Claims for Patent: 5,905,185

Summary for Patent: 5,905,185

Title:	Protein C production in non-human transgenic mammals
Abstract:	Methods for producing protein C in transgenic non-human mammals are disclosed. The protein C is modified at the two-chain cleavage site between the light and heavy chains of protein C from Lys-Arg to R.sub.1 -R.sub.2 -R.sub.3 -R.sub.4 where R.sub.1 through R.sub.4 are individually Arg or Lys. DNA segments encoding modified protein C are introduced into the germ line of a non-human mammals, and the mammal or its female progeny produces milk containing protein C expressed from the introduced DNA segments. The protein C expressed from the introduced DNA segments has anticoagulant activity when activated. Non-human mammalian embryos and transgenic non-human mammals carrying DNA segments encoding heterologous protein C are also disclosed.
Inventor(s):	Garner; Ian (Edinburgh, GB), Cottingham; Ian R. (Edinburgh, GB), Temperley; Simon M. (Edinburgh, GB), Foster; Donald C. (Seattle, WA), Sprecher; Cindy A. (Seattle, WA), Prunkard; Donna E. (Seattle, WA)
Assignee:	PPL Therapeutics (Edinburgh, GB) ZymoGenetics, Inc. (Seattle, WA)
Application Number:	08/756,506
Patent Claims:	1. A method for producing protein C in a transgenic non-human mammal comprising: providing a DNA construct comprising a first DNA segment encoding a secretion signal and a protein C propeptide operably linked to a second DNA segment encoding protein C, wherein the encoded protein C comprises a two-chain cleavage site modified from Lysine (Lys)-Arginine (Arg) to R.sub.1 -R.sub.2 -R.sub.3 -R.sub.4, and wherein each of R.sub.1, R.sub.2, R.sub.3, R.sub.4 is individually Lys or Arg, and wherein said first and second segments are operably linked to additional DNA segments required for expression of the protein C DNA in a mammary gland of a host female non-human mammal; introducing said DNA construct into a fertilized egg of a non-human mammalian species; inserting said egg into an oviduct or uterus of a female of said species to obtain offspring carrying said DNA construct; breeding said offspring to produce female progeny that express said first and second DNA segments and produce milk containing protein C encoded by said second segment, wherein said protein has anticoagulant activity upon activation; collecting milk from said female progeny; and recovering the protein C from the milk. 2. The method of claim 1, further comprising the step of activating the protein C. 3. The method of claim 1, wherein R.sub.1 -R.sub.2 -R.sub.3 -R.sub.4 is Arg-Arg-Lys-Arg (SEQ ID NO: 20). 4. The method of claim 1, wherein said species is selected from sheep, rabbits, cattle and goats. 5. The method of claim 1, wherein each of said first and second DNA segments comprises an intron. 6. The method of claim 1, wherein the second DNA segment comprises a DNA sequence of nucleotides as shown in Seq. ID NO: 1 or Seq. ID. NO: 3. 7. The method of claim 6, wherein the second DNA segment comprises the DNA sequence of nucleotides as shown in SEQ. ID. NO: 1. 8. The method of claim 1, wherein the additional DNA segments comprise a transcriptional promoter selected from the group consisting of casein, .beta.-lactoglobulin, .alpha.-lactalbumin and whey acidic protein gene promoters. 9. The method of claim 8, wherein the transcriptional promoter is the .beta.-lactoglobulin gene promoter. 10. A transgenic non-human female mammal whose genome comprises a DNA construct comprising a first EDNA segment encoding a secretion signal and a protein C propeptide operably linked to a second DNA segment encoding protein C, wherein the encoded protein C comprises a two-chain cleavage site modified from Lysine (Lys)-Arginine (Arg) to R.sub.1 -R.sub.2 -R.sub.3 -R.sub.4, and wherein each of R.sub.1, R.sub.2 , R.sub.3, R.sub.4 is individually Lys or Arg, and wherein said first and second segments are operably linked to additional DNA segments required for the expression of the protein C DNA in the mammary gland of the mammal such that expression of the DNA construct results in the production of recoverable amounts of human protein C in milk of the mammal wherein at least 90% of the human protein C in the milk is two chain protein C. 11. A process for producing a transgenic offspring of a non-human mammal comprising: providing a DNA construct comprising a first DNA segment encoding a secretion signal and a protein C propeptide operably linked to a second DNA segment encoding protein C, wherein the encoded protein C comprises a two-chain cleavage site modified from Lys-Arg to R.sub.1 -R.sub.2 -R.sub.3 -R.sub.4, and wherein each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, is individually Lys or Arg, and wherein said first and second segments are operably linked to additional DNA segments required for expression of the protein C DNA in the mammary gland of a host female mammal; introducing said DNA construct into a fertilized egg of a non-human mammalian species; and inserting said egg into an oviduct or uterus of a female of said species to obtain offspring carrying said DNA construct. 12. The process according to claim 11, wherein R.sub.1 -R.sub.2 -R.sub.3 -R.sub.4 is Arg-Arg-Lys-Arg (SEQ ID NO: 20). 13. The process according to claim 11, wherein the offspring is female. 14. The process according to claim 11, wherein the offspring is male. 15. A non-human mammal produced according to the process of claim 11, wherein expression of the DNA construct results in the production of recoverable amounts of human protein C in milk, wherein the protein C has anticoagulant activity upon activation. 16. A non-human mammal of claim 15, wherein the mammal is female. 17. A non-human mammalian embryo whose genome comprises a DNA construct comprising a first DNA segment encoding a secretion signal and a protein C propeptide operably linked to a second DNA segment encoding protein C, wherein the encoded protein C comprises a two-chain cleavage site modified from Lysine (Lys)-Arginine (Arg) to R.sub.1 -R.sub.2 -R.sub.3 -R.sub.4, and wherein each of R.sub.1, R.sub.2, R.sub.3, R.sub.4 is individually Lys or Arg, and wherein said first and second segments are operably linked to additional DNA segments required for the expression of the protein C DNA in the mammary gland of the mammal such that the embryo develops into a transgenic non-human mammal such that expression of the DNA construct in the transgenic non-human mammal results in the production of recoverable amounts of human protein C in milk of the mammal wherein at least 90% of the human protein C in the milk is two chain protein C.

Details for Patent 5,905,185

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2015-11-30
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2015-11-30
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2015-11-30