Transgenically produced non-secreted proteins

• US 6,528,699 B1
• Filed: 02/24/1998
• Issued: 03/04/2003
• Est. Priority Date: 02/25/1997
• Status: Expired due to Term

• Current Assignee: LFB USA, Inc. (Agence des Participations de l’Etat)
• Original Assignee: Genzyme Transgenics Corporation
• Inventors: DiTullio, Paul, Meade, Harry, Chen, Li-How
• Primary Examiner(s): Crouch, Deborah
• Assistant Examiner(s): TON, THAIAN N
• Application Number:
  US09/028,551
• Time in Patent Office: 1,834 Days
• Field of Search: 800/3, 800/7, 800/14, 800/21, 800/13, 800/24, 800/18, 800/22, 800/25, 435/320.1, 435/455, 435/463, 435/325, 435/69.1, 514/44, 536/23.1, 536/23.5
• 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:

introducing into a mammal selected from the group consisting of sheep, mice, pigs, cows, rabbits and goats 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 protein;

(c) a sequence other than a signal sequence which encodes a sufficient portion of the amino terminal coding region from the amino terminal end of a milk 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 operatively linked in the order recited;

expressing the protein, 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 sequence encoding the non-secreted protein is of a human origin.

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

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

7. The method of claim 1, wherein the milk protein is β

-casein.

8. The method of claim 1, wherein the amino terminal coding region comprises about 10% to 30% of the amino terminal coding region of a milk protein.

9. A method for providing a non-secreted protein in the milk of a transgenic mammal, comprising

obtaining milk from a transgenic mammal selected from the group consisting of sheep, mice, pigs, cows, rabbits and goats having introduced into its germline a nucleic acid construct comprising:

(a) a mammary epithelial specific promoter sequence,

(b) a milk protein specific signal sequence,

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

(d) a sequence encoding a non-secreted protein,

wherein elements (a), (b), (c), and (d) are operatively linked in the order recited and the heterologous non-secreted protein is expressed and secreted into the milk of the mammal, thereby providing a non-secreted protein in the milk of the mammal.

10. The method of claim 9, wherein the non-secreted protein is inactive.

11. The method of claim 9, wherein the mammary epithelial specific promoter is selected from the group consisting of a beta lactoglobulin promoter, whey acid protein promoter, β

-casein promoter and a lactalbumin promoter.

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

-casein signal sequence.

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

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

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

16. The method of claim 9, wherein the non-secreted protein-coding sequence codes for an inactive form of a glutamic acid decarboxylase.

17. The method of claim 9, wherein the non-secreted protein is purified from the milk of a transgenic mammal.

18. The method of claim 9, wherein the non-secreted protein codes for an inactive form of myelin basic protein.

19. The method of claim 9, wherein the amino terminal coding region is from a milk protein selected from the group consisting of caseins, beta lactoglobulin, whey acid protein, and lactalbumin.

20. 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 other than a signal sequence which encodes a sufficient portion of the amino terminal coding region from the amino terminal end of a milk 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 operatively linked in the order recited.

21. The construct of claim 20, wherein the mammary epithelial specific promoter sequence is the β

-casein promoter sequence.

22. The construct of claim 20, wherein the milk protein specific signal sequence is the β

-casein signal sequence.

23. The construct of claim 20, wherein the sequence encoding the non-secreted protein is of a human origin.

24. The construct of claim 20, wherein the sequence encoding the non-secreted protein codes for a truncated, nuclear, or a cytoplasmic polypeptide.

25. The construct of claim 20, wherein the sequence encoding the non-secreted protein codes for glutamic acid decarboxylase or myelin basic protein.

26. The construct of claim 20, wherein the milk protein is β

-casein.

27. The construct of claim 20, wherein the amino terminal coding region comprises about 10% to 30% of the amino terminal coding region of a milk protein.

28. A method of making and secreting a non-secreted protein comprising:

introducing into a mammal selected from the group consisting of sheep, mice, pigs, cows, rabbits and goats 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 protein;

(c) a sequence other than a signal sequence which encodes about 10% of the amino terminal coding region from the amino terminal end of a milk 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 operatively linked in the order recited; and

expressing the protein, thereby producing the non-secreted protein in the milk of the transgenic mammal.

29. The method of claim 28, wherein the amino terminal coding region comprises about 20% of the amino terminal coding region of a milk protein.

30. The method of claim 28, wherein the amino terminal coding region comprises about 30% of the amino terminal coding region of a milk protein.

31. A method for providing a non-secreted protein in the milk of a transgenic mammal, comprising

obtaining milk from a transgenic mammal selected from the group consisting of sheep, mice, pigs, cows, rabbits and goats having introduced into its germline a nucleic acid construct comprising:

(a) a mammary epithelial specific promoter sequence,

(b) a milk protein specific signal sequence,

(c) a sequence other than a signal sequence which encodes about 10% of the amino terminal coding region from the amino terminal end of a milk protein to allow secretion in the milk of a transgenic mammal of a non-secreted protein; and

(d) a sequence encoding a non-secreted protein,

wherein elements (a), (b), (c), and (d) are operatively linked in the order recited and the heterologous non-secreted protein is expressed and secreted into the milk of the mammal, thereby providing a non-secreted protein in the milk of the mammal.

32. The method of claim 31, wherein the amino terminal coding region comprises about 20% of the amino terminal coding region of a milk protein.

33. The method of claim 31, wherein the amino terminal coding region comprises about 30% of the amino terminal coding region of a milk protein.

34. 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 other than a signal sequence which encodes about 10% of the amino terminal coding region from the amino terminal end of a milk 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 operatively linked in the order recited.

35. The construct of claim 34, wherein the amino terminal coding region comprises about 20% of the amino terminal coding region of a milk protein.

36. The construct of claim 34, wherein the amino terminal coding region comprises about 30% of the amino terminal coding region of a milk protein.