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Targeted integration of nucleic acids

A targeting, nuclease technology, applied in nucleic acid vectors, biochemical equipment and methods, microorganisms, etc., can solve the problems of unstable cell growth and/or product expression, laborious, time-consuming, etc.

Pending Publication Date: 2021-11-09
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, such gene amplification may result in unwanted cellular phenotypes such as unstable cell growth and / or product expression
Third, due to the heterogeneity of integrated loci inherent in the random integration process, the process of screening thousands of clones after transfection to isolate cell lines exhibiting desired expression levels of the polypeptide of interest is time-consuming and laborious

Method used

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  • Targeted integration of nucleic acids
  • Targeted integration of nucleic acids
  • Targeted integration of nucleic acids

Examples

Experimental program
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preparation example Construction

[0064] 6. Preparation and use of TI host cells

[0065] 7. Product

[0066] 8. Exemplary non-limiting embodiments

[0067] 1. Definition

[0068] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of ambiguity, this patent specification (including definitions) shall prevail. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0069] As used herein, the terms "comprises," "including," "has," "has," "may," "containing," and variations thereof are intended to be op...

example

[0559] The following examples are merely illustrative of the presently disclosed subject matter and should not be considered limiting in any way.

example 1

[0560] Example 1: Discovery of High Yield Targeted Integration Sites in CHO Host Cells for Clinical and Commercial Cell Line Development

[0561] This example describes a method for identifying loci with high yields of targeted integration in the CHO genome. Conventional cell line development (CLD) relies on the random integration (RI) of plasmids carrying a sequence of interest (SOI). The process is unpredictable and time-consuming. Therefore, considerable effort is required to identify high-yielding RI clones. Unlike conventional RI CLD, targeted integration (TI) CLD introduces a transgene with a defined copy number (usually 1-2 copies) at a predetermined "hotspot" in the CHO genome. Given the low copy number and pretested integration sites, TI cell lines should have superior stability compared to RI cell lines. Furthermore, since selectable markers are only used to select cells with appropriate TIs and not cells with high levels of transgene expression, less mutagenic ma...

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Abstract

The presently disclosed subject matter relates to targeted integration (TI) host cells suitable for the expression of recombinant proteins, wherein those TI host cells have been subjected to supertransfection resulting in the random integration (RI) of exogenous nucleic acids encodes into their genome. The invention also relates to methods of producing and using said supertransfected TI host cells.

Description

[0001] Cross references to related patent applications [0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 784,019, filed December 21, 2018, the contents of which are hereby incorporated by reference in their entirety. technical field [0003] The subject matter of the present disclosure relates to targeted integration (TI) host cells suitable for expression of recombinant proteins, wherein those TI host cells have been supertransfected such that exogenous nucleic acid codes are randomly integrated (RI) into their genomes, and to the production and use of The method for supertransfecting TI host cells. [0004] sequence listing [0005] This description refers to the sequence listing (a .txt file named "00B2060866SEQ.txt" submitted electronically on December 19, 2019). The 00B2060866SEQ.txt file was generated on December 10, 2019, and is 1,251,200 bytes in size. The entire content of this Sequence Listing is hereby incorporated by ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63
CPCC12N15/63C12N15/85C07K16/00C12N15/907C12N2800/30C12N2800/90
Inventor A·沈S·米沙吉
Owner F HOFFMANN LA ROCHE & CO AG