Site-specific integration

Patent metadata

Field	Value
Publication number	US-10280436-B2
Application number	US-201314409283-A
Country	US
Kind code	B2
Filing date	Jun 20, 2013
Priority date	Jun 22, 2012
Publication date	May 7, 2019
Grant date	May 7, 2019

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. An isolated site-specific integration (SSI) host cell comprising:
an endogenous fer-1-like 4 (Ferl L4) gene; and
an exogenous nucleotide sequence having a 3′ end and a 5′ end integrated in said Fer1L4 gene, the exogenous nucleotide sequence comprising at least one gene coding sequence of interest flanked by at least two recombination target sites, wherein the exogenous nucleotide sequence is flanked at its 3′ end by at least one of the nucleotide sequences as set forth in SEQ ID NO: 7 and 8, and at its 5′ end by the nucleotide sequence as set forth in SEQ ID NO: 9.

2. The SSI host cell according to claim 1 , 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.

3. The SSI host cell according to claim 1 , wherein the recombination target site is a FRT site or a lox site.

4. The SSI host cell according to claim 2 , wherein the selection marker is a glutamine synthetase selection marker, a hygromycin selection marker, a puromycin selection marker or a thymidine kinase selection marker.

5. The SSI host cell according to 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.

6. The SSI host cell according to claim 3 , wherein the host cell comprises a FRT site which is a wild type FRT site or a mutant FRT site.

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

8. A method for producing the SSI host cell, the method comprising:
a) providing cells comprising the endogenous fer-1-like 4 (Fer1L4) gene, wherein the exogenous nucleotide sequence is integrated in said Fer1L4 gene, wherein the exogenous nucleotide sequence comprises at least two recombination target sites, flanking at least one first gene coding sequence of interest, and wherein the exogenous nucleotide sequence is flanked at its 3′ end by at least one of the nucleotide sequences as set forth in SEQ ID NO: 7 and 8, and at its 5′ end by the nucleotide sequence as set forth in SEQ ID NO: 9; subsequently
b) transfecting the cells provided in step a) with a vector comprising a first exchangeable cassette, the cassette comprising at least two matching recombinant target sites, flanking at least one second gene coding sequence of interest; subsequently
c) expressing a recombinase to achieve recombination between the at least two recombination target sites and the at least two matching recombinant target sites in the cells; and subsequently
d) selecting transfected cells expressing the second gene coding sequence of interest so as to obtain the SSI host cell comprising the first exchangeable cassette stably integrated in its genome.

9. The method of claim 8 , further comprising i) transfecting the SSI host cell with a vector comprising at least one second exchangeable cassette, which cassette comprises at least two matching recombination target sites flanking at least one third gene coding sequence of interest and at least one selection marker, ii) effecting a site-directed recombination-mediated cassette exchange by expression of a recombinase that achieves recombination between the at least two matching recombination target sites flanking the at least one third gene coding sequence of interest and the at least two matching recombinant target sites in the SSI host cell so as to obtain a SSI host cell comprising the third gene coding sequence of interest, iii) allowing the SSI host cell obtained in step ii) to express the third gene coding sequence of interest and iv) recovering the product of the third gene coding sequence of interest.

10. An isolated polynucleotide molecule comprising a nucleotide sequence that expresses a gene of interest flanked at the 5′ end by the nucleotide sequence as set forth in SEQ ID NO: 9 and at the 3′ end by at least one of the nucleotide sequences as set forth in SEQ ID NO: 7 and 8, for producing cell lines showing enhanced expression.

11. The isolated polynucleotide molecule according to claim 10 , wherein the polynucleotide is contained in a vector.

12. An isolated host cell, comprising: a) a polynucleotide molecule including a nucleotide sequence that expresses a gene of interest flanked at the 5′ end by the nucleotide sequence as set forth in SEQ ID NO: 9 and at the 3′ end by at least one of the nucleotide sequences as set forth in SEQ ID NO: 7, 8 and a vector containing said polynucleotide sequences.

13. A method for producing an isolated cell or an isolated cell line, in particular a high producer cell or cell line with a high productivity stability, wherein a fer-1-like 4 (Fer1L4) nucleotide sequence is integrated in a vector comprising at least one gene coding sequence of interest having at least two recombination target sites, wherein the Fer1L4 nucleotide sequence is a nucleotide sequence selected from the group consisting of the nucleotide sequences as set forth in SEQ ID NO: 7, 8, and 9, and wherein subsequently the vector is stably transfected into a cell or cell line and wherein subsequently stably transfected cells or cell lines are selected and obtained.

14. The method of claim 8 in combination with a method for the production of a product of a gene coding sequence of interest comprising cultivating a host cell or cell line in a suitable medium and recovering a product therefrom.

15. A method for producing an isolated SSI host cell, the method comprising introducing the exogenous nucleotide sequence into the endogenous Fer1L4 gene in a cell, wherein the exogenous nucleotide sequence comprises at least one gene coding sequence of interest and at least two recombination target sites, and wherein the exogenous nucleotide sequence is flanked at its 3′ end by at least one of the nucleotide sequences as set forth in SEQ ID NO: 7 and 8 and at its 5′ end by the nucleotide sequence as set forth in SEQ ID NO: 9.

16. The method of claim 15 , wherein the exogenous nucleotide sequence is introduced by homologous recombination between the Fer1L4 gene and a polynucleotide, wherein the polynucleotide comprises
a) a first nucleotide sequence homologous to a first portion of the Fer1L4 gene,
b) the exogenous nucleotide sequence, and
c) a second nucleotide sequence homologous to a second portion of the Fer1L4 gene.

17. The method of claim 15 , wherein introduction of the exogenous nucleotide sequence is facilitated using a viral vector or an exogenous nuclease.

18. The method of claim 15 , wherein introduction of the exogenous nucleotide sequence is facilitated using an adeno-associated virus vector which mediates homologous recombination.

19. The method of claim 15 , wherein introduction of the exogenous nucleotide sequence is facilitated using an exogenous nuclease selected from a group consisting of a zinc finger nuclease, a transcription activator-like effector nuclease, and an engineered meganuclease.

20. The method of claim 13 in combination with a method for the production of a product of a gene coding sequence of interest comprising cultivating a host cell or cell line in a suitable medium and recovering a product therefrom.

Assignees

• Lonza Biologics Plc
• Pfizer

Inventors

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

Classifications

• C12N15/861

  Adenoviral vectors · CPC title

• C12N15/907 Primary

  in mammalian cells · CPC title

• C12N15/90 Primary

  Stable introduction of foreign DNA into chromosome · CPC title

• C12P21/00

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• C07K2317/24

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