Targeted integration of nucleic acids

Abstract

The subject matter disclosed in this invention relates to targeted integration (TI) host cells suitable for expressing recombinant proteins, and to methods for producing and using such TI host cells.

Description

[Technical Field] The subject matter disclosed in this invention relates to targeted integration (TI) host cells suitable for expressing recombinant proteins, and methods for producing and using such TI host cells. [Previous Technology] Due to the rapid development of cell biology and immunology, there is an increasing demand in the industry for novel therapeutic recombinant proteins for a variety of diseases, including cancer, cardiovascular disease, and metabolic diseases. These biopharmaceutical candidates are typically manufactured using commercial cell lines capable of expressing the protein of interest. For example, Chinese hamster ovary (CHO) cells have been widely used to produce monoclonal antibodies. Conventional strategies for generating commercial cell lines involve the random integration of nucleotide sequences encoding the polypeptide of interest, followed by the selection and isolation of cell lines that produce the polypeptide of interest. However, this approach has several drawbacks. First, this integration is not only a rare event, but given the randomness of the nucleotide sequence integration location, these rare events can produce a variety of gene expressions and cell growth phenotypes. This variation, known as "position-effect variation," originates at least in part from the complex gene regulatory networks present in the eukaryotic genome and the accessibility of certain genome loci to integration and gene expression. Second, random integration strategies typically cannot control the number of gene copies integrated into the host cell genome. In practice, gene amplification methods are often used to achieve high cell yields. However, this gene amplification can lead to undesirable cell phenotypes, such as unstable cell growth and / or product expression. Third, due to the inherent heterogeneity of the integrating loci during random integration, screening thousands of pure lines after transfection to isolate cell lines exhibiting the desired expression level of the polypeptide of interest is time-consuming and labor-intensive. Even after isolating these cell lines, stable performance of the peptide of interest cannot be guaranteed, and further screening may be required to obtain stable commercial cell lines. Finally, peptides derived from randomly integrated cell lines exhibit high levels of sequence variation, which may be partly due to the mutagenicity of the selector used to select for high performance levels of the peptide of interest. [Summary of the Invention] The subject matter disclosed herein relates to host cells suitable for targeted integration (TI) expression of recombinant proteins. The subject matter disclosed herein not only provides host cell TI sites with high productivity, but also provides a novel method for introducing multiple sequences of interest into a single TI locus in a host cell via recombinase-mediated cassette exchange (RMCE). Advantages of the RMCE method include increased productivity and the flexibility to co-express multiple polypeptides from the same TI locus at different ratios. For example, but not limited to, the RMCE strategy disclosed herein allows for the insertion of one, two, three, four, five, six, seven, eight, or more sequences (e.g., antibody heavy chain (HC) or light chain (LC) sequences) into the TI locus. Because it can simultaneously target eight or more sequences, biplasm RMCE enables the modulation of the HC and LC chain ratio of mAbs to improve productivity, express complex molecules with multiple chains, and utilize antibody-targeted transgenic genes, endogenous genes, or RNAi to modify cellular pathways. In some embodiments, the TI host cell contains a foreign nucleotide sequence integrated at an integration site within a locus of the host cell genome, wherein the locus is at least about 90% homologous to sequences selected from SEQ ID No. 1-7. In some embodiments, the nucleotide sequence immediately following the integrated exogenous nucleotide sequence at 5' is selected from the group consisting of sequences that are at least about 90% homologous to the following: nucleotides 41190-45269 of NW_006884592.1, nucleotides 63590-207911 of NW_006881296.1, nucleotides 253831-491909 of NW_003616412.1, nucleotides 69303-79768 of NW_003615063.1, nucleotides 293481-315265 of NW_006882936.1, nucleotides 2650443-2662054 of NW_003615411.1, and nucleotides 82214-97705 of NW_003615411.1. In some embodiments, the nucleotide sequence immediately following the integrated exogenous nucleotide sequence at 3' is selected from the group consisting of sequences that are at least about 90% homologous to the following: nucleotides 45270-45490 of NW_006874047.1, nucleotides 207912-792374 of NW_006881296.1, nucleotides 491910-667813 of NW_003616412.1, nucleotides 79769-100059 of NW_003615063.1, nucleotides 315266-362442 of NW_006882936.1, nucleotides 2662055-2701768 of NW_003615411.1, or nucleotides 97706-105117 of NW_003615411.1. In some embodiments, the integrated exogenous nucleotide sequence is operatively linked to a nucleotide sequence selected from a group consisting of sequences that are at least about 90% homologous to sequences selected from SEQ ID No. 1-7. In some embodiments, the TI host cell contains a foreign nucleotide sequence integrated at an integration site within an endogenous gene selected from the group consisting of: LOC107977062 (SEQ ID NO. 1), LOC100768845 (SEQ ID NO. 2), ITPR2 (SEQ ID NO. 3), ERE67000.1 (SEQ ID NO. 4), UBAP2 (SEQ ID NO. 5), MTMR2 (SEQ ID NO. 6), XP_003512331.2 (SEQ ID NO. 7), and sequences that are at least about 90% homologous to them. In some embodiments, the exogenous nucleotide sequence is integrated at an integration site operatively linked to an endogenous gene selected from the group consisting of: LOC107977062 (SEQ ID NO. 1), LOC100768845 (SEQ ID NO. 2), ITPR2 (SEQ ID NO. 3), ERE67000.1 (SEQ ID NO. 4), UBAP2 (SEQ ID NO. 5), MTMR2 (SEQ ID NO. 6), XP_003512331.2 (SEQ ID NO. 7), and sequences at least about 90% homologous to them. In some embodiments, the integrated exogenous nucleotide is flanked by a nucleotide sequence selected from the group consisting of sequences at least about 90% homologous to sequences selected from SEQ ID NO. 1-7. In some embodiments, the integration site of the exogenous nucleotide sequence is adjacent to all or part of a sequence selected from a group consisting of sequences that are at least about 90% homologous to sequences selected from SEQ ID No. 1-7. In some embodiments, the TI host cell line is a mammalian host cell. In some embodiments, the TI host cell line is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell. In some embodiments, the TI host cell line is a Chinese hamster ovary (CHO) host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell. In some embodiments, the TI host cell contains an integrated exogenous nucleotide sequence, wherein the exogenous nucleotide sequence includes one or more recombination recognition sequences (RRS). In some embodiments, the exogenous nucleotide sequence contains at least two RRSs. The RRS can be recognized by a recombinase, such as Cre recombinase, FLP recombinase, Bxb1 integrase, or φC31 integrase. The RRS can be selected from the group consisting of: LoxP sequence, LoxP L3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, Lox66 sequence, FRT sequence, Bxb1 attP sequence, Bxb1 attB sequence, φC31 attP sequence, and φC31 attB sequence. In some embodiments, the exogenous nucleotide sequence comprises first and second RRSs and at least one selectable marker located between the first and second RRSs. In some embodiments, the exogenous nucleotide sequence further comprises a third RRS, wherein the third RRS is located between the first and second RRSs and is different from the first or second RRS. In some embodiments, the exogenous nucleotide sequence comprises first and second RRSs and a first selectable marker located between the first and second RRSs. In some embodiments, the exogenous nucleotide sequence further comprises a second selectable marker, and the first and second selectable markers are different. In some embodiments, the exogenous nucleotide sequence may further comprise a third selectable marker and an internal ribosome entry site (IRES), wherein the IRES is operatively linked to the third selectable marker. The third selectable marker may be different from the first or second selectable marker. The selected marker can be chosen from the following groups: aminoglycoside phosphotransferases (APHs) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamic acid synthase (GS), aspartic acid synthase, tryptophan synthase (indole), histamine dehydrogenase (histamine D), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid. The selected marker may also be selected from the group consisting of: green fluorescent protein (GFP) markers, enhanced GFP (eGFP) markers, synthetic GFP markers, yellow fluorescent protein (YFP) markers, enhanced YFP (eYFP) markers, cyan fluorescent protein (CFP) markers, mPlum markers, mCherry markers, tdTomato markers, mStrawberry markers, J-red markers, DsRed-monomer markers, mOrange markers, mKO markers, mCitrine markers, Venus markers, YPet markers, Emerald6 markers, CyPet markers, mCFPm markers, Cerulean markers, and T-Sapphire markers. In some embodiments, the selected marker is selected from the group consisting of: green fluorescent protein (GFP) markers, enhanced GFP (eGFP) markers, and synthetic GFP markers. In some embodiments, the exogenous nucleotide sequence comprises at least one select marker and at least one exogenous sequence of interest (SOI). In some embodiments, the exogenous nucleotide sequence comprises first and second RRS, at least one select marker, and at least one exogenous SOI located between the first and second RRS. In some embodiments, the exogenous nucleotide sequence comprises first, second, and third RRS, at least one select marker, and at least one exogenous SOI located between the first and third RRS, and at least one exogenous SOI located between the third and second RRS. The sequence of interest may encode any polypeptide of interest, including (but not limited to) the examples listed herein. These SOIs may be the same or different; for example, these SOIs may comprise coding sequences for both chains of an antibody. In cases representing two or more different polypeptides, the sequence of interest may comprise two polypeptides in various ratios, such as when eight coding sequences encode two different polypeptides (including (but not limited to) 1:7, 2:6, etc.). These SOIs may encode single-chain antibodies, antibody light chains, antibody heavy chains, single-chain Fv fragments (scFv), or Fc fusion proteins. The subject matter disclosed in this invention also provides methods for targeted integration of exogenous nucleic acids into host cells to promote the expression of peptides of interest. In some embodiments, these methods include targeted integration of exogenous nucleic acids into host cells via recombinase-mediated recombination. In some embodiments, these methods relate to cells containing exogenous nucleotide sequences integrated at sites within endogenous genes selected from the group consisting of: LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto. In some embodiments, these methods relate to cells containing exogenous nucleotide sequences integrated into loci within the host cell genome, wherein the locus contains nucleotide sequences at least about 90% homologous to sequences selected from SEQ ID Nos. 1-7. In some embodiments, this disclosure provides a method for preparing TI host cells expressing a polypeptide of interest, the method comprising: a) providing a TI host cell containing a foreign nucleotide sequence integrated at a locus site in the TI host cell genome, wherein the locus is at least about 90% homologous to sequences selected from SEQ ID No. 1-7, wherein the foreign nucleotide sequence contains two RRSs, the two RRSs being side-mounted with at least one first selectable marker; b) introducing a vector containing two RRSs into the cell provided in a) the two RRSs being matched to the two RRSs on the integrated foreign nucleotide sequence and side-mounted with at least one foreign SOI and at least one second selectable marker; c) introducing a recombinase, wherein the recombinase recognizes the RRSs; and d) selecting TI cells expressing the second selectable marker to thereby isolate TI host cells expressing the polypeptide of interest. In some embodiments, this disclosure provides a method for preparing TI host cells expressing first and second polypeptides of interest (wherein the first and second polypeptides may be the same or different), the method comprising: a) providing a TI host cell containing a foreign nucleotide sequence integrated at a locus site in the host cell genome, wherein the locus is at least about 90% homologous to sequences selected from SEQ ID No. 1-7, wherein the foreign nucleotide sequence contains first and second RRSs side-attached with at least one first selectable marker, and a third RRS located between the first and second RRSs, wherein the first, second, and third RRSs are xenogeneic specific, i.e., the first, second, and third RRSs are not matched RRSs and therefore any two of the first, second, and third RRSs would be unsuitable for recombinase-mediated recombination; b) introducing a first vector containing two RRSs into the cell provided in a) the two RRSs being matched with the first and third RRSs on the integrated foreign nucleotide sequence and side-attached with at least one first foreign SOI and at least one second selectable marker; c) Introduce a second vector containing two RRSs into the cell provided in a), the two RRSs matching the second and third RRSs on the integrated exogenous nucleotide sequence and side-attached to at least one second exogenous SOI; d) introduce one or more recombinases, wherein the one or more recombinases recognize the RRSs; and e) select TI cells expressing the second select marker to thereby isolate TI host cells expressing the first and second polypeptides of interest. In some embodiments, this disclosure provides a method for expressing a polypeptide of interest, the method comprising: a) providing a host cell comprising at least one exogenous SOI and at least one selectable marker, the at least one exogenous SOI and the at least one selectable marker being side-connected to two RRS and integrated into a locus in the host cell genome, wherein the locus is at least about 90% homologous to sequences selected from SEQ ID No. 1-7; and b) culturing the cell of a) under conditions suitable for expressing the SOI and recovering the polypeptide of interest from it. In some embodiments, this disclosure provides a method for expressing a polypeptide of interest, the method comprising: a) providing a host cell comprising at least two exogenous SOIs and at least one selectable marker, the at least two exogenous SOIs and at least one selectable marker being integrated into a locus in the host cell genome, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1-7, wherein at least one exogenous SOI and one selectable marker are flanked by first and third RRS and at least one exogenous SOI is flanked by second and the third RRS; and b) culturing the cell in a) under conditions suitable for expressing the SOI and recovering the polypeptide of interest therefrom. The subject matter disclosed in this invention also provides compositions and methods for generating polypeptides of interest using TI host cells. These compositions and methods include (but are not limited to) polynucleotides and vectors that facilitate the integration of exogenous nucleotide sequences into one or more TI host cells, compositions comprising exogenous nucleotide sequences, and methods of using them. In some embodiments, the vector may comprise a nucleotide sequence flanked by a DNA cassette that is at least 50% homologous to a sequence selected from SEQ ID Nos. 1-7, wherein the DNA cassette comprises at least one selectable marker flanked by two RRSs and at least one exogenous SOI. The vector may be selected from the group consisting of: adenoviral vectors, adeno-associated virus vectors, lentiviral vectors, retroviral vectors, integrative phage vectors, nonviral vectors, transposons and / or transloses, integrase receptors, and plastids. In some embodiments, the methods of this disclosure include targeted integration of exogenous nucleic acids encoding polypeptides of interest into host cells via homologous recombination, homologous targeted repair (HDR), and / or non-homologous end joining (NHEJ). In some embodiments, these methods involve integrating exogenous nucleic acids encoding polypeptides of interest into cells at sites within endogenous genes selected from the following groups: LOC107977062 (SEQ ID NO. 1), LOC100768845 (SEQ ID NO. 2), ITPR2 (SEQ ID NO. 3), ERE67000.1 (SEQ ID NO. 4), UBAP2 (SEQ ID NO. 5), MTMR2 (SEQ ID NO. 6), XP_003512331.2 (SEQ ID NO. 7), and sequences at least about 90% homologous to them. In some embodiments, these methods involve integrating an exogenous nucleic acid encoding a polypeptide of interest into a cell at a site within a locus of the host cell genome, wherein the locus contains a nucleotide sequence that is at least about 90% homologous to sequences selected from SEQ ID No. 1-7. In some embodiments, this disclosure provides a method for preparing TI host cells expressing a polypeptide of interest, the method comprising: a) providing a host cell containing a locus of the host cell genome, wherein the locus is at least about 90% homologous to sequences selected from SEQ ID No. 1-7; b) introducing a vector into the host cell, wherein the vector contains a nucleotide sequence flanked by a DNA cassette that is at least 50% homologous to sequences selected from SEQ ID No. 1-7, wherein the DNA cassette contains at least one selectable marker flanked by two RRSs and at least one exogenous SOI; and c) selecting against the selectable marker to isolate the TI host cell from which the SOI is integrated into the locus of the genome and expresses the polypeptide of interest. In some embodiments, these methods involve the use of exogenous nucleases to facilitate targeted integration. Exogenous nucleases can be selected from the following groups: zinc finger nucleases (ZFN), ZFN dimers, transcription activator effector nucleases (TALEN), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, modified large-scale nucleases, and clustered regularly spaced short palindromic repeats (CRISPR) associated proteins (Cas) endonucleases. In some embodiments, this disclosure provides a method for preparing TI host cells expressing a polypeptide of interest, the method comprising: a) providing a TI host cell containing at least one exogenous nucleotide sequence integrated at a site within one or more loci of the TI host cell genome, wherein the one or more loci are at least about 90% homologous to sequences selected from SEQ ID No. 1-7, wherein the exogenous nucleotide sequence contains one or more RRSs; b) introducing a vector containing one or more RRSs into the cell provided in a) wherein the one or more RRSs are matched to one or more RRSs on the integrated exogenous nucleotide sequence and are side-connected to at least one exogenous SOI operatively linked to a regulated promoter; c) introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRSs; and d) selecting TI cells expressing an exogenous SOI in the presence of an inducer to thereby isolate TI host cells expressing the polypeptide of interest. In some embodiments, this disclosure provides a method for expressing a polypeptide of interest, the method comprising: a) providing a host cell containing at least one exogenous SOI integrated into a locus of the host cell genome, the at least one exogenous SOI being side-connected to two RRS and operatively linked to a tunable promoter, wherein the locus is at least about 90% homologous to sequences selected from SEQ ID No. 1-7; and b) culturing the cell under conditions suitable for expressing the SOI and recovering the polypeptide of interest from it. In some embodiments, this disclosure provides a method for preparing TI host cells expressing first and second polypeptides of interest (wherein the first and second polypeptides may be the same or different), the method comprising: a) providing a TI host cell containing a foreign nucleotide sequence integrated at a locus site in the host cell genome, wherein the locus is at least about 90% homologous to sequences selected from SEQ ID No. 1-7, wherein the foreign nucleotide sequence comprises first and second RRSs and a third RRS located between the first and second RRSs, wherein the first, second, and third RRSs are xenogeneic specific, i.e., the first, second, and third RRSs are not matched RRSs and therefore any two of the first, second, and third RRSs would be unsuitable for recombinase-mediated recombination; b) introducing a first vector containing two RRSs into the cell provided in a) the two RRSs being matched to the first and third RRSs on the integrated foreign nucleotide sequence and side-attached to at least one first foreign SOI operatively linked to a regulated promoter; c) Introduce a second vector containing two RRSs into the cell provided in a), the two RRSs being matched with the second and third RRSs on the integrated exogenous nucleotide sequence and side-attached at least one second exogenous SOI operatively linked to a regulated promoter; d) introduce one or more recombinases, or one or more nucleic acids encoding one or more recombinases, wherein the one or more recombinases recognize the RRSs; and e) select TI cells expressing the exogenous SOI in the presence of an inducer to thereby isolate TI host cells expressing the first and second polypeptides of interest.

Implementation Method

Claims

1. A targeted integration (TI) of a foreign nucleotide sequence into a Chinese hamster ovary (CHO) host cell, wherein the foreign nucleotide sequence is integrated at an integration site at a specific locus in the CHO host cell genome, wherein the nucleotide sequence immediately following the integrated foreign nucleotide sequence at the 5' end is located at a locus that is at least 90% homologous to nucleotides 69303-79768 of NW_003616412.

1.

2. The TI CHO host cell of claim 1, wherein the nucleotide sequence immediately following the 5' of the integrated exogenous nucleotide sequence is within 3,000 base pairs from the 5' end of nucleotide 79768 of NW_003616412.

1.

3. The TI CHO host cell of claim 2, wherein the nucleotide sequence immediately following the 5' of the integrated exogenous nucleotide sequence is within 2,000 base pairs from the 5' end of nucleotide 79768 of NW_003616412.

1.

4. The TI CHO host cell of claim 2, wherein the nucleotide sequence immediately following the 5' of the integrated exogenous nucleotide sequence is within 1,500 base pairs from the 5' end of nucleotide 79768 of NW_003616412.

1.

5. The TI CHO host cell of claim 2, wherein the nucleotide sequence immediately following the 5' of the integrated exogenous nucleotide sequence is within 1,000 base pairs from the 5' end of nucleotide 79768 of NW_003616412.

1.

6. The TI CHO host cell of claim 2, wherein the nucleotide sequence immediately following the 5' of the integrated exogenous nucleotide sequence is within 500 base pairs from the 5' end of nucleotide 79768 of NW_003616412.

1.

7. The TI CHO host cell of claim 2, wherein the nucleotide sequence immediately following the 5' of the integrated exogenous nucleotide sequence is within 400 base pairs from the 5' end of nucleotide 79768 of NW_003616412.

1.

8. The TI CHO host cell of claim 2, wherein the nucleotide sequence immediately following the 5' of the integrated exogenous nucleotide sequence is within 300 base pairs from the 5' end of nucleotide 79768 of NW_003616412.

1.

9. The TI CHO host cell of claim 2, wherein the nucleotide sequence immediately following the 5' of the integrated exogenous nucleotide sequence is within 200 base pairs from the 5' end of nucleotide 79768 of NW_003616412.

1.

10. The TI CHO host cell of claim 2, wherein the nucleotide sequence immediately following the 5' of the integrated exogenous nucleotide sequence is within 150 base pairs from the 5' end of nucleotide 79768 of NW_003616412.

1.

11. The TI CHO host cell of claim 2, wherein the nucleotide sequence immediately following the 5' of the integrated exogenous nucleotide sequence is within 100 base pairs from the 5' end of nucleotide 79768 of NW_003616412.

1.

12. The TI CHO host cell of claim 2, wherein the nucleotide sequence immediately following the 5' of the integrated exogenous nucleotide sequence is within 75 base pairs from the 5' end of nucleotide 79768 of NW_003616412.

1.

13. The TI CHO host cell of claim 2, wherein the nucleotide sequence immediately following the 5' of the integrated exogenous nucleotide sequence is within 50 base pairs from the 5' end of nucleotide 79768 of NW_003616412.

1.

14. The TI CHO host cell of claim 2, wherein the nucleotide sequence immediately following the 5' of the integrated exogenous nucleotide sequence is within 40 base pairs from the 5' end of nucleotide 79768 of NW_003616412.

1.

15. The TI CHO host cell of claim 2, wherein the nucleotide sequence immediately following the 5' of the integrated exogenous nucleotide sequence is within 30 base pairs from the 5' end of nucleotide 79768 of NW_003616412.

1.

16. The TI CHO host cell of claim 2, wherein the nucleotide sequence immediately following the 5' of the integrated exogenous nucleotide sequence is within 20 base pairs from the 5' end of nucleotide 79768 of NW_003616412.

1.

17. The TI CHO host cell of claim 2, wherein the nucleotide sequence immediately following the 5' of the integrated exogenous nucleotide sequence is within 15 base pairs from the 5' end of nucleotide 79768 of NW_003616412.

1.

18. The TI CHO host cell of claim 1, wherein the nucleotide sequence immediately following the 3' of the integrated exogenous nucleotide sequence is at least 90% homologous to nucleotides 79769-100059 of NW_003616412.

1.

19. The TI CHO host cell of claim 1, wherein the nucleotide sequence immediately following the 3' of the integrated exogenous nucleotide sequence is within 3,000 base pairs from the 3' end of nucleotide 79769 of NW_003616412.

1.

20. The TI CHO host cell of claim 19, wherein the nucleotide sequence immediately following the 3' of the integrated exogenous nucleotide sequence is within 2,000 base pairs from the 3' end of nucleotide 79769 of NW_003616412.

1.

21. The TI CHO host cell of claim 19, wherein the nucleotide sequence immediately following the 3' of the integrated exogenous nucleotide sequence is within 1,500 base pairs from the 3' end of nucleotide 79769 of NW_003616412.

1.

22. The TI CHO host cell of claim 19, wherein the nucleotide sequence immediately following the 3' of the integrated exogenous nucleotide sequence is within 1,000 base pairs from the 3' end of nucleotide 79769 of NW_003616412.

1.

23. The TI CHO host cell of claim 19, wherein the nucleotide sequence immediately following the 3' of the integrated exogenous nucleotide sequence is within 500 base pairs from the 3' end of nucleotide 79769 of NW_003616412.

1.

24. The TI CHO host cell of claim 19, wherein the nucleotide sequence immediately following the 3' of the integrated exogenous nucleotide sequence is within 400 base pairs from the 3' end of nucleotide 79769 of NW_003616412.

1.

25. The TI CHO host cell of claim 19, wherein the nucleotide sequence immediately following the 3' of the integrated exogenous nucleotide sequence is within 300 base pairs from the 3' end of nucleotide 79769 of NW_003616412.

1.

26. The TI CHO host cell of claim 19, wherein the nucleotide sequence immediately following the 3' of the integrated exogenous nucleotide sequence is within 200 base pairs from the 3' end of nucleotide 79769 of NW_003616412.

1.

27. The TI CHO host cell of claim 19, wherein the nucleotide sequence immediately following the 3' of the integrated exogenous nucleotide sequence is within 150 base pairs from the 3' end of nucleotide 79769 of NW_003616412.

1.

28. The TI CHO host cell of claim 19, wherein the nucleotide sequence immediately following the 3' of the integrated exogenous nucleotide sequence is within 100 base pairs from the 3' end of nucleotide 79769 of NW_003616412.

1.

29. The TI CHO host cell of claim 19, wherein the nucleotide sequence immediately following the 3' of the integrated exogenous nucleotide sequence is within 75 base pairs from the 3' end of nucleotide 79769 of NW_003616412.

1.

30. The TI CHO host cell of claim 19, wherein the nucleotide sequence immediately following the 3' of the integrated exogenous nucleotide sequence is within 50 base pairs from the 3' end of nucleotide 79769 of NW_003616412.

1.

31. The TI CHO host cell of any one of claims 1 to 30, wherein the TI CHO host cell is a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

32. The TI CHO host cell of claim 1, wherein the foreign nucleotide sequence contains one or more recombination recognition sequences (RRS), wherein the RRS can be recognized by a recombinase.

33. The TI CHO host cell of claim 32, wherein the exogenous nucleotide sequence contains at least two RRSs.

34. The TI CHO host cell of claim 32, wherein the recombinase is Cre recombinase or FLP recombinase or Bxb1 integrase or φC31 integrase; and / or the RRS is selected from the group consisting of: LoxP sequence, LoxP L3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, Lox66 sequence, FRT sequence, Bxb1 attP sequence, Bxb1 attB sequence, φC31 attP sequence and φC31 attB sequence.

35. The TI CHO host cell of claim 33, wherein the exogenous nucleotide sequence comprises a first and a second RRS and at least one selectable marker located between the first and the second RRS, wherein the first selectable marker is selected from the group consisting of: aminoglycoside phosphotransferase (APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamic acid synthase (GS), aspartate synthase, tryptophan synthase, histidine dehydrogenase, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid.

36. The TI CHO host cell of claim 35, wherein the APH is hygromycin phosphotransferase (HYG), neomycin, or G418 APH.

37. The TI CHO host cell of claim 35, wherein the exogenous nucleotide sequence further comprises: (a) a second selective marker, wherein the first selective marker differs from the second selective marker, wherein the second selective marker is selected from the group consisting of: aminoglycoside phosphotransferase (APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamic acid synthase (GS), aspartate synthase, tryptophan synthase, histamine dehydrogenase, blastomycin, bleomycin, cymoxanil, chloramphenicol, giomycin, and mycophenolic acid; (b) the second selective marker and the third selective marker of (a) and an internal ribosome entry site (IRES), wherein the IRES is operatively linked to the third selective marker, and wherein the third selective marker differs from the first or the second selective marker and / or is selected from the group consisting of: green fluorescent protein (GFP) marker, enhanced GFP, etc. (eGFP) markers, synthetic GFP markers, yellow fluorescent protein (YFP) markers, enhanced YFP (eYFP) markers, cyan fluorescent protein (CFP) markers, mPlum markers, mCherry markers, tdTomato markers, mStrawberry markers, J-red markers, DsRed-monomer markers, mOrange markers, mKO markers, mCitrine markers, Venus markers, YPet markers, Emerald6 markers, CyPet markers, mCFPm markers, Cerulean markers, and T-Sapphire markers; and / or (c) a third RRS, wherein the third RRS is located between the first and the second RRS, and the third RRS is xenospecific relative to the first or the second RRS.

38. The TI CHO host cell of claim 37, wherein the APH is hygromycin phosphotransferase (HYG), neomycin, or G418 APH.

39. The TI CHO host cell of any one of claims 32 to 38, wherein the exogenous nucleotide sequence further comprises (a) at least one exogenous sequence of interest (SOI) located between the first and the second RRS; or (b) at least one exogenous SOI located between the first and the third RRS, and at least one exogenous SOI located between the third and the second RRS.

40. The TI CHO host cell of claim 39, wherein in (a) and (b) the SOI encodes a single-chain antibody, antibody light chain, antibody heavy chain, single-chain Fv fragment (scFv) or Fc fusion protein.

41. A method for preparing Chinese hamster ovary (CHO) host cells that express the targeted integration (TI) of a polypeptide of interest, comprising: (a) A TI CHO host cell containing a foreign nucleotide sequence integrated at a locus within the TI CHO host cell genome, wherein: (i) the nucleotide sequence immediately following the 5' end of the integrated foreign nucleotide sequence is within 3,000 base pairs from the 5' end of nucleotide 79768 of NW_003616412.1; or (ii) the nucleotide sequence immediately following the 3' end of the integrated foreign nucleotide sequence is within 3,000 base pairs from the 3' end of nucleotide 79769 of NW_003616412.1; wherein the foreign nucleotide sequence contains two recombination recognition sequences (RRS), each RRS flanked by at least one first selectable marker; (b) (a) Introducing a vector containing two RRSs into the cell provided in (a), the two RRSs matching two RRSs on the integrated exogenous nucleotide sequence and being side-attached to at least one exogenous sequence of interest (SOI) and at least one second selectable marker; (c) Introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRSs; and (d) Selecting TI CHO cells expressing the second selectable marker to thereby isolate TI CHO host cells expressing the polypeptide.

42. A method for preparing Chinese hamster ovary (CHO) host cells exhibiting at least the first and second peptides of interest with targeted integration (TI), comprising: (a) Providing a TI CHO host cell containing a foreign nucleotide sequence integrated at a locus within the host cell genome, wherein: (i) the nucleotide sequence immediately following the 5' end of the integrated foreign nucleotide sequence is within 3,000 base pairs from the 5' end of nucleotide 79768 of NW_003616412.1; and / or (ii) the nucleotide sequence immediately following the 3' end of the integrated foreign nucleotide sequence is within 3,000 base pairs from the 3' end of nucleotide 79769 of NW_003616412.1; wherein the foreign nucleotide sequence comprises a first and a second recombination recognition sequence (RRS) flanked by at least one first selectable marker, and a third RRS located between the first and the second RRS, and all RRS are xenogeneic specific; (b) (a) Introducing a first vector comprising two RRSs into the cell provided in (a), the two RRSs matching the first and third RRSs on the integrated exogenous nucleotide sequence and being side-attached with at least one first exogenous sequence of interest (SOI) and at least one second select marker; (c) Introducing a second vector comprising two RRSs into the cell provided in (a), the two RRSs matching the second and third RRSs on the integrated exogenous nucleotide sequence and being side-attached with at least one second exogenous SOI; (d) Introducing one or more recombinases, or one or more nucleic acids encoding one or more recombinases, wherein the one or more recombinases recognize the RRSs; and (e) Selecting TI CHO cells expressing the second select marker to thereby isolate TI CHO host cells expressing the first and second polypeptides of interest.

43. The method of claim 42, wherein the first vector further comprises a promoter sequence operatively linked to the codon ATG, the promoter sequence being positioned upstream of the first SOI and downstream of the RRS, and the second vector further comprises a selection marker lacking the ATG transcription start codon, the selection marker being upstream of the RRS and downstream of the second SOI.

44. The method of any one of claims 41 to 43, wherein (i) the recombinase is a Cre recombinase or an FLP recombinase or a Bxb1 integrase or a φC31 integrase; (ii) the first and / or second SOI encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv) or an Fc fusion protein; and / or (iii) the TI CHO host cell line is a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell or a CHO K1M host cell.

45. A method for representing a polypeptide of interest, comprising: (a) Providing a targeted integration (TI) Chinese hamster ovary (CHO) host cell comprising at least one exogenous sequence of interest (SOI) and at least one selectable marker, wherein the at least one exogenous SOI and at least one selectable marker are flanked by two recombination recognition sequences (RRS) integrated into a locus of the TI CHO host cell genome, wherein: (i) the nucleotide sequence immediately following the integrated exogenous SOI, the at least one selectable marker, and the two RRS is within 3,000 base pairs from the 5' end of nucleotide 79768 of NW_003616412.1; or (ii) the nucleotide sequence immediately following the integrated exogenous SOI, the at least one selectable marker, and the two RRS is within 3,000 base pairs from the 3' end of nucleotide 79769 of NW_003616412.1; and (b) culturing the cell in (a) under conditions suitable for expressing the polypeptide of interest and recovering the polypeptide of interest from it.

46. ​​A method for representing a polypeptide of interest, comprising: (a) Providing a targeted integration (TI) Chinese hamster ovary (CHO) host cell comprising at least two exogenous sequences of interest (SOIs) and at least one selectable marker, wherein the at least two exogenous SOIs and at least one selectable marker are integrated into a locus in the host cell genome, wherein: (i) the nucleotide sequence immediately following the 5' end of the integrated at least two exogenous SOIs and at least one selectable marker is located within 3,000 base pairs from the 5' end of nucleotide 79768 of NW_003616412.1; or (ii) the nucleotide sequence immediately following the 3' end of the integrated at least two exogenous SOIs and at least one selectable marker is located within 3,000 base pairs from the 3' end of nucleotide 79769 of NW_003616412.1; and wherein at least one exogenous SOI and one selectable marker are flanked by first and third recombination recognition sequences (RRS) and at least one exogenous SOI is flanked by a second and the third RRS; and (b) The cells in (a) are cultured under conditions suitable for expressing the polypeptide of interest, and the polypeptide of interest is recovered from them.

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