Transgenic proteins from multi-gene systems, methods, compositions, uses and the like relating thereto

Abstract

A non-human transgenic mammalian animal, as described above, contains one or more exogenous double stranded DNA sequence(s) stably integrated into the genome of the animal, which comprises trans-acting regulatory units controlling expression of DNA sequences encoding proteins to be secreted into the milk of transgenic mammals. The DNA sequence of the trans-regulatory gene encodes transcriptional activating proteins, which are not secreted but made in a temporally controlled and mammary tissue specific manner. The DNA sequence containing the protein to be secreted in the milk is constructed on a separate gene sequence under the regulation of a minimal promoter and a trans-activation binding domain. The transgenic mammals are preferably pigs, cows, sheep, goats and rabbits. A related composition and method for making transgenic proteins which require specialized propeptides for proper post-translational processing is also described.

1 - 95 . (canceled) 
     
     
         96 . A transgenic non-human mammal comprising multiple exogenous DNA sequences stably integrated in its genome, wherein said exogenous DNA sequences comprise:
 (A) an exogenous DNA sequence of type A, said type A DNA sequence comprising:
 (1) 5′ regulatory sequences including a milk protein promoter operably linked to the DNA of (b), 
 (2) DNA encoding a non-secreted transactivating protein, wherein said non-secreted transactivating protein contains a DNA binding domain which recognizes the DNA binding site in a type B DNA sequence, and possesses transactivating activity, and 
 (3) 3′ regulatory sequences active in the transgenic non-human mammal; and 
   (B) one or more exogenous DNA sequences of type B, said type B DNA sequence or each type B DNA sequence comprising:   (4) a DNA binding site recognized by the DNA binding domain of said non-secreted transactivating protein, operably linked to a minimal promoter operably linked to the DNA of (e),
 (5) DNA encoding a peptide or protein, and 
 (6) 3′ regulatory sequences active in the transgenic non-human mammal, wherein said multiple exogenous DNA sequences are effective in directing the secretion of the peptide or protein into milk of said transgenic non-human mammal, 
   
       wherein said milk protein promoter and said minimal promoter are different promoters. 
     
     
         97 . The mammal of  claim 96 , wherein said milk protein promoter is selected from the group consisting of whey acidic protein (WAP) promoter, α-casein promoter, β-casein promoter, κ-casein promoter, α-lactalbumin promoter, lactoferrin promoter and β-lactoglobulin promoter. 
     
     
         98 . The mammal of  claim 96 , wherein said non-human transgenic mammal is selected from the group consisting of mouse, rat, rabbit, pig, goat, sheep and cow. 
     
     
         99 . The mammal of  claim 96 , wherein said 3′ regulatory sequences are chosen to function efficiently with said milk protein promoter. 
     
     
         100 . The mammal of  claim 96 , wherein said minimal promoter contains a TATA box and a transcriptional start site. 
     
     
         101 . The mammal of  claim 96 , wherein said minimal promoter contains a TATA box, a transcriptional start site and other 5′ regulatory sequences to boost expression level. 
     
     
         102 . The mammal of  claim 96 , wherein said DNA binding site is the binding site of the GAL 4 DNA binding protein. 
     
     
         103 . The mammal of  claim 96 , wherein said protein encoded by DNA of (B)(5) is selected from the group consisting of:
 (a) a fusion protein containing the light chain of factor X (X) combined with the 1 st  Kunitz domain of Tissue Factor Plasminogen Inhibitor (TFPI-KI) designated XKI,   (b) a fusion protein containing the propeptide of factor IX (9) and the light chain of factor X (X) combined with the 1 st  Kunitz domain of Tissue Factor Plasminogen inhibitor (TFPI-KI) designated 9XKI,   (c) a fusion protein containing the propeptide of factor IX combined with the mature peptide of protein C, designated 9PC,   (d) a fusion protein containing the propeptide of factor VII combined with the mature peptide of protein C, designated 7PC, and   (e) a fusion protein containing the propeptide of factor X combined with the mature peptide of protein C, designated 10PC.   
     
     
         104 . The mammal of  claim 96 , wherein said non-secreted transactivating protein is a modified glucocorticoid receptor (GR) or a modified Stat5 transcription factor, containing a DNA binding domain which recognizes the DNA binding site in a type B gene and possesses transactivating activity. 
     
     
         105 . The mammal of  claim 104 , wherein said non-secreted transactivating protein is further modified to contain an enhanced transactivating activity by the addition of extra transactivation domains. 
     
     
         106 . The mammal of  claim 105  wherein said extra transactivation domain is selected from the group consisting of TAF-1, TAF-2, TAU-1, TAU-2, VP16 and NF K B-p65. 
     
     
         107 . The mammal of  claim 104 , wherein the said DNA binding site is the binding site of the GAL 4 DNA binding protein. 
     
     
         108 . The mammal of  claim 104 , wherein said milk protein promoter is selected from the group consisting of whey acidic protein (WAP) promoter, α-casein promoter, β-casein promoter, κ-casein promoter, α-lactalbumin promoter, lactoferrin promoter and β-lactoglobulin promoter. 
     
     
         109 . The mammal of  claim 108 , wherein said non-human transgenic mammal is selected from the group consisting of mouse, rat, rabbit, pig, goat, sheep and cow. 
     
     
         110 . The mammal of  claim 96 , wherein said protein encoded by the DNA of (B)(5) is a vitamin K dependent (VKD) protein. 
     
     
         111 . The mammal of  claim 110 , wherein the VKD protein is replaced with another non-native propeptide from a different VKD protein. 
     
     
         112 . A cell or more than one cells, which said comprises:
 (A) an exogenous DNA sequence of type A, said type A DNA sequence comprising:
 (1) 5′ regulatory sequences including a milk protein promoter operably linked to the DNA of (b), 
 (2) DNA encoding a non-secreted transactivating protein, wherein said non-secreted transactivating protein contains a DNA binding domain which recognizes the DNA binding site in a type B DNA sequence, and possesses transactivating activity, and 
 (3) 3′ regulatory sequences active in the transgenic non-human mammal; and 
   (B) one or more exogenous DNA sequences of type B, said type B DNA sequence or each type B DNA sequence comprising:
 (4) a DNA binding site recognized by the DNA binding domain of said non-secreted transactivating protein, operably linked to a minimal promoter operably linked to the DNA of (e), 
 (5) DNA encoding a peptide or protein, and 
 (6) 3′ regulatory sequences active in the transgenic non-human mammal, wherein said DNA sequences are stably integrated into the genome of said cell, wherein said milk protein promoter and said minimal promoter are different promoters. 
   
     
     
         113 . An embryo comprising:
 (A) an exogenous DNA sequence of type A, said type A DNA sequence comprising:
 (1) 5′ regulatory sequences including a milk protein promoter operably linked to the DNA of (b), 
 (2) DNA encoding a non-secreted transactivating protein, wherein said non-secreted transactivating protein contains a DNA binding domain which recognizes the DNA binding site in a type B DNA sequence, and possesses transactivating activity, and 
 (3) 3′ regulatory sequences active in the transgenic non-human mammal; and 
   (B) one or more exogenous DNA sequences of type B, said type B DNA sequence or each type B DNA sequence comprising:
 (4) a DNA binding site recognized by the DNA binding domain of said non-secreted transactivating protein, operably linked to a minimal promoter operably linked to the DNA of (e), 
 (5) DNA encoding a peptide or protein, and 
 (6) 3′ regulatory sequences active in the transgenic non-human mammal, wherein said DNA sequences are stably integrated into the genome of said cell, wherein said milk protein promoter and said minimal promoter are different promoters. 
   
     
     
         114 . A method of altering the efficiency of expression of a vitamin K dependent (VKD) protein in said transgenic non-human mammal of  claim 110 , wherein said transgenic non-human mammal further comprises replacing the DNA sequence encoding the native propeptide on a VKD protein with another non-native propeptide from a different VKD protein. 
     
     
         115 . The method of  claim 114 , wherein the frequency of post translational modification of a vitamin K dependant (VKD) protein is increased. 
     
     
         116 . The method of  claim 114 , wherein said mammal is selected from the group consisting of mouse, hamster, rat, rabbit, pig, goat, sheep and cow. 
     
     
         117 . The method of  claim 114 , wherein the VKD protein is selected from the group consisting of:
 (a) Factor X, and Factor X's propeptide is replaced with the propeptide from Factor IX;   (b) Protein C, and Protein C's propeptide is replaced with the propeptide from Factor VII;   (c) Protein C, and Protein C's propeptide is replaced with the propeptide from Factor IX and   (d) Protein C, and Protein C's propeptide is replaced with the propeptide from Factor X.