Methods and Products for Producing Engineered Mammalian Cell Lines With Amplified Transgenes

Inactive Publication Date: 2014-06-26
PRECISION BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012]The present invention depends, in part, upon the development of mammalian cell lines in which sequences of interest (e.g., exogenous, actively transcribed transgenes) are inserted proximal to an endogenous selectable gene in an amplifiable locus, and the discovery that (a) the insertion of such exogenous sequences of interest does not inhibit amplification of the endogenous selectable gene, (b) the exogenous sequence of interest can be co-amplified with the endogenous selectable gene, and (c) the resultant cell lines, with an amplified region comprising multiple copies of the endogenous selectable gene and the exogenous sequence of intere

Problems solved by technology

The unparalleled growth in market size, however, is driven primarily by skyrocketing demand for fully human and humanized monoclonal antibodies (Reichert, Curr Pharm Biotechnol 9, 423 (2008)).
This greatly complicates the manufacturing process and introduces significant heterogeneity into product formulations (Field, Recombinant Human IgG Production from Myeloma and Chinese Hamster Ovary Cells, in Cell Culture and Upstream Processing, Butler, ed., (Taylor and Francis Group, New York, 2007)).
In addition, protein drugs are typically required at unusually high doses, which necessitates highly scalable manufacturing processes and makes manufacturing input costs a major price determinant.
Many of the protein pharmaceuticals on the market are glycoproteins that cannot readily be produced in easy-to-manipulate biological systems such as bacteria or yeast.
A second and more problematic consequence of random gene integration is the phenomenon of transgene silencing, in which recombinant protein expression slows or ceases entirely over time (Collingwood and Urnov, Targeted Gene Insertion to Enhance Protein Production from C

Method used

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  • Methods and Products for Producing Engineered Mammalian Cell Lines With Amplified Transgenes
  • Methods and Products for Producing Engineered Mammalian Cell Lines With Amplified Transgenes
  • Methods and Products for Producing Engineered Mammalian Cell Lines With Amplified Transgenes

Examples

Experimental program
Comparison scheme
Effect test

example 1

Targeted Gene Insertion into the CHO DHFR Locus Using Engineered Meganucleases

[0136]The CHO genomic DNA sequence 10,000-55,000 base pairs downstream of the DHFR gene was searched to identify DNA sites amenable to targeting with engineered meganucleases. Two sites (SEQ ID NO: 7 and SEQ ID NO: 8) were selected which are, respectively, 35,699 and 15,898 base pairs downstream of the DHFR coding sequence (Table 2).

TABLE 2Example Recognition Sites For EngineeredMeganucleases in the CHO DHFR Locus.SEQLocation RelativeIDto CHO DHFRNO:Target Site SequencesCoding Sequence75′-TAAGGCCTCATATGAAAATATA-3′35,699 bp downstream85′-ATAGATGTCTTGCATACTCTAG-3′15,898 bp downstream

1. Meganucleases that Recognize SEQ ID NO: 7 and SEQ ID NO: 8

[0137]An engineered meganuclease (SEQ ID NO: 9) was produced which recognizes and cleaves SEQ ID NO: 7. This meganuclease is called “CHO-23 / 24”. A second engineered meganuclease (SEQ ID NO: 10) was produced which recognizes and cleaves SEQ ID NO: 8. This meganuclease is...

example 2

Insertion of an Engineered Target Sequence into the CHO DHFR or GS Gene Coding Regions

[0150]As diagrammed in FIG. 4, an alternative method for targeting a sequence of interest to an amplifiable locus involves the production of a cell line in which a portion of a selectable gene is replaced by an engineered target sequence. The advantage of this approach is that the subsequent insertion of a sequence of interest can be coupled with reconstitution of the selectable gene so that cell lines harboring the properly targeted sequence of interest can be selected using the appropriate media conditions. A cell line harboring such an engineered target sequence can be produced using nuclease-induced homologous recombination. In this case, a site-specific endonuclease which cuts a recognition sequence near or within the selectable gene sequence is preferred.

1. Engineered Meganucleases that Cut within the DHFR or GS Genes.

[0151]A meganuclease called “CHO-13 / 14” (SEQ ID NO: 12) was produced which ...

example 3

Meganucleases for Targeting Gene Insertion to the CHO GS Locus

[0155]1. Engineered Meganucleases that Cut Downstream of the CHO GS Gene.

[0156]An engineered meganuclease called “CHOX-45 / 46” (SEQ ID NO: 29) was produced which recognizes a DNA sequence (SEQ ID NO: 30) approximately 7700 base pairs downstream of the CHO GS coding sequence. CHO cells were transfected with mRNA encoding CHOX-45 / 46 as described in Example 2. 72 hours post transfection, genomic DNA was extracted from the transfected cell pool and the region downstream of the CHO GS gene was PCR amplified using a pair of primers (SEQ ID NO: 31 and SEQ ID NO: 32) flanking the CHOX-45 / 46 recognition sequence. PCR products were then cloned and 24 cloned products were sequenced. It was found that 14 of the 24 clones PCR products (58.3%) had large mutations in the sequence consistent with meganuclease-induced genome cleavage followed by mutagenic repair by non-homologous end-joining From these data, we conclude that the CHOX-45 / 46...

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Abstract

Methods of inserting genes into defined locations in the chromosomal DNA of cultured mammalian cell lines which are subject to gene amplification are disclosed. In particular, sequences of interest (e.g., genes encoding biotherapeutic proteins) are inserted proximal to selectable genes in amplifiable loci, and the transformed cells are subjected to selection to induce co-amplification of the selectable gene and the sequence of interest. The invention also relates to meganucleases, vectors and engineered cell lines necessary for performing the methods, to cell lines resulting from the application of the methods, and use of the cell lines to produce protein products of interest.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of International Application No. PCT / US2012 / 040599, filed Jun. 1, 2012, which claims priority to U.S. Provisional application No. 61 / 492,174 filed Jun. 1, 2011, the disclosures of all of which are hereby incorporated by reference in their entireties for all purposes.FIELD OF THE INVENTION[0002]The invention relates to the field of molecular biology and recombinant nucleic acid technology. In particular, the invention relates to methods of inserting genes into defined locations in the chromosomal DNA of cultured mammalian cell lines which are subject to gene amplification. The invention also relates to meganucleases, vectors and engineered cell lines necessary for performing the methods, cell lines resulting from the application of the methods, and use of the cell lines to produce protein products of interest.BACKGROUND OF THE INVENTION[0003]Therapeutic proteins are the primary growth driver in the global...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N9/22
CPCC12N9/22C12N15/907C12N9/003C12Y603/01002C12Y105/01003C12N9/93C12Y301/04
Inventor JANTZ, DEREKSMITH, JAMES JEFFERSONNICHOLSON, MICHAEL G.
Owner PRECISION BIOSCI
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