Production of heterologous protein in the avian oviduct

Abstract

The invention includes methods of making transgenic avians by introducing a nucleic acid sequence into the genome of an avian embryo wherein the nucleic acid sequence comprises a nucleotide sequence encoding a heterologous protein, developing the avian embryo to hatch, developing the hatched chick to sexual maturity and obtaining offspring from the sexually mature hatched chick which produce egg white containing the heterologous protein.

1 . A method comprising:
 introducing a non-retroviral nucleic acid sequence into an avian embryo wherein the nucleic acid sequence comprises a nucleotide sequence encoding a heterologous protein;   incubating the avian embryo until hatch wherein the avian embryo comprises the non-retroviral nucleic acid sequence in its genome;   growing a hatched chick obtained from the avian embryo to sexual maturity;   obtaining offspring from a sexually mature avian obtained from the hatched chick;   developing to sexual maturity a female offspring obtained from the sexually mature avian obtained from the hatched chick; and   the sexually mature female offspring producing egg white containing the heterologous protein.   
     
     
         2 . The method of  claim 1  comprising isolating the heterologous protein from the egg white. 
     
     
         3 . The method of  claim 1  wherein the egg white is contained in an egg laid by the avian. 
     
     
         4 . The method of  claim 3  wherein the egg contains greater than 50 ng of heterologous protein. 
     
     
         5 . The method of  claim 3  wherein the egg contains greater than 100 ng of heterologous protein. 
     
     
         6 . The method of  claim 3  wherein the egg contains greater than 250 ng of heterologous protein. 
     
     
         7 . The method of  claim 3  wherein the egg contains greater than 500 ng of heterologous protein. 
     
     
         8 . The method of  claim 1  wherein the protein is produced in a tubular gland cell. 
     
     
         9 . The method of  claim 1  wherein the nucleotide sequence comprises a tissue specific promoter. 
     
     
         10 . The method of  claim 1  wherein the avian is a chicken. 
     
     
         11 . The method of  claim 10  comprising isolating the heterologous protein from the egg white. 
     
     
         12 . A method comprising:
 introducing a linear nucleic acid sequence into an avian embryo wherein the nucleic acid sequence comprises a nucleotide sequence encoding a heterologous protein;   incubating the avian embryo until hatch wherein the avian embryo comprises the linear nucleic acid sequence in its genome;   growing a hatched chick obtained from the avian embryo to sexual maturity;   obtaining offspring from a sexually mature avian obtained from the hatched chick;   developing to sexual maturity a female offspring obtained from the sexually mature avian obtained from the hatched chick; and   the sexually mature female offspring producing egg white containing the heterologous protein.   
     
     
         13 . The method of  claim 12  comprising isolating the heterologous protein from the egg white. 
     
     
         14 . The method of  claim 12  wherein the heterologous protein is a therapeutic protein. 
     
     
         15 . The method of  claim 12  wherein the sexually mature female offspring lays an egg containing greater than 250 ng of heterologous protein in the egg white. 
     
     
         16 . The method of  claim 12  wherein the avian is a chicken. 
     
     
         17 . The method of  claim 16  comprising isolating the heterologous protein from the egg white. 
     
     
         18 . A method comprising:
 introducing a non-infective nucleic acid sequence into an avian embryo wherein the nucleic acid sequence comprises a nucleotide sequence encoding a therapeutic protein;   incubating the avian embryo until hatch wherein the avian embryo comprises the non-retroviral nucleic acid sequence in its genome;   growing a hatched chick obtained from the avian embryo to sexual maturity;   obtaining offspring from a sexually mature avian obtained from the hatched chick;   developing to sexual maturity a female offspring obtained from the sexually mature avian obtained from the hatched chick; and   the sexually mature female offspring producing egg white containing the therapeutic protein.   
     
     
         19 . The method of  claim 18  comprising isolating the therapeutic protein from the egg white. 
     
     
         20 . The method of  claim 18  wherein the therapeutic protein is selected from the group consisting of growth hormone, human growth hormone, cytokine, structural protein, enzyme, interferon, lysozyme, β-casein, albumin, α-1 antitrypsin, antithrombin III, collagen, factor VIII, factor IX, factor X, fibrinogen, insulin, lactoferrin, protein C, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), tissue-type plasminogen activator (tPA), somatotropin, and chymotrypsin. 
     
     
         21 . The method of  claim 18  wherein the therapeutic protein is an immunoglobulin polypeptide. 
     
     
         22 . The method of  claim 18  wherein the therapeutic protein is an antibody. 
     
     
         23 . The method of  claim 22  wherein the antibody is a monoclonal antibody. 
     
     
         24 . The method of  claim 22  wherein the antibody is humanized. 
     
     
         25 . The method of  claim 18  wherein the therapeutic protein comprises at least one of an antibody heavy chain and an antibody light chain. 
     
     
         26 . The method of  claim 18  wherein the nucleic acid sequence comprises an IRES. 
     
     
         27 . The method of  claim 18  wherein the sexually mature female offspring lays an egg containing greater than 50 ng of heterologous protein in the egg white. 
     
     
         28 . The method of  claim 18  wherein the sexually mature female offspring lays an egg containing greater than 500 ng of heterologous protein in the egg white. 
     
     
         29 . The method of  claim 18  wherein the protein is produced in a tubular gland cell. 
     
     
         30 . The method of  claim 18  wherein the nucleotide sequence comprises a tissue specific promoter. 
     
     
         31 . The method of  claim 18  comprising preparing a pharmaceutical composition containing the therapeutic protein. 
     
     
         32 . The method of  claim 18  wherein the therapeutic protein is a fusion protein. 
     
     
         33 . The method of  claim 18  wherein the therapeutic protein is selected from the group consisting of trastuzumab, anti-HER2 antibody, anti-glycoprotein IIb/IIIa receptor antibody, daclizumab, anti-CD25 antibody, anti-17-IA cell surface antigen antibody, IgG2a antibody, BEC2, anti-idiotype IgG antibody, IMC-C225, anti-EGFR antibody, anti-αVβ3 integrin antibody, IH/LDP-03, anti CD52 antibody, anti-CD33 antibody, anti-CD20, anti-CD22 antibody, ICM3, anti-ICAM3 antibody, IDEC-114, anti-CD80 antibody, anti-CD20 antibody, IDEC-131, anti-CD40L antibody, IDEC-151, anti-CD4 antibody, IDEC-152, anti-CD23 antibody, anti-CD3, 5G1.1, anti-complement factor 5 (C5) antibody, D2E7, anti-TNF-α antibody, CDP870, anti-TNF-α Fab fragment, anti-TNF-α antibody, IDEC-151, anti-CD4 antibody, MDX-CD4, anti-CD4 antibody, CDP571, anti-TNF-α antibody, LDP-02, anti-α4β7 antibody, OKT4A, anti-CD4 antibody, anti-CD40L IgG antibody, anti-VLA-4 antibody, CAT-152 and anti-TGF-β2 antibody. 
     
     
         34 . The method of  claim 18  wherein the avian is a chicken. 
     
     
         35 . The method of  claim 34  comprising isolating the heterologous protein from the egg white. 
     
     
         36 . A chicken produced by the method of  claim 18 .