Transgenically produced non-secreted proteins

• US 20030213003A1
• Filed: 02/04/2003
• Published: 11/13/2003
• Est. Priority Date: 02/25/1997
• Status: Abandoned Application

• Current Assignee: GTC Biotherapeutics, Inc.
• Original Assignee: GTC Biotherapeutics, Inc.
• Inventors: DiTullio, Paul, Meade, Harry, Chen, Li-How
• Application Number:
  US10/358,037
• Publication Number:
  US 20030213003A1
• Time in Patent Office: Days
• US Class Current: 800/7
• 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 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: C07K 14/4713 Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
• CPC Class Codes: C07K 2319/036 targeting to the medium outside of the cell, e.g. type III secretion
• 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 9/88 Lyases (4.)

Abstract

The invention provides a method of making and secreting a non-secreted protein. The method includes expressing the protein from a nucleic acid construct which includes: (a) a mammary epithelial specific promoter; (b) a milk protein specific signal sequence which can direct the secretion of a protein; (c) optionally, a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein to allow secretion in the milk of a transgenic mammal, of the non-secreted protein; and (d) a sequence which encodes a non-secreted protein, wherein elements (a), (b), optionally (c), and (d) are preferably operatively linked in the order recited. Both glutamic acid decarboxylase (GAD) and myelin basic protein (MBP), which are cytoplasmic proteins, have been produced by the methods of the present invention. The invention also provides methods for treating diabetes and multiple sclerosis using proteins produced by the methods of the present invention.

Claims

1. A method of making and secreting a non-secreted protein comprising expressing the protein from a nucleic acid construct which comprises:

(a) a mammary epithelial specific promoter;

(b) a milk protein specific signal sequence which can direct the secretion of a non-secreted protein;

(c) a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein to allow secretion in the milk of a transgenic mammal of a non-secreted protein; and

(d) a sequence which encodes a non-secreted protein,

wherein elements (a), (b),(c), and (d) are preferably operatively linked in the order recited, thereby producing the non-secreted protein in the milk of the transgenic mammal.

2. The method of claim 1, wherein the mammary epithelial specific promoter is the β

-casein promoter sequence.

3. The method of claim 1, wherein the milk protein specific signal sequence is the β

-casein signal sequence.

4. The method of claim 1, wherein the non-secreted protein-coding sequence is of a human origin.

5. The method of claim 1, wherein the non-secreted protein-coding sequence codes for a truncated, nuclear, or a cytoplasmic polypeptide.

6. The method of claim 1, wherein the non-secreted protein-coding sequence codes for glutamic acid decarboxylase or myelin basic protein.

7. The method of claim 1, wherein the non-secreted protein is a mutant protein which lacks a biological activity of the wild type protein.

8. The method of claim 1, wherein the transgenic mammal is selected from the group consisting of sheep, mice, pigs, cows and goats.

9. An isolated nucleic acid construct which comprises:

(a) a mammary epithelial specific promoter;

(b) a milk protein specific signal sequence which can direct the secretion of a protein;

(c) a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein to allow secretion in the milk of a transgenic mammal of a non-secreted protein; and

(d) a sequence which encodes a non-secreted protein,

wherein elements (a), (b), (c), and (d) are preferably operatively linked in the order recited, thereby producing the non-secreted protein in the milk of the transgenic mammal.

10. The method of claim 9, wherein the mammary epithelial specific promoter sequence is the β

-casein promoter sequence.

11. The method of claim 9, wherein the milk protein specific signal sequence is the β

-casein signal sequence.

12. The method of claim 9, wherein the non-secreted protein-coding sequence is of a human origin.

13. The method of claim 9, wherein the non-secreted protein-coding sequence codes for a truncated, nuclear, or a cytoplasmic polypeptide.

14. The method of claim 9, wherein the non-secreted protein-coding sequence codes for glutamic acid decarboxylase or myelin basic protein.

15. The method of claim 9, wherein the non-secreted protein is a mutant protein which lacks a biological activity of the wild type protein.

16. A method for providing a non-secreted protein in the milk of a transgenic mammal, comprising obtaining milk from a transgenic mammal having introduced into its germline a nucleic acid construct comprising a heterologous non-secreted protein-coding sequence operatively linked to a milk protein specific signal sequence and a mammary epithelial specific promoter sequence that result in the preferential expression of the protein-coding sequence in mammary gland epithelial cells, thereby secreting the heterologous non-secreted protein in the milk of the mammal

17. The method of claim 16, wherein the non-secreted protein is inactive.

18. The method of claim 16, wherein the transgenic mammal is selected from the group consisting of sheep, mice, pigs, cows and goats.

19. The method of claim 16, wherein the mammary epithelial specific promoter is selected from the group consisting of the beta lactoglobulin promoter, whey acid protein promoter, β

-casein promoter and the lactalbumin promoter.

20. The method of claim 16, wherein the milk protein specific signal sequence is β

-casein signal sequence.

21. The method of claim 16, wherein the non-secreted protein-coding sequence is of a human origin.

22. The method of claim 16, wherein the non-secreted protein-coding sequence codes for a truncated, nuclear, or a cytoplasmic polypeptide.

23. The method of claim 16, wherein the non-secreted protein-coding sequence codes for glutamic acid decarboxylase or myelin basic protein.

24. The method of claim 16, wherein the non-secreted protein-coding sequence codes for an inactive form of a glutamic acid decarboxylase.

25. The method of claim 16, wherein the non-secreted polypeptide is purified from the milk of a transgenic mammal.

26. A method of inducing tolerance in a subject to an antigen, comprising:

providing a tolerogen expressed in a transgenic mammal which comprises the antigen; and

administering the tolerogen to the subject in an amount sufficient to induce tolerance to the antigen.

27. The method of claim 26, wherein the tolerogen is administered orally to the subject in the milk of a transgenic mammal.

28. The method of claim 26, wherein the tolerogen is an inactive protein.

29. The method of claim 26, wherein the tolerogen is a non-secreted protein.

30. The method of claim 26, wherein the tolerogen is a protein antigen fused to all or part of a secreted protein.

31. The method of claim 26, wherein the antigen is an antigen which is characteristic of an autoimmune disorder selected from the group consisting of diabetes, lupus, multiple sclerosis and rheumatoid arthritis.

32. The method of claim 26, wherein the subject is at risk of developing, or has, an anutoimmune disorder selected from the group consisting of diabetes, lupus, multiple sclerosis and rheumatoid arthritis.

33. A method of treating insulin-dependent diabetes mellitus (IDDM) in a subject, comprising administering to the subject therapeutically effective amount of a transgenically produced tolerogen which comprises glutamic acid decarboxylase, or an effective amount of a fusion protein which comprises glutamic acid decarboxylase.

34. The method of claim 33, wherein the subject is orally administered milk from a transgenic mammal which expresses the transgenically produced tolerogen which comprises glutamic acid decarboxylase, or an effective amount of a fusion protein which comprises glutamic acid decarboxylase.

35. The method of claim 33, wherein the transgenically produced tolarogen is in inactive form.

36. A method for treating multiple sclerosis in a subject, comprising administering to the subject therapeutically effective amount of a transgenically produced tolerogen which comprises myelin basic protein, or an effective amount of a fusion protein which comprises myelin basic protein.

37. The method of claim 36, wherein the subject is orally administered milk from a transgenic mammal which expresses the transgenically produced tolerogen which comprises myelin basic protein, or an effective amount of a fusion protein which comprises myelin basic protein.

38. The method of claim 36, wherein the transgenically produced tolarogen is in inactive form.

39. A therapeutic composition which comprises a therapeutically effective amount of the transgenically produced myelin basic protein and a pharmaceutically-acceptable carrier or diluent.

40. The method of claim 39, wherein the pharmaceutically-acceptable carrier or diluent comprises the milk of a transgenic mammal.

41. A fusion protein which comprises:

a non-secreted protein;

a milk protein specific signal sequence which directs the secretion of the protein; and

a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein to allow secretion in the milk of a transgenic mammal, of the non-secreted protein.

42. A method of inducing tolerance in a transgenic mammal to an antigen,

comprising expressing a tolerogen in the milk of the transgenic mammal at a level sufficient to induce tolerance.

43. The method of claim 42, wherein the antigen is selected from the group consisting of a xenoantigen, an alloantigen and an autoantigen.

44. The method of claim 42, wherein the transgenic mammal is a transgenic dairy mammal.

45. The method of claim 44, wherein the transgenic dairy mammal is selected from the group consisting of goats, sheep and cows.

46. The method of claim 42, wherein the tolerogen is a non-secreted protein.

47. The method of claim 42, wherein the antigen is an antigen which is characteristic of an autoimmune disorder selected from the group consisting of diabetes, lupus, multiple sclerosis, and rheumatoid arthritis.

48. A transgenically produced non-secreted polypeptide, wherein the polypeptide is secreted.

49. The transgenically produced non-secreted polypeptide of claim 48, wherein the transgenically produced non-secreted polypeptide is secreted into the milk of a transgenic mammal.

50. The transgenically produced non-secreted polypeptide of claim 48, wherein the transgenically produced non-secreted polypeptide is selected from the group consisting of a truncated, nuclear, and cytoplasmic polypeptide.

53. The transgenically produced non-secreted polypeptide of claim 49, wherein the transgenic mammal is selected from the group consisting of sheep, mice, pigs, cows and goats.

51. The transgenically produced non-secreted polypeptide of claim 51, wherein the cytoplasmic polypeptide is glutamic acid decarboxylase or myelin basic protein.

52. The transgenically produced non-secreted polypeptide of claim 51, wherein glutamic acid decarboxylase is expressed in an inactive form.