DNA sequences to target proteins to the mammary gland for efficient secretion

• US 5,304,489 A
• Filed: 10/24/1990
• Issued: 04/19/1994
• Est. Priority Date: 02/17/1987
• Status: Expired due to Term

• Current Assignee: Pharming Intellectual Property B.V. (Pharming Group N.V.)
• Original Assignee: Genpharm Incorporated (Bristol-Myers Squibb Company)
• Inventors: Rosen, Jeffrey M.
• Primary Examiner(s): Chambers, Jasemine C.
• Application Number:
  US07/602,066
• Time in Patent Office: 1,273 Days
• Field of Search: 435/320.1, 435/317.1, 435/172.3, 536/27, 800/2, 800/DIG. 1, 935/111
• US Class Current: 435/320.1
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/20 Animal model comprising regulated expression system
• CPC Class Codes: A01K 2227/10 Mammal
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2227/108 Swine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/02 Breeding vertebrates
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A23C 2230/05 Milk or milk products from transgenic animals
• CPC Class Codes: A23C 9/20 Dietetic milk products not covered by groups A23C9/12 - A23C9/18
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/4732 Casein in foodstuffs A23J
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/61 containing an enzyme fusion for detection (lacZ, luciferase)
• 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 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12N 2830/42 : being an intron or intervening sequence for splicing and/or stability of RNA
• CPC Class Codes: C12N 2830/85 : mammalian
• CPC Class Codes: C12N 9/1033 : Chloramphenicol O-acetyltransferase (2.3.1.28)

Abstract

Described is a method of targeting specific genes to the mammary gland which results in the efficient synthesis and secretion of biologically important molecules. Further, there is described as a composition of matter, a transgenic mammal having the ability to reproduce itself and being suitable for the secretion of biologically active agents into its milk. Additionally there is disclosed as a composition of matter, recombinant DNA gene complexes designed to integrate into a mammalian genome and to synthesize and secrete biological active agents into the milk. Furthermore methods of producing and using altered milk are disclosed.

Claims

1. A recombinant DNA complex to provide preferential mammary gland expression and secretion of a selected polypeptide comprising:

(a) a casein promoter;

(b) a transcribable DNA segment coding for said selected polypeptide, wherein said promoter does not naturally control the transcription of said DNA segment; and

(c) secretory DNA encoding a signal peptide functional in mammary secretory cells operably linked between said promoter and said DNA segment to facilitate secretion of said selected polypeptide,

(d) a casein enhancer sequence capable of conferring enhanced preferential transcription of said recombinant DNA complex in mammary secretory cells when said complex is integrated into the genome of a non-human mammal, and

(e) an intron from a casein gene.

2. The recombinant DNA complex of claim 1 wherein said promoter is from a casein gene selected from the group consisting of α

-casein, β

-casein, γ

-casein and κ

-casein.

3. The recombinant DNA complex of claim 2 wherein said promoter is a β

-casein promoter.

4. The recombinant DNA complex of claim 3 wherein said casein enhancer is selected from the group consisting of α

-casein, β

-casein, γ

-casein and κ

-casein enhancers.

5. The recombinant DNA complex of claim 1 further comprising a mouse mammary tumor virus glucocorticoid response element.

6. The recombinant DNA complex of claim 1 wherein said secretory DNA encoding a signal peptide is from a mammary gland specific gene.

7. The recombinant DNA complex of claim 6 wherein said mammary gland specific gene is selected from the group consisting of whey acid protein and casein genes.

8. The recombinant DNA complex of claim 7 wherein said mammary gland specific gene is a casein gene.

9. The recombinant DNA complex of claim 6 wherein said intron is from a casein gene selected from the group consisting of α

-casein, β

-casein, and κ

-casein genes.

10. The recombinant DNA complex of claim 1 wherein said transcribable DNA segment encodes a normally secreted polypeptide and said secretory DNA encodes the endogenous signal peptide sequence of said secreted polypeptide.

11. The recombinant DNA complex of claim 1 further comprising:

(f) DNA encoding a 5'"'"' untranslated sequence from a mammary gland specific gene.

12. The recombinant DNA complex of claim 11 further comprising:

(g) DNA encoding a 3'"'"' untranslated sequence.

13. The recombinant DNA complex of claim 1, wherein said intron links said transcribable DNA segment to said promoter.

14. The recombinant DNA complex of claim 1, wherein said intron is located within said transcribable DNA segment.