Production of high levels of transgenic factor ix without gene rescue, and its therapeutic uses

Abstract

A non-human transgenic mammalian animal, as described above, contains an exogenous double stranded DNA sequence stably integrated into the genome of the animal, which comprises cis-acting regulatory units operably linked to a DNA sequence encoding human FIX protein without the benefit of the presence of a complete milk gene sequence for gene rescue, and a signal sequence is active in directing newly expressed Factor IX into the milk of the animal at levels in an unactivated form that is suitable for subsequent processing for therapeutic applications in treating Hemophilia B. The transgenic mammals are preferably pigs, cows, sheep, goats and rebbits. The application include milk derivatives for oral delivery and oral tolerization in the treatment of Hemophilia B.

What is claimed is:  
     
         1 . A non-human transgenic mammal containing an exogenous DNA molecule 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 the endogenous secretion-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 consists only of the DNA encoding sequence of Factor IX;    (c) 3′ regulatory sequences from a mammary gland-specific gene, which sequences are operatively linked to said Factor IX-encoding DNA sequence;    (d) the stably integrated exogenous DNA does not include a WAP milk protein gene for providing gene rescue to achieve expression of the Factor IX DNA; and    (e) said Factor IX is secreted into the milk at least 220 micrograms Factor IX per milliliter of milk that is not activated by the milk-environment and therefore useable for Factor IX therapeutic applications.    
     
     
         2 . The non-human transgenic mammal of  claim 1 , wherein said promoter is selected from the group consisting of rodent-derived whey acidic protein (WAP) promoters, 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, lactoferrin promoter and β-lactoglobulin promoter.  
     
     
         3 . The non-human transgenic mammal of  claim 2 , wherein said short WAP promoter is the 2.5 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 . The non-human transgenic mammal of  claim 1 , wherein said Factor IX-encoding DNA molecule further comprises a 5′-untranslated region that is not the 5′-untranslated region of the Factor IX gene.  
     
     
         6 . The non-human transgenic mammal of  claim 5 , wherein said 5′-untranslated region is the 5′-untranslated region of a rodent-derived WAP milk gene.  
     
     
         7 . The non-human transgenic mammal of  claim 1 , wherein said Factor IX-encoding DNA molecule further comprises a propeptide region that is not the propeptide region of the Factor IX gene.  
     
     
         8 . The non-human transgenic mammal of  claim 7 , wherein said Factor IX-encoding DNA molecule further comprises the propeptide from human Factor X and the encoding region of Factor IX beginning at native amino terminal residue of tyrosine.  
     
     
         9 . The non-human transgenic mammal of  claim 7 , wherein said Factor IX-encoding DNA molecule further comprises the propeptide from human Factor VII and the encoding region of Factor IX beginning at native amino terminal residue of tyrosine.  
     
     
         10 . 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.  
     
     
         11 . The non-human transgenic mammal of  claim 2 , 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 10 , wherein said transgenic mammal secretes from about 220 to about 1000 μg of human Factor IX per milliliter whole milk.  
     
     
         13 . The non-human transgenic mammal of  claim 11 , wherein said transgenic mammal secretes from about 220 to about 1000 μg of human Factor IX per milliliter whole milk.  
     
     
         14 . The non-human transgenic mammal of  claim 12 , wherein said transgenic mammal is a pig.  
     
     
         15 . The non-human transgenic mammal of  claim 13 , wherein said transgenic mammal is a pig.  
     
     
         16 . The non-human transgenic mammal of  claim 10 , wherein said transgenic mammal secretes from about 1000 to about 5000 μg of human Factor IX per milliliter whole milk.  
     
     
         17 . The non-human transgenic mammal of  claim 16 , wherein said transgenic mammal is a pig.  
     
     
         18 . The non-human transgenic mammal of  claim 11 , wherein said transgenic mammal secretes from about 1000 to about 5000 μg of human Factor IX per milliliter whole milk.  
     
     
         19 . The non-human transgenic mammal of  claim 18 , wherein said transgenic mammal is a pig.  
     
     
         20 . The non-human transgenic mammal of  claim 4 , wherein said transgenic mammal secretes from about 1000 to about 5000 μg of human Factor IX per milliliter whole milk.  
     
     
         21 . The non-human transgenic mammal of  claim 4 , wherein said transgenic mammal secretes from about 1000 to about 3000 μg of human Factor IX per milliliter whole milk.  
     
     
         22 . The non-human transgenic mammal of  claim 20 , wherein said transgenic mammal is a pig.  
     
     
         23 . The non-human transgenic mammal of  claim 21 , wherein said transgenic mammal is a pig.  
     
     
         24 . The non-human transgenic mammal of  claim 10 , wherein the active fractions of human Factor IX, when purified from the milk of said transgenic mammal, has a specific activity that is at least about 5-140% of that defined for the specific activity of human Factor IX isolated from human plasma, as determined by an activated partial tliromboplastin clotting time assay coagulation assay.  
     
     
         25 . The non-human transgenic mammal of  claim 11 , wherein the active fractions of human Factor IX purified from the milk of said transgenic mammal have a specific activity that is at least about 5-140% of that defined for the specific activity of human Factor IX isolated from human plasma as determined by an activated partial thromboplastin clotting time assay coagulation assay.  
     
     
         26 . The non-human transgenic mammal of  claim 24 , wherein said transgenic mammal is a pig.  
     
     
         27 . The non-human transgenic mammal of  claim 25 , wherein the active fractions of human Factor IX purified from the milk of said transgenic pig have a specific activity that is at least about 5-100% of that defined for the specific activity of human Factor IX isolated from human plasma as determined by an activated partial thromboplastin clotting time assay coagulation assay.  
     
     
         28 . The non-human transgenic mammal of  claim 27 , wherein said transgenic mammal is a pig.  
     
     
         29 . 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 LX-encoding DNA sequence that encodes the endogenous signal sequence of Factor IX, 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) the stably integrated exogenous DNA does not include a milk protein gene for providing gene rescue to achieve expression of the Factor IX DNA; and    (4) 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) processing said Factor IX from said milk so as to be appropriate for therapeutic purposes.    
     
     
         30 . The process of  claim 29 , wherein said promoter is selected from the group consisting of rodent-derived whey acidic protein (WAP) promoters, 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, lactoferrin promoter and β-lactoglobulin promoter.  
     
     
         31 . The process of  claim 30 , wherein said short WAP promoter is the 2.5 kb EcoRI-KpnI promoter of the mouse WAP gene.  
     
     
         32 . The process of  claim 30 , 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.  
     
     
         33 . The process of  claim 29 , wherein said Factor IX-encoding DNA molecule further comprises a 5′-untranslated region that is not the 5′-untranslated region of the Factor IX gene.  
     
     
         34 . The process of  claim 33 , wherein said 5′-untranslated region is the 5′-untranslated region of a rodent-derived WAP milk gene.  
     
     
         35 . The process of  claim 29 , The non-human transgenic mammal of  claim 1 , wherein said Factor IX-encoding DNA molecule further comprises a propeptide region that is not the propeptide region of the Factor IX gene.  
     
     
         36 . The process of  claim 35 , wherein said Factor IX-encoding DNA molecule further comprises a 5′-untranslated region located 5′ from said signal sequence DNA, wherein said Factor IX-encoding DNA molecule further comprises the propeptide from human Factor VII and not the propeptide of FIX and the encoding region of Factor IX beginning at native amino terminal residue of tyrosine.  
     
     
         37 . The process of  claim 29 , wherein said Factor IX is useful for treating hemophilia B.  
     
     
         38 . The process of  claim 29 , wherein said transgenic mammal is selected from the group consisting of mice, rats, rabbits, pigs, sheep, goats and cows.  
     
     
         39 . The process of  claim 38 , wherein said transgenic mammal secretes from about 220 to about 1000 μg of human Factor IX per milliliter whole milk.  
     
     
         40 . The process of  claim 39 , wherein said transgenic mammal is a pig.  
     
     
         41 . The process of  claim 38 , wherein said transgenic mammal secretes from about 1000 to about 5000 μg of human Factor IX per milliliter whole milk.  
     
     
         42 . The process of  claim 40 , wherein said transgenic mammal is a pig.  
     
     
         43 . The process of  claim 38 , wherein the active fractions of human Factor IX, when purified from the milk of said transgenic mammal, have a specific activity that is at least about 5%-140% of that defined for the specific activity of human Factor IX isolated from human plasma, as determined by an activated partial thromboplastin clotting time assay coagulation assay.  
     
     
         44 . The process of  claim 31 , wherein said transgenic mammal secretes from about 220 to about 1000 μg of human Factor IX per milliliter milk.  
     
     
         45 . The process of  claim 44 , wherein said transgenic mammal is a pig.  
     
     
         46 . The process of  claim 45 , wherein the active fractions of human Factor IX, when purified from the milk of said transgenic mammal, have a specific activity that is at least about 5%-140% of that defined for the specific activity of human Factor IX isolated from human plasma, as determined by an activated partial thromboplastin clotting time assay coagulation assay.  
     
     
         47 . The process of  claim 32 , wherein said transgenic mammal secretes from about 1000 to about 5000 μg of human Factor IX per milliliter milk.  
     
     
         48 . The process of  claim 47 , wherein said transgenic mammal is a pig.  
     
     
         49 . The process of  claim 48 , wherein the active fractions of human Factor IX, when purified from the milk of said transgenic mammal, have a specific activity that is at least about 5%-100% of that defined for the specific activity of human Factor IX isolated from human plasma, as determined by an activated partial thromboplastin clotting time assay coagulation assay.  
     
     
         50 . The process of  claim 49 , wherein said transgenic mammal is a pig.  
     
     
         51 . The process of  claim 47 , wherein the active fractions of human Factor IX purified from the milk of said transgenic mammal have a specific activity that is at least about 5-60% of that defined for the specific activity of human Factor Ix isolated from human plasma.  
     
     
         52 . The process of  claim 51 , wherein said transgenic mammal is a pig.  
     
     
         53 . The process of  claim 47 , wherein the active fractions of human Factor IX purified from the milk of said transgenic pig have a specific activity that is at least about 5-40% of that defined for the specific activity of human Factor IX isolated from human plasma.  
     
     
         54 . The process of  claim 53 , wherein said transgenic mammal is a pig.  
     
     
         55 . A method of treating a patient having hemophilia B comprising administering to said patient a hemophilia B symptom preventing or ameliorating amount of Factor LX produced by the process of  claim 38  and a pharmaceutically acceptable carrier.  
     
     
         56 . The method of treating a patient having hemophilia B according to  claim 55  where said method is oral delivery of FIX.  
     
     
         57 . The method of treating a patient having hemophilia B according to  claim 55  where said method is oral tolerization of FIX.  
     
     
         58 . The method of treating a patient having hemophilia B according to  claim 55  where said method is oral tolerization of FIX and milk proteins.  
     
     
         59 . The method of  claim 58 , wherein said transgenic mammal is a pig.  
     
     
         60 . The method of treating a patient having hemophilia B according to  claim 55  where the pharmaceutically acceptable carrier is a processed derivative of whole milk.  
     
     
         61 . The method of treating a patient having hemophilia B according to  claim 60  where said method is oral delivery of FIX.  
     
     
         62 . The method of treating a patient having hemophilia B according to  claim 61  where said method is oral tolerization of FIX.  
     
     
         63 . The method of treating a patient having hemophilia B according to  claim 62  where said method is both oral delivery and oral tolerization of FIX and milk proteins.  
     
     
         64 . The method of  claim 63 , wherein said transgenic mammal is a pig.  
     
     
         65 . The method of treating a patient having hemophilia B according to  claim 62  where said method is oral tolerization of FIX derived from recombinant cell culture.  
     
     
         66 . The method of treating a patient having hemophilia B according to  claim 62  where said method is oral tolerization of FIX derived from human plasma.  
     
     
         67 . A method of treating a patient having hemophilia B comprising administering to said patient a hemophilia B symptom preventing or ameliorating amount of Factor IX produced by the process of  claim 39  and a pharmaceutically acceptable carrier.  
     
     
         68 . The method of treating a patient having hemophilia B according to  claim 67  where said method is oral delivery of FIX.  
     
     
         69 . The method of treating a patient having hemophilia B according to  claim 68  where said method is oral tolerization of FIX.  
     
     
         70 . The method of treating a patient having hemophilia B according to  claim 69  where said method is oral tolerization of FIX and milk proteins.  
     
     
         71 . The method of  claim 70 , wherein said transgenic mammal is a pig.  
     
     
         72 . The method of treating a patient having hemophilia B according to  claim 67  where the pharmaceutically acceptable carrier is a processed derivative of whole milk.  
     
     
         73 . The method of treating a patient having hemophilia B according to  claim 72  where said method is oral delivery of FIX.  
     
     
         74 . The method of treating a patient having hemophilia B according to  claim 73  where said method is oral tolerization of FIX.  
     
     
         75 . The method of treating a patient having hemophilia B according to  claim 74  where said method is both oral delivery and oral tolerization of FIX and milk proteins.  
     
     
         76 . The method of  claim 75 , wherein said transgenic mammal is a pig.  
     
     
         77 . The method of treating a patient having hemophilia B according to  claim 74  where said method is oral tolerization of FIX derived from recombinant cell culture.  
     
     
         78 . The method of  claim 77 , wherein said transgenic mammal is a pig.  
     
     
         79 . The method of treating a patient having hemophilia B according to  claim 74  where said method is oral tolerization of FIX derived from human plasma.  
     
     
         80 . The method of  claim 79 , wherein said transgenic mammal is a pig.  
     
     
         81 . A method of treating a patient having hemophilia B comprising administering to said patient a hemophilia B symptom preventing or ameliorating amount of Factor IX produced by the process of  claim 41  and a pharmaceutically acceptable carrier.  
     
     
         82 . The method of treating a patient having hemophilia B according to  claim 81  where said method is oral delivery of FIX.  
     
     
         83 . The method of treating a patient having hemophilia B according to claim S2 where said method is oral tolerization of FIX.  
     
     
         84 . The method of treating a patient having hemophilia B according to  claim 83  where said method is oral tolerization of FIX and milk proteins.  
     
     
         85 . The method of  claim 84 , wherein said transgenic mammal is a pig.  
     
     
         86 . The method of treating a patient having hemophilia B according to  claim 81  where the pharmaceutically acceptable carrier is a processed derivative of whole milk.  
     
     
         87 . The method of treating a patient having hemophilia B according to  claim 86  where said method is oral delivery of FIX.  
     
     
         88 . The method of treating a patient having hemophilia B according to  claim 87  where said method is oral tolerization of FIX.  
     
     
         89 . The method of treating a patient having hemophilia B according to  claim 88  where said method is both oral delivery and oral tolerization of FIX and milk proteins.  
     
     
         90 . The method of  claim 89 , wherein said transgenic mammal is a pig.  
     
     
         91 . The method of treating a patient having hemophilia B according to  claim 88  where the said method is oral tolerization of FIX derived from recombinant cell culture.  
     
     
         92 . The method of  claim 91 , wherein said transgenic mammal is a pig.  
     
     
         93 . The method of treating a patient having hemophilia B according to claim SS where said method is oral tolerization of FIX derived from human plasma.  
     
     
         94 . The method of  claim 93 , wherein said transgenic mammal is a pig.  
     
     
         95 . A non-human transgenic mammal containing an exogenous DNA molecule stably integrated in its genome, wherein said exogenous DNA molecule comprises: 
 (a) a mammary gland-specific gene including a promoter;    (b) a Factor IX-encoding DNA sequence that encodes the endogenous secretion-signal sequence, a Factor IX pro-sequence and a Factor IX sequence;    (c) 3′ regulatory sequences from a mammary gland-specific gene, which sequences are operatively linked to said Factor IX-encoding DNA sequence;    (d) the stably integrated exogenous DNA does not include a WAP milk protein gene for providing gene rescue to achieve expression of the Factor 1×DNA; and    (e) said Factor IX is secreted into the milk at least 220 micrograms Factor IX per milliliter of milk that is not activated by the milk-environment and therefore useable for Factor IX therapeutic applications.