Expression of active human factor ix in mammary tissue of transgenic animals

Abstract

Recombinant Factor IX characterized by a high percentage of active protein can be obtained in the milk of transgenic animals that incorporate chimeric DNA molecules according to the present invention. Transgenic animals of the present invention are produced by introducing into developing embryos DNA that encodes Factor IX, such that the foreign DNA is stably incorporated in the DNA of germ line cells of the mature animal. Particularly efficient expression was accomplished using a chimeric construct comprising a mammary gland specific promoter, Factor IX cDNA that lacked the complete or any portion of the 5′-untranslated and 3′-untranslated region, which is substituted with a 5-′ and 3′-end of the mouse whey acidic protein gene. In vitro cell cultures of cells explanted from the transgenic mammal of the invention and methods of producing Factor IX from such said culture and methods of treating hemophilia B are also described.

1 . A non-human transgenic mammal containing an exogenous DNA molecule that is stably integrated in its genome, wherein said exogenous DNA molecule comprises:
 (a) 5′ regulatory sequences of a mammary gland-specific gene including a promoter;   (b) a Factor IX-encoding DNA sequence that encodes a signal sequence, a Factor IX pro-sequence, and a Factor IX sequence in a 5′ to 3′ direction, wherein said signal sequence is effective in directing the secretion of said Factor IX into the milk of said transgenic mammal and wherein said Factor IX sequence lacks at least a portion of the complete 5′-untranslated and 3′-untranslated regions of the Factor IX gene; and   (c) 3′ regulatory sequences from a mammary gland-specific gene or 3′ regulatory sequences active in a mammary gland;   wherein said 5′ and said 3′ regulatory sequences are operatively linked to said Factor IX-encoding DNA sequence.   
     
     
         2 . The non-human transgenic mammal of  claim 1 , wherein said promoter is selected from the group consisting of short whey acidic protein (WAP) promoter, long WAP promoter, short α-casein promoter, short β-casein promoter, short kappa-casein promoter, long α-casein promoter, long β-casein promoter, long kappa-casein promoter, α-lactalbumin promoter and β-lactoglobulin promoter. 
     
     
         3 . The non-human transgenic mammal of  claim 2 , wherein said short WAP promoter is the 2.6 kb EcoRI-KpnI promoter of the mouse WAP gene. 
     
     
         4 . The non-human transgenic mammal of  claim 2 , wherein said long WAP promoter is the 4.1 kb NotI-KpnI promoter or the 4.2 kb Sau3A-KpnI promoter of the mouse WAP gene. 
     
     
         5 - 10 . (canceled) 
     
     
         11 . The non-human transgenic mammal of  claim 1 , wherein said transgenic mammal is selected from the group consisting of mice, rats, rabbits, pigs, sheep, goats and cows. 
     
     
         12 . The non-human transgenic mammal of  claim 1 , wherein said transgenic mammal secretes from about 100 to about 220 μg of active human Factor IX per milliliter milk. 
     
     
         13 - 16 . (canceled) 
     
     
         17 . The non-human transgenic mammal of  claim 12 , wherein said transgenic mammal is a pig. 
     
     
         18 - 19 . (canceled) 
     
     
         20 . The non-human transgenic mammal of  claim 1 , wherein said active human Factor IX purified from the milk of said transgenic mammal has a specific activity that is at least about 15-50% greater than the specific activity of human Factor IX isolated from human plasma. 
     
     
         21 - 22 . (canceled) 
     
     
         23 . A process for producing Factor IX, comprising:
 (a) providing a non-human transgenic mammal having integrated into its genome an exogenous DNA molecule, wherein said exogenous DNA molecule comprises: (1) 5′ regulatory sequences of a mammary gland-specific gene including a promoter; (2) a Factor IX-encoding DNA sequence that encodes a signal sequence, a Factor IX pro-sequence and a Factor IX sequence in a 5′ to 3′ direction, wherein said signal sequence is effective in directing the secretion of said Factor IX into the milk of said transgenic mammal and wherein said Factor IX sequence lacks at least a portion of the complete or the complete 5′-untranslated and 3′-untranslated regions of the Factor IX gene; and (3) 3′ regulatory sequences from a mammary gland-specific gene or 3′ regulatory sequences active in a mammary gland; wherein said 5′ and said 3′ regulatory sequences are operatively linked to said Factor IX-encoding DNA sequence;   (b) allowing said DNA sequences encoding said Factor IX to be expressed and said Factor IX to be secreted into the milk of said transgenic mammal;   (c) collecting said milk from said mammal; and   (d) isolating said Factor IX from said milk.   
     
     
         24 . The process of  claim 23 , wherein said promoter is selected from the group consisting of short whey acidic protein (WAP) promoter, long WAP promoter, short α-casein promoter, short β-casein promoter, short kappa-casein promoter, long α-casein promoter, long β-casein promoter, long kappa-casein promoter, α-lactalbumin promoter and β-lactoglobulin promoter. 
     
     
         25 . The process of  claim 24 , wherein said short WAP promoter is the 2.6 kb EcoRI-KpnI promoter of the mouse WAP gene. 
     
     
         26 . The process of  claim 24 , wherein said long WAP promoter is the 4.1 kb NotI-KpnI promoter or the 4.2 kb Sau3A-KpnI promoter of the mouse WAP gene. 
     
     
         27 - 32 . (canceled) 
     
     
         33 . The process of  claim 23 , wherein said transgenic mammal is selected from the group consisting of mice, rats, rabbits, pigs, sheep, goats and cows. 
     
     
         34 . The process of  claim 23 , wherein said transgenic mammal secretes from about 100 to about 220 μg of active human Factor IX per milliliter milk. 
     
     
         35 - 44 . (canceled) 
     
     
         45 . A method of treating a patient having hemophilia B comprising administering to said patients a hemophilia B symptom preventing or ameliorating amount of Factor IX produced by the transgenic non-human mammal of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         46 . Mammary gland cells obtained from the non-human transgenic mammal of  claim 1 , wherein said cells produce said Factor IX. 
     
     
         47 - 49 . (canceled) 
     
     
         50 . A biologically active human Factor IX produced in the non-human transgenic mammal of  claim 1 , said Factor IX comprising a specific activity that is at least about 5-200% greater than the specific activity of human Factor IX isolated from human plasma. 
     
     
         51 . A biologically active human Factor IX of  claim 50 , wherein said non-human transgenic mammal is selected from the group consisting of mice, rats, rabbits, pigs, sheep, goats and cows.