Lysosomal proteins produced in the milk of transgenic animals

Abstract

The invention provides transgenic nonhuman mammals producing phosphorylated lysosomal proteins in their milk, and methods of generating the same. Phosphorylation occurs at the 6′ position of a mannose side chain residue. Also provided are methods of purifying lysosomal proteins from milk, and incorporating the proteins into pharmaceutical compositions for use in enzyme replacement therapy.

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 a lysosomal protein 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 descendent of the nonhuman mammal, is capable of expressing the secretory-recombinant DNA segment in the mammary secretory cells to produce a form of the lysosomal protein that is process and secreted by the mammary secretory cells into milk as a mannose 6-phosphate containing lysosomal protein.    
     
     
         2 . The transgenic nonhuman mammal of  claim 1 , wherein the lysosomal protein is an enzyme.  
     
     
         3 . The transgenic nonhuman mammal of  claim 2 , wherein the concentration of the mannose 6-phosphate containing lysosomal protein in the milk is at least 100 μg/ml.  
     
     
         4 . The nonhuman transgenic mammal of  claim 1 , wherein the lysosomal protein is human.  
     
     
         5 . The nonhuman transgenic mammal of  claim 4 , wherein the lysosomal protein is acid μ-glucosidase.  
     
     
         6 . The nonhuman transgenic mammal of  claim 5 , that is a mouse, rabbit, goat, sheep, porcine or bovine.  
     
     
         7 . The nonhuman transgenic mammal of  claim 6 , wherein the recombinant DNA segment is cDNA.  
     
     
         8 . The nonhuman transgenic mammal of  claim 6 , wherein the recombinant DNA segment is genomic.  
     
     
         9 . The nonhuman transgenic mammal of  claim 1 , wherein the recombinant DNA segment is a lysosomal protein secretory DNA segment.  
     
     
         10 . The nonhuman transgenic mammal of  claim 1 , wherein the secretory DNA segment is a lysosomal protein secretory DNA segment.  
     
     
         11 . A method for providing a mannose 6-phosphate containing lysosomal protein, the method comprising: 
 recovering milk comprising the mannose 6-phosphate containing lysosomal protein from an adult form of the transgenic nonhuman mammal or its female descendant of  claim 1 .    
     
     
         12 . The method of  claim 11 , further comprising incorporating the milk into a food product.  
     
     
         13 . The method of  claim 11 , further comprising purifying the mannose 6-phosphate containing lysosomal protein from the milk.  
     
     
         14 . The method of  claim 13 , wherein the mannose 6-phosphate containing lysosomal protein is purified to at least 95% pure from other components of the milk.  
     
     
         15 . The method of  claim 14 , further comprising mixing the mannose 6-phosphate containing lysosomal protein with a pharmaceutical carrier for intravenous, intradermal, intramuscular or oral administration.  
     
     
         16 . Milk from the transgenic nonhuman mammal of  claim 1 , the milk comprising the the mannose 6-phosphate containing lysosomal protein.  
     
     
         17 . The milk of  claim 16 , wherein the concentration of lysosomal protein is at least 100 μg/ml.  
     
     
         18 . A mannose 6-phosphate containing lysosomal protein produced by the method of  claim 11 .  
     
     
         19 . A pharmaceutical composition comprising the mannose 6-phosphate containing lysosomal protein of  claim 18  and a pharmaceutical carrier.  
     
     
         20 . The pharmaceutical composition of  claim 19 , wherein the pharmaceutical carrier is for intravenous administration.