Transgenic animals secreting proteins into milk

• US 7,045,676 B1
• Filed: 08/31/1992
• Issued: 05/16/2006
• Est. Priority Date: 04/09/1986
• Status: Expired due to Term

• Current Assignee: Genzyme Corporation (Sanofi )
• Original Assignee: GTC Biotherapeutics, Inc.
• Inventors: Gordon, Katherine, Groet, Suzanne
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US07/938,322
• Time in Patent Office: 5,006 Days
• Field of Search: 800/7, 800/14, 800/25
• 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 2227/10 Mammal
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/89 using microinjection
• CPC Class Codes: C12N 2730/10122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12N 2830/85 mammalian
• CPC Class Codes: C12N 9/6459 t-plasminogen activator (3.4.21.68), i.e. tPA
• CPC Class Codes: C12Y 304/21069 Protein C activated (3.4.21.69)

Abstract

A DNA sequence containing a gene encoding a protein, the gene being under the transcriptional control in the DNA sequence of a mammalian milk protein promoter which does not naturally control the transcription of the gene, such DNA sequence including DNA enabling secretion of the protein.

Claims

1. A method of producing a recombinant protein comprising the steps of:

(a) inserting into an embryo of a nonhuman mammal a DNA construct comprising a gene encoding said protein, said gene being under transcriptional control of a milk protein promoter sequence that does not naturally control transcription of said gene, said DNA construct further comprising a DNA sequence encoding a signal peptide for enabling secretion of said protein into the milk of said mammal,

(b) transferring the embryo to a female mammal of the same species,

(c) allowing said embryo to develop into an adult nonhuman mammal whose genome comprises said DNA construct,

(d) inducing lactation in said mammal or transgenic progeny of said mammal, wherein the progeny'"'"'s genome comprises said DNA construct,

(e) collecting milk from said lactating mammal or lactating progeny, and

(f) isolating said protein from said collected milk.

2. The method of claim 1 wherein said protein is human tissue plasminogen activator.

3. The method of claim 1 wherein the signal peptide comprises a secretion signal sequence which is cleaved from said protein after secretion into said milk.

4. The method of claim 1 wherein the signal peptide is the secretion signal peptide naturally associated with said protein.

5. The method of claim 1 wherein the signal peptide is a secretion signal peptide naturally associated with a milk serum protein.

6. The method of claim 1 wherein said milk protein is a milk serum protein.

7. The method of claim 6, wherein said milk serum protein is α

-lactalbumin.

8. The method of claim 1, wherein said gene is under the transcriptional control of a sequence upstream from the transcriptional start site of a mammalian milk protein which includes a milk protein promoter and which does not naturally control the transcription of said gene.

9. The method of claim 8, wherein said secretion-enabling DNA comprises a secretion signal-encoding sequence interposed between said gene and said promoter.

10. The method of claim 9, wherein said signal encoding sequence is the signal encoding sequence naturally associated with said gene encoding said protein.

11. The method of claim 9, wherein said signal encoding sequence is the signal encoding sequence naturally associated with said mammalian milk protein promoter.

12. The method of claim 8, wherein said milk protein is a milk serum protein.

13. The method of claim 12, wherein said milk serum protein is α

-lactalbumin.

14. The method of claim 8, wherein said DNA sequence includes a transcriptional stop sequence.

15. The method of claim 14 wherein said stop sequence is a SV40 virus DNA sequence.

16. The method of claim 15 wherein said stop sequence is contained in the polyadneylation sequence of SV40.

17. A method of producing a recombinant protein comprising the steps of:

(a) providing an adult nonhuman mammal developed from an embryo of a nonhuman mammal, said embryo comprising a DNA construct comprising a gene encoding said protein, said gene being under transcriptional control of a milk protein promoter sequence that does not naturally control transcription of said gene, said DNA construct further comprising a DNA sequence encoding a signal peptide for enabling secretion of said protein into the milk of said mammal, wherein the genome of said mammal comprises said DNA construct,

(b) inducing lactation in said mammal,

(c) collecting milk from said lactating mammal, and

(d) isolating said protein from said collected milk.

18. The method of claim 17, wherein said milk protein is a milk serum protein.

19. The method of claim 18, wherein said milk serum protein is α

-lactalbumin.

20. The method of claim 17, wherein said gene is under the transcriptional control of a sequence upstream from the transcriptional start site of a mammalian milk protein which includes a milk protein promoter and which does not naturally control the transcription of said gene.

21. The method of claim 20, wherein said secretion-enabling DNA comprises a secretion signal-encoding sequence interposed between said gene and said promoter.

22. The method of claim 21, wherein said signal encoding sequence is the signal encoding sequence naturally associated with said gene encoding said protein.

23. The method of claim 21, wherein said signal encoding sequence is the signal encoding sequence naturally associated with said mammalian milk protein promoter.

24. The method of claim 20, wherein said milk protein is a milk serum protein.

25. The method of claim 24, wherein said milk serum protein is α

-lactalbumin.

26. The method of claim 20, wherein said DNA sequence includes a transcriptional stop sequence.

27. The method of claim 26 wherein said stop sequence is derived from SV40 virus DNA.

28. The method of claim 27 wherein said stop sequence is contained in the polyadneylation sequence of SV40.

29. A method of producing a recombinant protein comprising the steps of:

(a) providing a lactating transgenic mammal whose genome comprises a DNA construct comprising a gene encoding said recombinant protein, said gene being under transcriptional control of a milk protein promoter sequence that does not naturally control transcription of said gene, said DNA construct further comprising a DNA sequence encoding a signal peptide for enabling secretion of said recombinant protein into the milk of said mammal,

(b) collecting milk from said lactating mammal, and

(c) isolating said recombinant protein from said collected milk.

30. The method of claim 29, wherein said recombinant protein is a human tissue plasminogen activator or a hepatitis B surface antigen.

31. The method of claim 29, wherein said milk protein is a milk serum protein.

32. The method of claim 31, wherein said milk serum protein is α

-lactalbumin.

33. The method of claim 29, wherein said gene is under the transcriptional control of a sequence upstream from the transcriptional start site of a mammalian milk protein which includes a milk protein promoter and which does not naturally control the transcription of said gene.

34. The method of claim 33, wherein said secretion-enabling DNA comprises a secretion signal-encoding sequence interposed between said gene and said promoter.

35. The method of claim 34, wherein said signal encoding sequence is the signal encoding sequence naturally associated with said gene encoding said protein.

36. The method of claim 34, wherein said signal encoding sequence is the signal encoding sequence naturally associated with said mammalian milk protein promoter.

37. The method of claim 33, wherein said milk protein is a milk serum protein.

38. The method of claim 37, wherein said milk serum protein is α

-lactalbumin.

39. The method of claim 33, wherein said DNA sequence includes a transcriptional stop sequence.

40. The method of claim 39, wherein said stop sequence is a SV40 virus DNA sequence.

41. The method of claim 40 wherein said stop sequence is contained in the polyadneylation sequence of SV40.