and (4) below:

[0253] [ka] Preferred are polymers comprising units independently selected from the formula: wherein one or more of the nitrogen atoms of repeat units (1), (2), (3) and / or (4) can be protonated to provide a positive charge on the polymer.

[0254] Four classes of polymers that contain multiple units that include amino groups are particularly preferred as cationic polymers:

[0255] A first preferred class includes poly(ethyleneimine) ("PEI"), including branched poly(ethyleneimine) ("brPEI").

[0256] A second preferred class of cationic polymers are polymers which contain, as side chains and / or as end groups, a plurality of groups of the following formula (c6-1), as disclosed as groups of formula (II) in WO 2014 / 207231 (applicant ethris GmbH):

[0257] [ka] where the variables a, b, p, m, n and R 2 ~R 6 is defined independently for each group of formula (c6-1) in such groups as follows: 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 is ≧2; R 2 ~R 5 are, independently of each other, hydrogen; the radical -CH 2 -CH(OH)-R 7 , -CH(R 7 )-CH 2 -OH, -CH 2 -CH 2 -(C=O)-OR 7 , -CH 2 -CH 2 -(C=O)-NH-R 7 or -CH 2 -R 7 Selected from R 7 is selected from a C3-C18 alkyl or a C3-C18 alkenyl having one C-C double bond; a protecting group for an amino group; and a poly(ethylene glycol) chain; R 6 is hydrogen; the group -CH 2 -CH(OH)-R 7 , -CH(R 7 )-CH-OH, -CH 2 -CH 2 -(C=O)-OR 7 , -CH 2 -CH2 -(C=O)-NH-R 7 or -CH 2 -R 7 Selected from R 7 is a C3-C18 alkyl or a 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; One or more of the nitrogen atoms shown in formula (c6-1) can be protonated to provide a cationic group of formula (c6-1).

[0258] With regard to these polymers and further preferred definitions of the variables contained in formula (c6-1) above, the respective disclosures in WO 2014 / 207231 regarding the groups of formula (II) also apply to the invention described herein.

[0259] A third preferred class of cationic polymers are polymers comprising, as repeat units, a plurality of groups of the following formula (c6-2), as disclosed as groups of formula (III) in WO 2014 / 207231 (applicant ethris GmbH):

[0260] [ka] where the variables a, b, p, m, n and R 2 ~R 5 is defined independently for each group of formula (6c-2) in such groups as follows: 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 is ≧2; R 2 ~R 5 are, independently of each other, hydrogen; the radical -CH 2 -CH(OH)-R 7 , -CH(R7 )-CH 2 -OH, -CH 2 -CH 2 -(C=O)-OR 7 , -CH 2 -CH 2 -(C=O)-NH-R 7 or -CH 2 -R 7 Selected from R 7 is a C3-C18 alkyl or a 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; One or more of the nitrogen atoms shown in formula (c6-2) may be protonated to provide a cationic group of formula (c6-2).

[0261] With regard to these polymers and further preferred definitions of the variables contained in formula (c6-2) above, the respective disclosures in WO 2014 / 207231 regarding their repeat units of formula (III) also apply to the invention described herein.

[0262] A fourth preferred class of cationic polymers is represented by the statistical copolymers disclosed in WO 2016 / 097377 (applicant ethris GmbH), which have the following formulae (a1) and (a2):

[0263] [ka] (a) a plurality of repeating units independently selected from the repeating units and the following formulas (b1) to (b4):

[0264] [ka] (b) a plurality of repeating units independently selected from the repeating units Including, The molar ratio of the sum of repeat units (a) to the sum of repeat units (b) is in the range of 0.7 / 1.0 to 1.0 / 0.7, and one or more nitrogen atoms of repeat units (a) and / or (b) contained in the copolymer can be protonated to provide a cationic copolymer.

[0265] With regard to further preferred definitions of this copolymer, the respective disclosures in WO 2016 / 097377 also apply to the invention described herein. As specified therein, particularly preferred copolymers are linear copolymers comprising or consisting of repeat units (a1) and (b1).

[0266] As an optional component of nanoparticle, polyanion component different from nucleic acid may also be included.Examples of such polyanion are polyglutamic acid and chondroitin sulfate.When such polyanion component different from nucleic acid is used in nanoparticle, its amount is preferably limited so that the amount of negative charge provided by polyanion component is not more than the amount of negative charge provided by nucleic acid.

[0267] As explained above, the lipid or lipidoid nanoparticles present in the suspension formulations and aerosols according to the invention comprise (a) a nucleic acid and (b) an ionizable lipid or an ionizable lipidoid. When a lipidoid is included, the nanoparticles shall be referred to herein as lipidoid nanoparticles.

[0268] Preferably, the nanoparticles are Nucleic acid (a), (b) an ionizable lipid or an ionizable lipidoid; and possibly one or more of the following: A non-ionizable lipid having a sterol structure (c1), Phosphoglyceride lipids (c2), PEG-conjugated lipids (c3), Polysarcosine-conjugated lipid (c4), PAS lipid (c5) and Cationic polymer (c6) and more preferably, consists of the above.

[0269] Exemplary suspension formulations comprising nanoparticles formed from the components listed above and also suitable for use in the context of the present invention include those disclosed by Patel et al., Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA, Nature Communications, 2020, 11:983.

[0270] It will be appreciated that the components of the nanoparticles, and in particular one or more of components (a) and (b), and optionally (c1)-(c6), will typically be included as a mixture in the nanoparticles.

[0271] In terms of the amounts of these components, the nanoparticles Nucleic acids and 30-65 mol % of an ionizable lipid or an ionizable lipidoid (b) and the following components: 10 to 50 mol% of a lipid having a sterol structure (c1), 4-50 mol% phosphoglyceride lipid (c2), 0.5 to 10 mol % of one of PEG-conjugated lipid (c3), polysarcosine-conjugated lipid (c4) and PAS-modified lipid (c5), or any combination thereof; 0.5 to 10 mol% of a cationic polymer (c6), It is further preferred that the nanoparticles comprise one or more of the following components: (b) and (c1) to (c6) in an amount such that the sum of (b) and (c1) to (c6) is 100 mol %, more preferably consisting of the above. As will be understood, the molar percentages for components (c1) to (c6) are given with the proviso that not all of these components need to be present in the nanoparticles. Thus, for example, a cationic polymer may or may not be present in the context of this preferred embodiment, but if present, it is used in an amount of 0.5 to 10 mol %. As further indicated above, in the context of this preferred embodiment, the amount of components (c1), (c2), (c3), (c4), (c5) and / or (c6) is such that the sum of (b) and (c1) to (c6) is 100 mol %.

[0272] Nanoparticles are Nucleic acid (a), (b) an ionizable lipid or an ionizable lipidoid; A non-ionizable lipid having a sterol structure (c1), phosphoglyceride lipids (c2), and PEG-conjugated lipids (c3) More preferably, it comprises or consists of the above.

[0273] In terms of the amounts of these components, the nanoparticles Nucleic acid (a) 30-65 mol % of an ionizable lipid or ionizable lipidoid (b); 10 to 50 mol% of a lipid having a sterol structure (c1), 4 to 50 mol% of a phosphoglyceride lipid (c2), and 0.5-10 mol% PEG-conjugated lipid (c3) It is further preferred that the total of (b) and (c1) to (c3) is 100 mol %, and more preferably that the composition consists of the above.

[0274] In accordance with the above information regarding preferred nucleic acids and preferred components of the lipid composition other than the nucleic acid, the suspension formulation according to the invention and the lipid nanoparticles contained in the aerosol according to the invention, respectively, (a) as a nucleic acid, mRNA; (b) an ionizable lipidoid of formula (b-1b)

[0275] [ka] (In the formula, R 1A ~R 6A is hydrogen and -CH 2 -CH(OH)-R 7A are independently selected from R 7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond, provided that R 1A ~R 6A At least two of the residues are -CH 2 -CH(OH)-R 7A and more preferably R 1A ~R 6A At least four of the residues are -CH 2 -CH(OH)-R 7A and R 7A is selected from C8-C18 alkyl, and C8-C18 alkenyl having one C-C double bond. or one or more of the nitrogen atoms shown in formula (b-1b) are protonated to provide a cationic lipidoid, in its protonated form. (c1) A non-ionizable lipid having a sterol structure of formula (c1-1)

[0276] [ka] (In the formula, R 1K is a C3 to C12 alkyl group. (c2) Phosphoglyceride of formula (c2-2)

[0277] [ka] (In the formula, R 1G and R 2G are independently selected from C8 to C18 alkyl groups and C8 to C18 alkenyl groups, preferably C12 to C18 alkyl groups and C12 to C18 alkenyl groups. or a pharma- ceutically acceptable salt thereof, and (c3) PEG-conjugated lipid of formula (c3-1)

[0278] [ka] (In the formula, R 1H and R 2H are independently selected from C8 to C18 alkyl groups and C8 to C18 alkenyl groups, preferably C12 to C18 alkyl groups and C12 to C18 alkenyl groups, and p is an integer of 5 to 200, preferably 10 to 100, more preferably 20 to 60. Preferably it comprises:

[0279] In the nanoparticles contained in the suspension formulation and aerosol according to the present invention, the composition of the nanoparticles is preferably such that the weight ratio of the total weight of components other than the nucleic acid in the nanoparticle to the weight of the nucleic acid is in the range of 30:1 to 1:1, more preferably 20:1 to 2:1, and most preferably 15:1 to 3:1.

[0280] The N / P ratio, i.e. the ratio of the number of amine nitrogen atoms contributed by the ionizable lipid or ionizable lipidoid to the number of phosphate groups contributed by the nucleic acid of the nanoparticle, is preferably in the range of 0.5-20, more preferably in the range of 0.5-10.

[0281] The lipid or lipidoid nanoparticles contained in the suspensions and aerosols according to the invention preferably have a Z-average diameter in the range of 10 to 500 nm, more preferably in the range of 10 to 250 nm, even more preferably in the range of 20 to 200 nm. The particle diameters indicated are the hydrodynamic diameters of the particles, as determined by dynamic light scattering (DLS). Measurements are generally carried out at 25°C.

[0282] The polydispersity index of the nanoparticles contained in the suspension formulation and aerosol according to the present invention is preferably in the range of 0.05 to 0.4, more preferably in the range of 0.05 to 0.2. The polydispersity index can be determined by dynamic light scattering (DLS). Measurements are generally carried out at 25°C.

[0283] It is possible to provide suspension formulations or aerosols containing different lipid or lipidoid nanoparticles as defined above, i.e. particles that differ with respect to their constituents, however preferably the nanoparticles contained in the suspension formulation according to the invention or in the aerosol according to the invention are of the same constituents.

[0284] Lipid nanoparticles can be conveniently prepared by mixing a solution containing nucleic acid in an aqueous solvent, for example, a citrate buffer having a pH of 4.5 and optionally containing a salt such as sodium chloride, with a solution containing ionizable lipids or ionizable lipidoids, for example, in ethanol. In addition, optional components can be combined, for example, by adding them to one of the two solutions. The lipid nanoparticles produced in this way can be further processed by chromatography and / or dialysis and / or tangential flow filtration to obtain lipid nanoparticles of the desired liquid composition. Before or during these downstream processing steps, additional excipients such as cryoprotectants and other excipients can be added to obtain the desired pharmaceutical composition. When nanoparticles are subjected to tangential flow filtration, for stability reasons, it is preferred to perform the filtration on a suspension of nanoparticles containing a triblock copolymer containing one poly(propylene oxide) block and two poly(ethylene oxide) blocks, defined herein as a component of the vehicle solution.

[0285] To that extent, the present invention further provides a method of preparing an aqueous suspension formulation for forming an aerosol comprising lipid or lipidoid nanoparticles suspended in an aqueous vehicle solution, the method comprising mixing a solution containing a nucleic acid (a) and a solution containing an ionizable lipid or ionizable lipidoid (b) to form a suspension comprising lipid or lipidoid nanoparticles. Further components, such as one or more of components (c1)-(c6), may be conveniently incorporated into the nanoparticles, for example, by adding them to the solution containing the ionizable lipid or ionizable lipidoid.

[0286] In a preferred embodiment, the present invention provides a method for preparing an aqueous suspension formulation for forming an aerosol, the aqueous suspension comprising lipid or lipidoid nanoparticles suspended in an aqueous vehicle solution, the method comprising the steps of: mixing a solution containing a nucleic acid (a) and a solution containing an ionizable lipid or ionizable lipidoid (b) to form a suspension containing lipid or lipidoid nanoparticles; adding to the above suspension a triblock copolymer containing one poly(propylene oxide) block and two poly(ethylene oxide) blocks, as defined herein; and subjecting said suspension to tangential flow filtration to obtain an aqueous suspension formulation according to the present invention. The present invention provides a method comprising:

[0287] Aqueous suspension formulations for forming an aerosol include the lipid or lipidoid nanoparticles discussed above together with an aqueous vehicle solution. As indicated by reference to a suspension formulation, the nanoparticles are suspended in the vehicle solution.

[0288] The vehicle solution is an aqueous solution, i.e. a solution in which the predominant solvent in relation to the total volume of solvent is water, preferably a solution containing more than 70% water, more preferably more than 90% water as solvent, expressed as a volume percentage of water in the total volume of solvent contained in the vehicle solution (at a temperature of 25° C.). Most preferably, water is the only solvent in the vehicle solution. Thus, the vehicle solution is liquid at room temperature (e.g., 25° C.).

[0289] The weight per volume ratio of nanoparticles in the vehicle solution in the composition is preferably in the range of 0.5 g / L to 100 g / L, preferably 10 g / L to 100 g / L, more preferably 10 g / L to 50 g / L, and most preferably 10 g / L to 75 g / L.

[0290] The concentration of the 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 0.02 to 5 mg / ml and most preferably 0.1 to 5 mg / ml relative to the total volume of the suspension.

[0291] As specified above, the lipid or lipidoid nanoparticles contained in the suspension formulations and aerosols according to the invention preferably have a Z-average diameter in the range of 10-500 nm, more preferably in the range of 10-250 nm, even more preferably in the range of 20-200 nm. The particle diameters indicated are the hydrodynamic diameters of the particles, as determined by dynamic light scattering (DLS). Measurements are generally carried out at 25° C.

[0292] The polydispersity index of the nanoparticles contained in the suspension formulation and aerosol according to the present invention is preferably in the range of 0.05 to 0.4, more preferably in the range of 0.05 to 0.2. The polydispersity index can be determined by dynamic light scattering (DLS). Measurements are generally carried out at 25°C.

[0293] The vehicle solution comprises a triblock copolymer containing one poly(propylene oxide) block and two poly(ethylene oxide) blocks.

[0294] Preferably, the triblock copolymer comprises one poly(propylene oxide) block B of formula (p-1):

[0295] [ka] (wherein s is an integer of 15 to 67, preferably 20 to 40), and Two poly(ethylene oxide) blocks A of formula (p-2)

[0296] [ka] (In the formula, r is independently an integer of 2 to 130, preferably 50 to 100, and more preferably 60 to 90 for each block.) It is an ABA triblock copolymer containing

[0297] More preferably, the triblock copolymer has the following structure:

[0298] [ka] (In the formula, r and t are each independently an integer of 2 to 130, preferably an integer of 50 to 100, and more preferably an integer of 60 to 90, s is an integer of 15 to 67, preferably 20 to 40. has.

[0299] Most preferably, poloxamer P188 is used as the triblock copolymer.

[0300] The vehicle solution generally contains the triblock copolymer dissolved therein, however, as will be appreciated by those skilled in the art, this does not exclude the possibility that a certain amount of the copolymer molecules may be adsorbed onto lipid or lipidoid nanoparticles contained in the composition.

[0301] Preferably, the aerosol-forming composition contains the triblock copolymer in a concentration of 0.05-5% w / v (ie grams per 100 mL), preferably 0.1-2%, based on the total volume of the composition.

[0302] In addition to the triblock copolymer, other excipients may be present in the vehicle solution. Preferably, the vehicle solution further comprises at least one of sucrose and NaCl, more preferably sucrose and NaCl.

[0303] Suspension formulations according to the invention can be conveniently prepared, for example, by a method comprising adding the triblock copolymer to a suspension comprising a vehicle solution and lipid or lipidoid nanoparticles, or adding lipid or lipidoid nanoparticles to a vehicle solution comprising the triblock copolymer.

[0304] The aqueous suspension formulation for forming aerosol according to the present invention can be nebulized to obtain the aerosol according to the present invention.Advantageously, the negative impact of the nebulization step on the nanoparticles and nucleic acid contained in the aqueous suspension formulation can be minimized or avoided in this way.Furthermore, nebulization can be efficiently carried out within a reasonable time, for example, 60 minutes or less, preferably 30 minutes or less, for a given dose of mRNA.

[0305] Thus, the aerosol that can be obtained by nebulization of the aqueous suspension formulation for forming aerosol according to the present invention comprises aerosol droplets dispersed in the gas phase.The aerosol droplets comprise lipid or lipidoid nanoparticles, including any preferred embodiment thereof, discussed above, and an aqueous vehicle solution for nanoparticles.The aqueous vehicle solution comprises a triblock copolymer containing one poly(propylene oxide) block and two poly(ethylene oxide) blocks, which is provided by the vehicle solution of the aqueous suspension formulation of the present invention, and is discussed in this context above.

[0306] As explained above, it has been found that the presence of the triblock copolymer makes it possible to retain the advantageous nanoparticulate characteristics exhibited by the nanoparticles in the aqueous suspension formulation prior to spraying, as discussed above.

[0307] Thus, the lipid or lipidoid nanoparticles contained in the aerosol droplets of the aerosol according to the invention preferably have a Z-average diameter in the range of 10 to 500 nm, more preferably in the range of 10 to 250 nm, even more preferably in the range of 20 to 200 nm. The particle diameters indicated are the hydrodynamic diameters of the particles, as determined by dynamic light scattering (DLS). Measurements are typically carried out at 25°C.

[0308] The polydispersity index of the lipid or lipidoid nanoparticles contained in the aerosol droplets of the aerosol according to the invention is preferably in the range of 0.05 to 0.4, more preferably in the range of 0.05 to 0.2. The polydispersity index can be determined by dynamic light scattering (DLS). Measurements are generally carried out at 25°C.

[0309] The vehicle solution in the aerosol droplets of the aerosol from the suspension formulation refers to an aqueous solution, i.e., a solution in which the main solvent is water with respect to the total volume of the solvent.Preferably, the vehicle solution contains more than 70% water as a solvent, more preferably more than 90% water, expressed as the volume percentage of water in the total volume of the solvent contained in the vehicle solution (at a temperature of 25°C).Most preferably, water is the only solvent in the vehicle solution.

[0310] As specified above, the aerosol according to the present invention comprises droplets dispersed in a gas phase, usually in air. The droplets can be obtained by nebulization of the aerosol-forming composition according to the present invention. They comprise a liquid phase derived from the vehicle solution of the composition detailed above, as well as lipid or lipidoid nanoparticles. Typically, the lipid or lipidoid nanoparticles are dispersed in the vehicle solution. Furthermore, the aerosol droplets usually comprise a plurality of lipid or lipidoid nanoparticles dispersed in a single droplet.

[0311] As further explained above, the aerosol according to the invention can be administered to a subject, in particular to or via the respiratory tract of the subject, preferably by pulmonary or nasal administration. Typically, administration is performed by inhalation of the aerosol by the subject.

[0312] An aerosol droplet can be characterized by its aerodynamic diameter, which takes into account its density and its shape. The aerodynamic diameter is the diameter of the droplet that has the same settling velocity in air as the droplet under consideration, 1 g / cm. 3(Luftbeschaffenheit - Festlegung von Partikelgrossenverteilungen fur die gesundheitsbezogene Schwebstaubprobenahme, (1995); Vincent JH. Aerosol Sampling - Science, Standards, Instrumentation and Applications. Chichester, England: John Wiley & Sons, Ltd.; 2007). The size distribution of aerodynamic diameters is often parameterized by the median aerodynamic diameter (MMAD), i.e. the aerodynamic diameter related to the median mass. The MMAD is therefore the diameter below which particles smaller or larger, respectively, contribute 50% of the total mass and is therefore a measure of the average size of the particles. MMAD can be measured using a cascade impactor or a next-generation impactor (Preparations for inhalation: Aerodynamic assessment of fine particles; European Pharmacopoeia 90; Volume I: EDQM Council of Europe; 2019). The median aerodynamic diameter (MMAD) of the aerosol droplets influences where in the respiratory tract the aerosol particles are deposited. Particles with an MMAD of 10 μm or more tend to be already deposited (impacted) in the pharynx due to their inertia, while particles between 0.1 μm and 1.0 μm tend to be too light and may be exhaled again due to diffusion processes caused by Brownian motion.

[0313] The aerosol droplets of the aerosol according to the invention preferably have an MMAD, as determined by measurement using a cascade impactor or a next generation impactor, of 2 to 10 μm, more preferably 3 to 8 μm.

[0314] The spray device (nebulizer) for forming aerosol from suspension formulations containing particles in vehicle solution is known in the art and is commercially available.A nebulizer is a device that converts liquid into a mist of fine droplets dispersed in a gas phase, i.e., an aerosol suitable for inhalation.Particularly, examples of suitable nebulizers for generating aerosols that can be used in the context of the present invention are as follows: - Jet nebulizer, e.g. Pari Boy (Pari) - vibrating mesh nebulizers, e.g. Pari eFlow (Pari), Aeroneb (Aerogen), Fox (Vectura) or Innospire GO (Philips); - Passive mesh nebulizer, e.g. MicroAir U22 (Omron) or Smarty (Flaem); - Ultrasonic nebulizer, e.g. My-520A (Fish), or Aerosonic Combineb (Flores) - A soft mist inhaler, such as Trachospray (MedSpray), Pulmospray (Medspray), or Respimat (Boehriner Ingelheim).

[0315] In the context of the present invention, suspension formulations for forming an aerosol are preferably nebulized using a vibrating mesh nebulizer or a soft mist inhaler, more preferably a soft mist inhaler.

[0316] In a further aspect, the present invention provides a method for preparing an aerosol according to the invention as discussed above, comprising the step of atomizing an aerosol-forming suspension formulation according to the invention.

[0317] It has been found that the suspension formulation according to the present invention can be efficiently and continuously nebulized for a long period of time without compromising the quality of the suspension formulation and the nanoparticles contained in the aerosol droplets (e.g., due to particle aggregation).Therefore, it can provide an effective dose of nucleic acid as an active agent contained in nanoparticles, and can be administered in the form of aerosol in a reasonable amount of time, such as 60 minutes or less, preferably 30 minutes or less.

[0318] The nucleic acids, such as RNA, preferably mRNA, present in the lipid or lipidoid nanoparticles used in the context of the present invention are particularly useful in medical situations and in the treatment of diseases and disorders, particularly in nucleic acid-based therapies. Thus, the suspension formulations for forming aerosols according to the present invention, and the aerosols, are generally provided or used as medicines or pharmaceutical compositions.

[0319] In particular, the suspension formulation for forming aerosol and the aerosol according to the present invention are suitable for administration to a subject.In this way, the nucleic acid, such as RNA, preferably mRNA, contained in the nanoparticles of the suspension formulation and the aerosol can also be administered to a subject.The preferred administration route for the composition is the administration of the aerosol produced by the spraying of the suspension formulation according to the present invention to or through the respiratory tract, particularly pulmonary administration or nasal administration.Usually, the aerosol is inhaled by the subject to be administered.

[0320] Upon administration to a subject, the nucleic acid contained in the lipid or lipidoid nanoparticle particles can be delivered to or via the airways to target cells. The term "delivered to target cells" preferably refers to the transfer of nucleic acid to cells.

[0321] Thus, the present invention also provides an aqueous suspension formulation for use as a medicament, the suspension formulation being nebulized and the aerosol delivered by nebulization being administered to a subject.Similarly, the present invention provides an aerosol according to the present invention for use as a medicament.

[0322] The aqueous suspension formulation or aerosol can be administered to the subject in a suitable dose. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical art, the dosage for any one subject depends on a number of factors, including the subject's size, body surface area, age, the specific compound administered, sex, time and route of administration, general health, and other drugs administered at the same time. A typical dose of a therapeutically active substance can be in the range of, for example, 1 ng to several grams. The dosage of a nucleic acid for expression or inhibition of expression should correspond to this range, although doses below or above this exemplary range are envisioned, taking into account, among other things, the factors mentioned above. In general, the regimen for regular administration of the pharmaceutical composition should be in the range of 0.01 μg to 10 mg units per kilogram of body weight per day. Progress can be monitored by regular evaluation. Dosages will vary, but a preferred dosage for administering a nucleic acid as a constituent of the composition of the invention is approximately 10 μg to 10 mg units per kilogram of body weight per day. 10 ~10 19 It is a nucleic acid molecule having a copy number of

[0323] Similarly, the present invention provides a method of treatment that includes administering the aerosol provided by spraying the aqueous suspension formulation according to the present invention to or through the subject's airways, preferably by pulmonary or nasal administration. Thus, the nucleic acid contained in said suspension formulation can cause a preventive or therapeutic effect. In particular, the term "subject" includes animals and humans. Similarly, the present invention provides a method of treatment that includes administering the aerosol according to the present invention to a subject, preferably by pulmonary or nasal administration, to or through the subject's airways. As specified above, the aerosol is usually inhaled by the subject to whom it is administered.

[0324] By administering the aqueous suspension formulation or aerosol of the present invention to a subject, disease or disorder can be treated or prevented. The term "disease" refers to any possible pathological condition that can be treated, prevented, or vaccinated by using the aqueous suspension formulation or aerosol of the present invention. Preferably, the disease to be treated or prevented is a lung disease. The disease can be, for example, inherited, acquired, infectious or non-infectious, age-related, cardiovascular, metabolic, intestinal, neoplastic (especially cancer) or genetic. The disease can be, for example, based on irregularities in physiological processes, molecular processes, biochemical reactions in an organism, which in turn can be, for example, based on the genetic equipment of the organism, behavioral factors, social factors, or environmental factors (such as exposure to chemicals or radiation).

[0325] Therefore, the present invention further provides the aqueous suspension formulation of the present invention for use in the treatment or prevention of disease or disorder by nucleic acid-based therapy, wherein the treatment or prevention comprises the administration of the aerosol provided by spraying the suspension formulation and spraying to or through the airways, preferably by pulmonary or nasal administration.Similarly, the present invention provides the aerosol of the present invention for use in the treatment or prevention of disease or disorder by nucleic acid-based therapy, wherein the treatment or prevention comprises the administration of the aerosol to or through the airways, preferably by pulmonary or nasal administration.As stated above, the aerosol is usually inhaled by the subject to whom it is administered.

[0326] In a preferred embodiment, the present invention further provides the aqueous suspension formulation of the present invention for use in the treatment or prevention of pulmonary disease, wherein the treatment or prevention comprises the administration of the aerosol provided by spraying the suspension formulation and spraying to or through the respiratory tract, preferably by pulmonary or nasal administration.Similarly, the present invention provides the aerosol disclosed above, including its preferred embodiment, for use in the treatment or prevention of pulmonary disease, wherein the treatment or prevention comprises the administration of the aerosol to or through the respiratory tract, preferably by pulmonary or nasal administration.As stated above, the aerosol is usually inhaled by the subject to whom it is administered.

[0327] The term "treatment" or "treating" as used herein generally means obtaining a desired pharmacological and / or physiological effect in the human or animal body. Thus, the treatment of the present invention can relate to the treatment of a (acute) state of a certain disease, but also to a prophylactic treatment, which relates to completely or partially preventing the disease or its symptoms. Preferably, the term "treatment" should be understood as therapeutic, which relates to partially or completely curing the disease and / or adverse effects and / or symptoms caused by the disease. In this respect, "acute" means that the subject shows symptoms of the disease. In other words, the subject to be treated is actually in need of treatment, and in the context of the present invention, the term "acute treatment" relates to the measures adopted to actually treat the disease after the onset of the disease or the sudden onset of the disease. Treatment can also be a preventive or prophylactic treatment, i.e. a measure adopted for disease prevention, for example to prevent infection and / or the onset of the disease. The therapeutic progress can be monitored by regular evaluation.

[0328] In general, the nucleic acid is contained in an effective amount in the suspension formulation and aerosol according to the present invention. The term "effective amount" refers to an amount sufficient to induce a detectable therapeutic response in the subject to which the pharmaceutical composition is to be administered. According to the above, the content of the nucleic acid is not limited as long as it is useful for the above treatment. As specified above, the composition for forming an aerosol or the aerosol containing particles containing nucleic acid preferably contains the particles in an amount to provide the nucleic acid contained in the particles at a concentration of 0.01 to 50 mg / ml, more preferably 0.02 to 30 mg / ml and most preferably 0.05 to 10 mg / ml, relative to the total volume of the composition.

[0329] 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 a most preferred embodiment, the subject is a human.

[0330] As stated above, the suspension formulations and aerosols according to the present invention can be for use in the treatment or prevention of nucleic acid-based therapy.For example, the nucleic acid-based therapy is for the treatment or prevention of a disease or disorder listed in Table A above.

[0331] The suspension formulations and aerosols of the present invention are particularly suitable for use in the treatment or prevention of pulmonary diseases. Exemplary diseases include asthma, surfactant metabolic dysfunction, surfactant protein B (SPB) deficiency, ATP-binding cassette subfamily A member 3 (ABCA3) deficiency, cystic fibrosis, alpha-1 antitrypsin (A1AT) deficiency; lung cancer, surfactant protein C (SPC) deficiency, pulmonary alveolar proteinosis, sarcoidosis, acute and chronic bronchitis, emphysema, McLeod syndrome, chronic obstructive pulmonary disease (COPD), bronchial asthma, bronchiectasis, pneumoconiosis, asbestosis, acute respiratory distress syndrome (ARDS), infantile respiratory distress syndrome (IRDS), pulmonary edema, eosinophilic pneumonia, Löffler pneumonia, Haemophilus influenzae, and pulmonary edema. These include, for example, Roman-Rich syndrome, idiopathic pulmonary fibrosis, interstitial lung disease, primary ciliary dyskinesia, pulmonary arterial hypertension (PAH) and STAT5b deficiency, coagulation disorders, especially hemophilia A and B; complement disorders, especially protein C deficiency, thrombotic thrombocytopenic purpura and congenital hemochromatosis, especially hepcidin deficiency; pulmonary infections, preferably respiratory syncytial virus (RSV) infection, parainfluenza virus (PIV) infection, influenza virus infection, rhinovirus infection, severe acute respiratory syndrome, coronavirus (SARS-CoV) infection, tuberculosis, Pseudomonas aeruginosa infection, Burkholderia cepacia infection, methicillin-resistant Staphylococcus aureus (MRSA) infection and Haemophilus influenzae infection.

[0332] However, it will be understood that the aqueous suspension formulation for the aerosol formulation may migrate from the respiratory tissue to other tissues or organs in the body and may transfect cells in said distant tissues or organs.Similarly, the protein encoded by the mRNA contained in the suspension formulation for the aerosol formulation may migrate from the respiratory tissue to other tissues or organs in the body and may have a therapeutic effect in said distant tissues or organs.

[0333] In other exemplary embodiments, compositions and aerosols of the invention may be for use in nucleic acid-based therapy in the treatment or prevention of lysosomal disorders such as Gaucher disease, Fabry disease, MPS I, MPS II (Hunter syndrome), MPS VI, and glycogen storage diseases such as, for example, glycogen storage diseases type I (von Gierecke disease), type II (Pompe disease), type III (Cori disease, type IV (Andersen disease), type V (McArdle disease), type VI (Haas disease), type VII (Tarui disease), type VII, type IX, type X, type XI (Fanconi-Bickel syndrome), type XI, or type 0. Transcript replacement therapy / enzyme replacement therapy does not beneficially affect the underlying genetic defect, but improves the concentration of an enzyme in which the subject has a deficiency. As an example, in Pompe disease, transcript replacement therapy / enzyme replacement therapy replaces the defective lysosomal enzyme acid alpha-glucosidase (GAA).

[0334] According to further examples, the nucleic acid-based therapy according to the present invention may be for use in treating cancer, cardiovascular disease, viral infection, immune dysfunction, autoimmune disease, neurological disorder, genetic metabolic or hereditary disorder, or any disease in which a protein or protein fragment produced in a cell can have a beneficial effect on a patent. Examples of cancer include head and neck cancer, breast cancer, kidney cancer, bladder cancer, lung cancer, prostate cancer, bone cancer, brain cancer, cervical cancer, anal cancer, colon cancer, colorectal cancer, appendix cancer, eye cancer, stomach cancer, leukemia, lymphoma, liver cancer, skin cancer, ovarian cancer, penile cancer, pancreatic cancer, testicular cancer, thyroid cancer, vaginal cancer, vulvar cancer, endometrial cancer, cardiac cancer, and sarcoma. Examples of cardiovascular disease include atherosclerosis, coronary heart disease, pulmonary heart disease, and cardiomyopathy. Examples of immune dysfunction and autoimmune disease include, but are not limited to, rheumatic disease, multiple sclerosis, and asthma. Examples of viral infections include, but are not limited to, infections with human immunodeficiency virus, herpes simplex virus, human papilloma virus, and hepatitis B and C viruses. Examples of neurological disorders include, but are not limited to, Parkinson's disease, multiple sclerosis, and dementia. Examples of genetic metabolic disorders include, but are not limited to, Gaucher's disease and phenylketonuria.

[0335] Several documents, including patent applications and manufacturer's guides, are cited herein. The disclosures of these documents are not believed to be relevant with respect to the patentability of the present invention, but are incorporated herein by reference in their entirety. More specifically, all of the referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0336] [Table 2]

[0337] 1. Experiment 1 - Quality of nanoparticles after nebulization with different excipients 1.1 Materials and Methods 1.1.1 Nanoparticle preparation Lipidoid nanoparticles were formulated from cationic lipidoid (dL_05(R), Scheme 1), helper lipids DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, Avanti Polar Lipids) and cholesterol (Avanti Polar Lipids) and PEG lipid DMG-PEG2k (1,2-dimyristoyl-sn-glycerol methoxy(polyethylene glycol)-2000, Avanti Polar Lipids) in molar ratios of 8 / 5.29 / 4.41 / 0.88, respectively. Appropriate amounts of stock solutions of lipids in HPLC grade ethanol at concentrations of 50, 20, 20 and 20 mg / mL, respectively, were combined. The formulation process was carried out by rapid solvent exchange. Using a NanoAssemblr benchtop (Precision NanoSystems), the lipid mixture in ethanol was combined with mRNA in citrate buffer (10 mM citric acid, 150 mM NaCl, pH 4.5) at a volume ratio of 1:4. The resulting formulation had an mRNA concentration of 0.2 mg / mL with an N / P ratio of 8. After 30 minutes of incubation at room temperature, the formulation was purified and concentrated by tangential flow filtration (KR2i TFF system, Repligen) using a 50 kDa filter module (mPES, Repligen) with 50 mM NaCl as diluent and diafiltration buffer. Bioburden reduction and final sterile filtration were performed using 0.8 μm and 0.2 μm syringe filters.

[0338] [ka]

[0339] 1.1.2 Mixing of nanoparticles with excipients The excipients used in this experiment are listed in Table 3. Excipient dilutions were prepared at 2% (w / v) excipient in 10% (w / v) sucrose, 50 mM NaCl. Subsequent serial dilutions in sucrose / NaCl buffer gave excipient concentrations of 0.2%, 0.02% and 0.002% (w / v). Nanoparticles were mixed with equal amounts of individual excipients immediately prior to nebulization.

[0340] [Table 3]

[0341] 1.1.3 Spraying Nebulization of nanoparticles was performed with an eFlow nebulizer (Pari). Complete nebulization was performed at room temperature and the time until complete nebulization was measured. Aerosols were collected by condensation in sample tubes at room temperature.

[0342] 1.1.4 Measurement of complex size and PdI The hydrodynamic diameter (Z-average, size) and polydisperity index (PdI) of the nanoparticles were measured by dynamic light scattering (DLS) using a Zetasizer Nano-ZS (Malvern Instruments) equipped with an automatic attenuator and reported as intensity particle size distribution. Samples were measured undiluted at 25°C.

[0343] 1.2 Results This experimental setup aimed to compare different classes of additives with respect to their ability to improve the particle quality of nanoparticles after nebulization. As data from the reference (without excipients, Figure 1 "w / o") demonstrate, nebulization of nanoparticles leads to an undesirable increase in size (hydrodynamic diameter, Z-average) and polydispersity index (PdI). Significant improvements in overall particle size and PdI were achieved with the addition of certain excipients. Figure 1 shows the size and PdI of nanoparticle formulations before and after nebulization of 1 mL at a concentration of 0.5 mg / mL mRNA in 1% excipients, 10% (w / v) sucrose, 50 mM NaCl.

[0344] 2. Experiment 2 - Stability of nanoparticles in the presence of excipients 2.1 Materials and Methods 2.1.1 Nanoparticle preparation Please see 1.1.1.

[0345] 2.1.2 Mixing of nanoparticles with excipients The excipients used in this experiment are listed in Table 4. Excipient dilutions were prepared at 20% (w / v) excipient in 10% (w / v) sucrose, 50 mM NaCl.

[0346] [Table 4]

[0347] 2.1.3 Measurement of encapsulation efficiency To determine the encapsulation efficiency, all samples were diluted to 4 μg / mL in water for injection. For "untreated samples", 50 μL of each sample and water as a blank control were incubated with 2.67 mg / mL heparin in 2% (v / v) Triton-X-100 in a 96-well plate at 70° C. for 15 minutes and then cooled to room temperature. For "untreated samples", 50 μL of each sample and water as a blank control were diluted with 50 μL of water for injection. 100 μL of RiboGreen (Quant-iT™ RiboGreen® RNA Assay Kit, ThermoFisher) reagent diluted 100 times in 1×TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5 in diethylpyrocarbonate (DEPC)-treated water) was added to each well and incubated for 5 minutes at room temperature protected from light. Fluorescence intensity was measured on a Tecan plate reader at excitation / emission wavelengths of 785 / 535 nm, respectively. Encapsulation efficiency was expressed as (100%-(([luminescence amount of "untreated sample"]-[luminescence amount of "untreated blank"]) / ([luminescence amount of "treated sample"]-[luminescence amount of "treated blank"])*100%).

[0348] 2.2 Results The excipients should not negatively affect the nanoparticles themselves, allowing their use as stabilizers in the spraying process. To test this, an in-use stability study was performed in the presence of various excipient concentrations. For this, the suspensions were stored at room temperature for 6 hours. The encapsulation efficiency, which indicates the particle integrity, was measured. The data are shown in Figure 2, which shows the encapsulation efficiency of a nanoparticle formulation at an mRNA concentration of 2.5 mg / mL in x% (w / v) excipients, 10% (w / v) sucrose, 50 mM NaCl. The data were recorded 6 hours after mixing.

[0349] 2.3 Discussion and Conclusions In the presence of tyloxapol, Tween-20 and Tween-80, encapsulation efficiency decreased with increasing excipient concentration, indicating a loss of nanoparticle integrity. In contrast, poloxamer is well tolerated by the formulation. Particles remain intact over 6 hours at all poloxamer concentrations tested.

[0350] 3. Experiment 3 - Nebulization of 2.5 mg / mL nanoparticles in the presence of poloxamer 3.1 Materials and Methods 3.1.1 Nanoparticle preparation Please see section 1.1.10.

[0351] 3.1.2 Addition of excipients Poloxamer was added immediately before spraying (stock solution concentration: 20% (w / v) P188 in 10% (w / v) sucrose in 50 mM NaCl).

[0352] 3.1.3 Spraying Please see section 1.1.3.

[0353] 3.1.4 Measurement of complex size and PdI Please see section 1.1.4.

[0354] 3.1.5 Measurement of Encapsulation Efficiency Please see section 2.1.3.

[0355] 3.1.6 Measurement of mRNA integrity The integrity of the mRNA in the nanoparticles was determined by capillary gel electrophoresis using a fragment analyzer (Agilent Technologies). Release of the mRNA from the nanoparticles was performed at an mRNA concentration of 0.05 mg / mL in 6 μg / μL heparin (Sigma-Aldrich), 0.2% (v / v) Triton-X-100, 50% (v / v) formamide. Samples were incubated for 15 min at 70 °C and 300 rpm (Thermomixer, Eppendorf). The mRNA reference was processed accordingly. For sample analysis, the treated nanoparticles and mRNA were diluted 1:4 in a dilution marker (Standard Sensitivity RNA Dilution Marker (15 nt), Agilent technologies).

[0356] 3.2 Results In experiment 1, it was demonstrated that 1% (v / v) of the excipient was able to stabilize the nanoparticles during nebulization. Experiment 2 revealed that, in contrast to the other tested excipients, the poloxamer did not have a negative effect on the nanoparticle stability. In this experiment, the poloxamer concentration required to stabilize a nanoparticle solution with an mRNA concentration of 2.5 mg / mL was evaluated, and P188 concentrations ranging from 1 to 5% (w / v) were tested. For that purpose, 1 mL samples were prepared at each concentration, nebulized by an eFlow nebulizer (Pari) and compared with an untreated sample of the same concentration. Figure 3 shows the biophysical characteristics of 1 mL of a 2.5 mg / mL nanoparticle suspension before (untreated) and after nebulization with various poloxamer concentrations: (a) size, (b): encapsulation efficiency, (c) relative mRNA integrity.

[0357] 3.3 Discussion and conclusions Results show that poloxamer stabilizes the formulation at a nucleic acid concentration of 2.5 mg / mL over a broad concentration range of 1-5% (w / v) excipient. The three most important quality attributes, particle size, encapsulation efficiency, and mRNA integrity, remain unaffected by the nebulization process.

[0358] 4. Experiment 4-25mg Nebulization 4.1 Materials and Methods 4.1.1 Nanoparticle preparation Please see section 1.1.1.

[0359] 4.1.2 Addition of poloxamer Please see section 3.1.2.

[0360] 4.1.3 Spraying Please see section 1.1.3.

[0361] 4.1.4 Measurement of complex size and PdI Please see section 1.1.4.

[0362] 4.1.5 Measurement of Encapsulation Efficiency Please see section 2.1.3.

[0363] 4.1.6 Measurement of mRNA integrity Please see section 3.1.6.

[0364] 4.2 Results During this experiment, nebulization of larger amounts of nanoparticle solutions was tested, since this is an additional challenge for drug nebulization. A volume of 10 mL of formulated mRNA at a concentration of 2.5 mg / mL (maximum loading volume of the device) was nebulized at a poloxamer concentration of 5% (w / v). The aerosol was collected and fractionated every 5 minutes. The biophysical properties of the fractions were analyzed. The volume was nebulized at an average exit rate of about 0.3 mL / min. Finally, about 500 μL of the formulation remained in the reservoir of the nebulizer. Figure 4 shows the results of biophysical characterization of fractionated aerosol of 10 mL of a formulation at a concentration of 2.5 mg / mL mRNA in the presence of 5% poloxamer (buffer: 5% (w / v) P188, 10% (w / v) sucrose, 50 mM NaCl): (a) size, (b) PdI, (c) encapsulation efficiency, and (d) relative mRNA integrity over the nebulization time.

[0365] 4.3 Discussion and conclusions Particle size and PdI remained stable in the collected fractions (Figures 4a and b). Encapsulation efficiency remained constant at >95% (Figure 4c) and mRNA integrity remained unaffected (Figure 4d). Thus, the addition of 5% (w / v) poloxamer allowed nebulization of 10 mL of concentrated formulation (2.5 mg / mL) within 30 min, which was not possible without poloxamer.

[0366] 5. Experiment 5 - Effect of poloxamer on the in vitro function of aerosol formulations 5.1 Materials and Methods 5.1.1 Nanoparticle preparation An mRNA encoding eGFP was used, see section 1.1.1.

[0367] 5.1.2 Addition of poloxamer Please see section 3.1.2.

[0368] 5.1.3 Spraying Please see section 1.1.3.

[0369] 5.1.4 Measurement of complex size and PdI Please see section 1.1.4.

[0370] 5.1.5 Measurement of Encapsulation Efficiency Please see section 2.1.3.

[0371] 5.1.6 Measurement of mRNA integrity Please see section 3.1.6.

[0372] 5.1.7 Cell culture and transfection The cells were cultured in MEM+ (Thermo Fisher Scientific) GlutaMax™ (Gibco™, Thermo Fisher Scientific) supplemented with heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific) and penicillin / streptomycin (Pen / Strep, Gibco™, Thermo Fisher Scientific) in collagen type I (Corning)-coated flasks (Corning) at 37°C and 5% CO. 2 To generate air-liquid interface cultures (ALI), 250 μL (2.4 × 10 ) of cells were plated onto the apical side of inserts coated with type I collagen (Corning) in a 24-well plate. 5 ALI cultures were seeded at 25 μL (cells / mL). 500 μL of medium was added to the basal side and cells were incubated for 72 h to allow attachment. 24 h prior to transfection, medium in the basal wells was replaced and medium on the apical side was removed. After washing with water for injection, transfection was performed and 25 μL of LNP doses were added to the apical side of the ALI cultures, incubated at 37° C., 5% CO 2 After completion of transfection, the cell layer was washed with 200 μL of PBS (- / -, Gibco™, Thermo Fisher Scientific) and further maintained as an ALI culture.

[0373] 5.1.8 Quantification of eGFP in ALI lysates 24 hours after transfection, cells were lysed to quantify intracellular EGFP. ALI inserts were washed with 200 μL apical and 500 μL basolateral PBS (- / -, Gibco™, Thermo Fisher Scientific). PBS was aspirated and replaced with 100 μL Triton X-100 lysis buffer (0.25 M TRIS-HCl (Carl Roth), 1% Triton-X-100 (Sigma-Aldrich), pH 7.8) supplemented with protease inhibitors (cOmplete™, EDTA-free protease inhibitor cocktail, Roche). Inserts were incubated at 600 rpm for 20 min at room temperature on a shaking platform. Lysates were collected by pipetting up and down several times and stored at -80°C until analysis. eGFP levels were quantified in cell lysates using the GFP SimpleStep ELISA® kit (Abcam ab171581).

[0374] 5.2 Results As an excipient that stabilizes the nanoparticles, the excipient should not negatively affect its efficiency in transporting mRNA into the cells leading to expression of the encoded protein. Therefore, the transfection efficiency of the nanoparticles was tested in the presence of poloxamer and Tween-80 before and after nebulization. mRNA encoding eGFP protein was used in the formulation to allow quantification of the produced protein. The results are shown in Figure 5, which shows the eGFP levels in cell lysates 24 hours after transfection of 16HBEo-ALI with nanoparticles encapsulating eGFP mRNA before (untreated) and after nebulization in the presence of 5% (w / v) excipient (poloxamer or Tween-80). Dotted line: baseline eGFP levels after transfection with nanoparticles without excipients.

[0375] 5.3 Discussion and conclusions Addition of Tween-80 as an excipient for nebulization leads to a strong decrease in transfection efficiency. This effect is unrelated to the nebulization process and can therefore be attributed to the presence of the excipient itself. In contrast, addition of poloxamer does not affect transfection efficiency. Protein levels before and after nebulization are comparable to those after transfection with the same nanoparticles in the absence of excipients.

[0376] 6. Experiment 6 - Effect of poloxamer on the in vivo efficiency of mRNA nanoparticles 6.1 Materials and Methods 6.1.1 Nanoparticle preparation Please see section 1.1.1.

[0377] 6.1.2 Addition of poloxamer Please see section 3.1.2.

[0378] 6.1.3 Spraying Please see section 1.1.3.

[0379] 6.1.4 Measurement of complex size and PdI Please see section 3.1.4.

[0380] 6.1.5 Measurement of Encapsulation Efficiency Please see section 2.1.3.

[0381] 6.1.6 Measurement of mRNA integrity Please see section 3.1.6.

[0382] 6.1.7 Animal husbandry Mice were housed in individual ventilated cages under a circadian light cycle (lights on from 7 am to 7 pm) and in specific pathogen-free conditions (the facility tested negative for any of the FELASA-listed pathogens during the 2017 Annual Health and Sanitation Survey). Food and drinking water were provided ad libitum. Animals were given at least 7 days for acclimatization after arrival before entering the study.

[0383] 6.1.8 Intratracheal injection The animals were anesthetized by inhalation of pure oxygen containing 4% isoflurane (Isothesia, Henry Shine). The unconscious animals were intubated using a 20G catheter shortened to 37 mm. A drop of the formulation was applied to the proximal end of the tube with a final volume of 50 μL, which was then inhaled during the animal's physiological inspiratory movement. Finally, 150 μL of air was applied, completely emptying the catheter.

[0384] 6.1.9 Euthanasia and Necropsy The animals were fixed under complete anesthesia by intraperitoneal injection of fentanyl / midazolam / medetomidine (0.05 / 5.0 / 0.5 mg / kg body weight). Mice were then sacrificed by cervical dislocation. The abdominal cavity was opened in the midline. For lung explantation, the microcirculation was flushed by injecting 5 mL of PBS through the right ventricle. The heart was then excised from the cardiopulmonary block. The lungs were explanted and flash frozen on dry ice.

[0385] 6.1.10 Quantification of eGFP in lung homogenates To quantify eGFP, frozen lungs were weighed and whole organs were homogenized. Lysis Matrix D (MP Biomedicals) homogenizer tubes were used filled with 500 μL of lysis buffer (0.25 M TRIS (Carl Roth), 0.1% Triton X-100 (Carl Roth), pH 7.8). Three homogenizations were performed for 20 seconds with a tissue homogenizer (MP FastPrep-24 tissue and cell homogenizer). Lysates were then incubated for 10 minutes on ice and centrifuged for 10 minutes at 20,000×g at 4° C. (Mikro 22R centrifuge, Hettich Zentrifugen). eGFP was quantified using the GFP SimpleStep ELISA® kit (Abcam ab171581). Levels of eGFP correlated with lung weight and were reported in ng protein / g tissue.

[0386] 6.2 Results The excipient should not adversely affect the transfection efficiency of such nanoparticles in vivo in order to act as an excipient to stabilize the nanoparticles used as drugs. During this study, the transfection efficiency of nanoparticles in the presence of excipients was measured in mice after intratracheal administration. Efficiency was determined by quantifying mRNA-encoded eGFP 24 hours after treatment. Figure 6 shows the eGFP levels upon intratracheal instillation of nanoparticles in 10% (w / v) sucrose, 50 mM NaCl, specifically without excipient (w / o) and with excipient, following treatment with three different doses in the presence and absence of poloxamer.

[0387] 6.3 Discussion and Conclusions The eGFP levels measured in the lungs of mice were comparable in the presence and absence of poloxamer at each dose level. Analysis of the resulting data leads to the conclusion that poloxamer does not negatively affect the transfection efficiency of the nanoparticles in vivo.

[0388] 7. Experiment 7 – Addition of poloxamer during downstream nanoparticle processing improves nanoparticle quality 7.1 Materials and Methods 7.1.1 Nanoparticle preparation Please see section 1.1.1.

[0389] 7.1.2 Measurement of complex size and PdI Please see section 1.1.4.

[0390] 7.2 Results After complexation by solvent exchange, it is necessary to remove the buffer components required for this step (EtOH and citric acid). The standard procedure for this step and for concentration adjustment of the formulation is tangential flow filtration. During this process, the nanoparticles undergo a stress state and lose particle quality (e.g., aggregation). During this experiment, poloxamer was used to stabilize the nanoparticles during this process step. For that purpose, the nanoparticles were formulated under standard conditions. In one setup, poloxamer was added at a concentration of 0.5% (w / v) before the TFF process. The quality of the particles was determined by measuring the particle size after mixing and after processing. Figure 7 shows the size and PdI of the nanoparticle formulation before and after the spraying process, in the presence or absence of poloxamer.

[0391] 7.3 Discussion and Conclusions As shown in Figure 7, in the absence of poloxamer, particle size and polydispersity increased during TFF processing, indicating particle aggregation. In the presence of poloxamer, particle size and polydispersity remained stable during processing. Thus, the addition of poloxamer clearly improves particle quality during nanoparticle processing.

[0392] 8. Experiment 8 - Nanoparticle quality of ICE-based nanoparticles after nebulization in the absence and presence of poloxamer 8.1 Materials and Methods 8.1.1 Preparation of nanoparticles Lipid nanoparticles were formulated from cationic lipid (ICE (imidazole cholesterol ester)), helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, Avanti Polar Lipids) and PEG lipid DMG-PEG2k (1,2-dimyristoyl-sn-glycerol methoxy(polyethylene glycol)-2000, Avanti Polar lipids) in a molar ratio of 60 / 35 / 5, respectively. Appropriate aliquots of lipid stock solutions in HPLC grade ethanol at 10 mg / mL were combined. The formulation process was carried out by rapid solvent exchange. The lipid mixture in ethanol was combined with mRNA in citrate buffer (10 mM citric acid, 150 mM NaCl, pH 4.5) in a volume ratio of 1:4 using a NanoAssemblr benchtop (Precision NanoSystems). The resulting formulation had an mRNA concentration of 0.2 mg / mL at an N / P ratio of 4. After 30 minutes of incubation at room temperature, the formulation was purified and concentrated by tangential flow filtration (KR2i TFF system, Repligen) using a 100 kDa filter module (mPES, Repligen) with 25 mM NaCl as diluent and diafiltration buffer. Bioburden reduction and final sterile filtration were performed using 0.8 μm and 0.2 μm syringe filters.

[0393] 8.1.2 Addition of excipients Poloxamer was added immediately before spraying (stock solution concentration: 20% (w / v) poloxamer 188 in 25 mM NaCl) to give an mRNA concentration of 0.5 mg / mL in 5% (w / v) poloxamer 188, 25 mM NaCl.

[0394] 8.1.3 Spraying Please see section 1.1.3.

[0395] 8.1.4 Measurement of complex size and PdI Please see section 1.1.4.

[0396] 8.2 Results Experiments 1 and 3 show that poloxamer stabilizes nanoparticles based on the cationic lipidoid dL_05(R) during nebulization. This experimental setup aimed to test whether the addition of poloxamer could also maintain the quality of other commonly used nanoparticles for different cationic lipids (ICE) during nebulization. Data from the reference (without excipients, Figure 8 "w / o") demonstrate that nebulization of nanoparticles leads to an increase in size (hydrodynamic diameter, Z-average) and polydispersity index (PdI). Addition of 5% (w / v) poloxamer prevents this effect and maintains particle quality. Figure 8 shows the size and PdI of 1 mL of nanoparticle suspension at an mRNA concentration of 0.5 mg / mL before and after nebulization, with and without poloxamer.

[0397] 8.3 Discussion and Conclusions The presence of poloxamer also stabilizes lipid nanoparticles based on the cationic lipid ICE during nebulization, preventing the increase in size (hydrodynamic diameter, Z-average) and polydispersity index (PdI).

[0398] 9. Experiment 9 - Nanoparticle quality of DLin-MC3-DMA based nanoparticles after nebulization in the absence and presence of poloxamer 9.1 Materials and Methods 9.1.1 Nanoparticle preparation Lipid nanoparticles were formulated from cationic lipid (DLin-MC3-DMA), helper lipids DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine, Avanti Polar Lipids) and cholesterol (Avanti Polar Lipids) and PEG lipid DMPE-PEG2k (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000, Avanti Polar Lipids) in a molar ratio of 50 / 10 / 38.5 / 1, respectively. Appropriate aliquots of lipid stock solutions in 10 mg / mL HPLC grade ethanol were combined. The formulation process was carried out by rapid solvent exchange. The lipid mixture in ethanol was combined with mRNA in citrate buffer (50 mM citric acid, 160 mM NaCl, pH 3) in a volume ratio of 1:3 using a NanoAssemblr benchtop (Precision NanoSystems). The resulting formulation had an mRNA concentration of 0.2 mg / mL with an N / P ratio of 3. After 30 min incubation at room temperature, the formulation was purified and concentrated by tangential flow filtration (KR2i TFF system, Repligen) using a 100 kDa filter module (mPES, Repligen) with PBS as the diluent and diafiltration buffer. Bioburden reduction and final sterile filtration were performed using 0.8 μm and 0.2 μm syringe filters.

[0399] 9.1.2 Addition of excipients Poloxamer was added immediately before nebulization (stock concentration: 20% (w / v) poloxamer 188 in PBS) to a concentration of 0.5 mg / mL in 5% (w / v) poloxamer 188 in PBS.

[0400] 9.1.3 Spraying Please see section 1.1.3.

[0401] 9.1.4 Measurement of complex size and PdI See section 1.1.4

[0402] 9.2 Results Experiments 1 and 3 show that poloxamer stabilizes nanoparticles based on the cationic lipidoid dL_05(R) during nebulization. This experimental setup aimed to test whether the addition of poloxamer could also maintain the particle quality of other commonly used nanoparticles during nebulization. Well-described nanoparticles based on the cationic lipid DLin-MC3-DMA were chosen as a model. In the absence of excipients (no poloxamer, Figure 9 "w / o"), nebulization of nanoparticles leads to an increase in size (hydrodynamic diameter, Z-average value) and polydispersity index (PdI). Addition of 5% (w / v) poloxamer prevents this effect and maintains particle quality. Figure 9 shows the size and PdI of 1 mL of a 0.5 mg / mL nanoparticle suspension before and after nebulization with and without poloxamer.

[0403] 9.3 Discussion and Conclusions The presence of poloxamer stabilizes lipid nanoparticles based on the cationic lipid DLin-MC3-DMA during nebulization: the addition of poloxamer prevents the increase in size (hydrodynamic diameter, Z-average value) and polydispersity index (PdI).

Claims

1. 1. An aqueous suspension formulation for forming an aerosol comprising lipid or lipidoid nanoparticles suspended in an aqueous vehicle, The lipid or lipidoid nanoparticles comprise the following components (a) and (b): (a) a nucleic acid, and (b) an ionizable lipid or an ionizable lipidoid wherein the aqueous vehicle solution comprises a triblock copolymer containing one poly(propylene oxide) block and two poly(ethylene oxide) blocks.

2. The suspension formulation described in claim 1, wherein the (a) nucleic acid includes mRNA.

3. The suspension formulation according to claim 1 or 2, wherein the concentration of the nucleic acid in the suspension formulation is in the range of 0.01 to 10 mg / mL with respect to the total volume of the suspension formulation.

4. 4. The suspension formulation of any of claims 1 to 3, wherein the nanoparticles have a Z-average diameter in the range of 10 to 500 nm as determined by dynamic light scattering.

5. The nanoparticles are comprised of the following components (c1) to (c6): (c1) a non-ionizable lipid having a sterol structure; (c2) phosphoglyceride lipids, (c3) PEG-conjugated lipids; (c4) polysarcosine-conjugated lipids; (c5) PAS lipid, and (c6) Cationic polymer The suspension formulation of any one of claims 1 to 4, further comprising one or more of:

6. The nanoparticles are 30-65 mol % of an ionizable lipid or ionizable lipidoid (b), and the following components: 10 to 50 mol% of a lipid having a sterol structure (c1); 4 to 50 mol % of the phosphoglyceride lipid (c2); 0.5 to 10 mol % of one of the PEG-conjugated lipid (c3), the polysarcosine-conjugated lipid (c4) and the PAS-conjugated lipid (c5), or any combination thereof; 0.5 to 10 mol % of said cationic polymer (c6); The suspension formulation according to any one of claims 1 to 5, comprising one or more of the following in an amount such that the sum of (b) and (c1) to (c6) is 100 mol %.

7. The nanoparticles are an ionizable lipidoid (b) of the following formula (b-1): 【Chemistry 1】 (In the formula, 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 is ≧2; R １Ａ ~R ６Ａ are each independently hydrogen; ２ -CH(OH)-R ７Ａ , -CH(R ７Ａ )-CH ２ -OH, -CH ２ -CH ２ -(C=O)-OR 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, R ７Ａ is selected from C3-C18 alkyl, and C3-C18 alkenyl having one C-C double bond; However, R １Ａ ~R ６Ａ At least two of the residues are -CH ２ -CH(OH)-R ７Ａ , -CH(R ７Ａ )-CH ２ -OH, -CH ２ -CH ２ -(C=O)-OR ７Ａ , -CH ２ -CH ２ -(C=O)-NH-R ７Ａ and -CH ２ -R ７Ａ is selected from R ７Ａ is selected from C3-C18 alkyl, or C3-C18 alkenyl having one C-C double bond. or a protonated form thereof, in which one or more of the nitrogen atoms contained in the compound of formula (b-1) are protonated to provide a compound having a positive charge. The suspension formulation according to any one of claims 1 to 6, comprising:

8. The triblock copolymer comprises one poly(propylene oxide) block B of formula (p-1): 【Chemistry 2】 where s is an integer from 15 to 67, and Two poly(ethylene oxide) blocks A of formula (p-2) 【Chemistry 3】 wherein r is, independently for each block, an integer from 2 to 130. The suspension formulation according to any one of claims 1 to 7, which is an A-B-A triblock copolymer comprising:

9. The suspension formulation according to claim 8, wherein s is an integer from 20 to 40.

10. The suspension formulation according to claim 8, wherein r is an integer of 50 to 100.

11. The suspension formulation according to claim 8, wherein r is an integer of 60 to 90.

12. The suspension formulation according to any one of claims 1 to 11, comprising the triblock copolymer in a concentration of 0.05 to 5% (w / v, at a temperature of 25°C) relative to the total volume of the suspension formulation.

13. A nebulizer comprising a compartment containing the aqueous suspension formulation for forming an aerosol according to any one of claims 1 to 12.

14. 1. An aerosol comprising aerosol droplets dispersed in a gas phase, said aerosol droplets comprising lipid or lipidoid nanoparticles and an aqueous vehicle solution for said nanoparticles, said lipid or lipidoid nanoparticles comprising the following components (a) and (b): (a) a nucleic acid, and (b) an ionizable lipid or an ionizable lipidoid Including, The aerosol, wherein the aqueous vehicle solution comprises a triblock copolymer containing one poly(propylene oxide) block and two poly(ethylene oxide) blocks.

15. The aerosol described in claim 14, wherein the (a) nucleic acid includes mRNA.

16. 16. The aerosol of claim 14 or 15, wherein the aerosol droplets have a mass median aerodynamic diameter (MMAD) in the range of 2 to 10 μm.

17. 17. The aerosol of any one of claims 14 to 16, wherein the nanoparticles have a Z-average diameter in the range of 10 to 500 nm as determined by dynamic light scattering.

18. The aerosol of claim 17, wherein the nanoparticles have a Z-average diameter in the range of 10 to 250 nm as determined by dynamic light scattering.

19. The aerosol of claim 17, wherein the nanoparticles have a Z-average diameter in the range of 20 to 200 nm as determined by dynamic light scattering.

20. An aerosol according to any one of claims 14 to 19, obtained by spraying an aqueous suspension formulation according to any one of claims 1 to 12.

21. 13. An aqueous suspension formulation according to any one of claims 1 to 12 for use as a medicament, wherein the aqueous suspension formulation is to be nebulized and the aerosol delivered by nebulization is to be administered to a subject.

22. 13. The aqueous suspension formulation of any of claims 1 to 12 for use in the treatment or prevention of a disease or disorder by nucleic acid based therapy, wherein the treatment or prevention comprises nebulization of the aqueous suspension formulation and administration of the aerosol provided by nebulization to or through the respiratory tract of a subject.

23. The aqueous suspension formulation of claim 22, wherein the administration is by pulmonary or nasal administration.

24. 24. The aqueous suspension formulation of claim 22 or 23, wherein the disease or disorder to be treated or prevented is a pulmonary disease.

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