Method for producing biologically active human factor VIII in the milk of transgenic animals driven by mammary-specific expression cassettes

• US 20050229261A1
• Filed: 04/09/2004
• Published: 10/13/2005
• Est. Priority Date: 04/09/2004
• Status: Active Grant

• Current Assignee: National Chung Hsing University (Government of The Republic of China)
• Original Assignee: National Chung Hsing University (Government of The Republic of China)
• Inventors: Cheng, Winston T.K., Wu, Shinn-Chih, Lin, Chih-Jen, Wang, Chih-Hong, Chen, Chuan-Mu, Lin, Shwu-Wha
• Granted Patent: US 7,667,089 B2
• Time in Patent Office: Days
• US Class Current: 800/7
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• 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: C07K 14/755 Factors VIII , e.g. factor VIII C (AHF), factor VIII Ag (VWF)
• 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

Abstract

Hemophilia A is one of the major inherited bleeding disorders caused by a deficiency or abnormality in coagulation factor VIII (FVIII). Hemophiliacs have been treated with whole plasma or purified FVIII concentrates. The risk of transmitting blood-borne viruses and the cost of highly purified FVIII are the major factors that restrict prophylaxis in hemophilia therapy. One of the challenges created by the biotechnology revolution is the development of methods for the economical production of highly purified proteins in large scales. The present invention provides improved mammary expression cassettes useful for the expression of genes at high levels in the milk of transgenic animals. In particular, the present invention provides recombinant signal peptide sequences derived from a-lactalbumin and aS1-casein milk genes suitable for leading protein secretion in the mammary gland. These gene cassettes are capable of delivering different transgenic constructs which result in the production of full-length or B domain-deleted therapeutic levels of biologically active human FVIII in the transgenic animals in vivo. Within the scope of the invention are also method for producing the transgenic non-human mammal, such as mouse, rat, rabbit, goat, sheep, pig and bovine species, capable of expressing human FVIII, and methods of making milk and methods of identifying protein from the transgenic milk.

Claims

1. A transgenic animal whose genome comprises a mammary-specific expression cassette, said expression cassette system comprising:

(a) a foreign DNA sequence encoding a mature polypeptide which contains full-coding region or recombinant gene structure;

(b) a second DNA sequence which is a secretion signal sequence preceding and operably linked to upstream of (a), said signal sequence encoding a secretional peptide, whereby said mature polypeptide is secreted of high levels into milk by said mammary gland cells;

(c) a third DNA sequence which is polyadenylation signal sequence preceding and operably linked to downstream of (a), said polyadenylation signal sequence can be recognized by poly(A)-polymerase for polyadenylation tail editing, whereby said polyadenylation tail acts for stablized the transgene mRNA molecules;

(d) a regulatory element of a gene encoding a milk protein of a mammal operably linked to the DNA sequences of (a), (b) and (c) above so as to form a hybrid gene which is expressible in the mammary gland of an adult lactating female of a transgenic animal whose genome comprises said hybrid gene;

so that the mature polypeptide is secreted at detectable levels into the milk of said mammal if said mammal is a lactating female.

2. The transgenic animal whose genome comprises a mammary-specific expression cassette according to claim 1, wherein the regulatory element is selected from the group consisting of alpha-lactalbumin, beta-lactoglobulin, why acidic protein and casein promoters, especially using the bovine alpha-lactalbumin promoter.

3. The transgenic animal whose genome comprises a mammary-specific expression cassette according to claim 1, wherein the foreign DNA sequence encoding the mature polypeptide is selected from the group consisting of full-length or B domain-deleted hFVIII polypeptide sequences.

4. The transgenic animal whose genome comprises a mammary-specific expression cassette according to claim 1, wherein the signal peptide is selected from the group consisting of alpha-lactalbumin, aS1-casein signal peptide and other milk protein signal peptides.

5. The transgenic animal whose genome comprises a mammary-specific expression cassette according to claim 1, wherein the signal peptide is an artificial synthetic sequence as SEQ ID:

NO. 1 which obtained from the bovine alpha-lactalbumin signal peptide and created a restriction enzyme, HpaI, cloning site in downstream sequence.

6. The transgenic animal whose genome comprises a mammary-specific expression cassette according to claim 1, wherein the signal peptide is an artificial synthetic sequence as SEQ ID:

NO. 2 which obtained from the bovine aS1-casein signal peptide and created a restriction enzyme, HpaI, cloning site in downstream sequence.

7. The transgenic animal whose genome comprises a mammary-specific expression cassette according to claim 1, wherein the polyadenylation signal sequence is comprised a bovine growth hormone polyadenylation sequence.

8. The transgenic animal according to claim 1, wherein the mammal is selected from the group consisting of mouse, goat, and pig species.

9. The transgenic animal according to claim 3, wherein said nucleotide sequence encoding the full-length human FVIII polypeptide comprises an intact A1-A2-B-A3-C1-C2 domain sequence, while the intrinsic 19-amino acids signal peptide was replaced by mammary gland-specific signal peptide sequence.

10. The transgenic animal according to claim 3, wherein said nucleotide sequence encoding the B-domain deleted human FVIII polypeptide comprises a light chain (A3-C1-C2 domain) and a heavy chain (A1-A2 domain) and wherein said light chain and heavy chain are operably linked by a junction.

11. The transgenic animal according to claim 9, wherein said the mammary gland-specific signal peptide sequences are SEQ ID:

NO.13 and SEQ ID;

NO.14 for 19-residue of alpha-lactalbumin and 15-residue of alpha-S1 casein signal peptides, respectively.

12. he transgenic animal according to claim 10, wherein said the B-domain deleted human FVIII polypeptide is SEQ ID:

NO.15 for recombinant FVIII construct, such that said created junctional amino acid sequence flanking in Ser-Leu.

13. A method for producing the transgenic animal of claim 1 comprising the steps of:

a. introducing into a mammalian embryo at least one expression cassette system comprising a DNA sequence encoding a mature polypeptide which intact human FVIII or B domain-deleted human FVIII operatively linked to mammary gland-specific regulatory sequences; and

b. implanting the embryos into a female of the same species, permitting the embryo to develop to full term and identifying those transgenic mammals which produce in their milk detectable quantities of a mature polypeptide which intact human FVIII or B domain-deleted human FVIII.

14. The method for producing the transgenic animal according to claim 13, wherein 1-50 copies of the mammary gland-specific expression cassette are introduced into transgenic mammalian genomes.

15. The method for producing the transgenic animal according to claim 13, wherein a plurality of different expression cassettes are introduced and these cassettes express at least two different mature polypeptides which intact human FVIII and B domain-deleted human FVIII.

16. The method of claim 13, wherein the expression level of human FVIII in the milk of said transgenic animals can reach 50 mg/L, and its clotting activity can reach 13-fold than that of normal human plasma.

17. The method of claim 13, wherein the purified human FVIII from the transgenic milk can be applied for supplementary therapy used.