Transgenically produced non-secreted proteins

Abstract

The invention provides a method of making and secreting a non-secreted protein. The method includes expressing the protein from a nucleic acid construct which includes: (a) a mammary epithelial specific promoter; (b) a milk protein specific signal sequence which can direct the secretion of a protein; (c) optionally, a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein to allow secretion in the milk of a transgenic mammal, of the non-secreted protein; and (d) a sequence which encodes a non-secreted protein, wherein elements (a), (b), optionally (c), and (d) are preferably operatively linked in the order recited. Both glutamic acid decarboxylase (GAD) and myelin basic protein (MBP), which are cytoplasmic proteins, have been produced by the methods of the present invention. The invention also provides methods for treating diabetes and multiple sclerosis using proteins produced by the methods of the present invention.

1 . A method of making and secreting a non-secreted protein comprising expressing the protein from a nucleic acid construct which comprises: 
 (a) a mammary epithelial specific promoter;    (b) a milk protein specific signal sequence which can direct the secretion of a non-secreted protein;    (c) a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein to allow secretion in the milk of a transgenic mammal of a non-secreted protein; and    (d) a sequence which encodes a non-secreted protein,    wherein elements (a), (b),(c), and (d) are preferably operatively linked in the order recited, thereby producing the non-secreted protein in the milk of the transgenic mammal.    
     
     
         2 . The method of  claim 1 , wherein the mammary epithelial specific promoter is the β-casein promoter sequence.  
     
     
         3 . The method of  claim 1 , wherein the milk protein specific signal sequence is the β-casein signal sequence.  
     
     
         4 . The method of  claim 1 , wherein the non-secreted protein-coding sequence is of a human origin.  
     
     
         5 . The method of  claim 1 , wherein the non-secreted protein-coding sequence codes for a truncated, nuclear, or a cytoplasmic polypeptide.  
     
     
         6 . The method of  claim 1 , wherein the non-secreted protein-coding sequence codes for glutamic acid decarboxylase or myelin basic protein.  
     
     
         7 . The method of  claim 1 , wherein the non-secreted protein is a mutant protein which lacks a biological activity of the wild type protein.  
     
     
         8 . The method of  claim 1 , wherein the transgenic mammal is selected from the group consisting of sheep, mice, pigs, cows and goats.  
     
     
         9 . An isolated nucleic acid construct which comprises: 
 (a) a mammary epithelial specific promoter;    (b) a milk protein specific signal sequence which can direct the secretion of a protein;    (c) a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein to allow secretion in the milk of a transgenic mammal of a non-secreted protein; and    (d) a sequence which encodes a non-secreted protein,    wherein elements (a), (b), (c), and (d) are preferably operatively linked in the order recited, thereby producing the non-secreted protein in the milk of the transgenic mammal.    
     
     
         10 . The method of  claim 9 , wherein the mammary epithelial specific promoter sequence is the β-casein promoter sequence.  
     
     
         11 . The method of  claim 9 , wherein the milk protein specific signal sequence is the β-casein signal sequence.  
     
     
         12 . The method of  claim 9 , wherein the non-secreted protein-coding sequence is of a human origin.  
     
     
         13 . The method of  claim 9 , wherein the non-secreted protein-coding sequence codes for a truncated, nuclear, or a cytoplasmic polypeptide.  
     
     
         14 . The method of  claim 9 , wherein the non-secreted protein-coding sequence codes for glutamic acid decarboxylase or myelin basic protein.  
     
     
         15 . The method of  claim 9 , wherein the non-secreted protein is a mutant protein which lacks a biological activity of the wild type protein.  
     
     
         16 . A method for providing a non-secreted protein in the milk of a transgenic mammal, comprising obtaining milk from a transgenic mammal having introduced into its germline a nucleic acid construct comprising a heterologous non-secreted protein-coding sequence operatively linked to a milk protein specific signal sequence and a mammary epithelial specific promoter sequence that result in the preferential expression of the protein-coding sequence in mammary gland epithelial cells, thereby secreting the heterologous non-secreted protein in the milk of the mammal  
     
     
         17 . The method of  claim 16 , wherein the non-secreted protein is inactive.  
     
     
         18 . The method of  claim 16 , wherein the transgenic mammal is selected from the group consisting of sheep, mice, pigs, cows and goats.  
     
     
         19 . The method of  claim 16 , wherein the mammary epithelial specific promoter is selected from the group consisting of the beta lactoglobulin promoter, whey acid protein promoter, β-casein promoter and the lactalbumin promoter.  
     
     
         20 . The method of  claim 16 , wherein the milk protein specific signal sequence is β-casein signal sequence.  
     
     
         21 . The method of  claim 16 , wherein the non-secreted protein-coding sequence is of a human origin.  
     
     
         22 . The method of  claim 16 , wherein the non-secreted protein-coding sequence codes for a truncated, nuclear, or a cytoplasmic polypeptide.  
     
     
         23 . The method of  claim 16 , wherein the non-secreted protein-coding sequence codes for glutamic acid decarboxylase or myelin basic protein.  
     
     
         24 . The method of  claim 16 , wherein the non-secreted protein-coding sequence codes for an inactive form of a glutamic acid decarboxylase.  
     
     
         25 . The method of  claim 16 , wherein the non-secreted polypeptide is purified from the milk of a transgenic mammal.  
     
     
         26 . A method of inducing tolerance in a subject to an antigen, comprising: 
 providing a tolerogen expressed in a transgenic mammal which comprises the antigen; and    administering the tolerogen to the subject in an amount sufficient to induce tolerance to the antigen.    
     
     
         27 . The method of  claim 26 , wherein the tolerogen is administered orally to the subject in the milk of a transgenic mammal.  
     
     
         28 . The method of  claim 26 , wherein the tolerogen is an inactive protein.  
     
     
         29 . The method of  claim 26 , wherein the tolerogen is a non-secreted protein.  
     
     
         30 . The method of  claim 26 , wherein the tolerogen is a protein antigen fused to all or part of a secreted protein.  
     
     
         31 . The method of  claim 26 , wherein the antigen is an antigen which is characteristic of an autoimmune disorder selected from the group consisting of diabetes, lupus, multiple sclerosis and rheumatoid arthritis.  
     
     
         32 . The method of  claim 26 , wherein the subject is at risk of developing, or has, an anutoimmune disorder selected from the group consisting of diabetes, lupus, multiple sclerosis and rheumatoid arthritis.  
     
     
         33 . A method of treating insulin-dependent diabetes mellitus (IDDM) in a subject, comprising administering to the subject therapeutically effective amount of a transgenically produced tolerogen which comprises glutamic acid decarboxylase, or an effective amount of a fusion protein which comprises glutamic acid decarboxylase.  
     
     
         34 . The method of  claim 33 , wherein the subject is orally administered milk from a transgenic mammal which expresses the transgenically produced tolerogen which comprises glutamic acid decarboxylase, or an effective amount of a fusion protein which comprises glutamic acid decarboxylase.  
     
     
         35 . The method of  claim 33 , wherein the transgenically produced tolarogen is in inactive form.  
     
     
         36 . A method for treating multiple sclerosis in a subject, comprising administering to the subject therapeutically effective amount of a transgenically produced tolerogen which comprises myelin basic protein, or an effective amount of a fusion protein which comprises myelin basic protein.  
     
     
         37 . The method of  claim 36 , wherein the subject is orally administered milk from a transgenic mammal which expresses the transgenically produced tolerogen which comprises myelin basic protein, or an effective amount of a fusion protein which comprises myelin basic protein.  
     
     
         38 . The method of  claim 36 , wherein the transgenically produced tolarogen is in inactive form.  
     
     
         39 . A therapeutic composition which comprises a therapeutically effective amount of the transgenically produced myelin basic protein and a pharmaceutically-acceptable carrier or diluent.  
     
     
         40 . The method of  claim 39 , wherein the pharmaceutically-acceptable carrier or diluent comprises the milk of a transgenic mammal.  
     
     
         41 . A fusion protein which comprises: 
 a non-secreted protein;    a milk protein specific signal sequence which directs the secretion of the protein; and    a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein to allow secretion in the milk of a transgenic mammal, of the non-secreted protein.    
     
     
         42 . A method of inducing tolerance in a transgenic mammal to an antigen, 
 comprising expressing a tolerogen in the milk of the transgenic mammal at a level sufficient to induce tolerance.    
     
     
         43 . The method of  claim 42 , wherein the antigen is selected from the group consisting of a xenoantigen, an alloantigen and an autoantigen.  
     
     
         44 . The method of  claim 42 , wherein the transgenic mammal is a transgenic dairy mammal.  
     
     
         45 . The method of  claim 44 , wherein the transgenic dairy mammal is selected from the group consisting of goats, sheep and cows.  
     
     
         46 . The method of  claim 42 , wherein the tolerogen is a non-secreted protein.  
     
     
         47 . The method of  claim 42 , wherein the antigen is an antigen which is characteristic of an autoimmune disorder selected from the group consisting of diabetes, lupus, multiple sclerosis, and rheumatoid arthritis.  
     
     
         48 . A transgenically produced non-secreted polypeptide, wherein the polypeptide is secreted.  
     
     
         49 . The transgenically produced non-secreted polypeptide of  claim 48 , wherein the transgenically produced non-secreted polypeptide is secreted into the milk of a transgenic mammal.  
     
     
         50 . The transgenically produced non-secreted polypeptide of  claim 48 , wherein the transgenically produced non-secreted polypeptide is selected from the group consisting of a truncated, nuclear, and cytoplasmic polypeptide.  
     
     
         51 . The transgenically produced non-secreted polypeptide of  claim 51 , wherein the cytoplasmic polypeptide is glutamic acid decarboxylase or myelin basic protein.  
     
     
         52 . The transgenically produced non-secreted polypeptide of  claim 51 , wherein glutamic acid decarboxylase is expressed in an inactive form.  
     
     
         53 . The transgenically produced non-secreted polypeptide of  claim 49 , wherein the transgenic mammal is selected from the group consisting of sheep, mice, pigs, cows and goats.