阅读说明：本技术 信使rna的干粉制剂 (Dry powder formulations of messenger RNA ) 是由 S·卡夫 F·德罗莎 M·哈特莱因 Z·帕特尔 A·萨罗德 于 2019-07-23 设计创作，主要内容包括：本发明提供了用于治疗用途的稳定的干粉信使RNA制剂,以及它们的制备和使用方法。(The present invention provides stable dry powder messenger RNA formulations for therapeutic use, as well as methods for their preparation and use.)

1. A dry powder formulation for delivery of cystic fibrosis conductance regulator (CFTR) messenger RNA (mRNA), the dry powder formulation comprising a plurality of spray-dried particles, the particles comprising

mRNA encoding CFTR protein;

one or more lipids, and

one or more polymers.

2. The dry powder formulation of claim 1, wherein the one or more lipids are present in one or more Lipid Nanoparticles (LNPs) encapsulating the mRNA encoding the CFTR protein.

3. The dry powder formulation of claim 1, wherein the one or more lipids and the one or more polymers are present in one or more Lipid Nanoparticles (LNPs) encapsulating the mRNA encoding the CFTR protein.

4. The dry powder formulation of any one of the preceding claims, wherein the CFTR mRNA has an integrity of 90% or greater.

5. The dry powder formulation of any one of the preceding claims, wherein the mRNA maintains 90% or greater integrity after 6 months or longer of storage at or below room temperature.

6. The dry powder formulation of any one of the preceding claims, wherein at least 20% of the plurality of spray-dried particles are fine particles.

7. The dry powder formulation as claimed in claim 6, wherein the fine particles have a volume median diameter of less than 5 μm.

8. A dry powder formulation as claimed in any one of the preceding claims, wherein the dry powder formulation is inhalable.

9. A dry powder formulation as claimed in any one of claims 1 to 7, wherein the formulation is aerosolizable on reconstitution.

10. The dry powder formulation of any one of the preceding claims, wherein the one or more polymers comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the combined weight of the lipid and polymer.

11. A dry powder formulation as claimed in any one of the preceding claims, wherein the one or more polymers comprise about 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, 10-40%, 10-30%, 10-20%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80% or 15-90% by weight of the combination of the lipid and polymer.

12. The dry powder formulation as claimed in any one of the preceding claims, wherein the one or more polymers constitute no more than 90%, 80%, 70%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20% by weight of the combined weight of the lipid and polymer.

13. The dry powder formulation of any one of the preceding claims, wherein the one or more polymers are selected from the group consisting of: chitosan, poly (lactic acid) (PLA), poly (lactic-co-glycolic acid) (PLGA), poly (q-caprolactone) (PCL), polyamidoamines, polyesters, polycarbonates, poly (hydroxyalkyl L-asparagine), poly (hydroxyalkyl L-glutamine), poly (2-alkyloxazoline) acrylates, modified acrylates and polymethacrylate-based polymers, poly-N- (2-hydroxy-propyl) methacrylamide, poly-2- (methacryloyloxy) ethylphosphocholine, poly (2- (methacryloyloxy) ethylphosphocholine), and poly (dimethylaminoethyl methacrylate) (pDMAEMA).

14. A dry powder formulation as claimed in any one of the preceding claims, wherein the one or more polymers comprise a polymethacrylate-based polymer.

15. The dry powder formulation of any one of the preceding claims, wherein the one or more polymers comprises Eudragit EPO.

16. The dry powder formulation of any one of claims 2-15, wherein the one or more mRNA-encapsulating LNPs have a lipid to mRNA (N/P) ratio in the range of 1-20, 1-15, 1-10, 2-8, 2-6, or 2-4.

17. The dry powder formulation of claim 16, wherein the one or more mRNA-encapsulating LNPs have a lipid to mRNA (N/P) ratio of 2 or 4.

18. The dry powder formulation of any one of claims 2-17, wherein the LNP has an encapsulation efficiency of 80% or more.

19. A dry powder formulation as claimed in any one of the preceding claims, wherein the one or more lipids comprise a cationic lipid.

20. The dry powder formulation of claim 19, wherein the cationic lipid is selected from the group consisting of: c12-200, DOTAP (1, 2-dioleoyl-3-trimethylammonium propane), DODAP (1, 2-dioleoyl-3-dimethylammonium propane), DOTMA (1, 2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA, DLin-KC2-DMA, HGT4003, cKK-E12, ICE, and combinations thereof.

21. The dry powder formulation of claim 20, wherein the cationic lipid is cKK-E12.

22. The dry powder formulation of claim 20, wherein the cationic lipid is ICE.

23. The dry powder formulation of any one of claims 19-22, wherein the cationic lipid comprises about 25% -50% of the total lipid in the LNP on a molar basis.

24. The dry powder formulation of any one of the preceding claims, wherein the one or more lipids comprise a PEG-modified lipid.

25. The dry powder formulation of claim 24, wherein the PEG-modified lipid comprises about 1% -15% of the total lipid in the LNP on a molar basis.

26. The dry powder formulation of claim 24, wherein the PEG-modified lipid comprises at least 1%, 2%, 3%, 4%, 5%, 6%, 8%, 10%, or 12% of the total lipid in the LNP on a molar basis.

27. The dry powder formulation of any one of claims 2-26, wherein the LNP is a dual lipid component LNP.

28. A dry powder formulation as claimed in any one of the preceding claims, wherein the one or more lipids do not include neutral lipids or cholesterol-based lipids.

29. A dry powder formulation as claimed in any one of the preceding claims, wherein the one or more lipids further comprise a neutral lipid or a cholesterol-based lipid.

30. A dry powder formulation as claimed in any one of the preceding claims, wherein the one or more lipids further comprise neutral lipids.

31. The dry powder formulation of claim 29 or 30, wherein the LNP is a tri-lipid component LNP.

32. A dry powder formulation as claimed in any preceding claim, further comprising at least one sugar selected from the group consisting of: monosaccharides, disaccharides, polysaccharides, glucose, fructose, galactose, mannose, sorbose, lactose, sucrose, cellobiose, trehalose, raffinose, starch, dextran, maltodextrin, cyclodextrin, inulin, xylitol, sorbitol, lactitol, and mannitol.

33. The dry powder formulation of claim 32, wherein the sugar is mannitol.

34. The dry powder formulation of any one of the preceding claims, further comprising a pharmaceutically acceptable excipient selected from the group consisting of: esters, carbamates, phosphate esters, phosphazenes, amino acids, collagen, chitosan, polysaccharides, albumin, surfactants, buffers, salts, and combinations thereof.

35. The dry powder formulation of claim 34, wherein the surfactant is selected from the group consisting of: CHAPS (3- [ (3-cholamidopropyl) dimethylammonio ] -1-propanesulfonate), phospholipids, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, octaethyleneglycol monododecyl ether, pentaethyleneglycol monododecyl ether, Triton X-100, cocamide monoethanolamine, cocamide diethanolamine, glycerol monostearate, glycerol monolaurate, sorbitan monostearate, Tween 20, Tween 40, Tween 60, Tween 80, alkylpolyglucoside, and poloxamer.

36. The dry powder formulation of claim 35, wherein the surfactant is a poloxamer.

37. The dry powder formulation of any one of the preceding claims, wherein the CFTR mRNA comprises about 1% -20%, 1% -15%, 1% -10%, 1% -8%, 1% -6%, 1% -5%, 5% -15%, or 5% -10% of the total weight of the spray-dried particles.

38. The dry powder formulation of any one of the preceding claims, wherein the CFTR mRNA comprises about 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 12.5%, or 15% of the total weight of the spray-dried particles.

39. A method of delivering cystic fibrosis conductance regulator (CFTR) messenger rna (mrna) for expression in vivo, the method comprising the step of administering the dry powder formulation of any one of the preceding claims to a subject in need thereof.

40. The method of claim 39, wherein the dry powder formulation is administered by pulmonary delivery.

41. The method of claim 39 or 40, wherein the dry powder formulation is administered by inhalation.

42. A method of delivering cystic fibrosis conductance regulator (CFTR) messenger rna (mrna) for expression in vivo, the method comprising the steps of:

reconstituting a dry powder formulation of any one of claims 1-38 into a liquid solution; and

administering the reconstituted liquid solution to a subject in need thereof.

43. The method of claim 42, wherein the reconstituted liquid solution is administered by nebulization.

44. The method of any one of claims 39-43, wherein the subject has cystic fibrosis.

45. A dry powder formulation for delivery of messenger RNA (mRNA), the dry powder formulation comprising a plurality of spray dried particles, the particles comprising

mRNA encoding a protein or peptide;

one or more lipids, and

one or more polymers.

46. A dry powder formulation for delivery of messenger RNA (mRNA), the dry powder formulation comprising a plurality of spray dried particles, the particles comprising

a. One or more Lipid Nanoparticles (LNPs) encapsulating mRNA encoding a peptide or polypeptide, and

b. one or more polymers.

47. A dry powder formulation for delivery of messenger RNA (mRNA), the dry powder formulation comprising a plurality of spray dried particles, the particles comprising one or more nanoparticles encapsulating mRNA encoding a peptide or polypeptide, the nanoparticles comprising

a. One or more lipids, and

b. one or more polymers.

48. The dry powder formulation of any one of claims 45-47, wherein the mRNA has an integrity of 90% or greater.

49. The dry powder formulation of any one of claims 45-48, wherein the mRNA maintains 90% or greater integrity after 6 months or longer of storage at or below room temperature.

50. The dry powder formulation of any one of claims 45-48, wherein the mRNA maintains 90% or greater integrity after storage at 4 ℃ or below for 6 months or longer.

51. The dry powder formulation of any one of claims 45-50, wherein at least 20% of the plurality of spray-dried particles are fine particles.

52. The dry powder formulation of claim 51, wherein the fine particles have a volume median diameter of less than 5 μm.

53. The dry powder formulation of any one of claims 45-52, wherein the dry powder formulation is inhalable.

54. The dry powder formulation of any one of claims 45-52, wherein the formulation is aerosolizable upon reconstitution.

55. The dry powder formulation of any one of claims 46-54, wherein the one or more mRNA-encapsulating LNPs have a lipid to mRNA (N/P) ratio in the range of 1-20, 1-15, 1-10, 2-8, 2-6, or 2-4.

56. The dry powder formulation of claim 55, wherein the one or more mRNA-encapsulating LNPs have a lipid to mRNA (N/P) ratio of 2 or 4.

57. The dry powder formulation of any one of claims 46-56, wherein the mRNA-loaded lipid nanoparticle has an encapsulation efficiency of 80% or greater.

58. The dry powder formulation of any one of claims 46-57, wherein the one or more mRNA-loaded lipid nanoparticles comprise one or more cationic lipids.

59. The dry powder formulation of claim 58, wherein the one or more cationic lipids are selected from the group consisting of: c12-200, DOTAP (1, 2-dioleoyl-3-trimethylammonium propane), DODAP (1, 2-dioleoyl-3-dimethylammonium propane), DOTMA (1, 2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA, DLin-KC2-DMA, HGT4003, cKK-E12, ICE, and combinations thereof.

60. The dry powder formulation of any one of claims 46-59, wherein the one or more mRNA-loaded lipid nanoparticles comprise one or more PEG-modified lipids.

61. The dry powder formulation of any one of claims 46-60, wherein the LNP is a dual lipid component LNP.

62. The dry powder formulation of any one of claims 46-60, wherein the one or more mRNA-loaded lipid nanoparticles further comprise one or more neutral lipids or one or more cholesterol-based lipids.

63. The dry powder formulation of claim 62, wherein the LNP is a tri-lipid component LNP.

64. The dry powder formulation of any one of claims 45-63, wherein the one or more polymers comprise less than 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, or 5% of the total weight.

65. The dry powder formulation of any one of claims 45-64, wherein the one or more polymers comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the combined weight of the lipid and polymer.

66. The dry powder formulation of any one of claims 45-65, wherein the one or more polymers comprise about 10% -90%, 10% -80%, 10% -70%, 10% -60%, 10% -50%, 10% -40%, 10% -30%, 10% -20%, 15% -25%, 15% -30%, 15% -35%, 15% -40%, 15% -45%, 15% -50%, 15% -55%, 15% -60%, 15% -65%, 15% -70%, 15% -75%, 15% -80%, or 15% -90% by combined weight of the lipid and polymer.

67. The dry powder formulation of any one of claims 45-66, wherein the one or more polymers comprise no more than 90%, 80%, 70%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% of the combined weight of the lipid and polymer.

68. The dry powder formulation of any one of claims 45-67, wherein the one or more polymers are selected from the group consisting of: chitosan, poly (lactic acid) (PLA), poly (lactic-co-glycolic acid) (PLGA), poly (q-caprolactone) (PCL), polyamidoamines, polyesters, polycarbonates, poly (hydroxyalkyl L-asparagine), poly (hydroxyalkyl L-glutamine), poly (2-alkyloxazoline) acrylates, modified acrylates and polymethacrylate-based polymers, poly-N- (2-hydroxy-propyl) methacrylamide, poly-2- (methacryloyloxy) ethylphosphocholine, poly (2- (methacryloyloxy) ethylphosphocholine), and poly (dimethylaminoethyl methacrylate) (pDMAEMA).

69. The dry powder formulation of any one of claims 45-68, wherein the one or more polymers comprises a polymethacrylate-based polymer.

70. The dry powder formulation of any one of claims 45-69, further comprising at least one sugar selected from the group consisting of: monosaccharides, disaccharides, polysaccharides, glucose, fructose, galactose, mannose, sorbose, lactose, sucrose, cellobiose, trehalose, raffinose, starch, dextran, maltodextrin, cyclodextrin, inulin, xylitol, sorbitol, lactitol, and mannitol.

71. The dry powder formulation of claim 70, wherein the sugar is mannitol.

72. The dry powder formulation of any one of claims 45-71, further comprising a pharmaceutically acceptable excipient selected from the group consisting of: esters, carbamates, phosphate esters, phosphazenes, amino acids, collagen, chitosan, polysaccharides, albumin, surfactants, buffers, salts, and combinations thereof.

73. The dry powder formulation of claim 72, wherein the surfactant is selected from the group consisting of: CHAPS (3- [ (3-cholamidopropyl) dimethylammonio ] -1-propanesulfonate), a phospholipid, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, octaethyleneglycol monododecyl ether, pentaethyleneglycol monododecyl ether, Triton X-100, cocamide monoethanolamine, cocamide diethanolamine, glycerol monostearate, glycerol monolaurate, sorbitan monostearate, tween 20, tween 40, tween 60, tween 80, an alkylpolyglucoside, a poloxamer and optionally poloxamer 407.

74. The dry powder formulation of claim 73, wherein the surfactant is a poloxamer.

75. The dry powder formulation of any one of claims 45-74, wherein the mRNA encodes a peptide.

76. The dry powder formulation of any one of claims 45-74, wherein the mRNA encodes a therapeutic protein.

77. The dry powder formulation of claim 76, wherein the therapeutic protein is CFTR.

78. The dry powder formulation of claim 76, wherein the therapeutic protein is OTC.

79. A method of delivering mRNA in vivo, comprising administering the dry powder formulation of any one of claims 45-78 to a subject in need thereof.

80. A method of treating a disease or disorder in a patient by administering to the patient an effective dose of mRNA in the dry powder formulation of any one of claims 45-78.

81. The method of claim 79 or 80, wherein the dry powder formulation is administered by inhalation.

82. The method of claim 79 or 80, wherein the dry powder formulation is administered by intranasal spray.

83. The method of claim 79 or 80, wherein the formulation is administered by a metered dose inhaler.

84. The method of any one of claims 80-83, wherein the disease or disorder is selected from the group consisting of cystic fibrosis; asthma; COPD; emphysema; primary ciliary dyskinesia (CILD1) with or without visceral inversion, or castalasner syndrome; pulmonary fibrosis; bort-hodge-dubu syndrome; hereditary hemorrhagic telangiectasia; alpha-1 antitrypsin deficiency; cytochrome b positive granulomatosis (CGD, X-linked); autosomal recessive cytochrome b-positive granulomatosis; surfactant deficiency, pulmonary surfactant metabolic dysfunction 1, pulmonary surfactant metabolic dysfunction 2, pulmonary surfactant metabolic dysfunction 3; premature respiratory distress syndrome; tuberculosis, pneumovirus diseases including influenza, Respiratory Syncytial Virus (RSV).

85. A method for manufacturing a dry powder formulation, the method comprising:

providing a mixture comprising mRNA, one or more lipids, and a polymer; and

spray drying the mixture to form a plurality of particles.

86. The method of claim 85, wherein the one or more lipids are first mixed with the mRNA to form mRNA-loaded lipid nanoparticles prior to adding the polymer.

87. The method of claim 85, wherein the one or more lipids are mixed with the mRNA and the polymer in a single step to form mRNA-loaded lipid-polymer nanoparticles.

88. The method of any one of claims 85-87, further comprising adding one or more excipients to the mixture prior to spray drying.

89. The method of any one of claims 85-88, wherein said plurality of spray dried particles are characterized by one or more of the following:

a) the water content is less than 10 percent;

b) a portion of the fine particles having a volume median diameter of less than 5 μm;

c) the range of Z-average particle size is 10-3000 nm;

d) the N/P ratio ranges from 1 to 20;

e) an mRNA encapsulation efficiency of 80% or more;

f) the mRNA integrity was 90% or higher.

90. The method of any one of claims 85-89, wherein the mRNA encodes a protein or peptide.

91. The method of any one of claims 85-90, wherein the mRNA encodes a peptide.

92. The method of any one of claims 85-90, wherein the mRNA encodes a therapeutic protein.

93. The method of claim 92, wherein the therapeutic protein is CFTR.

94. The method of claim 92, wherein the therapeutic protein is OTC.

95. A dry powder formulation made according to the method of any one of claims 85-94.

96. A method of treating a disease or disorder in a patient by administering to the patient an effective dose of mRNA in the dry powder formulation of claim 95.