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Integration sites in CHO cells

A technology of cells and Chinese hamsters, applied in the field of identification of genomic integration sites, can solve the problem of difficult prediction of the possibility of genomic loci

Pending Publication Date: 2020-04-10
BOEHRINGER INGELHEIM INT GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many of these elements within the genome are not known or characterized, and thus the likelihood of genomic loci during cell line development is difficult to predict

Method used

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  • Integration sites in CHO cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0151] CHO producer cell clones are usually obtained by random integration of heterologous polynucleotides into the host cell genome of CHO cells, ie by random integration (RI). The position effect results in a highly heterogeneous cell population consisting mainly of low producer cells with only a small subset of high producer cells. Additionally, high producer cells tend to be outnumbered by low producer cells. To assess the potential of the Chinese hamster S100A gene cluster as a reliable, high-level production site (i.e., a "hotspot") for heterologous protein production, DNA engineered to SEQ ID NO: 11 (ZFN 13) was used as described above. Sequence-specific pairs of zinc finger nucleases that stably integrate polynucleotides encoding IgG antibodies into the genomes of CHO-DG44 and CHOZN GS cells.

[0152] After confirmation of ZFN activity and preparation of donor plasmids, cells were co-transfected with a non-linear plasmid containing an expression cassette encoding an I...

Embodiment 2

[0157] Random integration results in cell repertoires that are highly heterogeneous in expression of heterologous proteins. To assess whether targeted integration within the Chinese hamster S100A gene cluster resulted in more uniform expression levels and thus greater predictability in terms of productivity, individual clones were selected from the TI and RI cell banks of Example 1.

[0158] Single cell clones (SCC) were obtained using targeted integration and random integration pools from CHOZN GS cells from Example 1. A single cloning process was accomplished by limiting the dilution of enriched TI and RI pools using conditioned medium. Prepare conditioned media by culturing cells at 0.3e6 cells / ml for 48 h in TPP tubes. Cells were pelleted and conditioned medium was sterile filtered. Inoculation was performed in an 80:20 mixture of cloning medium (SAFC fusion platform) and conditioned medium using the following procedure. Step 1: Place serial dilutions in a 96-well plate...

Embodiment 3

[0162] To validate the hotspot loci in the S100A gene cluster, a number of additional TI zinc finger nucleases were designed and generated as shown in Table 3 to create a production library as described in Example 1. image 3 A shows the positions of individual ZFNs and hotspot loci in the S100A gene cluster with NCBI standard sequence: NW_003613854.1. Shown are the integration sites of ZNF 7 to 14, categorized as "non-destructive and productive", "non-destructive and low / non-productive" and "destructive and low / non-productive" sites.

[0163] Data were generated using CHO-ZN GS cells as described in Example 1. Eight different genomic loci were tested to assess whether a region favors heterologous gene product production relative to the S100A3 / A4 / A5 / A6 major gene cluster. It was further tested whether integration into the S100A3 / A4 / A5 / A6 major gene cluster would result in reduced productivity ( image 3 B).

[0164] table 3:

[0165] zinc finger nuclease target...

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Abstract

The present invention relates to the identification of a genomic integration site for heterologous polynucleotides in Chinese Hamster Ovary (CHO) cells resulting in high RNA and / or protein production.More specifically it relates to CHO cells comprising at least one heterologous polynucleotide stably integrated into the S100A gene cluster of the CHO genome and to methods for the production of saidCHO cells. Further, the invention relates to a method for the production of a protein of interest using said CHO cell and to the use of said CHO cell for producing a protein of interest at high yield. Integration within these specific target regions leads to reliable, stable and high yielding production of an RNA and / or protein of interest, encoded by the heterologous polynucleotide.

Description

technical field [0001] The present invention relates to the identification of genomic integration sites of heterologous polynucleotides leading to high RNA and / or protein production in Chinese Hamster Ovary (CHO) cells. More specifically, the present invention relates to CHO cells comprising at least one heterologous polynucleotide stably integrated into the S100A gene cluster of the CHO genome, and to methods for producing said CHO cells. Furthermore, the present invention relates to a method for producing a protein of interest using the CHO cell, and to a use of the CHO cell for producing the protein of interest in high yield. Integration within these specific target regions results in reliable, stable and high-yield production of the RNA and / or protein of interest encoded by the heterologous polynucleotide. Background technique [0002] Chinese hamster ovary (CHO) cells are the most prevalent host cell for recombinant production of therapeutic proteins. Classic cell lin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C07K16/00C12N9/22
CPCC07K16/00C12N9/22C12N15/907C07K2319/81C12N2800/30C07K2317/14C12N2310/20C12N5/0682C12N15/63C12N2510/02
Inventor 马库斯·穆勒约亨·肖布克里斯蒂安·伯尔勒赫詹妮弗·柯尼泽
Owner BOEHRINGER INGELHEIM INT GMBH
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