Method to alter chinese hamster ovary cell line stability

Abstract

The present invention provides a recombinant eukaryotic cell expressing one or more heterologous double strand break (DSB) repair proteins in an amount effective for enhancing DSB repair in the cell. The recombinant eukaryotic cell may express a recombinant product of interest. Also provided are methods for enhancing double strand break (DSB) repair in eukaryotic cells, establishing host cells for production of a recombinant product of interest, producing a recombinant product of interest, improving production of a recombinant product of interest by eukaryotic cells, and/or investigating suitability of eukaryotic cells as host cells for producing a recombinant product of interest.

What is claimed: 
     
         1 . A recombinant eukaryotic cell expressing a heterologous double strand break (DSB) repair protein in an amount effective for enhancing DSB repair in the cell. 
     
     
         2 . The recombinant eukaryotic cell of  claim 1 , wherein the heterologous DSB repair protein is expressed in an amount effective for enhancing stability of the cell for at least 1 month. 
     
     
         3 . The recombinant eukaryotic cell of  claim 1  or  2 , wherein the heterologous DSB repair protein may be selected from the group consisting of DNA ligase IV (LIG4), x-ray repair cross complementing 6 (XRCC6), partner and localizer of BRCA2 (PALB2), and PARP1 binding protein which is encoded by the PARPBP gene (PARI). 
     
     
         4 . The recombinant eukaryotic cell of  claim 3 , wherein the heterologous DSB repair protein is LIG4 or XRCC6. 
     
     
         5 . The recombinant eukaryotic cell of any one of  claims 1 - 4 , wherein the heterologous DSB repair protein is expressed transiently. 
     
     
         6 . The recombinant eukaryotic cell of any one of  claims 1 - 4 , wherein the heterologous DSB repair protein is expressed stably. 
     
     
         7 . The recombinant eukaryotic cell of any one of  claims 1 - 6 , wherein the recombinant eukaryotic cell is a mammalian cell. 
     
     
         8 . The recombinant eukaryotic cell of  claim 7 , wherein the mammalian cell is selected from the group consisting of a rodent cell, a mouse cell and a Chinese hamster cell. 
     
     
         9 . The recombinant eukaryotic cell of any one of  claims 1 - 8 , wherein the mammalian cell is a Chinese hamster ovary (CHO) cell. 
     
     
         10 . The recombinant eukaryotic cell of any one of  claim 1 - 9 , wherein the heterologous DSB repair protein is from a Chinese hamster cell. 
     
     
         11 . The recombinant eukaryotic cell of any one of  claims 1 - 9 , wherein the heterologous DSB repair protein is from a Chinese hamster ovary (CHO) cell. 
     
     
         12 . The recombinant eukaryotic cell of  claim 1 , wherein the heterologous DSB repair protein comprises an amino acid sequence at least 70% identical to the amino acid sequence of SEQ ID No: 1, 2, 3 or 4. 
     
     
         13 . The recombinant eukaryotic cell of any one of  claims 1 - 12 , wherein the recombinant eukaryotic cell comprises a heterologous DSB repair gene encoding the heterologous DSB repair protein. 
     
     
         14 . The recombinant eukaryotic cell of  claim 13 , wherein the heterologous DSB repair gene comprises a nucleic acid sequence at least 70% identical to the nucleic acid sequence of SEQ ID No: 5, 6, 7 or 8. 
     
     
         15 . The recombinant eukaryotic cell of  claim 13  or  14 , wherein the heterologous DSB repair gene is integrated into the genome of the recombinant eukaryotic cell. 
     
     
         16 . The recombinant eukaryotic cell of any one of  claims 1 - 15 , comprising a heterologous nucleic acid sequence encoding a recombinant product of interest and expressing the recombinant product of interest. 
     
     
         17 . The recombinant eukaryotic cell of  claim 16 , wherein the recombinant product of interest is a protein or a polypeptide. 
     
     
         18 . The recombinant eukaryotic cell of  claim 17 , wherein the protein is a monoclonal antibody. 
     
     
         19 . The recombinant eukaryotic cell of  claim 16 , wherein the heterologous nucleic acid sequence encoding the recombinant product of interest is integrated into the genome of the recombinant eukaryotic cell. 
     
     
         20 . The recombinant eukaryotic cell of  claim 19 , further comprising a heterologous nucleic acid sequence encoding a selection marker integrated into the genome of the recombinant eukaryotic cell. 
     
     
         21 . The recombinant eukaryotic cell of any one of  claims 16 - 20 , wherein the recombinant product of interest is a secreted embryonic alkaline phosphate (SEAP). 
     
     
         22 . A method for enhancing double strand break (DSB) repair in eukaryotic cells, comprising expressing an effective amount of a heterologous DSB repair protein in the eukaryotic cells. 
     
     
         23 . The method of  claim 22 , further comprising enhancing stability of the eukaryotic cells over time. 
     
     
         24 . The method of  claim 22  or  23 , further comprising introducing into the eukaryotic cells a heterologous nucleic acid gene encoding the heterologous DSB repair protein. 
     
     
         25 . The method of  claim 24 , wherein the heterologous nucleic acid sequence encoding the heterologous DSB repair protein is introduced into the eukaryotic cells by overexpression, transgene expression, gene knock-in, gene activation, transcription activation, translation activation, gene mutation or a combination thereof. 
     
     
         26 . A method for establishing host cells for production of a recombinant product of interest, comprising:
 (a) expressing a heterologous double strand break (DSB) repair protein in the eukaryotic cells;   (b) determining DSB repair in the eukaryotic cells of step (a); and   (c) isolating eukaryotic cells in which the DSB repair is enhanced as host cells.   
     
     
         27 . The method of  claim 26 , further comprising editing the genome of the host cells to improve DSB repair in the host cells. 
     
     
         28 . A method for producing a recombinant product of interest, comprising:
 (a) growing eukaryotic cells in a culture medium, wherein the recombinant eukaryotic cells comprise a heterologous nucleic acid sequence encoding a recombinant product of interest;   (b) expressing a heterologous double strand break (DSB) repair protein in the eukaryotic cells; and   (c) expressing the recombinant product of interest by the eukaryotic cells.   
     
     
         29 . The method of  claim 28 , wherein the average productivity of the recombinant product of interest by the eukaryotic cells drops less than 30% over a period of at least 8 weeks. 
     
     
         30 . The method of  claim 28 , wherein the eukaryotic cells retain at least 70% of the copy number of the heterologous nucleic acid sequence encoding the recombinant product of interest over a period of at least 8 weeks. 
     
     
         31 . The method of any one of  claims 28 - 30 , further comprising editing the genome of the eukaryotic cells to improve DSB repair in the eukaryotic cells. 
     
     
         32 . A method of improving production of a recombinant product of interest by eukaryotic cells, wherein the eukaryotic cells comprise a heterologous nucleic acid sequence encoding the recombinant product of interest and produce the recombinant product of interest, comprising expressing a heterologous double strand break (DSB) repair protein by the recombinant eukaryotic cells. 
     
     
         33 . The method of  claim 32 , further comprising enhancing DSB repair in the eukaryotic cells. 
     
     
         34 . The method of  claim 32 , further comprising enhancing stability of the eukaryotic cells over time. 
     
     
         35 . A method of investigating suitability of eukaryotic cells as host cells for producing a recombinant product of interest, wherein the eukaryotic cells comprise a heterologous nucleic acid sequence encoding the recombinant product of interest, comprising:
 (a) expressing a heterologous double strand break (DSB) repair protein by the eukaryotic cells; and   (b) determining DSB repair in the eukaryotic cells, wherein an improvement of the DSB repair indicates that the eukaryotic cells are suitable as host cells for producing a recombinant product of interest.   
     
     
         36 . The method of any one of  claims 22 - 35 , wherein the DSB repair protein is from the group consisting of DNA ligase IV (LIG4), x-ray repair cross complementing 6 (XRCC6), partner and localizer of BRCA2 (PALB2), and PARP1 binding protein which is encoded by the PARPBP gene (PARI). 
     
     
         37 . The method of  claim 36 , wherein the heterologous DSB repair protein is LIG4 or XRCC6. 
     
     
         38 . The method of any one of  claims 22 - 37 , wherein the heterologous DSB repair protein is expressed transiently. 
     
     
         39 . The method of any one of  claims 22 - 37 , wherein the heterologous DSB repair protein is expressed stably. 
     
     
         40 . The method of any one of  claims 22 - 39 , wherein the eukaryotic cells are mammalian cells. 
     
     
         41 . The method of  claim 40 , wherein the mammalian cells are selected from the group consisting of rodent cells, mouse cells and Chinese hamster cells. 
     
     
         42 . The method of  claim 40 , wherein the mammalian cells are CHO cells. 
     
     
         43 . The method of any one of 22-42, wherein the heterologous DSB repair protein is from a Chinese hamster (CH) cell. 
     
     
         44 . The method of any one of  claims 22 - 42 , wherein the heterologous DSB repair protein is from a Chinese hamster ovary (CHO) cell. 
     
     
         45 . The method of any one of  claims 22 - 42 , wherein the heterologous DSB repair protein comprises an amino acid sequence at least 70% identical to the amino acid sequence of SEQ ID No: 1, 2, 3 or 4. 
     
     
         46 . The method of any one of  claims 22 - 45 , wherein the eukaryotic cell comprises a heterologous DSB repair gene encoding the heterologous DSB repair protein. 
     
     
         47 . The method of  claim 46 , wherein the heterologous DSB repair gene comprises a nucleic acid sequence at least 70% identical to the nucleic acid sequence of SEQ ID No: 5, 6, 7 or 8. 
     
     
         48 . The method of  claim 46  or  47 , wherein the heterologous DSB repair gene is integrated into the genome of the eukaryotic cell. 
     
     
         49 . The method of any one of  claims 28 - 48 , further comprising expressing a selection maker by the eukaryotic cells, wherein the eukaryotic cells further comprises a heterologous nucleic acid sequence encoding the selection marker, and wherein the heterologous nucleic acid sequence encoding the recombinant product of interest and the heterologous nucleic acid sequence encoding the selection marker are integrated into the genome of the eukaryotic cells. 
     
     
         50 . The method of any one of  claims 28 - 49 , further comprising growing the eukaryotic cells under a condition that induces DNA damage. 
     
     
         51 . The method of any one of  claims 28 - 50 , wherein the recombinant product of interest is a protein or a polypeptide. 
     
     
         52 . The method of  claim 51 , wherein the protein is a monoclonal antibody. 
     
     
         53 . The method of  claim 35 , wherein the heterologous DSB repair protein is LIG4 or XRCC6, further comprising quantifying the expression of the LIG4 or XRCC6 in the eukaryotic cells. 
     
     
         54 . The method of  claim 35  or  53 , further comprising quantifying the expression of the recombinant product of interest by the eukaryotic cells. 
     
     
         55 . The method of  claim 54 , further comprising identifying eukaryotic cells into whose genome the heterologous nucleic acid sequence encoding the recombinant product of interest is integrated. 
     
     
         56 . The method of  claim 54 , further comprising identifying eukaryotic cells producing the recombinant product of interest in an amount greater than 100 mg per liter for recombinant eukaryotic cells. 
     
     
         57 . Host cells established according to the method of  claim 26  or  27 .