SUPER-ENCHANCERS FOR RECOMBINANT GENE EXPRESSION IN CHO CELLS

Abstract

Abstract: The present invention belongs to the field of biotechnology, and specifically relates to recombinant gene expression. The invention concerns a method of recombinant gene expression from a Chinese Hamster Ovary (CHO) cell, by using super-enhancer sequences for increased gene expression. Thus, the invention provides a method for producing an engineered CHO cell by introducing into the cell an exogenous nucleic acid molecule into or within 500 kb upstream or downstream of a super-enhancer as expression-enhancing sequence. The invention further provides an engineered CHO cell produced by the method. The invention further provides a method of producing a recombinant polypeptide. The invention is further directed to the use of a super-enhancer for transgene expression.

Description

Claims

1. A method of producing an engineered Chinese Hamster Ovary (CHO) cell, the method comprising: introducing into a CHO cell a construct for integration of an exogenous nucleic acid molecule into or within 500 kb upstream or downstream of an expression-enhancing sequence in the genome of the cell, the expression-enhancing sequence being at least 90% identical to a sequence selected from any one of SEQ ID NOs: 1-47.

2. The method according to claim 1, wherein the expression-enhancing sequence is selected from any one of SEQ ID NOs: 1-47.

3. The method according to claim 1, wherein integration of the exogenous nucleic acid molecule is within 100 kb upstream or downstream of the expression-enhancing sequence.

4. The method according to claim 1, wherein integration of the exogenous nucleic acid molecule is achieved by the CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 method, TALEN (Transcription Activator-Like Effector Nuclease)-based method or ZFN (zinc-finger nuclease)-based method.

5. The method according to claim 4, wherein integration of the exogenous nucleic acid molecule is achieved by the CRISPR /Cas9 method.

6. The method according to claim 1, wherein the exogenous nucleic acid molecule integrates at two or more integration sites in the genome of the cell.

7. The method according to claim 1, wherein the exogenous nucleic acid encodes a polypeptide.

8. An engineered cell produced by the method according to claim 1.

9. A method of producing a recombinant polypeptide, the method comprising: (i) introducing into a CHO cell a construct for integration of an exogenous nucleic acid molecule into or within 500 kb upstream or downstream of an expression-enhancing sequence in the genome of the cell, the expression-enhancing sequence being at least 90% identical to a sequence selected from any one of SEQ ID NOs: 1-47, to produce an engineered cell,(ii) culturing the engineered cell to recombinantly express the exogenous nucleic acid to produce a recombinant polypeptide encoded by the exogenous nucleic acid, and(iii) isolating the recombinant polypeptide.

10. Use of a nucleic acid sequence being at least 90% identical to a sequence selected from any one of SEQ ID NOs: 1-47 for transgene expression.