Means and methods for regulating gene expression

Abstract

Described are means and methods for regulating gene expression and production of proteinaceous molecules. Described are methods for producing a proteinaceous molecule in a cell comprising selecting a cell for its suitability for producing the proteinaceous molecule, providing a nucleic acid encoding the proteinaceous molecule with a nucleic acid comprising a STAR (STabilizing Anti-Repression) sequence, expressing the resulting nucleic acid in the cell and collecting the proteinaceous molecule. Providing at least one STAR sequence to a nucleic acid encoding a proteinaceous molecule enhances production (yield) of the proteinaceous molecule by the host cell, increases the proportion of host cells with acceptable expression levels, and/or increases the stability of a gene expression level.

1 . A method for producing a proteinaceous molecule in a cell, the method comprising:
 providing a cell selected from the group consisting of a cell having an adenovirus Early Region 1 (E1) sequence, a HuNS-1 myeloma cell, a 293 cell, a CHO cell, a Vero cell, a WERI-Rb-1 retinoblastoma cell, a BHK cell, a non-secreting mouse myeloma Sp2/0-Ag 14 cell, a non-secreting mouse myeloma NSO cell, and an NCI-H295R adrenal gland carcinoma cell;   wherein the cell comprises an anti-repressor activity sequence operably linked to a nucleic acid sequence encoding a heterologous proteinaceous molecule, wherein the anti-repressor activity sequence comprises SEQ ID NO: 17;   expressing the proteinaceous molecule in the cell; and   harvesting material comprising the thus expressed proteinaceous molecule.   
     
     
         2 . The method according to  claim 1 , wherein the anti-repressor activity sequence consists of SEQ ID NO: 17. 
     
     
         3 . The method according to  claim 1 , wherein the cell comprises an adenovirus Early Region 1 (E1) sequence. 
     
     
         4 . The method according to  claim 1 , wherein the proteinaceous molecule is secreted by the cell. 
     
     
         5 . The method according to  claim 2 , wherein the proteinaceous molecule is secreted by the cell. 
     
     
         6 . The method according to  claim 1 , wherein the cell comprises a plurality of said anti-repressor activity sequence operably linked to the nucleic acid sequence encoding the heterologous proteinaceous molecule. 
     
     
         7 . The method according to  claim 6 , wherein at least one anti-repressor activity sequence is positioned 5′ of the sequence encoding the proteinaceous molecule and at least one anti-repressor activity sequence is positioned 3′ of the sequence encoding the proteinaceous molecule. 
     
     
         8 . The method according to  claim 1 , wherein the cell is a CHO cell. 
     
     
         9 . The method according to  claim 6 , wherein the cell is a CHO cell. 
     
     
         10 . The method according to  claim 7 , wherein the cell is a CHO cell. 
     
     
         11 . A method for producing a proteinaceous molecule in a cell, the method comprising:
 providing a cell selected from the group consisting of a cell having an adenovirus Early Region 1 (E1) sequence, a HuNS-1 myeloma cell, a 293 cell, a CHO cell, a Vero cell, a WERI-Rb-1 retinoblastoma cell, a BHK cell, a non-secreting mouse myeloma Sp2/0-Ag 14 cell, a non-secreting mouse myeloma NSO cell, and an NCI-H295R adrenal gland carcinoma cell; wherein the cell comprises an anti-repressor activity sequence operably linked to a nucleic acid sequence encoding a heterologous proteinaceous molecule, wherein the anti-repressor activity sequence comprises at least 50 bases of SEQ ID NO: 17;   expressing the proteinaceous molecule in the cell; and   harvesting material comprising the thus expressed proteinaceous molecule.   
     
     
         12 . A recombinant host cell line comprising:
 a cell selected from the group consisting of a cell line comprising an adenovirus Early Region 1 (E1) sequence, a HuNS-1 myeloma cell line, a 293 cell line, a CHO cell line, a Vero cell line, a WERI-Rb-1 retinoblastoma cell line, a BHK cell line, a non-secreting mouse myeloma Sp2/0-Ag 14 cell line, a non-secreting mouse myeloma NSO cell line, and an NCI-H295R adrenal gland carcinoma cell line;   the cell comprising an anti-repressor activity sequence operably linked to a nucleic acid sequence encoding a heterologous proteinaceous molecule, wherein the anti-repressor activity sequence comprises SEQ ID NO: 17.   
     
     
         13 . The cell line of  claim 12 , wherein the anti-repressor activity sequence consists of SEQ ID NO: 17. 
     
     
         14 . The cell line of  claim 12 , wherein the cell line comprises an adenovirus Early Region 1 sequence. 
     
     
         15 . The cell line of  claim 12 , wherein the cell comprises a plurality of the anti-repressor activity sequence operably linked to said nucleic acid sequence encoding the heterologous proteinaceous molecule. 
     
     
         16 . The cell line of  claim 12 , wherein at least one anti-repressor activity sequence is positioned 5′ of the sequence encoding the proteinaceous molecule and at least one anti-repressor activity sequence is positioned 3′ of the sequence encoding the proteinaceous molecule. 
     
     
         17 . The cell line of  claim 12 , wherein the cell line is a CHO cell line. 
     
     
         18 . The cell line of  claim 15 , wherein the cell line is a CHO cell line. 
     
     
         19 . The cell line of  claim 16 , wherein the cell line is a CHO cell line. 
     
     
         20 . A recombinant host cell line comprising:
 a cell selected from the group consisting of a cell line comprising an adenovirus Early Region 1 (E1) sequence, a HuNS-1 myeloma cell line, a 293 cell line, a CHO cell line, a Vero cell line, a WERI-Rb-1 retinoblastoma cell line, a BHK cell line, a non-secreting mouse myeloma Sp2/0-Ag 14 cell line, a non-secreting mouse myeloma NSO cell line, and an NCI-H295R adrenal gland carcinoma cell line;   wherein the cell comprises an anti-repressor activity sequence operably linked to a nucleic acid sequence encoding a heterologous proteinaceous molecule, wherein the anti-repressor activity sequence comprises at least 50 bases of SEQ ID NO: 17.