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KR-20260064879-A - Novel Hotspots in the CHO cells and Uses Thereof

KR20260064879AKR 20260064879 AKR20260064879 AKR 20260064879AKR-20260064879-A

Abstract

The present invention relates to isolated CHO (Chinese hamster ovary) cells in which a gene sequence encoding a target protein (GOI) is inserted into one or more of five novel hot spots within the genome of CHO cells, and to a method for enhancing the expression of a target protein or a method for producing a target protein, comprising the step of inserting a gene sequence encoding the target protein into said hot spots. According to the present invention, a CHO cell line capable of expressing a target protein with high efficiency can be secured, and there is an advantage of increasing efficiency compared to cell lines developed by classical methods and reducing resources such as time and cost invested in the development process by up to about 50%.

Inventors

  • 윤성태
  • 염진호
  • 경민지
  • 한성혁
  • 이동엽
  • 박서영
  • 김동석
  • 이민욱

Assignees

  • 주식회사 녹십자
  • 성균관대학교산학협력단

Dates

Publication Date
20260508
Application Date
20241030

Claims (9)

  1. As isolated CHO cells in which a gene sequence encoding a target protein (GOI) is inserted into one or more hot spots within the genome of CHO (Chinese hamster ovary) cells, Isolated CHO cells characterized in that the above hotspot is a genomic region selected from the group consisting of NCBI Accession No. NC_048596.1, NC_048597.1, NC_048599.1 and NW_023276806.1.
  2. In claim 1, the isolated CHO cells are characterized in that the hotspot is selected from the following group: i) Areas from location 50477240 to 50482240 of NCBI Accession No. NC_048596.1; ii) Areas from location 50456217 to 50461217 of NCBI Accession No. NC_048596.1; iii) Areas from location 173447274 to 173452274 of NCBI Accession No. NC_048597.1; iv) the region of positions 55234551 to 55239551 of NCBI Accession No. NC_048599.1; and v) Areas 229708919 to 229713919 of NCBI Accession No. NW_023276806.1.
  3. An isolated CHO cell according to claim 1, characterized in that the gene sequence encoding the target protein comprises one or more of genes encoding bacterial or viral immunogens, antibodies, toxin proteins, enzymes, hormones, growth factors, receptors, transport proteins, immune proteins, signal transduction proteins, or biological constituent proteins.
  4. A method for enhancing the expression of a target protein or a method for producing a target protein, comprising the step of inserting a gene sequence encoding a target protein (GOI) into one or more hot spots within the genome of a CHO (Chinese hamster ovary) cell, A method characterized in that the above hotspot is a genomic region selected from the group consisting of NCBI Accession No. NC_048596.1, NC_048597.1, NC_048599.1 and NW_023276806.1.
  5. A method for enhancing the expression of a target protein or a method for producing, wherein, in paragraph 4, the hotspot is selected from the group below: i) Areas from location 50477240 to 50482240 of NCBI Accession No. NC_048596.1; ii) Areas from location 50456217 to 50461217 of NCBI Accession No. NC_048596.1; iii) Areas from location 173447274 to 173452274 of NCBI Accession No. NC_048597.1; iv) the region of positions 55234551 to 55239551 of NCBI Accession No. NC_048599.1; and v) Areas 229708919 to 229713919 of NCBI Accession No. NW_023276806.1.
  6. In claim 4, a method for enhancing the expression of a target protein or a method for producing a target protein, characterized by including the following steps: a) a step of inserting a landing pad vector into the genome of a CHO cell; and b) A step of replacing the landing pad vector with an RMCE (recombinase-mediated cassette exchange) vector.
  7. A method for enhancing the expression of a target protein or a method for producing, wherein, in claim 6, the insertion of the landing pad vector is characterized by using CRISPR/Cas9 gene scissors.
  8. A method for enhancing the expression of a target protein or a method for producing one, wherein, in claim 6, the landing pad vector comprises 5' and 3' homology arms; a promoter region; a recombinase recognition sequence; and a selection marker.
  9. A method for enhancing the expression of a target protein or a method for producing a target protein, wherein the RMCE vector comprises: a recombinase recognition sequence; a selection marker; a promoter region; and a gene sequence encoding a target protein.

Description

Novel Hotspots in the CHO cells and Uses Thereof The present invention relates to isolated CHO (Chinese hamster ovary) cells in which a gene sequence encoding a target protein (Gene of Interest: GOI) is inserted into one or more novel hot spots within the genome of CHO cells. More specifically, the invention relates to isolated CHO cells in which a gene sequence encoding a target protein is inserted into one or more of five novel hot spots newly discovered through the present invention, and to a method for enhancing the expression of a target protein or a method for producing a target protein comprising the step of inserting a gene sequence encoding a target protein into said hot spots. The first step in the development of biopharmaceuticals using animal cells is cell line development. Classical cell line development involves a process of screening various cell lines created by randomly transforming a host cell line with a gene expressing a target protein, and then selecting specific cell lines that exhibit the desired productivity and quality profiles. Securing superior cell lines during development consumes significant time and resources in this screening process. This is an inevitable step in the development process to increase the probability of obtaining cell lines with desired characteristics. One proposed method to address this is the development of cell lines using site-specific integration (SSI). This approach requires information regarding specific regions within the host cell genome known as "hotspots" that guarantee high expression, as well as genome editing technology to specifically insert genes into these regions. Under this technical background, the inventors utilized major genetic information on CHO (Chinese hamster ovary) cells and gene high-expression induction hotspot library information obtained based on big data to select and verify novel hotspots with increased expression levels compared to the control group through cell lines produced by a site-specific insertion method based on RMCE (recombinase-mediated cassette exchange) technology, thereby completing the present invention. The information described above in the background section is intended solely to enhance understanding of the background of the present invention and may not include information that forms prior art already known to those skilled in the art to which the present invention belongs. Figure 1 is a flowchart for selecting target high-expression genes and obtaining candidate gene sequences as GOI insertion sites. Figure 2 is a flowchart for evaluating selected new hotspot candidates. Figure 3 is a schematic diagram showing a method for securing a cell line inserted into a landing pad. Figure 4 is a diagram showing the Junction PCR results of a landing pad-inserted cell line. Figure 5 is a schematic diagram showing the method for obtaining RMCE cell lines. Figure 6 is a diagram showing the Junction PCR results of the RMCE cell line. Figure 7 is a diagram showing the results of the copy number analysis of the RMCE cell line. Figure 8 is a diagram showing the Junction PCR results of the control cell line. Figure 9 is a diagram showing the results of the copy number analysis of the control cell line. Figure 10 is a figure showing the growth curve results of the RMCE cell line, Figure 10a is a graph of the growth curve results of the enzyme expression cell line, and Figure 10b is a graph of the growth curve results of the antibody expression cell line. Figure 11 is a diagram showing the results of a comparison of productivity of RMCE cell lines, Figure 11A is a graph showing productivity per volume of culture medium, and Figure 11B is a graph showing the results of a comparison of productivity per copy number. Figure 12 is a diagram showing the productivity ratio of the RMCE cell line, Figure 12A is a graph showing the results of comparing enzyme productivity with control 1, and Figure 12B is a graph showing the results of comparing antibody productivity with control 1. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art. The present invention relates to novel hotspots within CHO cells to be utilized in the development of cell lines using a site-specific insertion method. In one embodiment of the present invention, in order to secure novel hotspots, data on CHO host cell lines was first obtained through whole-genome and transcriptome data analysis. Through the analysis of these results, information on genomic mutations, information on high-expression genes, and target hotspot sites were selected as candidates. After applying recombinant technology based on RMCE (recombinase-mediated cassette exchange) technology and landing pad insertion to the selected hotspot candidates, expression evaluations of model enzymes