Transgenic ungulates having reduced prion protein activity and uses thereof

• US 20050097627A1
• Filed: 11/10/2003
• Published: 05/05/2005
• Est. Priority Date: 11/08/2002
• Status: Active Grant

• Current Assignee: SAB LLC (W.P. Carey, Inc. (Real Estate))
• Original Assignee: Kyowa Hakko Kirin Company Limited (Kirin Holdings Company Limited)
• Inventors: Kuroiwa, Yoshimi, Tomizuka, Kazuma, Ishida, Isao, Robl, James M.
• Granted Patent: US 7,429,690 B2
• Time in Patent Office: Days
• US Class Current: 800/15
• 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 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2227/101 Bovine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 2267/02 Animal zootechnically ameliorated
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2317/12 isolated from milk
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Abstract

The invention provides cloned transgenic ungulates (e.g., bovines) in which prion protein activity is reduced by one or more genetically engineered mutations. Desirably, these transgenic bovines are also genetically modified to express xenogenous (e.g., human) antibodies. Because of their resistance to prion-related diseases such as bovine spongiform encephalopy (also known as mad cow disease), these bovines are a safer source of human antibodies for pharmaceutical uses and a safer source of agricultural products.

Claims

1. A bovine comprising a non-naturally occurring mutation in one or both alleles of an endogenous prion nucleic acid

2. The bovine of claim 1, wherein said mutation reduces the expression of functional prion protein.

3. The bovine of claim 2, wherein said mutation substantially eliminates the expression of functional prion protein.

4. The bovine of claim 1, wherein said mutation is hemizygous.

5. The bovine of claim 1, wherein said mutation is homozygous.

6. The bovine of claim 1, wherein said mutation comprises an insertion of a positive selection marker into a prion nucleic acid.

7. The bovine of claim 1, wherein said mutation comprises an insertion of a STOP codon into a prion nucleic acid.

8. The bovine of claim 1, wherein said mutation comprises a deletion of one or more nucleotides in a prion nucleic acid.

9. The bovine of claim 1, comprising one or more nucleic acids comprising one or more transgenes and expressing an mRNA or protein encoded by said transgene(s).

10. The bovine of claim 1, comprising one or more nucleic acids comprising all or part of a xenogenous immunoglobulin (Ig) gene which undergoes rearrangement and expresses more than one xenogenous Ig molecule.

11. The bovine of claim 10, comprising one or more nucleic acids encoding a xenogenous antibody.

12. The bovine of claim 11, wherein said xenogenous antibody is a human antibody.

13. The bovine of claim 12, wherein said antibody is expressed in serum and/or milk.

14. The bovine of claim 1, comprising a mutation that reduces the expression of an endogenous antibody.

15. The bovine of claim 14, wherein said mutation reduces the expression of functional IgM heavy chain.

16. The bovine of claim 15, wherein said mutation substantially eliminates the expression of functional IgM heavy chain.

17. The bovine of claim 14, wherein said mutation reduces the expression of functional Ig light chain.

18. The bovine of claim 17, wherein said mutation substantially eliminates the expression of functional Ig light chain.

19. The bovine of claim 14, wherein said mutation reduces the expression of functional IgM heavy chain and functional Ig light chain.

20. The bovine of claim 19, wherein said mutation substantially eliminates the expression of functional IgM heavy chain and functional Ig light chain.

21. The bovine of claim 1, comprising a mutation in one or both alleles of an endogenous nucleic acid encoding alpha-(1,3)-galactosyltransferase.

22. The bovine of claim 1, comprising a mutation in one or both alleles of an endogenous nucleic acid encoding J chain.

23. The bovine of claim 1, comprising a nucleic acid encoding an exogenous J chain.

24. The bovine of claim 23, wherein said J chain is a human J chain.

25. A bovine cell comprising a non-naturally occurring mutation in one or both alleles of an endogenous prion nucleic acid.

26. The cell of claim 25, wherein said mutation reduces the expression of functional prion protein.

27. The cell of claim 26, wherein said mutation substantially eliminates the expression of functional prion protein.

28. The cell of claim 25, wherein said mutation is hemizygous.

29. The cell of claim 25, wherein said mutation is homozygous.

30. The cell of claim 25, wherein said cell is a fetal fibroblast.

31. The cell of claim 25, wherein said cell is a B-cell.

32. A method for producing a transgenic bovine cell having reduced expression of functional prion protein, comprising introducing a first prion gene targeting vector into a bovine cell under conditions that allow homologous recombination between said first vector and a first allele of an endogenous prion nucleic acid in said cell, thereby introducing a hemizygous mutation in said cell.

33. The method of claim 32, further comprising introducing said first vector into said cell under conditions that allow homologous recombination between said first vector and a second allele of an endogenous prion nucleic acid in said cell, thereby introducing homozygous mutation in said cell.

34. The method of claim 32, further comprising introducing a second prion gene targeting vector that has a different antibiotic resistance gene than said first vector into said cell under conditions that allow homologous recombination between said second vector and a second allele of an endogenous prion nucleic acid in said cell, thereby introducing a homozygous mutation in said cell.

35. The method of claim 32, wherein said cell is a bovine fibroblast.

36. The method of claim 35, wherein said cell is a bovine fetal fibroblast.

37. A method for producing a transgenic bovine having reduced expression of functional prion protein, said method comprising the steps of:

(a) inserting a cell, a chromatin mass from a cell, or a nucleus from a cell into an oocyte, wherein said cell comprises a first mutation in an endogenous prion nucleic acid; and

(b) transferring said oocyte or an embryo formed from said oocyte into the uterus of a host bovine under conditions that allow said oocyte or said embryo to develop into a fetus.

38. The method of claim 37, wherein said fetus develops into a viable offspring.