Dually derivatized chitosan nanoparticles and methods of making and using the same for gene transfer in vivo

• US 10,456,478 B2
• Filed: 04/17/2017
• Issued: 10/29/2019
• Est. Priority Date: 03/21/2012
• Status: Active Grant

• Current Assignee: enGene, Inc.
• Original Assignee: enGene, Inc.
• Inventors: Gao, Jun, Hsu, Eric, Cheung, Anthony
• Primary Examiner(s): McDonald, Jennifer Pitrak
• Application Number:
  US15/489,199
• Publication Number:
  US 20180008720A1
• Time in Patent Office: 925 Days
• Field of Search: None
• CPC Class Codes: A61K 47/61 the organic macromolecular compound being a polysaccharide or a derivative thereof
• CPC Class Codes: A61K 47/6939 the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
• CPC Class Codes: A61K 9/5161 Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C07H 13/04 having the esterifying carboxyl radicals attached to acyclic carbon atoms
• CPC Class Codes: C07H 5/06 Aminosugars
• CPC Class Codes: C08B 37/003 Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
• CPC Class Codes: C08L 5/08 Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 2310/14 interfering N.A.
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific

Abstract

Provided herein is chitosan dually derivatized with arginine and gluconic acid; and methods of making and using the same, e.g., for gene delivery in vivo.

Claims

1. A method of delivering a therapeutic nucleic acid to a subject in need thereof comprising administering to the subject a therapeutically effective amount of a dually derivatized (DD) chitosan nucleic acid polyplex, wherein said DD chitosan nucleic acid polyplex comprises a chitosan-derivative nanoparticle comprising chitosan coupled with gluconic acid and arginine and said therapeutic nucleic acid.

2. The method according to claim 1, wherein said nanoparticle comprises arginine at a concentration of about 10% to about 55%.

3. The method according to claim 2, wherein said nanoparticle comprises gluconic acid at an initial concentration of about 8% to about 30%.

4. The method according to claim 3, wherein said nanoparticle comprises gluconic acid at a final functionalization of about 3% to about 10%.

5. The method according to claim 1, wherein said DD-chitosan nucleic acid polyplex has a combined degree of functionalization with said arginine and said gluconic acid of 1-60%.

6. The method according to claim 5, wherein said DD-chitosan nucleic acid polyplex has a combined degree of functionalization with said arginine and said gluconic acid of 1-30%.

7. The method according to claim 1, wherein the amine to phosphate ratio of said DD-chitosan nucleic acid polyplex is between 2to 100.

8. The method according to claim 7, wherein the amine to phosphate ratio of said DD-chitosan nucleic acid polyplex is between 2 to50.

9. The method according to claim 8, wherein the amine to phosphate ratio of said DD-chitosan nucleic acid polyplex is between 2 to30.

10. The method according to claim 9, wherein the amine to phosphate ratio of said DD-chitosan nucleic acid polyplex is between 2 to 15.

11. The method according to claim 1, wherein said DD-chitosan nucleic acid polyplex has a molar ratio of said arginine to said gluconic acid of between 100:

1 and 1;

100.

12. The method according to claim 11, wherein said DD-chitosan nucleic acid polyplex has a molar ratio of said arginine to said gluconic acid of between 50:

1 and 1;

50.

13. The method according to claim 12, wherein said DD-chitosan nucleic acid polyplex has a molar ratio of said arginine to said gluconic acid of between 10:

1 and 1;

10.

14. The method according to claim 13, wherein said DD-chitosan nucleic acid polyplex has a molar ratio of said arginine to said gluconic acid of between 5:

1 and 1;

5.

15. The method according to claim 14, wherein said DD-chitosan nucleic acid polyplex has a molar ratio of said arginine to said gluconic acid of between 2:

1 and 1;

2.

16. The method according to claim 1, wherein said DD-chitosan nucleic acid polyplex is isotonic.

17. The method according to claim 1, wherein said DD-chitosan nucleic acid polyplex comprises chitosan molecules having an average molecular weight of less than 110 kDa before functionalization with said gluconic acid and said arginine.

18. The method according to claim 17, wherein said DD-chitosan nucleic acid polyplex comprises chitosan molecules having an average molecular weight of less than 50 kDa before functionalization with said gluconic acid and said arginine.

19. The method according to claim 18, wherein said DD-chitosan nucleic acid polyplex comprises chitosan molecules having an average molecular weight of less than 30 kDa before functionalization with said gluconic acid and said arginine.

20. The method according to claim 19, wherein said DD-chitosan nucleic acid polyplex comprises chitosan molecules having an average molecular weight of less than 10 kDa before functionalization with said gluconic acid and said arginine.

21. The method according to claim 1, wherein said DD-chitosan nucleic acid polyplex has an average polydispersity index (PDI) of less than 0.5.

22. The method according to claim 1, wherein said DD-chitosan nucleic acid polyplex increases in average diameter by less than 100% at room temperature for 6 hours.

23. The method according to claim 1, wherein said DD-chitosan nucleic acid polyplex has a therapeutic nucleic acid concentration between about 0.5 mg/ml and about 1.5 mg/ml, and is substantially free of precipitated polyplex.

24. The method according to claim 1, wherein said therapeutic nucleic acid isDNA.

25. The method according to claim 1, wherein said therapeutic nucleic acid is RNA.

26. The method according to claim 25, wherein said RNA is selected from the group consisting of antisense RNA, siRNA, short hairpin RNA, micro RNA, and enzymatic RNA.

27. The method according to claim 1, wherein said therapeutic nucleic acid encodes insulin, leptin, a glucagon antagonist, GLP-1, GLP-2, ghrelin, cholecystokinin, a growth hormone, a clotting factor, PYY, erythropoietin, an inhibitor of inflammation, IL-10, an IL-17 antagonist, a TNFα

antagonist, growth hormone releasing hormone, or parathyroid hormone.

28. The method according to claim 1, wherein said therapeutic nucleic acid encodes IL-10.

29. The method according to claim 1, wherein said therapeutic nucleic acid encodes leptin, cholecystokinin, PYY, or GLP-1.

30. The method according to claim 1, wherein said therapeutic nucleic acid encodes an IL-1 antagonist.

31. The method according to claim 1, wherein said therapeutic nucleic acid encodes an IL-17 antagonist.

32. The method according to claim 1, wherein said therapeutic nucleic acid encodes a TNFα

antagonist.

33. The method according to claim 29, wherein said therapeutic nucleic acid encodes GLP-1.