Self-assembling polynucleotide delivery system

• US 6,300,317 B1
• Filed: 06/06/1995
• Issued: 10/09/2001
• Est. Priority Date: 04/03/1992
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Haensler, Jean, Szoka, Francis C. Jr.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/469,899
• Time in Patent Office: 2,317 Days
• Field of Search: 435/172.3, 435/440, 435/443, 435/455, 435/458, 514/44, 536/23.1
• US Class Current: 514/44.00R
• CPC Class Codes: A61K 47/54 the modifying agent being an organic compound
• CPC Class Codes: A61K 47/543 Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• CPC Class Codes: A61K 47/55 the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
• CPC Class Codes: A61K 47/645 Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• CPC Class Codes: A61K 47/6949 inclusion complexes, e.g. clathrates, cavitates or fullerenes
• 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: A61K 9/1271 Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers liposome as conjugate A61K47/6911
• 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: 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 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07D 401/14 containing three or more hetero rings
• CPC Class Codes: C07F 9/10 Phosphatides, e.g. lecithin
• CPC Class Codes: C08G 2261/312 Non-condensed aromatic systems, e.g. benzene
• CPC Class Codes: C08G 83/003 Dendrimers
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Abstract

This invention provides a self-assembling polynucleotide delivery system comprising components aiding in the delivery of the polynucleotide to the desired address which are associated via noncovalent interactions with the polynucleotide. The components of this system include DNA-masking components, cell recognition components, charge-neutralization and membrane-permeabilization components, and subcellular localization components. Specific compounds useful in this system are also provided.

Claims

1. A composition for presenting a polynucleotide to a subcellular component of a eukaryotic cell comprising:

a) a polynucleotide; and

b) three or more functional agents associated with the polynucleotide, the agents being selected from the group consisting of;

i) a cell recognition agent capable of recognizing the eukaryotic cell, said agent comprising a ligand for a receptor located on the eukaryotic cell surface and a DNA-associating moiety being coupled thereto, the DNA-associating moiety being selected from the group consisting of a single stranded polynucleotide linker, a dendrimer polycation, a major-or minor groove binder, and an intercalating agent;

ii) a membrane-permeabilizing agent capable of transporting the polynucleotide across the cytoplasmic membrane of the eukaryotic cell;

iii) a subcellular-localization agent capable of delivering the polynucleotide from the cytoplasm of the eukaryotic cell to a subcellular component of the eukaryotic cell, said agent further comprising a DNA-associating moiety being coupled thereto, the DNA-associating moiety being selected from the group consisting of a single stranded polynucleotide linker, a dendrimer polycation, a major-or minor groove binder, and an intercalating agent; and

iv) a polynucleotide-masking agent capable of increasing the circulatory half-life of the desired polynucleotide.

65. The composition of claim 1 wherein one or more of the functional agents is operatively coupled to the desired polynucleotide by a polynucleotide-associating moiety.

66. The composition of claim 65 wherein the polynucleotide-associating moiety is selected from the group consisting of a single stranded polynucleotide linker, a dendrimer polycation, a major- or minor-groove binder, and an intercalator.

2. A composition for presenting a polynucleotide to a subcellular component of a eukaryotic cell comprising:

a) a polynucleotide;

b) an agent operatively coupled to the polynucleotide, the agent being selected from the group consisting of

i) a cell recognition agent capable of recognizing the eukaryotic cell, said agent comprising a ligand for a receptor located on the eukaryotic cell surface and a DNA-associating moiety being coupled thereto, the DNA-associating moiety being selected from the group consisting of a single stranded polynucleotide linker, a dendrimer polycation, a major-or minor-groove binder, and an intercalating agent;

ii) a membrane-permeabilizing agent capable of transporting the polynucleotide across the cytoplasmic membrane of the eukaryotic cell; and

iii) a subcellular-localization agent capable of delivering the polynucleotide from the cytoplasm of the eukaryotic cell to a subcellular component of the eukaryotic cell; and

c) a DNA masking agent capable of increasing the circulatory half-life of the polynucleotide, wherein the DNA-masking agent has the chemical formula

wherein

n is an integer of 6 to 24;

Y is selected from the group consisting of hydroxy, ethanolamine, choline, glycerol, serine and inositol;

R¹ is (C₆-C₂₄) is alkyl or (C₆-C₂₄) alkenyl;

R³ is H, or (C₁-C₁₀) alkyl or (C₁-C₁₀) alkylamine; and

R⁴ is a positively charged linear or branched (C₁-C₃₀) alkyl or (C₁-C₃₀) alkylamine, wherein one or more of the carbon atoms may be substituted with NR′

, wherein R′

is H or (C₁-C₁₀) alkyl or (C₁-C₁₀) alkylamine.

3. A composition for presenting a polynucleotide to a subcellular component of a eukaryotic cell comprising

a) a polynucleotide;

b) an agent operatively coupled to the polynucleotide, the agent being selected from the group consisting of

i) a cell recognition agent capable of recognizing the eukaryotic cell, said agent comprising a ligand for a receptor located on the eukaryotic cell surface and a DNA-associating moiety being coupled thereto, the DNA-associating moiety being selected from the group consisting of a single stranded polynucleotide linker, a dendrimer polycation, a major-or minor groove binder, and an intercalating agent;

ii) a membrane-permeabilizing agent capable of transporting the polynucleotide across the cytoplasmic membrane of the eukaryotic cell; and

iii) a subcellular-localization agent capable of delivering the polynucleotide from the cytoplasm of the eukaryotic cell to a subcellular component of the eukaryotic cell; and

c) a DNA masking agent capable of increasing the circulatory half-life of the polynucleotide, wherein the masking agent comprises polyethylene glycol (PEG) covalently linked to a DNA-associating moiety.

4. A composition for presenting a polynucleotide to a subcellular component of a eukaryotic cell comprising

a polynucleotide;

a membrane-permeabilizing agent capable of transporting the polynucleotide across the cytoplasmic membrane of the eukaryotic cell operatively coupled to the polynucleotide, wherein the membrane-permeabilizing agent comprises an amphipathic peptide; and

a lipid or a polyamine.

5. The composition of claim 4, wherein the amphipathic peptide comprises GALA/SEQ ID NO:

10.

6. The composition of claim 4, wherein the peptide comprises a cyclic peptide.

7. The composition of claim 6, wherein the cyclic peptide is selected from the group consisting of gramicidin S and tyrocidines.

8. The composition of claim 7, wherein the cyclic peptide comprises gramicidin S.

56. The composition of claim 4 wherein the amphipathic peptide comprises a cyclic peptide.

57. The composition of claim 56 wherein the cyclic peptide is selected from the group consisting of tyrocidines and gramicidin S.

58. The composition of claim 56 wherein the cyclic peptide comprises gramicidin S.

59. The composition of claim 4 comprising gramicidin S and dioleoylphosphatidylethanolamine.

60. The composition of claim 59 comprising gramicidin S and dioleoylphosphatidylethanolamine in a molar ratio of greater than about 1:

1.

61. The composition of claim 60 comprising gramicidin S and dioleoylphosphatidylethanolamine in a molar ratio of greater than about 5:

1.

9. A composition for presenting a polynucleotide to a subcellular component of a eukaryotic cell comprising

a polynucleotide;

and

a cell recognition agent operatively coupled to the polynucleotide and capable of recognizing the eukaryotic cell, said agent comprising a ligand for a receptor located on the eukaryotic cell surface and a DNA-associating moiety being coupled thereto, the DNA-associating moiety comprising an intercalating agent, wherein the intercalating agent has the formula

wherein

Z comprises a bond, a reactive group selected from the group consisting of N-hydroxysuccinimide, maleimide, maleimidophenyl, pyridyl disulfide, hydrazide, and phenylglyoxal, or ZY, wherein Y is selected from the group consisting of cell surface receptor ligands, nuclear localization sequences, and membrane permeabilizing components;

n and m are independently an integer of 1 to 20;

p is an integer of 0 to 20; and

Ar₁ and Ar₂ are independently selected from the group consisting of ethidium bromide, acridine, mitoxanotrone, oxazolopyridocarbazole, ellipticine, N-methyl-2, 7-diazapyrenium, and derivatives capable of intercalating a polynucleotide.

10. The composition of claim 9, wherein the intercalating agent is coupled to a plurality of ligands.

11. A composition for presenting a polynucleotide to a subcellular component of a eukaryotic cell comprising

a polynucleotide;

and

a cell recognition agent operatively coupled to the polynucleotide and capable of recognizing the eukaryotic cell, said agent comprising a ligand for a receptor located on the eukaryotic cell surface and a DNA-associating moiety being coupled thereto, the DNA-associating moiety comprising an intercalating agent, wherein the intercalating agent comprises the trigalactosylated spermidine bis-acridine compound (26) of FIG. 13.

12. A composition for presenting a polynucleotide to a subcellular component of a eukaryotic cell comprising

a polynucleotide;

a subcellular-localization agent operatively coupled to the polynucleotide and capable of delivering the polynucleotide from the cytoplasm of the eukaryotic cell to a subcellular component of the eukaryotic cell comprising a nuclear localization segment coupled to a DNA-associating moiety, wherein the DNA-associating moiety comprises an intercalating agent having the chemical formula

wherein

comprises a bond, a reactive group selected from the group consisting of N-hydroxysuccinimide, maleimide, maleimidophenyl, pyridyl disulfide, hydrazide, and phenylglyoxal, or ZY, wherein Y is selected from the group consisting of cell surface receptor ligands, nuclear localization sequences, and membrane permeabilizing components;

n and m are independently an integer of 1 to 20;

p is an integer of 0 to 20; and

Ar₁ and Ar₂ are independently selected from the group consisting of etuidium bromide, acridine, mitoxantrone, oxazolopyridocarbazole, ellipticine, N-methyl-2, 7-diazapyrenium, and derivatives capable of intercalating a polynucleotide.

13. The composition of claim 12, wherein Ar₁ and Ar₂ comprise acridine.

14. The composition of claim 13, wherein Z comprises the reactive group maleimidophenyl.

16. The composition of claim 12, wherein Z comprises the group

15. A composition for presenting a polynucleotide to a subcellular component of a eukaryotic cell comprising

a polynucleotide;

a subcellular-localization agent operatively coupled to the polynucleotide and capable of delivering the polynucleotide from the cytoplasm of the eukaryotic cell to a subcellular component of the eukaryotic cell comprising a nuclear localization segment coupled to a DNA-associating moiety, wherein the DNA-associating moiety comprises an intercalating agent having the chemical formula

17. A composition for presenting a desired polynucloetide to a subcellular component of a eukaryotic cell comprising:

a) a desired polynucleotide operatively coupled to a polynucleotide-associating moiety selected from the group consisting of;

i) an intercalator;

ii) a linker strand comprising a single stranded polynucleotide;

iii) a dendrimer polycation; and

b) three functional agents operatively coupled to the polynucleotide, the function agents selected from the group consisting of;

i) a cell recognition agent capable of recognizing the eukaryotic cell;

ii) membrane-permeabilizing agent capable of transporting the desired polynucleotide across the cytoplasmic membrane of the eukaryotic cell;

iii) a subcellular-localization agent capable of delivering the desired polynucleotide from the cytoplasm of the eukaryotic cell to a subcellular component of the eukaryotic cell;

iv) a polynucleotide-masking agent capable of increasing the circulatory half-life of the desired polynucleotide; and

v) a group to link with one of more of the functional agents i)-iv) by a covalent bond.

18. The composition of claim 17 wherein the polynucleotide-associating moiety comprises an intercalator.

19. The composition of claim 18 wherein the intercalator is coupled to a plurality of the functional agents i)-iv).

20. The composition of claim 18 wherein the intercalator has the formula

wherein

Z comprises a reactive group selected from the group consisting of N-hydroxysuccinimide, maleimide, maleimidophenyl, pyridyl disulfide, hydrazide, and phenylglyoxal, or ZY, wherein Y is selected from the group consisting of functional agents i)-iv);

n and m are independently an integer of 1 to 20;

p is an integer of 0 to 20; and

Ar₁ and Ar₂ are independently selected from the group consisting of ethidium bromide, acridine, mitoxanotrone, oxazolopyridocarbazole, ellipticine, N-methyl-2, 7-diazapyrenium, and derivatives capable of intercalating a polynucleotide.

21. The composition of claim 20 wherein Ar₁ and Ar₂ each comprise acridine.

22. The composition of claim 21 wherein the intercalator comprises the trigalactosylated spermidine bis-acridine compound (26) of FIG. 13.

23. The composition of claim 20 wherein Z comprises the group maleimidophenyl.

24. The composition of claim 20 wherein Z comprises the group

25. The composition of claim 18 wherein the intercalator has the chemical formula

26. The composition of claim 18 wherein the intercalator is biodegradable.

27. The composition of claim 26 wherein the intercalator comprises a peptide sequence and intercalating groups linked to the peptide sequence.

28. The composition of claim 27 wherein the peptide sequence comprises KK.

29. The composition of claim 18 wherein the intercalator has the formula

wherein

Ar₁ and Ar₂ are independently selected from the group consisting of ethidium bromide, acridine, mitoxanotrone, oxazolopyridocarbazole, ellipticine, N-methyl-2, 7-diazapyrenium, and derivatives capable of intercalating a polynucleotide;

each aa is independently an amino acid;

x and z are integers independently selected from 1 to 100;

y is an integer from 0 to 5;

aa₁ and aa₂ are lysine residues; and

N¹ and N² are nitrogens from the ε

-amino groups of the lysine residues aa₁ and aa₂.

30. The composition of claim 17 wherein the polynucleotide-associating moiety comprises a linker strand complementary to the desired polynucleotide.

31. The composition of claim 30 wherein substantially the entire linker strand sequence is complementary to the desired polynucleotide.

32. The composition of claim 30 wherein the linker strand comprises an extension of the desired polynucleotide.

33. The composition of claim 30 wherein the linker strand has a sequence with a plurality of regions, each region complementary to a portion of the desired polynucleotide.

34. The composition of claim 30 wherein the polynucleotide-associating moiety comprises a plurality of linker strands.

35. The composition of claim 30 wherein the polynucleotide-associating moiety comprises a first linker complementary to the desired polynucleotide and a second linker complementary to the desired polynucleotide.

36. The composition of claim 17 wherein the polynucleotide-associating moiety comprises a dendrimer polycation.

37. The composition of claim 17 wherein the polynucleotide-associating moiety comprises a major- or minor-groove binder.

38. The composition of claim 37 wherein the major- or minor-groove binder is selected from the group consisting of distamycin A and Hoechst dye 33258.

39. The composition of claim 17 wherein the functional agent comprises a cell recognition agent.

40. The composition of claim 17 wherein the functional agent comprises a membrane-permeabilization agent.

41. The composition of claim 17 further comprising a membrane-permeabilizer selected from the group consisting of polylysine, polyarginine, poly (lysine-arginine), polyamines, dendrimer polycations, cationic bile salts and amphipathic peptides.

42. The composition of claim 17 wherein the functional agent comprises a subcellular-localization agent selected from the group consisting of a nuclear-localization component, a lysosomal-localization component and a mitochondrial-localization component.

43. The composition of claim 42 wherein the subcellular-localization agent is selected from the group consisting of the sequence PKKKRKV/SEQ ID NO. 1, the sequence AAFEDLRVLS/SEQ ID NO. 4, the sequence KRPRP/SEQ ID NO. 5, the sequence KFERQ/SEQ ID NO. 6 and the sequence MLSLRQSIRFFKPATR/SEQ ID NO 7.

44. The composition of claim 17 wherein the functional agent comprises a polynucleotide-masking agent.

45. The composition of claim 44 wherein the polynucleotide-masking agent comprises polyethylene glycol linked to the polynucleotide-associating moiety.

46. The composition of claim 45 comprising polyethylene glycol-bis-acridine.

47. A composition for presenting a desired polynucleotide to a subcellular component of a eukaryotic cell comprising a desired polynucleotide associated with a cationic bile salt having the formula

wherein

X and Y are independently H or OH;

R³ is hydrogen (C₁-C₁₀) alkyl or (C₁-C₁₀) alkylamine; and

R⁴ is a positively charged linear or branched (C₁-C₃₀) alkyl or (C₁-C₃₀) alkylamine, wherein one or more of the carbon atoms may be substituted with NR′

, wherein R′

is H, (C₁-C₁₀)alkyl or (C₁-C₁₀) alkylamine.

48. The composition of claim 47 further comprising a lipid.

67. The composition of claim 47 further comprising dioleoylphosphatidylethanolamine.

49. A composition for presenting a desired polynucleotide to a subcellular component of a eukaryotic cell comprising a desired polynucleotide associated with a polynucleotide-masking agent having the chemical formula

wherein

n is an integer of 6 to 24;

Y is selected from the group consisting of hydroxy, ethanolamine, choline, glycerol, serine and inositol;

R¹ is (C₆-C₂₄) alkyl or (C₆-C₂₄) alkenyl;

R³ is H, or (C₁-C₁₀) alkyl or (C₁-C₁₀) alkylamine; and

R⁴ is a positively charged linear or branched (C₁-C₃₀) alkyl or (C₁-C₃₀) alkylamine, wherein one or more of the carbon atoms may be substituted with NR′

, wherein R′

is H or (C₁-C₁₀) alkyl or (C₁-C₁₀) alkylamine.

50. The composition of claim 49 wherein the lipid comprises lecithin acyl amine.

51. A composition for presenting a desired polynucleotide to a subcellular component of a eukaryotic cell comprising a desired polynucleotide associated with an amphipathic peptide capable of assuming a pH dependent α

-helix conformation.

52. The composition of claim 51 further comprising a lipid.

53. The composition of claim 51 further comprising a polyamine.

62. The composition of claim 51 wherein the amphipathic peptide α

-helix comprises a first and a second axial face such that the first face is substantially charged and the second face is substantially neutral.

63. The composition of claim 62 wherein the first face is negatively charged.

64. The composition of claim 63 wherein the amphipathic peptide comprises the sequence GALA/SEQ ID NO. 10.

54. The composition of 4 wherein the amphipathic peptide comprises an amphipathic peptide capable of assuming a β

-pleated sheet conformation.

55. The composition of 54 wherein the β

-pleated sheet amphipathic peptide has a first and second face such that the first face is positively charged and the second face is substantially neutral.