Site-specific integration

Patent metadata

Field	Value
Publication number	US-11326185-B2
Application number	US-201816176525-A
Country	US
Kind code	B2
Filing date	Oct 31, 2018
Priority date	Jun 22, 2012
Publication date	May 10, 2022
Grant date	May 10, 2022

Abstract

The present invention relates to stable and high-producing site-specific integration (SSI) host cells, e. g. Chinese hamster ovary (CHO)-derived host cells, methods to produce and to use them.

First claim

The invention claimed is:

1. A site-specific integration (SSI) host cell comprising:
an endogenous Fer1L4 gene; and
an exogenous nucleotide sequence integrated in said Fer1L4 gene, the exogenous nucleotide sequence comprising at least two recombination target sites,
wherein the exogenous nucleotide sequence is integrated in a region spanning and including exon 28 to exon 40 of the endogenous Fer1L4 gene.

2. The SSI host cell of claim 1 , wherein the exogenous nucleotide sequence comprises a gene coding sequence of interest.

3. The SSI host cell of claim 2 , wherein the at least two recombination target sites flank the gene coding sequence of interest,
wherein an integration site of one of the recombination target sites that flank the gene coding sequence of interest is located between exon 39 and 40 of the endogenous Fer1L4 gene and an integration site of the other recombination target site that flanks the gene coding sequence of interest is located between exon 28 and 29 of the endogenous Fer1L4 gene.

4. The SSI host cell of claim 2 , wherein the gene coding sequence of interest comprises one or more of a gene encoding a selection marker, a detectable protein, an antibody, a peptide antigen, an enzyme, a hormone, a growth factor, a receptor, a fusion protein or other biologically active protein.

5. The SSI host cell of claim 1 , wherein the recombination target site is a FRT site or a lox site.

6. The SSI host cell of claim 4 , wherein the selection marker is a glutamine synthase selection marker, a hygromycin selection marker, a puromycin selection marker or a thymidine kinase selection marker.

7. The SSI host cell of claim 1 , wherein the host cell is a mouse cell, a human cell or a CHO host cell, a CHOK1 host cell or a CHOK1SV host cell.

8. The SSI host cell of claim 3 , wherein the nucleotide sequence of the Fer1L4 gene flanking the integrated exogenous nucleotide sequence is selected from the group consisting of SEQ ID No. 7, 8, 9 and homologous sequences thereof.

9. The SSI host cell of claim 1 , wherein the exogenous nucleotide sequence replaces a portion of the Fer1L4 gene.

10. The SSI host cell of claim 5 , wherein the host cell comprises a FRT site which is a wild type FRT site or a mutant FRT site.

11. The SSI host cell of claim 1 , wherein the recombination target sites comprise at least one wild type FRT site and at least one mutant FRT site.

Assignees

• Lonza Biologics Plc
• Pfizer

Inventors

• Rance James
• Young Robert
• Agostino Michael J
• Moffat Mark
• Zhang Lin
• Zhang Baohong

Classifications

• C12N15/907 Primary

  in mammalian cells · CPC title

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title

• C12N2800/30

  Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT · CPC title

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C07K2317/24

  containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered · CPC title