Delivery and formulation of engineered nucleic acids

• US 9,950,068 B2
• Filed: 12/14/2016
• Issued: 04/24/2018
• Est. Priority Date: 03/31/2011
• Status: Active Grant

• Current Assignee: Moderna TX, Inc. (Moderna, Inc.)
• Original Assignee: Moderna TX, Inc. (Moderna, Inc.)
• Inventors: De Fougerolles, Antonin, Elbashir, Sayda M.
• Primary Examiner(s): Kelly, Robert M
• Application Number:
  US15/379,284
• Publication Number:
  US 20170258915A1
• Time in Patent Office: 496 Days
• Field of Search: None
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• CPC Class Codes: A61K 31/7115 Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
• CPC Class Codes: A61K 38/193 Colony stimulating factors [CSF]
• CPC Class Codes: A61K 38/4846 Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
• CPC Class Codes: A61K 47/18 Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
• CPC Class Codes: A61K 47/22 Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
• CPC Class Codes: A61K 47/28 Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/00 Drugs for disorders of the urinary system
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 : Immunomodulators
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/00 : Drugs for disorders of the endocrine system
• CPC Class Codes: A61P 7/00 : Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/535 : Granulocyte CSF; Granulocyte-macrophage CSF
• CPC Class Codes: C12N 15/67 : General methods for enhancing the expression
• CPC Class Codes: C12N 15/87 : Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 2310/335 : Modified T or U
• CPC Class Codes: C12N 9/644 : Coagulation factor IXa (3.4.21.22)

Abstract

Provided are formulations, compositions and methods for delivering biological moieties such as modified nucleic acids into cells to modulate protein expression. Such compositions and methods include the delivery of biological moieties, and are useful for production of proteins.

Claims

1. A pharmaceutical composition, comprising:

a modified messenger RNA (mRNA) encoding a polypeptide of interest, wherein the modified mRNA comprises one or more uridines and one or more cytidines and wherein substantially all uridines are modified, and

the modified mRNA is in a lipid nanoparticle formulation comprising a lipid selected from the group consisting of DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12-200;

a cholesterol; and

a PEG-lipid.

2. The pharmaceutical composition of claim 1, wherein the modified uridine is a pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy uridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, or pseudouridine.

3. The pharmaceutical composition of claim 1, wherein the one or more uridines are modified on the major groove face of the uridine.

4. The pharmaceutical composition of claim 1, wherein the modified uridine is pseudouridine or 1-methyl-pseudouridine.

5. The pharmaceutical composition of claim 4, wherein the modified uridine is 1-methyl-pseudouridine.

6. The pharmaceutical composition of claim 2, wherein the modified mRNA further comprises one or more modified cytidines.

7. The pharmaceutical composition of claim 6, wherein the modified cytidine is 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 5-methyl-cytidine, or 4-methoxy-1-methyl-pseudoisocytidine.

8. The pharmaceutical composition of claim 7, wherein the modified cytidine is 5-methyl-cytidine.

9. The pharmaceutical composition of claim 7, wherein at least about 95% of the uridines are modified and at least about 95% of the cytidines are modified.

10. The pharmaceutical composition of claim 7, wherein about 100% of the uridines are modified and about 100% of the cytidines are modified.

11. The pharmaceutical composition of claim 1, wherein the modified mRNA comprises a 5′

Cap1 structure and a polyA tail of 160 nucleotides in length.

12. The pharmaceutical composition of claim 1, wherein the modified mRNA and lipid nanoparticle are formulated at a total lipid to mRNA weight ratio of 10:

1, 15;

1, 20;

1, or 30;

1.

13. The pharmaceutical composition of claim 1, wherein the lipid is DLin-KC2-DMA or 98N12-5.

14. The pharmaceutical composition of claim 1, wherein the lipid nanoparticle formulation comprises about 42% lipid, about 48% cholesterol, and about 10% PEG-lipid.

15. The pharmaceutical composition of claim 1, wherein the lipid nanoparticle formulation comprises about 50% lipid, about 38.5% cholesterol, and about 1.5% PEG-lipid.

16. The pharmaceutical composition of claim 1, wherein the modified mRNA further comprises an operably-linked signal sequence, wherein the signal sequence is that of either (i) the polypeptide of interest or (ii) a heterologous protein.

17. The pharmaceutical composition of claim 1, wherein the lipid nanoparticle has a mean particle size between 86 nm and 155 nm.

18. The pharmaceutical composition of claim 1, wherein the lipid nanoparticle has a polydisperity index between 0.02 and 0.17.

19. The pharmaceutical composition of claim 1 further comprising a formulation buffer for in vivo delivery, wherein the formulation buffer has a pH of 6.5 and comprises sodium chloride, calcium chloride, and Na+-phosphate.

20. The pharmaceutical composition of claim 19, wherein the formulation buffer comprises 150 mM sodium chloride, 2 mM calcium chloride, and 2 mM Na+-phosphate.