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

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...

AI Technical Summary

Benefits of technology

The present invention is about creating mammalian cell lines that can produce proteins of interest. These cell lines have a specific gene that is constantly working and can be used to produce the protein. Additionally, the cell lines have a space where a new gene can be inserted to produce the protein of interest. These cell lines are stable and can be used for a long time without needing any special treatment. This invention can help create new cell lines that can produce proteins in large quantities.

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 Cell Lines, in Cell Culture and Upstream Processing, Butler, ed.

This large number of screening and expansion steps results in a very lengthy and expensive process to simply generate the cell line that will, ultimately, produce the therapeutic of interest.

If one takes into account lost time on market for a blockbuster protein therapeutic, inefficiencies in cell line production can cost biopharmaceutical manufacturers hundreds of millions of dollars (Seth et al., Curr Opin Biotechnol 18, 557 (2007)).

Much of the time and expense of bioproduction cell line creation can be attributed to random genomic integration of the bioproduct gene resulting in clone-to-clone variability in genotype and, hence, variability in gene expression.

The principal drawback to recombinase-based gene targeting systems is that the recombinase recognition sites (loxP, FRT, or attB / attP sites) do not naturally occur in mammalian genomes.

In addition, the biomanufacturing industry is notoriously hesitant to adopt “new” cell lines, such as those that have been engineered to carry a recombinase site, that do not have a track record of FDA approval.

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