Methods of enhancing SPLP-mediated transfection using endosomal membrane destabilizers

• US 20020072121A1
• Filed: 04/20/2001
• Published: 06/13/2002
• Est. Priority Date: 04/20/2000
• Status: Active Grant

• Current Assignee: Angela M.I. Lam, Lorne R. Palmer, Pieter R. Cullis
• Original Assignee: Angela M.I. Lam, Lorne R. Palmer, Pieter R. Cullis
• Inventors: Lam, Angela M.I., Palmer, Lorne R., Cullis, Pieter R.
• Granted Patent: US 7,189,705 B2
• Time in Patent Office: Days
• US Class Current: 435/458
• 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 novel and surprisingly effective methods for delivering nucleic acids to cells. These methods are based upon the discovery that the presence of endosomal membrane destabilizers (e.g., calcium) leads to a dramatic increase in the transfection efficiency of plasmids formulated as SPLP, or “stabilized plasmid-lipid particles.”

Claims

1. A nucleic acid-lipid particle composition for introducing a nucleic acid into a cell, said particle comprising:

a cationic lipid, a conjugated lipid that inhibits aggregation of particles, a nucleic acid and an endosomal membrane destabilizer.

2. The nucleic acid-lipid particle composition of claim 1, wherein said endosomal membrane destabilizer is outside said nucleic acid-lipid particle.

3. The nucleic acid-lipid particle composition of claim 1, wherein said endosomal membrane destabilizer is both outside and inside said nucleic acid-lipid particle.

4. The nucleic acid-lipid particle composition of claim 1, wherein said endosomal membrane destabilizer is Ca⁺⁺ ion.

5. The nucleic acid-lipid particle composition of claim 4, wherein the concentration of Ca⁺⁺ ion is from about 0.1 mM to about 100 mM.

6. The nucleic acid-lipid particle composition of claim 5, wherein the concentration of Ca⁺⁺ ion is from about 1 mM to about 20 mM.

7. The nucleic acid-lipid particle composition of claim 1, wherein said particle has a median diameter of less than about 150 nm.

8. The nucleic acid-lipid particle composition of claim 1, wherein said cationic lipid is a member selected from the group consisting of N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), and N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), and combinations thereof.

9. The nucleic acid-lipid particle composition of claim 1, wherein said particle further comprises an additional noncationic lipid.

10. The nucleic acid-lipid particle composition of claim 9, wherein said noncationic lipid is selected from the group consisting of DOPE, POPC, and EPC.

11. The nucleic acid-lipid particle composition of claim 1, wherein said particle comprises a functional group that facilitates Ca⁺⁺ ion chelation.

12. The nucleic acid-lipid particle composition of claim 1, wherein said conjugated lipid that inhibits aggregation of particles has the formula

A-W-Y  

 

I

wherein;

A is a lipid moiety;

W is a hydrophilic polymer; and

Y is a polycationic moiety.

13. The nucleic acid-lipid particle composition of claim 12, wherein W is a polymer selected from the group consisting of PEG, polyamide, polylactic acid, polyglycolic acid, polylactic acid/polyglycolic acid copolymers and combinations thereof, said polymer having a molecular weight of about 250 to about 7000 daltons.

14. The nucleic acid-lipid particle composition of claim 12, wherein Y has at least 4 positive charges at a selected pH.

15. The nucleic acid-lipid particle composition of claim 12, wherein Y is a member selected from the group consisting of lysine, arginine, asparagine, glutamine, derivatives thereof and combinations thereof.

16. The nucleic acid-lipid particle composition of claim 12, wherein A is a member selected from the group consisting of a diacylglycerolyl moiety, a dialkylglycerolyl moiety, a N-N-dialkylamino moiety, a 1 ,2-diacyloxy-3-aminopropane moiety and a 1,2-dialkyl-3-aminopropane moiety.

17. The nucleic acid-lipid particle composition of claim 12, wherein W is PEG.

18. The nucleic acid-lipid particle composition of claim 12, wherein W is a polyamide polymer.

19. The nucleic acid-lipid particle composition of claim 12, wherein W has a molecular weight of about 250 to about 2000 daltons.

20. The nucleic acid-lipid particle composition of claim 17, having the general structure of Formula II:

21. The nucleic acid-lipid particle composition of claim 20, wherein

X is a member selected from the group consisting of a single bond, phosphatidylethanolamino, phosphatidylethanolamido, phosphoro, phospho, phosphoethanolamino, phosphoethanolamido, carbonyl, carbamate, carboxyl, carbonate, amido, thioamido, oxygen, sulfur and NR, wherein R is a hydrogen or alkyl group.

22. The nucleic acid-lipid particle composition of claim 20, wherein

Z is a member selected from the group consisting of a single bond, phosphatidylethanolamino, phosphatidylethanolamido, phosphoro, phospho, phosphoethanolamino, phosphoethanolamido, carbonyl, carbamate, carboxyl, carbonate, amido, thioamido, oxygen, sulfur and NR, wherein R is a hydrogen or alkyl group.

23. The nucleic acid-lipid particle composition of claim 20, wherein

A is a diacylglycerolyl moiety;

X is phosphoethanolamido;

Z is NR, wherein R is a hydrogen atom; and

Y is a member selected from the group consisting of about 1 to about 10 basic amino acids or derivatives thereof.

24. The nucleic acid-lipid particle composition of claim 23, wherein

A is a diacylgercerolyl moiety having 2 fatty acyl chains, wherein each acyl chain is independently between 2 and 30 carbons in length and is either saturated or has varying degrees of saturation.

25. The nucleic acid-lipid particle composition of claim 23, wherein

Y is a member selected from the group consisting of lysine, arginine, asparagine, glutamine, derivatives thereof and combinations thereof.

26. The nucleic acid-lipid particle composition of claim 23, wherein

A is a diacylgercerolyl moiety having 2 fatty acyl chains, wherein each acyl chain is a saturated C-1 8 carbon chain;

and

Y is a cationic group having 4 lysine residues or derivatives thereof.

27. The nucleic acid-lipid particle composition of claim 1, wherein said conjugated lipid that inhibits aggregation of particles is a PEG-lipid.

28. The nucleic acid-lipid particle composition of claim 27, wherein said PEG-lipid is PEG-ceramide.

29. The nucleic acid-lipid particle composition of claim 28, wherein the ceramide of said PEG-ceramide comprises a fatty acid group having about 8 to about 20 carbon atoms.

30. The nucleic acid-lipid particle composition of claim 28, wherein said PEG-lipid is PEG-phosphatidylethanolamine.

31. The nucleic acid-lipid particle composition of claim 1, wherein said conjugated lipid that inhibits aggregation of particles is an ATTA-lipid.

32. The nucleic acid-lipid particle composition of claim 1, wherein said nucleic acid is selected from the group consisting of a plasmid, an antisense oligonucleotide, and a ribozyme.

33. A method of introducing a nucleic acid into a cell, said method comprising:

contacting said cell with a nucleic acid-lipid particle composition, said particle comprising a cationic lipid, a conjugated lipid that inhibits aggregation of particles, and a nucleic acid; and

an endosomal membrane destabilizer.

34. The method of introducing a nucleic acid into a cell of claim 33, wherein said endosomal membrane destabilizer is outside said nucleic acid-lipid particle.

35. The method of introducing a nucleic acid into a cell of claim 33, wherein said endosomal membrane destabilizer is Ca⁺⁺ ion.

36. The method of introducing a nucleic acid into a cell of claim 35, wherein the concentration of Ca⁺⁺ ion is from about 0.1 mM to about 100 mM.

37. The method of introducing a nucleic acid into a cell of claim 36, wherein the concentration of Ca⁺⁺ ion is from about 1 mM to about 20 mM.

38. The method of introducing a nucleic acid into a cell of claim 33, wherein said particle has a median diameter of less than about 150 nm.

39. The method of introducing a nucleic acid into a cell of claim 33, wherein said cationic lipid is a member selected from the group consisting of N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), and N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), and combinations thereof.

40. The method of introducing a nucleic acid into a cell of claim 33, wherein said particle further comprises an additional noncationic lipid.

41. The method of introducing a nucleic acid into a cell of claim 40, wherein said noncationic lipid is selected from the group consisting of DOPE, POPC, and EPC.

42. The method of introducing a nucleic acid into a cell of claim 33, wherein said particle comprises a finctional group that facilitates Ca⁺⁺ ion chelation.

43. The method of introducing a nucleic acid into a cell of claim 33, wherein said conjugated lipid that inhibits aggregation of particles has the formula

A-W-Y  

 

I

wherein;

A is a lipid moiety;

W is a hydrophilic polymer; and

Y is a polycationic moiety.

44. The method of introducing a nucleic acid into a cell of claim 43, wherein W is a polymer selected from the group consisting of PEG, polyamide, polylactic acid, polyglycolic acid, polylactic acid/polyglycolic acid copolymers and combinations thereof, said polymer having a molecular weight of about 250 to about 7000 daltons.

45. The method of introducing a nucleic acid into a cell of claim 43, wherein Y has at least 4 positive charges at a selected pH.

46. The method of introducing a nucleic acid into a cell of claim 43, wherein Y is a member selected from the group consisting of lysine, arginine, asparagine, glutamine, derivatives thereof and combinations thereof.

47. The method of introducing a nucleic acid into a cell of claim 43, wherein A is a member selected from the group consisting of a diacylglycerolyl moiety, a dialkylglycerolyl moiety, a N-N-dialkylamino moiety, a 1,2-diacyloxy-3-aminopropane moiety and a 1,2-dialkyl-3-aminopropane moiety.

48. The method of introducing a nucleic acid into a cell of claim 43, wherein W is PEG.

49. The method of introducing a nucleic acid into a cell of claim 43, wherein W is a polyamide polymer.

50. The method of introducing a nucleic acid into a cell of claim 43, wherein W has a molecular weight of about 250 to about 2000 daltons.

51. The method of introducing a nucleic acid into a cell of claim 48, having the general structure of Formula II:

52. The method of introducing a nucleic acid into a cell of claim 51, wherein

X is a member selected from the group consisting of a single bond, phosphatidylethanolamino, phosphatidylethanolamido, phosphoro, phospho, phosphoethanolamino, phosphoethanolamido, carbonyl, carbamate, carboxyl, carbonate, amido, thioamido, oxygen, sulfur and NR, wherein R is a hydrogen or alkyl group.

53. The method of introducing a nucleic acid into a cell of claim 51, wherein

Z is a member selected from the group consisting of a single bond, phosphatidylethanolamino, phosphatidylethanolamido, phosphoro, phospho, phosphoethanolamino, phosphoethanolamido, carbonyl, carbamate, carboxyl, carbonate, amido, thioamido, oxygen, sulfur and NR, wherein R is a hydrogen or alkyl group.

54. The method of introducing a nucleic acid into a cell of claim 51, wherein

A is a diacylglycerolyl moiety;

X is phosphoethanolamido;

Z is NR, wherein R is a hydrogen atom; and

Y is a member selected from the group consisting of about 1 to about 10 basic amino acids or derivatives thereof.

55. The method of introducing a nucleic acid into a cell of claim 54, wherein

A is a diacylgercerolyl moiety having 2 fatty acyl chains, wherein each acyl chain is independently between 2 and 30 carbons in length and is either saturated or has varying degrees of saturation.

56. The method of introducing a nucleic acid into a cell of claim 54, wherein

Y is a member selected from the group consisting of lysine, arginine, asparagine, glutamine, derivatives thereof and combinations thereof.

57. The method of introducing a nucleic acid into a cell of claim 54, wherein

A is a diacylgercerolyl moiety having 2 fatty acyl chains, wherein each acyl chain is a saturated C-1 8 carbon chain;

and

Y is a cationic group having 4 lysine residues or derivatives thereof.

58. The method of introducing a nucleic acid into a cell of claim 33, wherein said conjugated lipid that inhibits aggregation of particles is a PEG-lipid.

59. The method of introducing a nucleic acid into a cell of claim 58, wherein said PEG-lipid is PEG-ceramide.

60. The method of introducing a nucleic acid into a cell of claim 59, wherein the ceramide of said PEG-ceramide comprises a fatty acid group having about 8 to about 20 carbon atoms.

61. The method of introducing a nucleic acid into a cell of claim 59, wherein said PEG-lipid is PEG-phosphatidylethanolamine.

62. The method of introducing a nucleic acid into a cell of claim 33, wherein said conjugated lipid that inhibits aggregation of particles is an ATTA-lipid.

63. The method of introducing a nucleic acid into a cell of claim 33, wherein said nucleic acid is selected from the group consisting of a plasmid, an antisense oligonucleotide, and a ribozyme.

64. A method for inducing H_II phase structure in a lipid bilayer, said method comprising:

contacting said lipid bilayer with an endosomal membrane destabilizer, thereby inducing H_II phase structure in a lipid bilayer.

65. The method for inducing H_II phase structure of claim 64, wherein said lipid bilayer comprises DOPC:

DOPE;

DOPS;

Chol.

66. The method for inducing H_II phase structure of claim 64, wherein said endosomal membrane destabilizer is Ca⁺⁺ ion.

67. The method for inducing H_II phase structure of claim 66, wherein Ca⁺⁺ ion acts in concert with low levels of the cationic lipid to trigger H_II phase formation.

68. Use of nucleic acid-lipid particle composition for introducing a nucleic acid into a cell, said particle comprising:

a cationic lipid, a conjugated lipid that inhibits aggregation of particles, a nucleic acid and an endosomal membrane destabilizer.