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.

1 - 33 . (canceled)  
     
     
         34 . A procollagen or collagen protein, wherein 7-27% of proline residues of the protein are hydroxylated, and/or 0-5% of lysine residues of the protein are hydroxylated.  
     
     
         35 . The protein of  claim 34  in purified form.  
     
     
         36 . The protein of  claim 34  that is procollagen.  
     
     
         37 . The protein of  claim 34  that is collagen.  
     
     
         38 . The protein of  claim 34 , wherein 7-27% of proline residues of the protein are hydroxylated and 0-5% of lysine residues of the protein are hydroxylated.  
     
     
         39 . The protein of  claim 34 , wherein the protein is hydroxylated at 0-5% of lysine residues.  
     
     
         40 . The protein of  claim 34 , wherein the protein is hydroxylated at 0% of lysine residues.  
     
     
         41 . The protein of  claim 34 , wherein the protein is hydroxylated at 2% of lysine residues.  
     
     
         42 . The protein of  claim 34 , wherein the protein is hydroxylated at 4% of lysine residues.  
     
     
         43 . The protein of  claim 34 , wherein the protein is hydroxylated at 5% of lysine residues.  
     
     
         44 . The protein of  claim 34 , wherein 7-27% of proline residues in the protein are hydroxylated.  
     
     
         45 . The procollagen or collagen of  claim 34  in trimeric form.  
     
     
         46 . The procollagen or collagen of  claim 34  in homotrimeric form.  
     
     
         47 . The procollagen or collagen protein of  claim 46  associated with another type of procollagen or collagen protein in heterotrimeric form.  
     
     
         48 . The protein of  claim 34 , wherein the protein is human.  
     
     
         49 . The protein of  claim 34  that is proα1(I).  
     
     
         50 . The protein of  claim 34  produced by 
 (a) providing a transgenic nonhuman mammal having a transgene comprising: 
 (i) a mammary-gland specific promoter;  
 (ii) a mammary-gland specific enhancer;  
 (iii) a secretory DNA segment encoding a signal peptide functional in mammary secretory cells of the transgenic nonhuman mammal; and  
 (iv) 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;    (c) 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    (d) purifying the procollagen or collagen from the milk.    
     
     
         51 . A method of producing an underhydroxylated procollagen or collagen polypeptide comprising: 
 (a) providing a transgenic nonhuman mammal having a transgene comprising: 
 (i) a mammary-gland specific promoter;  
 (ii) a mammary-gland specific enhancer;  
 (iii) a secretory DNA segment encoding a signal peptide functional in mammary secretory cells of the transgenic nonhuman mammal; and  
 (iv) 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;    (b) 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    (d) purifying procollagen or collagen from the milk, wherein 7-27% of proline residues of the protein are hydroxylated, and/or 0-5% of lysine residues of the protein are hydroxylated.    
     
     
         52 . The method of  claim 51  wherein the polypeptide is procollagen, further comprising the step of contacting the procollagen with a proteolytic enzyme to convert the procollagen to collagen.  
     
     
         53 . The method of  claim 51 , wherein the transgenic nonhuman mammal is a bovine.  
     
     
         54 . The method of  claim 51 , wherein the transgenic nonhuman mammal is a mouse.  
     
     
         55 . The method of  claim 51 , wherein the transgenic nonhuman mammal is a sheep.  
     
     
         56 . Milk produced by the method of  claim 51 , the milk comprising the procollagen or collagen polypeptide wherein 7-27% of proline residues of the protein are hydroxylated, and/or 0-5% of lysine residues of the protein are hydroxylated.  
     
     
         57 . The milk of  claim 56 , wherein the procollagen or collagen is in trimeric form.  
     
     
         58 . The milk of  claim 56 , wherein the concentration of procollagen or collagen is at least 100 μg/ml.  
     
     
         59 . The milk of  claim 56 , wherein the concentration of the procollagen or collagen in the milk is at least 1 mg/ml.  
     
     
         60 . In a method of treating a patient using procollagen or collagen protein, the improvement wherein 7-27% of proline residues of the protein are hydroxylated, and/or 0-5% of lysine residues of the protein are hydroxylated.  
     
     
         61 . The method of  claim 60 , wherein the polypeptide is crosslinked before administration into the patient.  
     
     
         62 . The method of  claim 60 , wherein the polypeptide is crosslinked after administration into the patient.  
     
     
         63 . The method of  claim 60  comprising injecting the procollagen or collagen into the patient to correct a defect in a soft tissue.  
     
     
         64 . The method of  claim 60 , wherein the method is selected from the group consisting of correcting defects in soft tissues, reconstructive surgery, restoring defects in bone, enhancing bone growth, repairing cartilage damage, inducing tolerance to rheumatoid arthritis, cardiovascular surgery, thoracic surgery, neurosurgery, and stabilizing an agent in a drug delivery system.