Claims for Patent: 6,713,662

Summary for Patent: 6,713,662

Title:	Production of collagen in the milk of transgenic mammals
Abstract:	The invention provides transgenic nonhuman mammals capable secreting exogenous procollagen or collagen into their milk. The mammals are healthy and capable of producing procollagen or collagen at high levels, usually in trimeric form. Suitable transgenes for incorporation into the mammals are also provided.
Inventor(s):	Karatzas; Costas N. (Beaconsfield, CA), Pieper; Frank (Utrecht, NL), De Wit; Ineke (Leiden, NL), Berg; Richard (Los Altos, CA), Platenburg; Gerard (Voorschoten, NL), Toman; Paul David (Mountain View, CA)
Assignee:	Pharming Intellectual Property B.V. (Leiden, NL) Cohesion Technologies (Palo Alto, CA)
Application Number:	08/482,173
Patent Claims:	1. A transgenic nonhuman mammal having a transgene comprising: a mammary-gland specific promoter; a mammary-gland specific enhancer a secretory DNA segment encoding a signal peptide functional in mammary secretory cells of the transgenic nonhuman mammal; and a recombinant DNA segment encoding an exogenous procollagen polypeptide operably linked to the secretory DNA segment to form a secretory-recombinant DNA segment, the secretory-recombinant DNA segment being operably linked to the promoter and to the enhancer; wherein the transgene, in an adult form of the nonhuman mammal or a female descendant of the nonhuman mammal, expresses the secretory-recombinant DNA segment in the mammary secretory cells to produce a form of the exogenous procollagen polypeptide that is processed and secreted by the mammary secretory cells into milk as exogenous procollagen or collagen in a detectable amount; provided the recombinant DNA segment comprises a genomic DNA segment from the .alpha.1(I) collagen gene and is without nucleotides 1-114 from the first exon of the gene, and/or without a segment from the first intron of the gene. 2. The transgenic nonhuman mammal of claim 1, wherein the exogenous procollagen or collagen is in trineric form. 3. The transgenic nonhuman mammal of claim 2, wherein the transgene comprises: a casein promoter; a casein enhancer; and a genomic DNA segment comprising a segment from a signal peptide coding sequence to a 3' flanking region of a procollagen a1(I) or a2(I) gene, operably linked to the promoter and the enhancer and the concentration of the exogenous collagen or collagen in the milk is at least 100 .mu.g/ml. 4. The transgenic nonhuman mammal of claim 3, wherein the genomic segment further comprises a 5' untranslated region from the procollagen .alpha.1(I) or .alpha.2(I) gene. 5. The transgenic nonhuman mammal of claim 3, wherein the genomic DNA segment is from the .alpha.1(I) gene and is without nucleotides 1-114 from the first exon of the gene. 6. The transgenic nonhuman mammal of claim 5, wherein the genomic DNA segment is without a segment from the first intron of the gene. 7. The transgenic nonhuman mammal of claim 3, wherein the concentration of the exogenous collagen or collagen in the milk is at least 1 mg/ml. 8. The nonhuman transgenic mammal of claim 7, wherein the exogenous procollagen or collagen polypeptide is human. 9. The nonhuman transgenic mammal of claim 8, wherein the exogenous procollagen or collagen polypeptide is proa1(I). 10. The nonhuman transgenic mammal of claim 1, wherein the recombinant DNA segment is a cDNA-genomic DNA hybrid. 11. The nonhuman mammal of claim 1, wherein the genomic segment comprises a contiguous segment from the 5' untranslated region to the 3' untranslated region of a human pro.alpha.1(I) gene. 12. The nonhuman transgenic mammal of claim 1, wherein the genomic segment lacks a segment from the first intron of a human pro.alpha.1(I) gene. 13. The nonhuman transgenic mammal of claim 1, wherein the first transgene encodes .alpha.1(I) procollagen and the nonhuman transgenic mammal has a second transgene comprising: a second mammary-gland specific promoter, the same or different from the mammary-gland specific promoter; a second mammary-gland specific enhancer, the same or different from the mammary-gland specific enhancer; a second secretory DNA segment encoding a signal peptide functional in mammary secretory cells of the transgenic nonhuman mammal, the same or different from the secretory DNA segment; and a second recombinant DNA segment comprising a genomic DNA segment comprising a segment from a signal peptide coding sequence to a 3' flanking region of a procollagen a2(I) gene, operably linked to the promoter and the enhancer encoding a human a2(I) procollagen polypeptide operably linked to the second secretory DNA segment to form a second secretory-recombinant DNA segment, said secretory-recombinant DNA segment being operably linked to, the second promoter and to the second enhancer; wherein the first and second transgenes, in the adult form of the nonhuman mammal or the female descendant, express the first and second secretory-recombinant DNA segments in the mammary secretory cells to produce forms of a1(I) and a2(I) procollagen that are processed and secreted by the mammary secretory cells into milk as a trimer comprising at least one chain of a1(I) procollagen or collagen and at least one chain of a2(I) procollagen or collagen. 14. The transgenic nonhuman mammal of claim 1 that is an embryo. 15. The transgenic nonhuman mammal of claim 1, that is a bovine. 16. The transgenic nonhuman mammal of claim 1, that is a mouse. 17. The transgenic nonhuman mammal of claim 1, wherein the trimer is a homotrimer. 18. The transgenic nonhuman mammal of claim 1, further comprising a second transgene comprising: a second mammary-gland specific promoter, the same or different from the mammary-gland specific promoter; a second mammary-gland specific enhancer, the same or different from the mammary-gland specific enhancer; a second DNA segment encoding a second signal sequence capable of targeting the expressing of a polypeptide operably linked to the signal sequence to the endoplasmic reticulum of a cell; a second recombinant DNA segment encoding a second human procollagen polypeptide operably linked to the second secretory DNA segment to form a second secretory-recombinant DNA segment, said secretory-recombinant DNA segment being operably linked to the second promoter and to the second enhancer, wherein the first and second transgenes, in the adult form of the nonhuman mammal or the female descendant, express the first and second secretory-recombinant DNA segments in the mammary secretory cells to produce forms of procollagen that are processed and secreted by the mammary secretory cells into milk as a detectable amount of a heterotrimer comprising at least one procollagen or collagen chain expressed from each transgene. 19. A method for preparing procollagen or collagen, the method comprising: providing a transgenic nonhuman mammal having a transgene comprising: a mammary-gland specific promoter; a mammary-gland specific enhancer a secretory DNA segment encoding a signal peptide functional in mammary secretory cells of the transgenic nonhuman mammal; and a recombinant DNA segment encoding an exogenous procollagen polypeptide operably linked to the secretory DNA segment to form a secretory-recombinant DNA segment, the secretory-recombinant DNA segment being operably linked to the promoter and to the enhancer; wherein the transgene, in an adult form of the nonhuman mammal or a female descendant of the nonhuman mammal, expresses the secretory-recombinant DNA segment in the mammary secretory cells to produce a form of the exogenous procollagen polypeptide that is processed and secreted by the mammary secretory cells into milk as exogenous procollagen or collagen in a detectable amount; supplementing the diet of the transgenic nonhuman mammal or its female descendant with Vitamin c at a level of 50-1000 mg/kg food; recovering milk from the adult form of the transgenic nonhuman mammal or its female descendant, wherein the milk comprises exogenous procollagen or collagen in a recoverable amount; and purifying procollagen or collagen from the milk. 20. The method of claim 19, further comprising the step of contacting the procollagen with a proteolytic enzyme to convert the procollagen to collagen. 21. Milk from the transgenic nonhuman mammal of claim 1, the milk comprising the exogenous procollagen or collagen polypeptide. 22. The milk of claim 21, wherein the procollagen or collagen is in trimeric form. 23. The milk of claim 22, wherein the concentration of procollagen or collagen is at least 100 .mu.g/ml. 24. A transgene for expressing procollagen or collagen, the transgene comprising: a casein promoter; a casein enhancer; a genomic DNA segment comprising a segment from a signal peptide coding sequence to a 3' flanking region of a procollagen .alpha.2(I) gene, operably linked to the promoter and the enhancer. 25. The transgene of claim 24, further comprising a casein 5' untranslated sequence between the promoter and the genomic DNA segment. 26. A stable mammary-gland derived cell line, having a transgene comprising: a mammary-gland specific promoter; a mammary-gland specific enhancer; a secretory DNA segment encoding a signal peptide functional in the cell line; and a recombinant DNA segment encoding an exogenous procollagen polypeptide operably linked to the secretory DNA segment to form a secretory-recombinant DNA segment, the secretory-recombinant DNA segment being operably linked to the promoter and to the enhancer; wherein the cell line is induced by a lactogenic hormone to express the transgene to produce a form of the exogenous procollagen polypeptide that is processed and secreted by the cell lines as exogenous procollagen or collagen in trimeric form; provided the recombinant DNA segment comprises a genomic DNA segment from the .alpha.1(I) collagen gene and is without nucleotides 1-114 from the first exon of the gene, and/or without a segment from the first intron of the gene.

Details for Patent 6,713,662

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		2021-03-30
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2021-03-30
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2021-03-30