Production of hSA-linked butyrylcholinesterases in transgenic mammals

• US 20060253913A1
• Filed: 04/10/2006
• Published: 11/09/2006
• Est. Priority Date: 12/21/2001
• Status: Abandoned Application

• Current Assignee: PharmAthene, Inc. (Altimmune, Inc.)
• Original Assignee: PharmAthene, Inc. (Altimmune, Inc.)
• Inventors: Huang, Yue-Jin, Lazaris, Anthoula, Karatzas, Costas N.
• Application Number:
  US11/401,390
• Publication Number:
  US 20060253913A1
• Time in Patent Office: Days
• US Class Current: 800/14
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/00 Genetically modified animals
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/102 Caprine
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12N 2830/40 being an insulator
• CPC Class Codes: C12N 2830/85 mammalian
• CPC Class Codes: C12N 2840/20 translation of more than one cistron

Abstract

The present invention provides methods for the large-scale production of recombinant butyrylcholinesterase fused to human serum albumin in cell culture, and in the milk and/or urine of transgenic mammals. The recombinant butyrylcholinesterase-albumin fusion protein of this invention can be used to treat and/or prevent organophosphate pesticide poisoning, nerve gas poisoning, cocaine intoxication, and succinylcholine-induced apnea.

Claims

1. A method for producing a fusion protein that comprises an enzymatically active butyrylcholinesterase (BChE) enzyme and a human serum albumin (hSA), comprising expressing said fusion protein in a recombinant cell that comprises a polynucleotide encoding said fusion protein operably linked to a promoter sequence.

2. The method of claim 1, wherein said promoter sequence is the cytomegalovirus (CMV) promoter.

3. The method of claim 1, wherein said polynucleotide and operably linked promoter are part of a plasmid.

4. The method of claim 1, wherein said BChE and said hSA are separated by an oligopeptide linker that permits independent folding and activity of said BChE.

5. The method of claim 4, wherein said oligopeptide linker is a polyglycine and serine linker.

6. The method of claim 1, wherein said cell is a BHK cell.

7. The method of claim 1, wherein said BChE is human BChE.

8. The method of claim 1, wherein said polynucleotide further comprises a signal sequence that directs secretion of said fusion protein by the cell.

9. The method of claim 1, wherein said BChE-hSA fusion protein comprises the amino acid sequence of SEQ ID NO:

50.

10. The method of claim 1, wherein said polynucleotide comprises the nucleotide sequence of SEQ ID NO:

49

11. The method of claim 1, wherein said cell further comprises a DNA sequence encoding a glycosyltransferase, operably linked to a promoter.

12. The method of claim 11, wherein said glycosyltransferase is expressed by said cell and glycosylates the BChE portion of the fusion protein.

13. An isolated fusion protein, comprising an enzymatically active BChE enzyme and a human serum albumin.

14. The isolated fusion protein of claim 13, wherein said fusion protein further comprises a signal sequence that directs secretion of said fusion protein from a cell.

15. The isolated fusion protein of claim 13, wherein said fusion protein further comprises a linker located between said BChE enzyme and said hSA protein and wherein said linker permits independent folding and activity of said BChE.

16. The isolated fusion protein of claim 13, wherein said linker comprises an amino acid sequence.

17. The isolated fusion protein of claim 16, wherein said amino acid sequence comprises at least 7 amino acid residues.

18. The isolated fusion protein of claim 17, wherein said amino acid residues are glycine and serine residues.

19. The isolated fusion protein of claim 13, wherein said BChE is a human BChE.

20. The isolated fusion protein of claim 19, wherein said human BChE-hSA fusion protein comprises the amino acid sequence of SEQ ID NO:

50.

21. The isolated fusion protein of claim 13, wherein the BChE portion of said fusion protein comprises one or more glycosyl residues.

22. The isolated fusion protein of claim 14, wherein said signal sequence directs secretion into milk.

23. The isolated fusion protein of claim 14, wherein said signal sequence directs secretion into urine.

24. An isolated polynucleotide, comprising:

(i) a nucleotide sequence encoding the fusion protein of claim 13, (ii) a promoter that directs expression of the fusion protein, and (iii) at least one signal sequence that directs secretion of the expressed fusion protein from a cell.

25. The isolated polynucleotide of claim 24, wherein the encoded fusion protein further comprises an oligopeptide linker.

26. The isolated polynucleotide of claim 25, wherein said oligopeptide linker comprises a polyglycine and a serine sequence.

27. The isolated polynucleotide of claim 24, wherein the BChE portion of said fusion protein is a human BChE.

28. The isolated polynucleotide of claim 27, wherein said human BChE-hSA fusion protein comprises the amino acid sequence of SEQ ID NO:

50.

29. The isolated polynucleotide of claim 24, wherein said polynucleotide comprises the nucleotide sequence of SEQ ID NO:

49.

30. The isolated polynucleotide of claim 24 wherein said signal sequence directs secretion into milk.

31. The isolated polynucleotide of claim 24, wherein said signal sequence directs secretion into urine.

32. The isolated polynucleotide of claim 24, wherein the promoter is a mammary gland-specific promoter selected from the group consisting of a WAP (whey acidic protein) promoter and a casein promoter.

33. The isolated polynucleotide of claim 24, wherein the promoter is a urinary endothelium-specific promoter selected from the group consisting of a uroplakin promoter or a uromodulin promoter.

34. A recombinant cell that comprises the isolated polynucleotide of claim 24.

35. The recombinant cell of claim 34, wherein the cell is a MAC-T (mammary epithelial) cell.

36. The recombinant cell of claim 24, wherein the cell is a BHK (baby hamster kidney) cell.

37. The recombinant cell of claim 24, wherein the cell is selected from the group of embryonic stem cells, embryonal carcinoma cells, primordial germ cells, oocytes, or sperm.

38. A non-human mammalian embryo that comprises the isolated polynucleotide of claim 24 as part of its genome.

39. A non-human mammalian embryo which comprises the polynucleotide of claim 32 as part of its genome.

40. A non-human mammalian embryo which comprises the polynucleotide of claim 33.

41. A non-human transgenic mammal that upon lactation, expresses in its milk the fusion protein of claim 13.

42. The non-human transgenic mammal of claim 41, wherein the linker portion of said fusion protein comprises at least 7 amino acid residues.

43. The non-human transgenic mammal of claim 42, wherein said amino acid residues are glycine and serine residues.

44. The non-human transgenic mammal of claim 41, wherein said mammal is a mouse.

45. The non-human transgenic mammal of claim 41, wherein said mammal is a goat.

46. A non-human transgenic mammal that upon urination, expresses in its urine the fusion protein of claim 13.

47. The non-human transgenic mammal of claim 46, wherein the linker portion of said fusion protein comprises at least 7 amino acid residues.

48. The non-human transgenic mammal of claim 47, wherein said amino acid residues are glycine and serine residues.

49. The non-human transgenic mammal of claim 46, wherein said mammal is a mouse.

50. The non-human transgenic mammal of claim 46, wherein said mammal is a goat.

51. A non-human transgenic mammal whose genome comprises the polynucleotide of claim 24.

52. The non-human transgenic mammal of claim 51, wherein the promoter is a mammary gland-specific promoter selected from the group consisting of a WAP (whey acidic protein) promoter and a casein promoter.

53. The non-human transgenic mammal of claim 51, wherein the genome of the mammal further comprises a DNA sequence encoding a glycosyltransferase, operably linked to a mammary gland-specific promoter, and a signal sequence that provides secretion of the glycosyltransferase.

54. The non-human transgenic mammal of claim 53, wherein the mammary gland-specific promoter is a casein promoter or a whey acidic protein (WAP) promoter.

55. The non-human transgenic mammal of claim 51, wherein the promoter is a urinary endothelium-specific promoter selected from the group consisting of a uroplakin promoter or a uromodulin promoter.

56. The non-human transgenic mammal of claim 51, wherein the genome of the mammal further comprises a DNA sequence encoding a glycosyltransferase, operably linked to a urinary gland-specific promoter, and a signal sequence that provides secretion of the glycosyltransferase.

57. The isolated polynucleotide of claim 56, wherein the promoter is a urinary endothelium-specific promoter selected from the group consisting of a uroplakin promoter or a uromodulin promoter.

58. The non-human transgenic mammal of claim 51, wherein said mammal is a mouse.

59. The non-human transgenic mammal of claim 51, wherein said mammal is a goat.

60. A method for producing a transgenic mammal that upon lactation secretes the fusion protein of claim 13 in its milk, which method comprises allowing an embryo, into which at least one genetically-engineered DNA sequence, comprising (i) a sequence encoding said fusion protein;

(ii) a mammary gland-specific promoter; and

(iii) a signal sequence that provides secretion of the fusion protein into the milk of the mammal, has been introduced, to grow when transferred into a recipient female mammal, resulting in the recipient female mammal giving birth to the transgenic mammal.

61. The method of claim 60, further comprising introducing the genetically-engineered DNA sequence into a cell of the embryo, or into a cell that will form at least part of the embryo.

62. The method of claim 61, wherein introducing the genetically-engineered DNA sequence comprises pronuclear or cytoplasmic microinjection of the DNA sequence.

63. The method of claim 61, wherein introducing the genetically-engineered DNA sequence comprises combining a mammalian cell stably transfected with the DNA sequence with a non-transgenic mammalian embryo.

64. The method of claim 61, wherein introducing the genetically-engineered DNA sequence comprises the steps of (a) introducing the DNA sequence into a non-human mammalian oocyte;

and (b) activating the oocyte to develop into an embryo.

65. A method for producing a transgenic mammal that upon lactation secretes the fusion protein of claim 13, in its milk, which method comprises cloning or breeding of a transgenic mammal, the genome of which comprises a DNA sequence encoding said fusion protein, operably linked to a mammary gland-specific promoter, wherein the sequence further comprises a signal sequence that provides secretion of the fusion protein into the milk of the mammal.

66. A method for producing a transgenic mammal that secretes the fusion protein of claim 13, in its urine, which method comprises allowing an embryo, into which at least one genetically-engineered DNA sequence, comprising (i) a sequence encoding said fusion protein;

(ii) a urinary endothelium-specific promoter; and

(iii) a signal sequence that provides secretion of the fusion protein into the urine of the mammal, has been introduced, to grow when transferred into a recipient female mammal, resulting in the recipient female mammal giving birth to the transgenic mammal.

67. The method of claim 66, which further comprises introducing the genetically-engineered DNA sequence into a cell of the embryo, or into a cell that will form at least part of the embryo.

68. The method of claim 67, wherein introducing the genetically-engineered DNA sequence comprises pronuclear or cytoplasmic microinjection of the DNA sequence.

69. The method of claim 67, wherein introducing the genetically-engineered DNA sequence comprises combining a mammalian cell stably transfected with the DNA sequence with a non-transgenic mammalian embryo.

70. The method of claim 67 wherein introducing the genetically-engineered DNA sequence comprises the steps of (a) introducing the DNA sequence into a non-human mammalian oocyte;

and (b) activating the oocyte to develop into an embryo.

71. A method for producing a transgenic mammal that secretes the fusion protein of claim 13, in its urine, which method comprises cloning or breeding of a transgenic mammal, the genome of which comprises a DNA sequence encoding said fusion protein, operably linked to a urinary endothelium-specific promoter, wherein the sequence further comprises a signal sequence that provides secretion of the fusion protein into the urine of the mammal.

72. A method for producing the fusion protein of claim 13, comprising:

(a) inducing or maintaining lactation of a transgenic mammal, the genome of which comprises a DNA sequence encoding said fusion protein, operably linked to a mammary gland-specific promoter, wherein the sequence further comprises a signal sequence that provides secretion of the fusion protein into the milk of the mammal; and

(b) extracting milk from the lactating mammal.

73. The method according to claim 72, which comprises the additional step of isolating the fusion protein from the extracted milk.

74. The method according to claim 73, further comprising purifying the fusion protein.

75. The milk of a non-human mammal comprising the fusion protein of claim 13.

78. A method for producing the fusion protein of claim 13, comprising extracting urine from a transgenic mammal, the genome of which comprises a DNA sequence encoding said fusion protein, operably linked to a urinary endothelium-specific promoter, where the sequence further comprises a signal sequence that provides secretion of said fusion protein into the urine of the mammal.

79. The method according to claim 78, comprising the additional step of isolating the fusion protein from the extracted urine.

80. The method according to claim 79, further comprising purifying the fusion protein.

81. Urine of a non-human mammal comprising the fusion protein of claim 13.

82. A composition comprising the fusion protein of claim 13 in a pharmaceutically acceptable carrier.

83. A method for treating organophosphate poisoning, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of claim 82.

84. A method for the treatment of post-surgical, succinyl choline-induced apnea, which comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of claim 82.

85. A method for the treatment of cocaine intoxication, which comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of claim 82.

76. The milk of claim 76, where the milk is whole milk.

77. The milk of claim 76, where the milk is defatted milk.