Compositions and methods for efficient delivery of molecules to cells

• US 10,195,280 B2
• Filed: 07/15/2015
• Issued: 02/05/2019
• Est. Priority Date: 07/15/2014
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: de Mollerat du Jeu, Xavier, Andronikou, Nektaria
• Primary Examiner(s): Burkhart, Michael D
• Application Number:
  US14/800,565
• Publication Number:
  US 20160045600A1
• Time in Patent Office: 1,301 Days
• Field of Search: None
• CPC Class Codes: A61K 31/7105 Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• CPC Class Codes: A61K 47/186 Quaternary ammonium compounds, e.g. benzalkonium chloride or cetrimide
• CPC Class Codes: A61K 9/1272 with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids with cholesterol as the only non-phosphatidyl surfactant A61K9/127; lipids as modifying agent A61K47/543
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle

Abstract

The present invention provides lipid aggregate compositions having at least two cationic lipids in combination with one or optionally more than one non-cationic lipids for the in vitro and/or in vivo delivery of biologically active molecules including nucleic acids, specifically DNA and RNA, and proteins into cells and tissues. Methods are also provided that use the compounds of the present invention to deliver biologically active molecules, into cells and tissues to facilitate the expression of target proteins therein. In some non-limiting embodiments, the subject lipid aggregate compositions can be used to deliver nucleic acid molecules to facilitate the expression of proteins involved in cellular reprogramming and genome editing applications.

Claims

1. A lipid aggregate comprising dihydroxyl-dimyristylspermine (DHDMS) or a salt thereof and hydroxyl-dimyristylspermine (HDMS) or a salt thereof, a first neutral lipid and optionally a second neutral lipid, wherein said lipid aggregate is suitable for forming a cationic complex with a nucleic acid under aqueous conditions, wherein said DHDMS has the structure:

2. The lipid aggregate of claim 1, wherein the molar ratio of DHDMS is from about 0.1 to about 0.4.

3. The lipid aggregate of claim 1, wherein the molar ratio of HDMS is from about 0.1 to about 0.4.

4. The lipid aggregate of claim 1, wherein the neutral lipid is selected from the group DOPE, DOPC and cholesterol.

5. The lipid aggregate of claim 1, wherein said second neutral lipid is selected from the group DOPE, DOPC and cholesterol, wherein said second neutral lipid is different from said first neutral lipid.

6. The lipid aggregate of claim 1, wherein said first neutral lipid is cholesterol.

7. The lipid aggregate of claim 1, wherein said second neutral lipid is DOPE.

8. The lipid aggregate of claim 6, wherein the molar ratio of cholesterol is about 0.1 to about 0.4.

9. The lipid aggregate of claim 7, wherein the molar ratio of DOPE is about 0.1 to about 0.4.

10. The lipid aggregate of claim 1, wherein the first neutral lipid is cholesterol and the second neutral lipid is DOPE.

11. The lipid aggregate of claim 10, wherein the molar ratio of each of cholesterol and DOPE is about 0.1 to about 0.4.

12. A lipid aggregate-nucleic acid complex comprising the lipid aggregate of claim 1 contacted with a nucleic acid molecule.

13. A lipid aggregate-nucleic acid complex comprising the lipid aggregate of claim 1 contacted with an RNA molecule.

14. The lipid aggregate-nucleic acid complex of claim 13, wherein the RNA molecule comprises at least one expressible sequence encoding a protein.

15. A lipid aggregate-nucleic acid complex comprising the lipid aggregate of claim 1 contacted with an mRNA molecule.

16. The lipid aggregate-nucleic acid complex according to claim 12, wherein the lipid aggregate-nucleic acid complex is stable for up to 1 hour.

17. The lipid aggregate-nucleic acid complex according to claim 12, wherein the lipid aggregate-nucleic acid complex is stable for up to 4 hours.

18. The lipid aggregate-nucleic acid complex according to claim 12, wherein the lipid aggregate-nucleic acid complex is stable for up to 10 hours.

19. The lipid aggregate-nucleic acid complex according to claim 12, wherein the lipid aggregate-nucleic acid complex is stable for up to 24 hours.

20. A lipid aggregate-RNA complex comprising about 0.1-0.4 molar ratio of each of DHDMS, HDMS, cholesterol and DOPE and an RNA molecule, said lipid aggregate-RNA complex being cationic in an aqueous solution at pH about 6 to about 8.

21. The lipid aggregate-RNA complex of claim 20, wherein said RNA molecule is from about 0.2 kb up to about 0.5 kb, Up to about 1 kb, up to about 1.25 kb, up to about 1.5 kb, up to about 1.75 kb, up to about 2 kb, up to about 2.25 kb, up to about 2.5 kb, up to about 2.75 kb, up to about 3 kb, up to about 3.25 kb, up to about 3.5 kb, up to about 3.75 kb, up to about 4 kb, up to about 4.25 kb, up to about 4.5 kb, up to about 4.75 kb, up to about 5 kb, up to about 5.25 kb, up to about 5.5 kb, up to about 5.75 kb, up to about 6 kb, up to about 6.5 kb, up to about 7 kb, up to about 7.5 kb, up to about 8 kb, up to about 8.5 kb, up to about 9 kb, up to about 9.5 kb, up to about 10 kb, up to about 10.5 kb, up to about 11 kb, up to about 12 kb, up to about 13 kb, up to about 14 kb, or up to about 15 kb.

22. The lipid aggregate-RNA complex of claim 20, wherein said RNA molecule comprises at least one expressible sequence encoding a protein.

23. The lipid aggregate-RNA complex of claim 20, wherein said molar ratio of DHDMS is about 0.1, 0.2, 0.25, 0.3, or 0.4.

24. The lipid aggregate-RNA complex of claim 20, wherein said molar ratio of HDMS is about 0.1, 0.2, 0.25, 0.3, or 0.4.

25. The lipid aggregate-RNA complex of claim 20, wherein said molar ratio of cholesterol is about 0.1, 0.2, 0.25, 0.3, or 0.4.

26. The lipid aggregate-RNA complex of claim 20, wherein said molar ratio of DOPE is about 0.1, 0.2, 0.25, 0.3, or 0.4.

27. The lipid aggregate-RNA complex of claim 20, wherein said molar ratio of DHDMS is about 0.1, 0.2, 0.25, 0.3, or 0.4 and molar ratio of cholesterol is about 0.1, 0.2, 0.25, 0.3, or 0.4.

28. The lipid aggregate-RNA complex of claim 20, wherein said molar ratio of HDMS is about 0.1, 0.2, 0.25, 0.3, or 0.4 and molar ratio of DOPE is about 0.1, 0.2, 0.25, 0.3, or 0.4.

29. The lipid aggregate-RNA complex of claim 20, wherein said molar ratio of DHDMS is about 0.1, the molar ratio of HDMS is about 0.4, the molar ratio of DOPE is about 0.1 and the molar ratio of cholesterol is about 0.4.

30. The lipid aggregate-RNA complex of claim 20, wherein said molar ratio of DHDMS is about 0.25, the molar ratio of HDMS is about 0.25, the molar ratio of DOPE is about 0.4 and the molar ratio of cholesterol is about 0.1.

31. The lipid aggregate-RNA complex of claim 20, wherein said molar ratio of DHDMS is about 0.3, the molar ratio of HDMS is about 0.1, the molar ratio of DOPE is about 0.3 and the molar ratio of cholesterol is about 0.3.

32. The lipid aggregate-RNA complex of claim 20, wherein said molar ratio of DHDMS is about 0.4, the molar ratio of HDMS is about 0.1, the molar ratio of DOPE is about 0.4 and the molar ratio of cholesterol is about 0.1.

33. The lipid aggregate-RNA complex of claim 20, wherein said molar ratio of DHDMS is about 0.3, the molar ratio of HDMS is about 0.3, the molar ratio of DOPE is about 0.1 and the molar ratio of cholesterol is about 0.3.

34. The lipid aggregate-RNA complex of claim 20, wherein said molar ratio of DHDMS is about 0.4, the molar ratio of HDMS is about 0.2, the molar ratio of DOPE is about 0.2 and the molar ratio of cholesterol is about 0.2.

35. The lipid aggregate-RNA complex of claim 20, wherein said molar ratio of DHDMS is about 0.4, the molar ratio of HDMS is about 0.25, the molar ratio of DOPE is about 0.1 and the molar ratio of cholesterol is about 0.25.