Exogenous proteins expressed in avians and their eggs

Abstract

This invention provides vectors and methods for the stable introduction of exogenous nucleic acid sequences into the genome of avians in order to express the exogenous sequences to alter the phenotype of the avians or to produce desired proteins. In particular, transgenic avians are produced which express exogenous sequences in their oviducts and which deposit exogenous proteins into their eggs. Avian eggs that contain exogenous proteins are encompassed by this invention. The instant invention further provides novel forms of interferon and erythropoietin which are efficiently expressed in the oviduct of transgenic avians and deposited into avian eggs.

What is claimed is:  
     
         1 . A vector comprising a coding sequence and a promoter in operational and positional relationship to express said coding sequence in an avian oviduct, wherein said coding sequence is selected from the group consisting of recombinant transgenic poultry derived interferon-α 2b and erythropoietin coding sequence.  
     
     
         2 . The vector of  claim 1 , wherein said promoter is selected from the group consisting of a cytomegalovirus (CMV) promoter, a MDOT promoter, a rous-sarcoma virus (RSV) promoter, a murine leukemia virus (MLV) promoter, a mouse mammary tumor virus (MMTV) promoter, an ovalbumin promoter, a lysozyme promoter, a conalbumin promoter, an ovomucoid promoter, an ovomucin promoter, an ovotransferrin promoter, and segments thereof, wherein said promoter is sufficient for effecting expression of the coding sequence in the avian oviduct.  
     
     
         3 . The vector of  claim 1 , wherein the vector is retroviral and wherein said coding sequence and said promoter are both positioned between the 5′ and 3′ LTRs of the retroviral vector.  
     
     
         4 . The vector of  claim 3 , wherein the retroviral vector is derived from the avian leukosis virus (ALV), murine leukemia virus (MLV), or lentivirus.  
     
     
         5 . The vector of  claim 1 , further comprising a signal peptide coding sequence which is operably linked to said coding sequence, so that upon translation in a cell, the signal peptide will direct secretion of the transgenic poultry derived interferon-α 2b or erythropoietin expressed by the vector into the egg white of a hard shell egg.  
     
     
         6 . The vector of  claim 1 , further comprising a marker gene, wherein said marker gene is operably linked to a promoter selected from the group consisting of a cytomegalovirus (CMV) promoter, a MDOT promoter, a rous-sarcoma virus (RSV) promoter, a murine leukemia virus (MLV) promoter, a mouse mammary tumor virus (MMTV) promoter, an ovalbumin promoter, a lysozyme promoter, a conalbumin promoter, an ovomucoid promoter, an ovomucin promoter, an ovotransferrin promoter, and segments thereof.  
     
     
         7 . A transgenic avian having a transgene in the genetic material of its germ-line tissue, wherein the transgene comprises an exogenous gene and a promoter in operational and positional relationship to express transgenic poultry derived interferon-α 2b or erythropoietin, wherein said exogenous gene is expressed in an avian oviduct of said transgenic avian.  
     
     
         8 . The transgenic avian of  claim 7 , wherein the promoter is selected from the group consisting of a cytomegalovirus (CMV) promoter, a MDOT promoter, a rous-sarcoma virus (RSV) promoter, a murine leukemia virus (MLV) promoter, a mouse mammary tumor virus (MMTV) promoter, an ovalbumin promoter, a lysozyme promoter, a conalbumin promoter, an ovomucoid promoter, an ovomucin promoter, an ovotransferrin promoter, and segments thereof.  
     
     
         9 . The transgenic avian of  claim 7 , wherein the transgenic avian is selected from the group consisting of chicken and turkey.  
     
     
         10 . An egg of an avian species containing protein exogenous to the avian species, wherein said protein is selected from the group consisting of transgenic poultry derived interferon-α 2b and erythropoietin.  
     
     
         11 . A method for the stable introduction of an exogenous coding sequence into the genome of an avian, comprising: 
 introducing the vector of  claim 1  into avian embryonic blastodermal cells, wherein the vector is randomly inserted into the avian genome.    
     
     
         12 . A method for producing transgenic poultry derived interferon-α 2b or erythropoietin protein in an avian oviduct, comprising: 
 providing a vector that comprises a coding sequence and a promoter operably linked to said coding sequence, wherein said promoter can effect expression of the coding sequence in an avian oviduct;  
 creating transgenic cells or tissue by introducing said vector into avian embryonic blastodermal cells, wherein the vector sequence is randomly inserted into the avian genome; and  
 deriving a mature transgenic avian from said transgenic cells or tissue, wherein the transgenic avian expresses the transgenic poultry derived interferon-α 2b or erythropoietin protein in its oviduct and in its blood.  
 
     
     
         13 . The method of  claim 12 , wherein said promoter is selected from the group consisting of a cytomegalovirus (CMV) promoter, a MDOT promoter, a rous-sarcoma virus (RSV) promoter, a murine leukemia virus (MLV) promoter, a mouse mammary tumor virus (MMTV) promoter, an ovalbumin promoter, a lysozyme promoter, a conalbumin promoter, an ovomucoid promoter, an ovomucin promoter, an ovotransferrin promoter, and segments thereof.  
     
     
         14 . The method of  claim 12 , wherein the introduction of the vector into the blastodermal cells is mediated by a retrovirus.  
     
     
         15 . The method of  claim 14 , wherein the step of introducing said vector to the embryonic blastodermal cells includes administering helper cells to an embryonic blastoderm, wherein said helper cells produce the retrovirus.  
     
     
         16 . A method for producing an avian egg which contains transgenic poultry derived interferon-α 2b or erythropoietin protein, comprising: 
 providing a vector that comprises a coding sequence and a promoter operably linked to said coding sequence, wherein said promoter can effect expression of the coding sequence in an avian oviduct;  
 creating transgenic cells or tissue by introducing said vector into avian embryonic blastodermal cells, wherein the vector sequence is randomly inserted into the avian genome; and  
 deriving a mature transgenic avian from said transgenic cells or tissue, wherein the transgenic avian expresses the coding sequence in its oviduct, and a resulting protein is secreted into the oviduct lumen, so that the transgenic poultry derived interferon-α 2b or erythropoietin protein is deposited into the egg white of a hard shell egg.  
 
     
     
         17 . An isolated polynucleotide comprising the optimized polynucleotide sequence of human interferon-α 2b (SEQ ID NO: 1).  
     
     
         18 . An isolated protein comprising the polypeptide sequence of transgenic poultry derived interferon-α 2b, wherein said protein is O-glycosylated at Thr-106 with Gal-NAcGal-, SA- Gal-NAcGal-,  
                         
Gal-Gal-NAcGal-, and  
       
         
           
           
               
               
           
         
       
     
     
         19 . The isolated protein of  claim 18 , wherein Gal-NAcGal- is 20%, SA-Gal-NAcGal- is 29%,  
                         
is 9%,  
                         
is 6%, Gal-Gal-NAcGal- is 7%, and  
       
         
           
           
               
               
           
         
       
     
     
         20 . A pharmaceutical composition comprising the polypeptide sequence of transgenic poultry derived interferon-α 2b, wherein said protein is O-glycosylated at Thr-106 with Gal-NAcGal-, SA-Gal-NAcGal-,  
                         
Gal-Gal-NAcGal-, and  
       
         
           
           
               
               
           
         
       
     
     
         21 . The isolated pharmaceutical composition of  claim 20 , wherein Gal-NAcGal- is 20%, SA-Gal-NAcGal- is 29%,  
                         
is 9%,  
                         
is 6%, Gal-Gal-NAcGal- is 7%, and  
       
         
           
           
               
               
           
         
       
     
     
         22 . An isolated polynucleotide comprising the optimized polynucleotide sequence of human erythropoietin (SEQ ID NO: 3).  
     
     
         23 . An MDOT promoter, comprised of elements from the ovomucoid (MD) and ovotransferrin (TO) promoters.  
     
     
         24 . A vector comprising a first and second coding sequence and a promoter in operational and positional relationship to said first and second coding sequence to express said first and second coding sequence in an avian oviduct; and an internal ribosome entry site element positioned between the first and second coding sequence, wherein said first coding sequence codes for protein X and said second coding sequence codes for protein Y, and wherein said protein X and protein Y are deposited into the egg white of a hard shell egg.  
     
     
         25 . The vector of  claim 24 , wherein protein X is a light chain (LC) of a monoclonal antibody and protein Y is a heavy chain (HC) of a monoclonal antibody.  
     
     
         26 . The vector of  claim 24 , further comprising at least one additional coding sequence and at least one additional internal ribosome entry site element, wherein said internal ribosome entry site element is further positioned between two coding sequences, such that each coding sequence in the vector is separated from another coding sequence by an internal ribosome entry site element.  
     
     
         27 . The vector of  claim 24 , wherein the protein encoded by the second coding sequence is capable of providing post-translational modification of the protein encoded by the first coding sequence.  
     
     
         28 . A method for producing an avian egg which contains a protein, comprising: 
 providing the vector of  claim 24;     creating transgenic cells or tissue by introducing said vector into avian embryonic blastodermal cells, wherein the vector sequence is randomly inserted into the avian genome; and    deriving a mature transgenic avian from said transgenic cells or tissue, wherein the transgenic avian expresses the coding sequences in its oviduct, and the resulting protein is secreted into the oviduct lumen, so that the protein is deposited into the egg white of a hard shell egg.    
     
     
         29 . The method of  claim 28 , wherein the protein is a monoclonal antibody.  
     
     
         30 . A method for producing an avian egg which contains a protein, comprising: 
 providing the vector of  claim 26;     creating transgenic cells or tissue by introducing said vector into avian embryonic blastodermal cells, wherein the vector sequence is randomly inserted into the avian genome; and    deriving a mature transgenic avian from said transgenic cells or tissue, wherein the transgenic avian expresses the coding sequences in its oviduct, and the resulting protein is secreted into the oviduct lumen, so that the protein is deposited into the egg white of a hard shell egg.    
     
     
         31 . The method of  claim 30 , wherein the protein is a monoclonal antibody.