Retrovirus-like particle-reduced chinese hamster ovary production cell line

Abstract

The presently disclosed subject matter relates to reduced retrovirus-like particle (RVLP) Chinese hamster ovary (CHO) cells suitable for production of recombinant proteins, and methods of producing and using such reduced RVLP CHO production cells.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 544,782, filed October 18, 2023, the contents of which are incorporated herein by reference in their entirety, and claims priority thereto.

[0003] sequence list

[0004] This specification references the sequence list (an XML file named "00B206_1461_SL.xml" submitted electronically on October 18, 2024). The 00B206_1461_SL.xml file was generated on October 8, 2024, and is 6,639,945 bytes in size. The entire contents of this sequence list are hereby incorporated by reference. Technical Field

[0005] The subject of this disclosure relates to Chinese hamster ovary (CHO) cells with reduced retrovirus-like particle (RVLP) content suitable for producing recombinant proteins, and methods for producing and using such RVLP-reduced CHO cells. Background Technology

[0006] Due to the rapid advancements in cell biology and immunology, there is a growing need to develop novel therapeutic recombinant proteins for a wide range of diseases, including cancer, cardiovascular disease, and metabolic disorders. These candidate biopharmaceuticals are typically produced using commercially available cell lines capable of expressing the target protein. For example, CHO cells have been widely used to produce monoclonal antibodies.

[0007] CHO cells are a preferred mammalian expression system for biomanufacturing, partly due to their safety profile compared to other mammalian expression systems. Compared to other mammalian cells, CHO cells are not only less susceptible to certain viral infections, but studies have repeatedly shown that CHO cells cannot produce infectious virus-like particles (VLPs) capable of replicating in human cells, such as infectious RVLPs. However, CHO cells do produce VLPs, and these VLPs have been identified both intracellularly and extracellularly in cell culture media. Since these VLPs (e.g., RVLPs) are thought to arise from the presence of endogenous retroviruses (ERVs) rather than newly acquired retroviral infections, there remains a need in the art for CHO cells with reduced RVLPs suitable for facilitating the commercial production of recombinant proteins. Summary of the Invention

[0008] In some embodiments, the subject matter of this disclosure relates to modified CHO cells, wherein prior to modification, the CHO cells comprise two or more ERV loci selected from:

[0009] (a) CHERV-1b (Genbank accession number MN527960, SEQ ID NO. 8);

[0010] (b) CHERV-2g (Genbank accession number MN527961, SEQ ID NO. 9);

[0011] (c) ETC109F (SEQ ID NO. 10, 30021-39247);

[0012] (d) CHERV-3g (Genbank accession number MN527962, SEQ ID NO. 11); and

[0013] (e) An ERV locus containing a sequence that has at least 90% identity with any of (a) to (d).

[0014] The modification includes the inactivation of two or more of the ERV loci (a) to (e) present in CHO cells prior to such modification. In some embodiments, the inactivation of two or more of the ERV loci (a) to (e) includes the knockout of the corresponding ERV GAG coding sequence. For example, but not by limitation, the knockout of the corresponding ERV GAG coding sequence may include introducing an insertion or deletion into the ERV GAG coding sequence. In some non-limiting embodiments, the knockout of the corresponding ERV GAG coding sequence may include introducing a base edit into the ERV GAG coding sequence. In some non-limiting embodiments, the knockout of the corresponding ERV GAG coding sequence may include introducing a deletion flanking the ERV GAG coding sequence.

[0015] In some embodiments, the subject matter of this disclosure relates to modified CHO cells, wherein prior to modification, the CHO cells comprise two or more ERV loci selected from:

[0016] (a) CHERV-1b (Genbank accession number MN527960, SEQ ID NO. 8);

[0017] (b) CHERV-2g (Genbank accession number MN527961, SEQ ID NO. 9);

[0018] (c) ETC109F (30021-39247 of SEQ ID NO. 10);

[0019] (d) CHERV-3g (Genbank accession number MN527962, SEQ ID NO. 11); and

[0020] (e) An ERV locus containing a sequence that has at least 90% identity with any of (a) to (d).

[0021] The modification includes the inactivation of two or more of the ERV loci (a) to (e) present in CHO cells prior to such modification, and the modified cells express the desired recombinant product. In some embodiments, the modified cells are generated from recombinant cells expressing the desired recombinant product. In some embodiments, the desired recombinant product includes a recombinant protein. In some embodiments, the recombinant protein is an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody is a multispecific antibody or an antigen-binding fragment thereof. In some embodiments, the antibody may consist of a single heavy chain sequence and a single light chain sequence or an antigen-binding fragment thereof. In some embodiments, the antibody is a chimeric antibody, a human antibody, or a humanized antibody. In some embodiments, the antibody is a monoclonal antibody.

[0022] In some embodiments, the target recombinant product expressed by the modified CHO cells of this disclosure is encoded by an exogenous nucleic acid sequence integrated into the cellular genome of the modified CHO cells at one or more target sites. In some embodiments, the target site is a portion of the contiguous sequence of one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to 7.

[0023] In some embodiments, the modified CHO cells of this disclosure include gene knockouts selected from: a) BAX; BAK; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; b) BAX; BAK; ICAM-1; SIRT-1; GGTA1; CMAH; LPL, LPLA2; and PPT1; c) BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; d) BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; e) BAX; BAK; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; f) BAX; BAK; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; g) BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; h) BAX; BAK; ICAM-1; LPL; LPLA2; and PPT1; i) BAX; BAK; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; j) BAX; BAK; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1; k) BAX; BAK; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA; l) BAX; BAK; ICAM-1; SIRT-1; and MYC; m) BAX; BAK; ICAM-1; PERK; SIRT-1; and MYC; n) BAX; BAK; ICAM-1; SIRT-1; PERK; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; o) BAX; BAK; LPL; LPLA2; GGTA1; and CMAH; p) and CMAH; q) BAX; BAK; MYC; LPL; LPLA2; GGTA1; and CMAH; r) BAX; BAK; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH; s) BAX; BAK; LPL; LPLA2; GGTA1; CMAH; and PPT1; t) BAX; BAK; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; u) BAX; BAK; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1;v) BAX; BAK; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; w) BAX; BAK; ICAM-1; and SIRT-1; x) BAX; BAK; and ICAM-1; y) BAX; BAK; BCKDHA; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; z) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; aa) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; bb) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; cc) BAX; BAK; BCKDHA; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; dd) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ee) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ff) BAX; BAK; BCKDHA; ICAM-1; LPL; LPLA2; and PPT1; gg) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; hh) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1; ii) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA; jj) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; and MYC; kk) BAX; BAK; BCKDHA; ICAM-1; PERK; SIRT-1; and MYC; ll) BAX; BAK; BCKDHA; ICAM-1; PERK; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; mm) BAX; BAK; BCKDHA; LPL; LPLA2; GGTA1; and CMAH; nn) BAX; BAK; BCKDHA; PERK; LPL; LPLA2; GGTA1; and CMAH; oo) BAX; BAK; BCKDHA; MYC;LPL; LPLA2; GGTA1; and CMAH; pp) BAX; BAK; BCKDHA; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH; qq) BAX; BAK; BCKDHA; LPL; LPLA2; GGTA1; CMAH; and PPT1; rr) BAX; BAK; BCKDHA; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; ss) BAX; BAK; BCKDHA; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1; tt) BAX; BAK; BCKDHA; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; uu) BAX; BAK; BCKDHA; ICAM-1; and SIRT-1; vv) BAX; BAK; BCKDHA; and ICAM-1; ww) BAX; BAK; BCKDHB; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; xx) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; yy) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; zz) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; aaa) BAX; BAK; BCKDHB; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; bbb) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ccc) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ddd) BAX; BAK; BCKDHB; ICAM-1; LPL; LPLA2; and PPT1; eee) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; fff) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1; ggg) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA; hhh) BAX; BAK;BCKDHB; ICAM-1; SIRT-1; and MYC; iii) BAX; BAK; BCKDHB; ICAM-1; PERK; SIRT-1; and MYC; jjj) BAX; BAK; BCKDHB; ICAM-1; PERK; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; kkk) BAX; BAK; BCKDHB; LPL; LPLA2; GGTA1; and CMAH; lll) BAX; BAK; BCKDHB; PERK; LPL; LPLA2; GGTA1; and CMAH; mmm) BAX; BAK; BCKDHB; MYC; LPL; LPLA2; GGTA1; and CMAH; nnn) BAX; BAK; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH; ooo) BAX; BAK; BCKDHB; LPL; LPLA2; GGTA1; CMAH; and PPT1; ppp) BAX; BAK; BCKDHB; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; qqq) BAX; BAK; BCKDHB; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1; rrr) BAX; BAK; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; sss) BAX; BAK; BCKDHB; ICAM-1; and SIRT-1; ttt) BAX; BAK; BCKDHB; and ICAM-1; uuu) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; vvv) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; www) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; xxx) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; yyy) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; zzz) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1;GGTA1; CMAH; LPLA2; PPT1; and LIPA; aaaa) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; bbbb) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; LPL; LPLA2; and PPT1; cccc) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; dddd) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1; eeee) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA; ffff) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; and MYC; gggg) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; PERK; SIRT-1; and MYC; hhhh) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; PERK; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; iiii) BAX; BAK; BCKDHA; BCKDHB; LPL; LPLA2; GGTA1; and CMAH; jjjj) BAX; BAK; BCKDHA; BCKDHB; PERK; LPL; LPLA2; GGTA1; and CMAH; kkkk) BAX; BAK; BCKDHA; BCKDHB; MYC; LPL; LPLA2; GGTA1; and CMAH; llll) BAX; BAK; BCKDHA; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH; mmmm) BAX; BAK; BCKDHA; BCKDHB; LPL; LPLA2; GGTA1; CMAH; and PPT1; nnnn) BAX; BAK; BCKDHA; BCKDHB; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; oooo) BAX; BAK; BCKDHA; BCKDHB; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1; pppp) BAX; BAK; BCKDHA; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1;qqqq) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; and SIRT-1; or rrrr) BAX; BAK; BCKDHA; BCKDHB; and ICAM-1. ;

[0024] In some embodiments, this disclosure relates to compositions comprising the modified CHO cells disclosed herein.

[0025] In some embodiments, this disclosure relates to a method for producing a recombinant product for a specific purpose, the method comprising:

[0026] a) Culturing the modified CHO cells of this disclosure; and

[0027] b) Recover the target recombinant product from the culture medium or modified CHO cells.

[0028] In some embodiments, the method for producing the desired recombinant product of this disclosure includes purifying the desired recombinant product, harvesting the desired recombinant product, and / or formulating the desired recombinant product.

[0029] In some embodiments, this disclosure relates to a method for producing modified CHO cells, the method comprising:

[0030] (a) Contacting cells with a nuclease-assisted gene-targeting system and / or nucleic acids that target at least two endogenous ERVs, wherein the endogenous ERVs are selected from:

[0031] (i) CHERV-1b (Genbank accession number MN527960, SEQ ID NO. 8);

[0032] (ii) CHERV-2g (Genbank accession number MN527961, SEQ ID NO. 9);

[0033] (iii) ETC109F (SEQ ID NO. 10, 30021-39247);

[0034] (iv) CHERV-3g (Genbank accession number MN527962, SEQ ID NO. 11); and

[0035] (v) An ERV locus containing a sequence that has at least 90% identity with any of (i) to (iv), and

[0036] (b) Select modified CHO cells in which the expression of the ERV has been reduced or eliminated compared with unmodified CHO cells.

[0037] In some embodiments, a method of producing modified CHO cells of the present disclosure includes introducing a foreign nucleic acid encoding a desired recombinant product into the CHO cells after modification, wherein the expression of the ERV is reduced or eliminated compared to unmodified CHO cells. In some embodiments, a method of producing modified CHO cells of the present disclosure includes introducing a foreign nucleic acid encoding a desired recombinant product into the CHO cells before modification, wherein the expression of the ERV is reduced or eliminated compared to unmodified CHO cells.

[0038] In some embodiments, the exogenous nucleic acid encoding the desired recombinant product introduced into the modified cells of this disclosure before or after the modification is integrated at one or more target sites in the cellular genome of the CHO cells. In some embodiments, the target site is a portion of the contiguous sequence of one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to 7.

[0039] In some embodiments, exogenous nucleic acids encoding a desired recombinant product introduced into the modified cells of this disclosure before or after the modification are randomly integrated into the cellular genome of the modified CHO cells.

[0040] In some embodiments, the target recombinant product encoded by an exogenous nucleic acid introduced into the modified cells of this disclosure before or after the modification comprises a recombinant protein. In some embodiments, the recombinant protein is an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody is a multispecific antibody or an antigen-binding fragment thereof. In some embodiments, the antibody may consist of a single heavy chain sequence and a single light chain sequence or an antigen-binding fragment thereof. In some embodiments, the antibody is a chimeric antibody, a human antibody, or a humanized antibody. In some embodiments, the antibody is a monoclonal antibody.

[0041] In some embodiments, the nucleic acid sequence encoding the desired recombinant product is introduced into the modified cells of this disclosure before or after the modification using a transposase-mediated gene integration system.

[0042] In some embodiments, the nuclease-assisted gene targeting system for reducing ERV expression in modified CHO cells of this disclosure is selected from the group consisting of CRISPR / Cas9, CRISPR / Cpf1, zinc finger nucleases, TALEN, or meganucleases.

[0043] In some embodiments, the reduction in ERV expression in the modified CHO cells of this disclosure is mediated by RNA silencing. In some embodiments, RNA silencing is selected from the group consisting of siRNA gene targeting and knockdown, shRNA gene targeting and knockdown, and miRNA gene targeting and knockdown.

[0044] In some embodiments, a method of producing the modified CHO cells of this disclosure includes using CHO cells comprising gene knockouts selected from: a) BAX; BAK; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; b) BAX; BAK; ICAM-1; SIRT-1; GGTA1; CMAH; LPL, LPLA2; and PPT1; c) BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; d) BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; e) BAX; BAK; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; f) BAX; BAK; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; g) BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; h) BAX; BAK; ICAM-1; LPL; LPLA2; and PPT1; i) BAX; BAK; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; j) BAX; BAK; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1; k) BAX; BAK; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA; l) BAX; BAK; ICAM-1; SIRT-1; and MYC; m) BAX; BAK; ICAM-1; PERK; SIRT-1; and MYC; n) BAX; BAK; ICAM-1; SIRT-1; PERK; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; o) BAX; BAK; LPL; LPLA2; GGTA1; and CMAH; p) and CMAH; q) BAX; BAK; MYC; LPL; LPLA2; GGTA1; and CMAH; r) BAX; BAK; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH; s) BAX; BAK; LPL; LPLA2; GGTA1; CMAH; and PPT1; t) BAX; BAK; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; u) BAX; BAK; MYC; LPL; LPLA2;GGTA1; CMAH; and PPT1; v) BAX; BAK; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; w) BAX; BAK; ICAM-1; and SIRT-1; x) BAX; BAK; and ICAM-1; y) BAX; BAK; BCKDHA; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; z) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; aa) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; bb) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; cc) BAX; BAK; BCKDHA; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; dd) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ee) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ff) BAX; BAK; BCKDHA; ICAM-1; LPL; LPLA2; and PPT1; gg) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; hh) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1; ii) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA; jj) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; and MYC; kk) BAX; BAK; BCKDHA; ICAM-1; PERK; SIRT-1; and MYC; ll) BAX; BAK; BCKDHA; ICAM-1; PERK; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; mm) BAX; BAK; BCKDHA; LPL; LPLA2; GGTA1; and CMAH; nn) BAX; BAK; BCKDHA; PERK; LPL; LPLA2; GGTA1; and CMAH;oo) BAX; BAK; BCKDHA; MYC; LPL; LPLA2; GGTA1; and CMAH; pp) BAX; BAK; BCKDHA; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH; qq) BAX; BAK; BCKDHA; LPL; LPLA2; GGTA1; CMAH; and PPT1; rr) BAX; BAK; BCKDHA; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; ss) BAX; BAK; BCKDHA; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1; tt) BAX; BAK; BCKDHA; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; uu) BAX; BAK; BCKDHA; ICAM-1; and SIRT-1; vv) BAX; BAK; BCKDHA; and ICAM-1; ww) BAX; BAK; BCKDHB; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; xx) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; yy) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; zz) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; aaa) BAX; BAK; BCKDHB; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; bbb) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ccc) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ddd) BAX; BAK; BCKDHB; ICAM-1; LPL; LPLA2; and PPT1; eee) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; fff) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1; ggg) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2;PPT1; and LIPA; hhh) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; and MYC; iii) BAX; BAK; BCKDHB; ICAM-1; PERK; SIRT-1; and MYC; jjj) BAX; BAK; BCKDHB; ICAM-1; PERK; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; kkk) BAX; BAK; BCKDHB; LPL; LPLA2; GGTA1; and CMAH; lll) BAX; BAK; BCKDHB; PERK; LPL; LPLA2; GGTA1; and CMAH; mmm) BAX; BAK; BCKDHB; MYC; LPL; LPLA2; GGTA1; and CMAH; nnn) BAX; BAK; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH; ooo) BAX; BAK; BCKDHB; LPL; LPLA2; GGTA1; CMAH; and PPT1; ppp) BAX; BAK; BCKDHB; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; qqq) BAX; BAK; BCKDHB; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1; rrr) BAX; BAK; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; sss) BAX; BAK; BCKDHB; ICAM-1; and SIRT-1; ttt) BAX; BAK; BCKDHB; and ICAM-1; uuu) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; vvv) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; www) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; xxx) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; yyy) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; zzz) BAX; BAK;BCKDHA; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; aaaa) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; bbbb) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; LPL; LPLA2; and PPT1; cccc) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; dddd) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1; eeee) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA; ffff) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; and MYC; gggg) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; PERK; SIRT-1; and MYC; hhhh) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; PERK; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; iiiii) BAX; BAK; BCKDHA; BCKDHB; LPL; LPLA2; GGTA1; and CMAH; jjjj) BAX; BAK; BCKDHA; BCKDHB; PERK; LPL; LPLA2; GGTA1; and CMAH; kkkk) BAX; BAK; BCKDHA; BCKDHB; MYC; LPL; LPLA2; GGTA1; and CMAH; llll) BAX; BAK; BCKDHA; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH; mmmm) BAX; BAK; BCKDHA; BCKDHB; LPL; LPLA2; GGTA1; CMAH; and PPT1; nnnn) BAX; BAK; BCKDHA; BCKDHB; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; oooo) BAX; BAK; BCKDHA; BCKDHB; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1; pppp) BAX; BAK; BCKDHA; BCKDHB; MYC; PERK; LPL;LPLA2; GGTA1; CMAH; and PPT1; qqqq) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; and SIRT-1; or rrrr) BAX; BAK; BCKDHA; BCKDHB; and ICAM-1. ; Attached Figure Description

[0045] Figures 1A-1D depict the distribution of expression of the three ERV substances in RVLP Gag knockout clones and controls (Figure 1A); total RVLP titer (the sum of the three ERVs) in Gag knockout clones and controls (Figure 1B); production culture integral viable cell concentration (IVCC) in Gag knockout clones and controls (Figure 1C); and RVLP titer assessed by TEM in Gag knockout clones and controls (Figure 1D).

[0046] Figures 2A-2C depict the knockout strategy for producing large amounts of deletions using dual CRISPR / Cas9-targeted nucleases targeting CHERV-3g and CHERV-1b (Figure 2A): and RVLP titers assessed by RT-ddPCR (2B) and TEM (2C) in the deletion knockout clones and controls. Detailed Implementation

[0047] In some embodiments, the subject matter of this disclosure relates to RVLP-reduced CHO cells suitable for producing recombinant proteins, and methods for producing and using such RVLP-reduced CHO production cells. For example, but not as a limitation, part of the subject matter of this disclosure relates to modified CHO cells and methods for producing such modified CHO cells, wherein prior to modification, the CHO cells contain two or more ERV loci selected from:

[0048] (a) CHERV-1b (Genbank accession number MN527960, SEQ ID NO. 8);

[0049] (b) CHERV-2g (Genbank accession number MN527961, SEQ ID NO. 9);

[0050] (c) ETC109F (SEQ ID NO. 10, 30021-39247);

[0051] (d) CHERV-3g (Genbank accession number MN527962, SEQ ID NO. 11); and

[0052] (e) An ERV locus containing a sequence that has at least 90% identity with any of (a) to (d).

[0053] The modifications include the inactivation of two or more of the ERV loci (a) to (e) present in CHO cells prior to such modifications, i.e., reduced or eliminated expression compared to unmodified CHO cells.

[0054] For clarity rather than for limitation, the detailed description is divided into the following sections:

[0055] 1. Definition

[0056] 2. Inactivation of endogenous retroviral loci

[0057] 3. Host cells

[0058] 4. Integration of exogenous nucleic acids

[0059] 5. Preparation and use of RVLP-reduced host cells

[0060] 6. Products

[0061] 7. Examples

[0062] 1. Definition

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of any ambiguity, this patent specification (including definitions) shall prevail. Preferred methods and materials are described below, although similar or equivalent methods and materials may be used to practice or test the subject matter currently disclosed. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0064] As used herein, the terms “comprising,” “including,” “having,” “having,” “may,” “containing,” and variations thereof are intended as open-ended transitional phrases, terms, or words and do not exclude the possibility of other actions or structures. Unless expressly stated otherwise, the singular forms “a,” “an,” and “the / described” include a plural referent. This disclosure also contemplates other embodiments that, whether or not expressly stated, “include,” “comprise,” and “substantially constitute” the embodiments or elements presented herein.

[0065] To enumerate the numerical ranges in this article, each intermediate number with the same precision between them is explicitly considered. For example, for the range of 6–9, the numbers 7 and 8 are considered in addition to 6 and 9; and for the range of 6.0–7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly considered.

[0066] As used herein, the term "about" or "approximately" refers to an acceptable range of error for a particular value as determined by one of ordinary skill in the art, the acceptable range of error depending in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" may mean three or more standard deviations. Alternatively, "about" may indicate a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within a certain order of magnitude of a value, preferably within 5 times, more preferably within 2 times.

[0067] As used herein, the term "selection marker" can be a gene that allows for the specific selection or exclusion of cells carrying that gene in the presence of a suitable selector. For example, but not as a limitation, a selection marker can allow for positive selection of host cells transfected with the selection marker gene in the presence of that gene; untransformed host cells will not be able to grow or survive under that selection condition. Selection markers can be positive, negative, or bifunctional. Positive selection markers can allow for the selection of cells carrying the marker, while negative selection markers can allow for the selective elimination of cells carrying the marker. Selection markers can confer resistance to drugs or compensate for metabolic or catabolistic defects in host cells. In prokaryotic cells, genes conferring resistance to ampicillin, tetracycline, kanamycin, or chloramphenicol, as well as other genes, can be used. Resistance genes that can be used as selection markers in eukaryotic cells include, but are not limited to, genes targeting aminoglycoside phosphotransferases (APHs) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthase (GS), asparagine synthase, tryptophan synthase (indole), histamine dehydrogenase (histamine D), and genes encoding resistance to puromycin, blastomycin, bleomycin, humicin, chloramphenicol, zeocin, and mycophenolic acid. Additional marker genes are described in WO 92 / 08796 and WO 94 / 28143.

[0068] In addition to aiding selection in the presence of appropriate selectants, selection markers can alternatively provide genes encoding molecules not normally present in cells, such as green fluorescent protein (GFP), enhanced GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), enhanced YFP (eYFP), cyan fluorescent protein (CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire. Cells carrying such genes can be distinguished from those not carrying them, for example, by detecting the fluorescence emitted by the encoded polypeptide.

[0069] As used herein, the term "operably linked" refers to the juxtaposition of two or more components in a manner that allows them to function in a desired way. For example, if a promoter and / or enhancer are used to regulate transcription of a coding sequence, then the promoter and / or enhancer are operably linked to the coding sequence. In some embodiments, the DNA sequences of "operably linked" sequences are linked and adjacent on a single chromosome. In some embodiments, for example, when two protein-coding regions (such as a secretory leader region and a polypeptide) must be joined, these sequences are linked, adjacent, and within the same reading frame. In some embodiments, the operably linked promoter is located upstream of the coding sequence and may be adjacent to it. In some embodiments, for example, with respect to enhancer sequences regulating the expression of a coding sequence, the two components are operably linked but not adjacent. If the enhancer increases transcription of the coding sequence, then the enhancer is operably linked to the coding sequence. Operatically linked enhancers may be located upstream, inside, or downstream of the coding sequence and may be located at a considerable distance from the promoter of the coding sequence. Operable ligation can be accomplished using recombination methods known in the art, such as PCR and / or by ligation at a convenient restriction site. If a convenient restriction site is not available, synthetic oligonucleotide adaptors or linkers can be used according to standard practice. An internal ribosome entry site (IRES) can be operably ligated to an open reading frame (ORF) if it allows for the initiation of ORF translation at an internal location in a manner independent of the 5' end.

[0070] As used herein, the term "expression" refers to transcription and / or translation. In some embodiments, the transcriptional level of the desired product can be determined based on the amount of the corresponding mRNA present. For example, mRNA transcribed from the target sequence can be quantified by PCR or RNA blot hybridization. In some embodiments, the protein encoded by the target sequence can be quantified by a variety of methods, such as by ELISA, by measuring the protein's biological activity, or by using an assay unrelated to this activity, such as Western blotting or radioimmunoassay, using antibodies that recognize and bind to the protein.

[0071] The term “antibody” is used in the broadest sense and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), haptens, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0072] As used herein, the term "antibody fragment" refers to a molecule other than a complete antibody that comprises a portion of the complete antibody and binds to an antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bisomatic antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0073] As used herein, the term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies bound to a specific antigen can be isolated using the VH or VL domains of the antibody bound to that antigen to screen libraries of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0074] As used in this article, the term "heavy chain" refers to the immunoglobulin heavy chain.

[0075] As used in this article, the term "light chain" refers to the immunoglobulin light chain.

[0076] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0077] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, i.e., individual antibodies comprising this group are identical and / or bind to the same epitopes, except for possible variant antibodies (e.g., those containing naturally occurring mutations or generated during the production of the monoclonal antibody formulation, such variants typically exist in small quantities). In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies according to the invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0078] A "multispecific antibody" is a monoclonal antibody that has binding specificity to at least two different sites (i.e., different epitopes on different antigens or different epitopes on the same antigen). In some respects, multispecific antibodies have three or more binding specificities. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0079] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably herein to refer to antibodies having a structure substantially similar to that of natural antibodies or having a heavy chain containing an Fc region as defined herein.

[0080] "Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody and binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bisomatic antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For a review of some antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0081] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0082] A "human antibody" is an antibody whose amino acid sequence corresponds to that of an antibody produced by a human or human cell, or to a non-human antibody derived from a complete library of human antibodies or other antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0083] "Humanized" antibodies refer to chimeric antibodies that contain amino acid residues from a non-human CDR and amino acid residues from a human FR. In some respects, humanized antibodies will substantially contain at least one, typically two, variable domains, where all or substantially all CDRs correspond to the CDRs of the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Humanized antibodies may optionally contain at least a portion of the antibody constant region derived from a human antibody. Antibodies in a "humanized form," such as non-human antibodies, refer to antibodies that have undergone humanization. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, i.e., individual antibodies comprising this group are identical and / or bind the same epitopes, except for possible variant antibodies (e.g., containing naturally occurring mutations or generated during the production of the monoclonal antibody formulation, such variants are typically present in small quantities). In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier “monoclonal” indicates that the antibody is characterized by being obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method.

[0084] The term "therapeutic antibody" refers to an antibody used to treat a disease. Therapeutic antibodies can have various mechanisms of action. Therapeutic antibodies can bind to and neutralize the normal function of a target associated with an antigen. For example, a monoclonal antibody that blocks the activity of a protein required for cancer cell survival leads to cell death. Another type of therapeutic monoclonal antibody can bind to and activate the normal function of a target associated with an antigen. For example, a monoclonal antibody can bind to a protein on a cell and trigger apoptosis signals. Yet another type of monoclonal antibody can bind to a target antigen expressed only in diseased tissue; conjugating a toxic payload (effective agent), such as a chemotherapeutic agent or a radioactive agent, to a monoclonal antibody can form a mechanism for specifically delivering the toxic payload to diseased tissue, reducing damage to healthy tissue. A "biological functional fragment" of a therapeutic antibody will exhibit at least one (if not part or all) of the biological functions attributable to the complete antibody, including at least specific binding to a target antigen.

[0085] The term "diagnostic antibody" refers to an antibody used as a diagnostic reagent for a disease. Diagnostic antibodies bind to target antigens that are specifically associated with or exhibit increased expression therein, for example, a specific disease. Diagnostic antibodies can be used, for example, to detect targets in biological samples from a patient, or for diagnostic imaging of disease sites such as tumors in a patient. A "biological functional fragment" of a diagnostic antibody will exhibit at least one (if not part or all) biological function attributable to the complete antibody, including at least specific binding to a target antigen.

[0086] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including progeny cells. Host cells include “transformations” and “transformed cells,” which include primary transformed cells and progeny derived from those primary transformed cells, regardless of passage number. Progeny cells may not have completely identical nucleic acid contents to the parent cells and may contain mutations. This article includes mutant progeny with the same function or biological activity as those screened or selected from the original transformed cells.

[0087] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance comprising a nucleotide polymer. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate ester group. Typically, nucleic acid molecules are described by a base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. Base sequences are typically represented from 5' to 3'. In this document, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mRNA)), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone bonds or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for the direct expression of antibodies used in this invention in vitro and / or in vivo (e.g., in a host or patient). Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoding molecule, enabling the mRNA to be injected into a subject to generate in vivo antibodies (see, for example, Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP2 101 823 B1).

[0088] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are contained in cells that normally contain nucleic acid molecules, but which are located outside the chromosome or at a chromosomal location different from their natural chromosomal location.

[0089] As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell into which they have been introduced. In some embodiments, the vector directs the expression of the nucleic acid operatively linked to it. Such vectors are referred to herein as "expression vectors."

[0090] As used herein, the term "homologous sequence" refers to sequences that share significant sequence similarity, as determined by sequence alignment. For example, two sequences may be approximately 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 99.9% homologous. Alignment is performed using algorithms and computer programs (including, but not limited to, BLAST, FASTA, and HMME) that compare sequences and calculate the statistical significance of a match based on factors such as sequence length, sequence identity and similarity, and the presence and length of sequence mismatches and gaps. Homologous sequences can refer to both DNA and protein sequences.

[0091] As used herein, the term "sidejoint" refers to a first nucleotide sequence located at the 5' or 3' end, or both ends, of a second nucleotide sequence. The sidejoint nucleotide sequence can be adjacent to or at a defined distance from the second nucleotide sequence. There is no specific limit to the length of the sidejoint nucleotide sequence. For example, a sidejoint sequence can have a few base pairs or thousands of base pairs. In some embodiments, the length of the side-attached nucleotide sequence may be at least 15 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 75 base pairs, at least 100 base pairs, at least 150 base pairs, at least 200 base pairs, at least 300 base pairs, at least 400 base pairs, at least 500 base pairs, at least 1,000 base pairs, at least 1,500 base pairs, at least 2,000 base pairs, at least 3,000 base pairs, at least 4,000 base pairs, at least 5,000 base pairs, at least 6,000 base pairs, at least 7,000 base pairs, at least 8,000 base pairs, at least 9,000 base pairs, or at least 10,000 base pairs.

[0092] As used herein, the term "exogenous" refers to a nucleotide sequence that is not derived from the host cell but is introduced into the host cell through conventional DNA delivery methods (e.g., transfection, electroporation, transformation). The term "endogenous" refers to a nucleotide sequence that is derived from the host cell. Exogenous nucleotide sequences may have identical base compositions to their endogenous counterparts, wherein the exogenous sequence is introduced into the host cell via, for example, recombinant DNA technology.

[0093] 2. Inactivation of endogenous retroviral loci

[0094] In some embodiments, the subject matter of this disclosure relates to RVLP-reduced CHO cells suitable for producing recombinant proteins. In some embodiments, such RVLP-reduced CHO cells can be produced via inactivation of ERV loci. In some embodiments, multiple ERV loci are inactivated. For example, but not as a limitation, part of the subject matter of this disclosure relates to modified CHO cells and methods for producing such modified CHO cells, wherein prior to modification, the CHO cells comprise two or more ERV loci selected from:

[0095] (a) CHERV-1b (Genbank accession number MN527960, SEQ ID NO. 8);

[0096] (b) CHERV-2g (Genbank accession number MN527961, SEQ ID NO. 9);

[0097] (c) ETC109F (SEQ ID NO. 10, 30021-39247);

[0098] (d) CHERV-3g (Genbank accession number MN527962, SEQ ID NO. 11); and

[0099] (e) An ERV locus containing a sequence that has at least 90% identity with any of (a) to (d).

[0100] The modifications include the inactivation of two or more of the ERV loci (a) to (e) present in CHO cells prior to such modifications, i.e., reduced or eliminated expression compared to unmodified CHO cells.

[0101] Regarding ETC109F, it corresponds to SEQ ID NO. 10, 30021-39247 (% 'LTR to 3' LTR). The remainder of SEQ ID NO. 10 corresponds to: the CHO-K1 genome sequence upstream of the 5' end of ETC109F (SEQ ID NO. 10, 1-30020); and the CHO-K1 genome sequence downstream of the 3' end of ETC109F (SEQ ID NO. 10, 39248-59558). Other CHERV sequences presented against the background of its genomic loci include CHERV-3g allele A presented as SEQ ID NO. 12, CHERV-3g allele B presented as SEQ ID NO. 13, and CHERV-1b presented as SEQ ID NO. 14.

[0102] In some embodiments, this disclosure relates to methods for inactivating two or more ERV loci, i.e., reducing or eliminating expression compared to unmodified CHO cells, wherein such inactivation includes: (1) modifying a gene encoding an ERV protein, for example by introducing deletion, insertion, substitution, or a combination thereof into the gene; (2) reducing or eliminating transcription and / or stability of mRNA encoding an ERV protein; and (3) reducing or eliminating translation of mRNA encoding an ERV protein. In some embodiments, the reduction or elimination of ERV protein expression is achieved through targeted genome editing. For example, RNA-guided nuclease-based genome editing systems, such as CRISPR / Cas9-based genome editing systems, can be used to modify one or more target genes, resulting in a reduction or elimination of expression of one or more targeted ERV genes. Other editing systems, such as TALENS, meganucleases, and zinc finger nucleases, can also be used in methods for editing ERV genes.

[0103] In some embodiments, inactivating two or more ERV loci (i.e., reduced or eliminated expression compared to unmodified CHO cells) includes knocking out the corresponding ERV GAG coding sequence. For example, but not as a limitation, knocking out the corresponding ERV GAG coding sequence may include introducing an insertion or deletion into the ERV GAG coding sequence via CRISPR / Cas9-based genome editing. In some non-limiting embodiments, knocking out the corresponding ERV GAG coding sequence may include introducing a base edit into the ERV GAG coding sequence via CRISPR / Cas9-based base editing. In some non-limiting embodiments, knocking out the corresponding ERV GAG coding sequence may include introducing a deletion flanking the ERV GAG coding sequence via CRISPR / Cas9-based genome editing.

[0104] In some embodiments of this disclosure, inactivating two or more ERV loci (i.e., reducing or eliminating expression compared to unmodified CHO cells) includes reducing the protein expression of ERV protein products to less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the corresponding ERV protein expression in a reference cell (e.g., a CHO host cell expressing an inactivated ERV). In some embodiments, the expression of one or more ERV proteins in cells modified to reduce or eliminate the expression of the ERV protein is less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%.

[0105] 3. Host cells

[0106] In some embodiments, the subject matter of this disclosure relates to CHO host cells. In some embodiments, the CHO host cell is a CHO K1 host cell. In some embodiments, the CHO host cell is a CHO K1SV host cell. In some embodiments, the CHO host cell is a DG44 host cell. In some embodiments, the CHO host cell is a DUKXB-11 host cell. In some embodiments, the CHO host cell is a CHOK1S host cell. In some embodiments, the CHO host cell is a CHO K1M host cell.

[0107] 3.1 Suitable host cells for integrating exogenous nucleotide sequences

[0108] In some embodiments, the subject matter of this disclosure provides modified CHO host cells suitable for targeted integration of exogenous nucleotide sequences. In some embodiments, the modified CHO host cells comprise exogenous nucleotide sequences integrated at integration sites on the genome of the host cell, i.e., the host cell is a TI host cell.

[0109] An "integration site" comprises a nucleic acid sequence within the host cell genome into which a foreign nucleotide sequence is inserted. In some embodiments, the integration site is located between two adjacent nucleotides in the host cell genome. In some embodiments, the integration site includes a nucleotide segment into which a foreign nucleotide sequence can be inserted between any nucleotides. In some embodiments, the integration site is located within a gene locus in the genome of a specific TI host cell. In some embodiments, the integration site is located within an endogenous gene within the TI host cell.

[0110] In some embodiments, the exogenous nucleotide sequence is integrated into a specific locus within the genome of the TI host cell. In some embodiments, the locus into which the exogenous nucleotide sequence is integrated is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to a sequence selected from SEQ ID No. 1 to 7.

[0111] In some embodiments, the locus in which the exogenous nucleotide sequence is integrated is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to all or a portion of sequences selected from contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1.

[0112] In some embodiments, the exogenous nucleotide sequence is integrated at an integration site located at a position selected from the following nucleotides in SEQ ID No. 1: 1-1,000 bp; 1,000-2,000 bp; 2,000-3,000 bp; 3,000-4,000 bp; and 4,000-4,301 bp. In some embodiments, the exogenous nucleotide sequence is integrated at an integration site located at a position selected from the following nucleotides numbered in SEQ ID No. 2: 1-100,000 bp; 100,000-200,000 bp; 200,000-300,000 bp; 300,000-400,000 bp; 400,000-500,000 bp; 500,000-600,000 bp; 600,000-700,000 bp; and 700,000-728785 bp. In some embodiments, the exogenous nucleotide sequence is integrated at an integration site located at a position selected from the following nucleotides in SEQ ID No. 3: 1-100,000 bp; 100,000-200,000 bp; 200,000-300,000 bp; 300,000-400,000 bp; and 400,000-413,983 bp. In some embodiments, the exogenous nucleotide sequence is integrated at an integration site located at a position selected from the following nucleotides in SEQ ID No. 4: 1-10,000 bp; 10,000-20,000 bp; 20,000-30,000 bp; and 30,000-30,757 bp. In some embodiments, the exogenous nucleotide sequence is integrated at an integration site located at a position selected from the following nucleotides numbered in SEQ ID No. 5: 1-10,000 bp; 10,000-20,000 bp; 20,000-30,000 bp; 30,000-40,000 bp; 40,000-50,000 bp; 50,000-60,000 bp; and 60,000-68,962 bp. In some embodiments, the exogenous nucleotide sequence is integrated at an integration site located at a position selected from the following nucleotides numbered in SEQ ID No. 6: 1-10,000 bp; 10,000-20,000 bp; 20,000-30,000 bp; 30,000-40,000 bp; 40,000-50,000 bp; and 50,000-51,326 bp.In some embodiments, the exogenous nucleotide sequence is integrated at an integration site located at a position selected from the following nucleotides numbered in SEQ ID No. 7: 1-10,000 bp; 10,000-20,000 bp; and 20,000-22,904 bp.

[0113] In some embodiments, the nucleotide immediately adjacent to the 5' end of the integrated exogenous sequence is a nucleotide within the sequence of the following nucleotides: nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides 69303-79768 of NW_003616412.1, nucleotides 293481-315265 of NW_003615063.1, nucleotides 2650443-2662054 of NW_006882936.1, or nucleotides 82214-97705 of NW_003615411.1. In some embodiments, the nucleotide immediately adjacent to the 5' end of the integrated exogenous sequence is a nucleotide within the sequence that is at least about 50% homologous to the following nucleotides: 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. Nucleotide 82214-97705. In some embodiments, the nucleotide immediately adjacent to the 5' end of the integrated exogenous sequence is homologous to at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% of the following nucleotides: 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 or NW_003615411.1 nucleotide 82214-97705.

[0114] In some embodiments, the nucleotide immediately adjacent to the 3' end of the integrated exogenous sequence is a nucleotide within the sequence of the following nucleotides: 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 nucleotide immediately adjacent to the 3' end of the integrated exogenous sequence is a nucleotide within the sequence that is at least about 50% homologous to the following nucleotides: 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. Nucleotides 97706-105117. In some embodiments, the nucleotide immediately adjacent to the 3' end of the integrated exogenous sequence is homologous to at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% of the following nucleotides: nucleotides 45270-45490 of NW_006884592.1, 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, and nucleotides 1. Nucleotides 2662055-2701768 or NW_003615411.1 nucleotides 97706-105117.

[0115] In some embodiments, the integrated exogenous nucleotide sequence is operatively linked to a nucleotide sequence selected from the group consisting of SEQ ID No. 1 to 7 and sequences at least 50% homologous thereto. In some embodiments, the nucleotide sequence operatively linked to the exogenous nucleotide sequence is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to the sequences selected from SEQ ID No. 1 to 7.

[0116] In some embodiments, the integrated exogenous sequence is joined at its 5' end with a nucleotide sequence selected from the group consisting of: 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, and at least 50% thereof. Homologous sequences, and joined at the 3' end with nucleotide sequences selected from the group consisting of: nucleotides 45270-45490 of NW_006884592.1, 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, and nucleotides 97706-105117 of NW_003615411.1, and at least 50% thereof. Homologous sequences.In some embodiments, the 5' end nucleotide sequence of the side-joined integrated exogenous nucleotide sequence is homologous to at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% of the following nucleotides: 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, and the 3' end nucleotide sequence of the integrated foreign nucleotide sequence is homologous to at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% of the following nucleotides: nucleotides 45270-45490 of SEQ ID No. NW_006874047.1, nucleotides 207912-792374 of NW_006884592.1, nucleotides 491910-667813 of NW_006881296.1, and nucleotides 491910-667813 of NW_003616412.1. Nucleotides 79769-100059, 315266-362442 of NW_003615063.1, 2662055-2701768 of NW_006882936.1, and 97706-105117 of NW_003615411.1.

[0117] In some embodiments, the integrated exogenous nucleotide sequence is integrated into 20 nucleotide sequences that are homologous to at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% of the following: inclusion position 45269 of NW_006874047.1; inclusion position 207911 of NW_006884592.1; inclusion position 491909 of NW_006881296.1; inclusion position 79768 of NW_003616412.1; inclusion position 315265 of NW_003615063.1; inclusion position 2662054 of NW_006882936.1; and inclusion position 97705 of NW_003615411.1. A nucleotide sequence.

[0118] In some embodiments, the integrated exogenous nucleotide sequence is integrated into 50 nucleotide sequences that are homologous to at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% of the following: inclusion position 45269 of NW_006874047.1; inclusion position 207911 of NW_006884592.1; inclusion position 491909 of NW_006881296.1; inclusion position 79768 of NW_003616412.1; inclusion position 315265 of NW_003615063.1; inclusion position 2662054 of NW_006882936.1; and inclusion position 97705 of NW_003615411.1. A nucleotide sequence.

[0119] In some embodiments, the integrated exogenous nucleotide sequence is integrated into 100 nucleotide sequences that are homologous to at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% of the following: inclusion position 45269 of NW_006874047.1; inclusion position 207911 of NW_006884592.1; inclusion position 491909 of NW_006881296.1; inclusion position 79768 of NW_003616412.1; inclusion position 315265 of NW_003615063.1; inclusion position 2662054 of NW_006882936.1; and inclusion position 97705 of NW_003615411.1. A nucleotide sequence.

[0120] In some embodiments, the integrated exogenous nucleotide sequence is integrated into 200 nucleotide sequences that are homologous to at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% of the following: inclusion position 45269 of NW_006874047.1; inclusion position 207911 of NW_006884592.1; inclusion position 491909 of NW_006881296.1; inclusion position 79768 of NW_003616412.1; inclusion position 315265 of NW_003615063.1; inclusion position 2662054 of NW_006882936.1; and inclusion position 97705 of NW_003615411.1. A nucleotide sequence.

[0121] In some embodiments, the integrated exogenous nucleotide sequence is integrated into 500 nucleotide sequences that are homologous to at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% of the following: inclusion position 45269 of NW_006874047.1; inclusion position 207911 of NW_006884592.1; inclusion position 491909 of NW_006881296.1; inclusion position 79768 of NW_003616412.1; inclusion position 315265 of NW_003615063.1; inclusion position 2662054 of NW_006882936.1; and inclusion position 97705 of NW_003615411.1. A nucleotide sequence.

[0122] In some embodiments, the integrated exogenous nucleotide sequence is integrated into 1000 nucleotide sequences that are homologous to at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% of the following sequences: inclusion position 45269 of NW_006874047.1; inclusion position 207911 of NW_006884592.1; inclusion position 491909 of NW_006881296.1; inclusion position 79768 of NW_003616412.1; inclusion position 315265 of NW_003615063.1; inclusion position 2662054 of NW_006882936.1; and inclusion position 97705 of NW_003615411.1. The 1000 nucleotide sequence.

[0123] In some embodiments, the integrated exogenous nucleotide sequence is integrated into a locus adjacent to all or part of a sequence selected from the group consisting of: sequences that are at least about 90% homologous to the sequences of SEQ ID No. 1 to 7.

[0124] In some embodiments, the integrated exogenous nucleotide sequence is adjacent to a nucleotide sequence selected from the group consisting of: sequences of SEQ ID No. 1 to 7 and sequences that are at least 50% homologous to them. In some embodiments, the integrated exogenous nucleotide sequence is within about 100 bp, about 200 bp, about 500 bp, or about 1 kb of a sequence selected from the group consisting of: SEQ ID No. 1 to 7 and sequences that are at least 50% homologous to them. In some embodiments, the nucleotide sequence adjacent to the exogenous nucleotide sequence is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to a sequence selected from SEQ ID No. 1 to 7.

[0125] In some embodiments, the exogenous nucleotide sequence is integrated at an integration site adjacent to a position of a nucleotide selected from the following nucleotides numbered in SEQ ID No. 1: 1-1,000 bp; 1,000-2,000 bp; 2,000-3,000 bp; 3,000-4,000 bp; and 4,000-4,301 bp. In some embodiments, the exogenous nucleotide sequence is integrated at an integration site adjacent to a position of a nucleotide selected from the following nucleotides numbered in SEQ ID No. 2: 1-100,000 bp; 100,000-200,000 bp; 200,000-300,000 bp; 300,000-400,000 bp; 400,000-500,000 bp; 500,000-600,000 bp; 600,000-700,000 bp; and 700,000-728785 bp. In some embodiments, the exogenous nucleotide sequence is integrated at an integration site adjacent to a nucleotide selected from the following nucleotides numbered in SEQ ID No. 3: 1-100,000 bp; 100,000-200,000 bp; 200,000-300,000 bp; 300,000-400,000 bp; and 400,000-413,983 bp. In some embodiments, the exogenous nucleotide sequence is integrated at an integration site adjacent to a nucleotide selected from the following nucleotides numbered in SEQ ID No. 4: 1-10,000 bp; 10,000-20,000 bp; 20,000-30,000 bp; and 30,000-30,757 bp. In some embodiments, the exogenous nucleotide sequence is integrated at an integration site adjacent to a position of a nucleotide selected from the following nucleotides numbered in SEQ ID No. 5: 1-10,000 bp; 10,000-20,000 bp; 20,000-30,000 bp; 30,000-40,000 bp; 40,000-50,000 bp; 50,000-60,000 bp; and 60,000-68,962 bp. In some embodiments, the exogenous nucleotide sequence is integrated at an integration site adjacent to a position of a nucleotide selected from the following nucleotides numbered in SEQ ID No. 6: 1-10,000 bp; 10,000-20,000 bp; 20,000-30,000 bp; 30,000-40,000 bp; 40,000-50,000 bp; and 50,000-51,326 bp.In some embodiments, the exogenous nucleotide sequence is integrated at an integration site adjacent to a position of a nucleotide selected from the following nucleotides numbered in SEQ ID No. 7: 1-10,000 bp; 10,000-20,000 bp; and 20,000-22,904 bp.

[0126] In some embodiments, the locus containing the integration site of the foreign nucleotide sequence does not encode an open reading frame (ORF). In some embodiments, the locus containing the integration site of the foreign nucleotide sequence includes cis-acting elements, such as promoters and enhancers. In some embodiments, the locus containing the integration site of the foreign nucleotide sequence does not contain any cis-acting elements, such as promoters and enhancers that enhance gene expression.

[0127] In some embodiments, the exogenous nucleotide sequence is integrated at an integration site within an endogenous gene selected from the group consisting of: LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2. The endogenous genes LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 include the wild-type and all homologous sequences of the genes LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2. In some embodiments, the homologous sequences of the LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes may be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to the wild-type LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes. In some embodiments, the LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes are wild-type mammal genes. In some embodiments, the LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes are wild-type human LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes.In some embodiments, the LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes are wild-type hamster genes.

[0128] In some embodiments, the integration site is operatively linked to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences with at least about 90% homology thereto. In some embodiments, the integration site is flanked by an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences with at least about 90% homology thereto.

[0129] Table 1 provides exemplary TI host cell integration sites:

[0130] Table 1 - TI Host Cell Integration Sites

[0131]

[0132] In some embodiments, the integration site and / or the nucleotide sequence flanking the integration site can be identified experimentally. In some embodiments, the integration site and / or the nucleotide sequence flanking the integration site can be identified by whole-genome screening to isolate host cells that express at desired levels a target polypeptide encoded by one or more SOIs integrated into one or more exogenous nucleotide sequences, wherein the exogenous sequence itself is integrated into one or more loci in the host cell's genome. In some embodiments, the integration site and / or the nucleotide sequence flanking the integration site can be identified by whole-genome screening following a transposase-based cassette integration event. In some embodiments, the integration site and / or the nucleotide sequence flanking the integration site can be identified by strong random integration screening. In some embodiments, the integration site and / or the nucleotide sequence flanking the integration site can be determined by conventional sequencing methods such as target locus amplification (TLA) followed by next-generation sequencing (NGS) and whole-genome NGS. In some embodiments, the location of the integration site on the chromosome can be determined by conventional cell biology methods such as fluorescence in situ hybridization (FISH) analysis.

[0133] In some embodiments, the TI host cell comprises: a first exogenous nucleotide sequence integrated at a first integration site within a specific first locus in the genome of the TI host cell; and a second exogenous nucleotide sequence integrated at a second integration site within a specific second locus in the genome. In some embodiments, the TI host cell comprises multiple exogenous nucleotide sequences integrated at multiple integration sites in the genome of the TI host cell.

[0134] In some embodiments, the TI host cell of this disclosure comprises at least two distinct exogenous nucleotide sequences, such as an exogenous nucleotide sequence comprising at least one RRS. In some embodiments, two or more exogenous nucleotide sequences may be targeted when one or more SOIs are introduced. In some embodiments, the SOIs are identical. In some embodiments, the SOIs are different. In some embodiments, the parental TI host cell comprising a first exogenous nucleotide sequence may comprise a second exogenous nucleotide sequence, the integration site of which is different from the integration site of the first exogenous nucleotide sequence.

[0135] In some embodiments, the integration site may be on the same chromosome. In some embodiments, the integration sites are located 1-1,000 nucleotides, 1,000-100,000 nucleotides, 100,000-1,000,000 nucleotides or more apart within the same chromosome. In some embodiments, the integration site is on a different chromosome. In some embodiments, a TI host cell containing a foreign nucleotide sequence at one integration site can be used to insert at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or more foreign nucleotide sequences at the same or different integration sites.

[0136] In some embodiments, the feasibility of recombinase-mediated cassette exchange (RMCE) occurring at at least two integration sites at each site can be evaluated individually. In some embodiments, the feasibility of RMCE occurring at at least two integration sites can be evaluated simultaneously. The feasibility of RMCE occurring at multiple sites can be evaluated using methods known in the art, such as measuring peptide titer or peptide-specific production. In some embodiments, this evaluation can be performed using methods known in the art, such as assessing the titer and / or specific productivity of cultures of TI host cells expressing one or more SOIs. Exemplary culture strategies include, but are not limited to, batch-fed shake-flask culture and bioreactor batch-fed culture. The titer and specific productivity of TI host cells expressing the target peptide can be evaluated using methods known in the art, such as, but not limited to, ELISA, FACS, fluorescence microwell assay (FMAT), protein A affinity chromatography, and Western blot analysis.

[0137] 3.2 Decreased or eliminated expression of endogenous host cell proteins

[0138] In some embodiments, this disclosure relates to modified CHO cells, such as CHO cells, wherein the expression of one or more CHO cell endogenous proteins is reduced or eliminated. For example, but not limited to, methods for reducing or eliminating the expression of endogenous proteins in CHO cells include: (1) modifying a gene encoding an endogenous protein or a component thereof, for example, by introducing deletion, insertion, substitution, or a combination thereof into the gene; (2) reducing or eliminating transcription and / or stability of mRNA encoding an endogenous protein or a component thereof; and (3) reducing or eliminating translation of mRNA encoding an endogenous protein or a component thereof. In some embodiments, the reduction or elimination of protein expression is achieved through targeted genome editing. For example, CRISPR / Cas9-based genome editing can be used to modify one or more target genes, thereby reducing or eliminating the expression of one (or more) genes targeted for editing.

[0139] In some embodiments, one or more of the CHO cell endogenous proteins whose expression is targeted for reduction or elimination are selected based on their role in promoting apoptosis. Since apoptosis can reduce culture viability and productivity, reducing or eliminating the expression of such proteins can have a positive impact on culture viability and productivity. For example, but not limited to, CHO cell proteins selected based on their role in promoting apoptosis are BCL2-associated X, apoptosis regulator (BAX), or BCL2 antagonist / killer factor 1 (BAK). In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of BAX. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of BAK. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of both BAX and BAK.

[0140] In some embodiments, the targeted reduction or elimination of expression of CHO cell endogenous products is selected based on their role in promoting aggregation and / or aggregation during cell culture. When CHO cells are used to produce the desired recombinant protein, such aggregation and / or aggregation during cell culture can lead to reduced protein titers due to the negative impact of aggregation and / or aggregation on CHO cell viability. For example, but not limited to, an endogenous CHO cell protein selected based on its role in promoting aggregation and / or aggregation during cell culture is intercellular adhesion molecule 1 (ICAM-1). In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of ICAM-1.

[0141] In some embodiments, the CHO cell endogenous proteins targeted for reduced or eliminated expression are selected based on their role in regulating the unfolded protein response (UPR). For example, but not limited to, cellular proteins selected based on their role in regulating UPR include inositol demand enzyme 1 (IRE1), protein kinase R-like ER kinase (PERK), or activating transcription factor 6 (ATF6). In some embodiments, the modified cells of this disclosure exhibit reduced or eliminated expression of PERK. In some embodiments, as used herein, PERK refers to eukaryotic PERK cell proteins, such as CHO PERK cell proteins (Gene ID: 100765343; GenBank: EGW03658.1; and isotype NCBI reference sequences: XP_027285344.2 and XP_016831844.1) and their functional variants. In some embodiments, as used herein, functional variants of PERK encompass PERK sequences having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the wild-type PERK sequence of the modified cells used to produce the recombinant protein for the purpose.

[0142] In some embodiments, one or more of the CHO cell endogenous proteins whose expression is targeted for reduction or elimination are selected based on their role in promoting inefficient cell growth. CHO cells express many endogenous proteins that are not essential for cell growth, survival, and / or productivity. Since the expression of these endogenous proteins consumes significant amounts of cellular energy and DNA / protein structural units, reducing or eliminating the expression of such endogenous proteins can make cell growth more efficient, and, where the cells are used to produce the desired recombinant protein, those cellular sources can be converted to achieve higher productivity of the desired recombinant protein. For example, but not limited to, CHO cell endogenous proteins selected based on their role in promoting efficient cell growth and higher productivity of the desired recombinant protein are BAX, BAK, ICAM-1, PERK, Sirtuin 1 (SIRT-1), or the MYC proto-oncogene, BHLH transcription factor (MYC). In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of SIRT-1. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of PERK. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of MYC. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of SIRT-1 and MYC. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of BAX and MYC. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of BAK and MYC. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of ICAM-1 and MYC. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of BAX and SIRT-1. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of BAK and SIRT-1. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of ICAM-1 and SIRT-1. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of BAX, SIRT-1, and MYC. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of BAK, SIRT-1, and MYC. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of ICAM-1, SIRT-1, and MYC. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of BAX, BAK, SIRT-1, and MYC.In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of BAX, ICAM-1, SIRT-1, and MYC. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of BAK, ICAM-1, SIRT-1, and MYC. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of BAX, BAK, MYC, SIRT-1, and ICAM. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of BAX, BAK, ICAM-1, PERK, SIRT-1, and / or MYC.

[0143] In some embodiments, the CHO cell endogenous proteins targeted for reduced or eliminated expression are, for example, endogenous proteins that can promote non-human glycosylation patterns in recombinant protein products when the cells are used for recombinant protein production. Such non-human glycosylation patterns may include the addition of galactose-α-1,3-galactose (αGAL) and / or N-hydroxyacetylneuraminic acid (NGNA). For example, but not limited to, CHO cell proteins selected based on their role in promoting non-human glycosylation patterns are glycoprotein α-galactosyltransferase 1 (GGTA1), which promotes αGAL addition, or cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), which promotes NGNA addition. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of GGTA1. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of CMAH. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of both GGTA1 and CMAH.

[0144] In some embodiments, the CHO cell endogenous proteins targeted for reduction or elimination are endogenous proteins that promote the catabolism of branched-chain amino acids (BCAAs). While branched-chain amino acids (e.g., leucine, isoleucine, and valine) are essential amino acids and are therefore typically included in the chemically defined culture media used in CHO cell culture, the catabolism of BCAAs can result in toxic intermediates and metabolites that reduce cell growth, productivity, and protein quality. For example, CHO cell proteins selected based on their role in promoting BCAA catabolism are branched-chain ketoacid dehydrogenase E1α subunit (BCKDHA) or branched-chain α-ketoacid dehydrogenase E1β subunit (BCKDHB).

[0145] Furthermore, when cells are used to produce the desired recombinant protein, certain CHO cell endogenous proteins can be co-purified with the target protein, leading to increased costs associated with additional purification processes and / or a shortened shelf life of the resulting recombinant product. For example, certain residual host cell proteins co-purified with the target recombinant protein may degrade polysorbate used as a surfactant in the final pharmaceutical product and result in particle formation. Therefore, in some embodiments, targeted reduction or elimination of expressed CHO cell endogenous host cell proteins based on their potential to co-purify with the target recombinant protein and degrade polysorbate used as a surfactant in the final pharmaceutical product include lipoprotein lipase (LPL), also known as LPL1; phospholipase A2 group (LPLA2), also known as PLA2G7; palmitoyl protein thioesterase 1 (PPT1); or lipase A (lysosomal acid lipase / cholesterol ester hydrolase, lipase) (LIPA). In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of PPT1. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of PPT1 and LPL. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of LPLA2. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of PPT1 and LIPA. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of PPT1, LPL, and LPLA2. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of PPT1, LPL, and LIPA. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of PPT1, LIPA, and LPLA2. In some embodiments, the CHO cells of this disclosure exhibit reduced or eliminated expression of PPT1, LPL, LIPA, and LPLA2.

[0146] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of one or more endogenous proteins in order to facilitate the purification of the target recombinant protein by reducing the total amount of host cell endogenous protein produced during cell culture. Such a reduction in overall host cell endogenous protein production can alleviate the burden on chromatographic and other materials and systems used in the purification process, thereby reducing the overall cost of purification and increasing the efficiency of the purification process. For example, but not as a limitation, the host cell endogenous proteins whose expression is targeted to be reduced or eliminated based on the total amount of endogenous proteins produced during cell culture are selected from the following endogenous products: MYC proto-oncogene, BHLH transcription factor (MYC); BCL2-associated X, apoptosis regulator (BAX); BCL2 antagonist / killer factor 1 (BAK); intercellular adhesion molecule 1 (ICAM-1); protein kinase R-like ER kinase (PERK); silencing regulator protein 1 (SIRT-1); glycoprotein α-galactosyltransferase 1 (GGTA1); cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH); lipoprotein lipase (LPL); phospholipase A2 group (LPLA2); palmitoyl protein thioesterase 1 (PPT1); branched-chain keto acid dehydrogenase E1α subunit (BCKDHA); branched-chain keto acid dehydrogenase E1β Subunit (BCKDHB); and lipase A (lysosomal acid lipase / cholesterol ester hydrolase, lipase) (LIPA).

[0147] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1.

[0148] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; ICAM-1; SIRT-1; GGTA1; CMAH; LPL; LPLA2; and PPT1.

[0149] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1.

[0150] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA.

[0151] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA.

[0152] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA.

[0153] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA.

[0154] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; ICAM-1; LPL; LPLA2; and PPT1.

[0155] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1.

[0156] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1.

[0157] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA.

[0158] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; ICAM-1; PERK; SIRT-1; and MYC.

[0159] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: MYC; BAX; BAK; ICAM-1; PERK; SIRT-1; GGTA1; CMAH; LPL; LPLA2; PPT1 and LIPA.

[0160] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; and PERK.

[0161] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; MYC; SIRT-1; and ICAM.

[0162] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; LPL; LPLA2; GGTA1; and CMAH.

[0163] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; PERK; LPL; LPLA2; GGTA1; and CMAH.

[0164] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; MYC; LPL; LPLA2; GGTA1; and CMAH.

[0165] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH.

[0166] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; LPL; LPLA2; GGTA1; CMAH; and PPT1.

[0167] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1.

[0168] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1.

[0169] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1.

[0170] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1.

[0171] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; ICAM-1; SIRT-1; GGTA1; CMAH; LPL; LPLA2; and PPT1.

[0172] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1.

[0173] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA.

[0174] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA.

[0175] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA.

[0176] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA.

[0177] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; ICAM-1; LPL; LPLA2; and PPT1.

[0178] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1.

[0179] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1.

[0180] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA.

[0181] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; ICAM-1; PERK; SIRT-1; and MYC.

[0182] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: MYC; BAX; BAK; BCKDHA; ICAM-1; PERK; SIRT-1; GGTA1; CMAH; LPL; LPLA2; PPT1 and LIPA.

[0183] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; and PERK.

[0184] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; MYC; SIRT-1; and ICAM.

[0185] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; LPL; LPLA2; GGTA1; and CMAH.

[0186] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; PERK; LPL; LPLA2; GGTA1; and CMAH.

[0187] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; MYC; LPL; LPLA2; GGTA1; and CMAH.

[0188] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH.

[0189] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; LPL; LPLA2; GGTA1; CMAH; and PPT1.

[0190] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1.

[0191] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1.

[0192] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1.

[0193] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1.

[0194] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1.

[0195] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPL; LPLA2; and PPT1.

[0196] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1.

[0197] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA.

[0198] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA.

[0199] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA.

[0200] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA.

[0201] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; ICAM-1; LPL; LPLA2; and PPT1.

[0202] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1.

[0203] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1.

[0204] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA.

[0205] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; ICAM-1; PERK; SIRT-1; and MYC.

[0206] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: MYC; BAX; BAK; BCKDHB; ICAM-1; PERK; SIRT-1; GGTA1; CMAH; LPL; LPLA2; PPT1 and LIPA.

[0207] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; and PERK.

[0208] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; MYC; SIRT-1; and ICAM.

[0209] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; LPL; LPLA2; GGTA1; and CMAH.

[0210] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; PERK; LPL; LPLA2; GGTA1; and CMAH.

[0211] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; MYC; LPL; LPLA2; GGTA1; and CMAH.

[0212] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH.

[0213] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; LPL; LPLA2; GGTA1; CMAH; and PPT1.

[0214] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1.

[0215] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1.

[0216] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1.

[0217] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1.

[0218] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1.

[0219] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPL; LPLA2; and PPT1.

[0220] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1.

[0221] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA.

[0222] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA.

[0223] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA.

[0224] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA.

[0225] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; ICAM-1; LPL; LPLA2; and PPT1.

[0226] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1.

[0227] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1.

[0228] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA.

[0229] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; ICAM-1; PERK; SIRT-1; and MYC.

[0230] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: MYC; BAX; BAK; BCKDHA; BCKDHB; ICAM-1; PERK; SIRT-1; GGTA1; CMAH; LPL; LPLA2; PPT1 and LIPA.

[0231] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; and PERK.

[0232] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; MYC; SIRT-1; and ICAM.

[0233] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; LPL; LPLA2; GGTA1; and CMAH.

[0234] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; PERK; LPL; LPLA2; GGTA1; and CMAH.

[0235] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; MYC; LPL; LPLA2; GGTA1; and CMAH.

[0236] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH.

[0237] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; LPL; LPLA2; GGTA1; CMAH; and PPT1.

[0238] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1.

[0239] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHB; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1.

[0240] In some embodiments, the host cells of this disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX; BAK; BCKDHA; BCKDHA; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1.

[0241] In some embodiments, the host cells of this disclosure are modified to reduce or eliminate the expression of one or more host cell endogenous proteins relative to the expression of one or more host cell endogenous proteins in an unmodified (i.e., "reference") host cell. In some embodiments, the reference host cell is a host cell in which the expression of one or more specific endogenous products is not reduced or eliminated, such endogenous products being, for example, BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK polypeptides. In some embodiments, the reference host cell is a cell containing at least one or two wild-type alleles of a gene encoding BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK. For example, but not limited to, a reference host cell is a host cell possessing two wild-type alleles of a gene encoding BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK. In some embodiments, the reference host cell is a WT host cell. In some embodiments, modifications to reduce or eliminate the expression of one or more endogenous host cell proteins are performed before the introduction of exogenous nucleic acids encoding the target recombinant protein. In some embodiments, modifications to reduce or eliminate the expression of one or more endogenous host cell proteins are performed after the introduction of exogenous nucleic acids encoding the target recombinant protein.

[0242] In some embodiments, the expression of one or more endogenous proteins (e.g., BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK peptides) in cells modified to reduce or eliminate the expression of the endogenous product is used as a reference cell (e.g., WT host cells) to represent the expression of the corresponding endogenous protein at less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%. In some embodiments, the expression of one or more endogenous proteins in cells that have been modified to reduce or eliminate the expression of the endogenous protein is less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the corresponding endogenous protein expression in reference cells (e.g., WT host cells).

[0243] In some embodiments, the expression of one or more endogenous proteins (e.g., BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK peptides) in host cells modified to reduce or eliminate the expression of the endogenous protein is at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 10%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, or at least about 1% of the corresponding endogenous protein expression in a reference host cell (e.g., WT host cell). In some embodiments, the expression of one or more endogenous proteins in host cells that have been modified to reduce or eliminate the expression of the endogenous product is at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 10%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, or at least about 1% of the corresponding endogenous protein expression in reference cells (e.g., WT CHO cells).

[0244] In some embodiments, the expression of one or more specific endogenous proteins (e.g., BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK peptides) in cells modified to reduce or eliminate the expression of the endogenous protein is no more than about 90%, no more than about 80%, no more than about 70%, no more than about 60%, no more than about 50%, no more than about 40%, no more than about 30%, no more than about 20%, no more than about 10%, no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, or no more than about 1% of the corresponding endogenous protein expression in a reference host cell (e.g., WT host cell). In some embodiments, the expression of one or more endogenous proteins (e.g., BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK peptides) in cells modified to reduce or eliminate the expression of the endogenous protein is no more than about 40% in reference cells (e.g., WT CHO cells). In some embodiments, the expression of one or more endogenous proteins in cells modified to reduce or eliminate the expression of the endogenous protein is no more than about 90%, no more than about 80%, no more than about 70%, no more than about 60%, no more than about 50%, no more than about 40%, no more than about 30%, no more than about 20%, no more than about 10%, no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, or no more than about 1% in reference cells (e.g., WT host cells).

[0245] In some embodiments, the expression of one or more endogenous proteins (e.g., BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK peptides) in cells that have been modified to reduce or eliminate the expression of such endogenous proteins is used as a reference cell (e.g.,The percentages of corresponding endogenous protein expression in WT host cells are approximately 1% to 90%, 10% to 90%, 20% to 90%, 25% to 90%, 30% to 90%, 40% to 90%, 50% to 90%, 60% to 90%, 70% to 90%, 80% to 90%, 85% to 90%, 1% to 80%, 10% to 80%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, and 60%. Between approximately 80%, between approximately 70% and approximately 80%, between approximately 75% and approximately 80%, between approximately 1% and approximately 70%, between approximately 10% and approximately 70%, between approximately 20% and approximately 70%, between approximately 30% and approximately 70%, between approximately 40% and approximately 70%, between approximately 50% and approximately 70%, between approximately 60% and approximately 70%, between approximately 65% ​​and approximately 70%, between approximately 1% and approximately 60%, between approximately 10% and approximately 60%, between approximately 20% and approximately 60%, between approximately 30% and approximately 60%, between approximately 40% and approximately 60%, between approximately 50% and approximately 60%, between approximately 55% and approximately 60%, between approximately 1% and approximately 50%. Between, approximately 10% and approximately 50%, approximately 20% and approximately 50%, approximately 30% and approximately 50%, approximately 40% and approximately 50%, approximately 45% and approximately 50%, approximately 1% and approximately 40%, approximately 10% and approximately 40%, approximately 20% and approximately 40%, approximately 30% and approximately 40%, approximately 35% and approximately 40%, approximately 1% and approximately 30%, approximately 10% and approximately 30%, approximately 20% and approximately 30%, approximately 25% and approximately 30%, approximately 1% and approximately 20%, approximately 5% and approximately 20%, approximately 10% and approximately 20%, approximately 15% and approximately 20%, approximately 1% and approximately 10%, approximately 5% Between approximately 10%, between approximately 5% and approximately 20%, between approximately 5% and approximately 30%, and between approximately 5% and approximately 40%. In some embodiments, one or more endogenous proteins (e.g.,The expression of BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK peptides in cells that have been modified to reduce or eliminate the expression of this endogenous protein is used as a reference cell (e.g.,The percentages of corresponding endogenous protein expression in WT host cells are approximately 1% to 90%, 10% to 90%, 20% to 90%, 25% to 90%, 30% to 90%, 40% to 90%, 50% to 90%, 60% to 90%, 70% to 90%, 80% to 90%, 85% to 90%, 1% to 80%, 10% to 80%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, and 60%. Between approximately 80%, between approximately 70% and approximately 80%, between approximately 75% and approximately 80%, between approximately 1% and approximately 70%, between approximately 10% and approximately 70%, between approximately 20% and approximately 70%, between approximately 30% and approximately 70%, between approximately 40% and approximately 70%, between approximately 50% and approximately 70%, between approximately 60% and approximately 70%, between approximately 65% ​​and approximately 70%, between approximately 1% and approximately 60%, between approximately 10% and approximately 60%, between approximately 20% and approximately 60%, between approximately 30% and approximately 60%, between approximately 40% and approximately 60%, between approximately 50% and approximately 60%, between approximately 55% and approximately 60%, between approximately 1% and approximately 50%. Between, approximately 10% and approximately 50%, approximately 20% and approximately 50%, approximately 30% and approximately 50%, approximately 40% and approximately 50%, approximately 45% and approximately 50%, approximately 1% and approximately 40%, approximately 10% and approximately 40%, approximately 20% and approximately 40%, approximately 30% and approximately 40%, approximately 35% and approximately 40%, approximately 1% and approximately 30%, approximately 10% and approximately 30%, approximately 20% and approximately 30%, approximately 25% and approximately 30%, approximately 1% and approximately 20%, approximately 5% and approximately 20%, approximately 10% and approximately 20%, approximately 15% and approximately 20%, approximately 1% and approximately 10%, approximately 5% Between approximately 10%, between approximately 5% and approximately 20%, between approximately 5% and approximately 30%, and between approximately 5% and approximately 40%.

[0246] In some embodiments, the expression of one or more endogenous proteins (e.g., BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK peptides) in cells modified to reduce or eliminate the expression of the endogenous protein is between about 5% and about 40% of the corresponding endogenous protein expression in reference cells (e.g., WT host cells).

[0247] In some embodiments, the expression levels of one or more endogenous proteins (e.g., BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK peptides) can vary in different reference cells (e.g., cells containing at least one or two wild-type alleles of the corresponding gene).

[0248] In some embodiments, genetic engineering systems are employed to reduce or eliminate the expression of one or more specific endogenous proteins (e.g., BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK expression). Various genetic engineering systems known in the art can be used in the methods disclosed herein. Non-limiting examples of such systems include CRISPR / Cas systems, zinc finger nuclease (ZFN) systems, transcription activator-like effector nuclease (TALEN) systems, and other tools that reduce or eliminate protein expression through gene silencing, such as small interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA). Any CRISPR / Cas system known in the art, including conventional, enhanced, or modified Cas systems, as well as other bacterial genome excision tools such as Cpf-1, can be used in conjunction with the methods disclosed herein.

[0249] In some embodiments, a portion of one or more genes (e.g., genes encoding endogenous proteins such as BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK peptides) is deleted to reduce or eliminate the expression of the corresponding endogenous protein in the host cell. In some embodiments, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% of the gene is deleted. In some embodiments, the deletion of the gene is no more than about 2%, no more than about 5%, no more than about 10%, no more than about 15%, no more than about 20%, no more than about 25%, no more than about 30%, no more than about 35%, no more than about 40%, no more than about 45%, no more than about 50%, no more than about 55%, no more than about 60%, no more than about 65%, no more than about 70%, no more than about 75%, no more than about 80%, no more than about 85%, or no more than about 90%.In some embodiments, the percentages are between approximately 2% and approximately 90%, between approximately 10% and approximately 90%, between approximately 20% and approximately 90%, between approximately 25% and approximately 90%, between approximately 30% and approximately 90%, between approximately 40% and approximately 90%, between approximately 50% and approximately 90%, between approximately 60% and approximately 90%, between approximately 70% and approximately 90%, between approximately 80% and approximately 90%, between approximately 85% and approximately 90%, between approximately 2% and approximately 80%, between approximately 10% and approximately 80%, between approximately 20% and approximately 80%, between approximately 30% and approximately 80%, between approximately 40% and approximately 80%, between approximately 50% and approximately 80%, between approximately 60% and approximately 80%, and between approximately 70% and approximately 80%. Between, approximately 75% and approximately 80%, approximately 2% and approximately 70%, approximately 10% and approximately 70%, approximately 20% and approximately 70%, approximately 30% and approximately 70%, approximately 40% and approximately 70%, approximately 50% and approximately 70%, approximately 60% and approximately 70%, approximately 65% ​​and approximately 70%, approximately 2% and approximately 60%, approximately 10% and approximately 60%, approximately 50% and approximately 60%, approximately 55% and approximately 60%, approximately 2% and approximately 50%, approximately 10% and approximately 50%, approximately 20% and approximately 50% The percentages of individuals with this gene deletion are between approximately 30% and approximately 50%, between approximately 40% and approximately 50%, between approximately 45% and approximately 50%, between approximately 2% and approximately 40%, between approximately 10% and approximately 40%, between approximately 20% and approximately 40%, between approximately 30% and approximately 40%, between approximately 35% and approximately 40%, between approximately 2% and approximately 30%, between approximately 10% and approximately 30%, between approximately 20% and approximately 30%, between approximately 25% and approximately 30%, between approximately 2% and approximately 20%, between approximately 5% and approximately 20%, between approximately 10% and approximately 20%, between approximately 15% and approximately 20%, between approximately 2% and approximately 10%, between approximately 5% and approximately 10%, or between approximately 2% and approximately 5%.

[0250] In some embodiments, at least one exon of the gene encoding BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK peptide is at least partially deleted in the host cell. As used herein, "partially missing" means, for example, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, not exceeding about 2%, not exceeding about 5%, not exceeding about 10%, not exceeding about 15%, not exceeding about 20%, not exceeding about 25%, not exceeding about 30%, not exceeding about 35%, not exceeding about 40%, not exceeding about 45%, not exceeding about 50%, not exceeding about 55%, not exceeding about 60%, not exceeding about 65%, not exceeding about 70%, not exceeding about 75%, not exceeding about 80%, not exceeding about 85%, not exceeding about 90%, not exceeding about 95%, between about 2% and about 90%, between about 10% and about 90%, and about 20%. Between approximately 90%, approximately 25% and approximately 90%, approximately 30% and approximately 90%, approximately 40% and approximately 90%, approximately 50% and approximately 90%, approximately 60% and approximately 90%, approximately 70% and approximately 90%, approximately 80% and approximately 90%, approximately 85% and approximately 90%, approximately 2% and approximately 80%, approximately 10% and approximately 80%, approximately 20% and approximately 80%, approximately 30% and approximately 80%, approximately 40% and approximately 80%, approximately 50% and approximately 80%, approximately 60% and approximately 80%, approximately 70% and approximately 80%, approximately 75% and approximately 80%, approximately 2% and approximately 70%, approximately 10%. Between approximately 70%, approximately 20% and approximately 70%, approximately 30% and approximately 70%, approximately 40% and approximately 70%, approximately 50% and approximately 70%, approximately 60% and approximately 70%, approximately 65% ​​and approximately 70%, approximately 2% and approximately 60%, approximately 10% and approximately 60%, approximately 20% and approximately 60%, approximately 30% and approximately 60%, approximately 40% and approximately 60%, approximately 50%Between approximately 60%, approximately 55% and approximately 60%, approximately 2% and approximately 50%, approximately 10% and approximately 50%, approximately 20% and approximately 50%, approximately 30% and approximately 50%, approximately 40% and approximately 50%, approximately 45% and approximately 50%, approximately 2% and approximately 40%, approximately 10% and approximately 40%, approximately 20% and approximately 40%, approximately 30% and approximately 40%, approximately 35% and approximately 40%, approximately 2% and approximately 30%, approximately 10% and approximately 30%, approximately 20% and approximately 30%, approximately 25% and approximately 30%, approximately 2% and approximately 20%, approximately 5% and approximately 20%, approximately 10% and approximately 20%. The regions between approximately 15% and approximately 20%, between approximately 2% and approximately 10%, between approximately 5% and approximately 10%, or between approximately 2% and approximately 5% are missing.

[0251] In some non-limiting embodiments, a CRISPR / Cas9 system is employed to reduce or eliminate the expression of one or more endogenous proteins (e.g., BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK peptides) in host cells. The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system is a genome editing tool found in prokaryotic cells. When used for genome editing, this system comprises Cas9 (a protein capable of using crRNA as its guide to modify DNA), CRISPR RNA (crRNA, which contains: RNA used by Cas9 to guide it to the correct segment of host DNA; and a region that binds to tracrRNA (typically in the form of a hairpin loop), forming an active complex with Cas9), and trans-activated crRNA (tracrRNA, which binds to crRNA and forms an active complex with Cas9). The terms “guide RNA” and “gRNA” refer to any nucleic acid that facilitates the specific association (or “targeting”) of RNA-guided nucleases (such as Cas9) with target sequences (such as genomic or appendage sequences in cells). gRNAs can be monomolecules (containing a single RNA molecule and alternatively referred to as chimeric) or modular (containing more than one, and usually two, separate RNA molecules, such as crRNA and tracrRNA, which often associate with each other, for example, through duplication).

[0252] The CRISPR / Cas9 strategy can employ vectors to transfect CHO cells. Guide RNA (gRNA) can be designed for each application, as this is the sequence that Cas9 uses to identify and directly bind to target DNA in CHO cells. Multiple crRNAs and this tracrRNA can be packaged together to form a single guide RNA (sgRNA). The sgRNA can be linked to the Cas9 gene and made into a vector for transfection into CHO cells.

[0253] In some embodiments, the CRISPR / Cas9 system for reducing or eliminating the expression of one or more endogenous proteins (e.g., BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK peptides) comprises a Cas9 molecule and one or more gRNAs containing a targeting domain complementary to a target sequence of a gene encoding an endogenous protein or a component thereof. In some embodiments, the target gene is a gene region encoding an endogenous product (e.g., BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK peptides). The target sequence can be any exon or intron region within a gene.

[0254] In some embodiments, gRNA is administered to CHO cells in a single vector, and the Cas9 molecule is administered to the host cell in a second vector. In some embodiments, gRNA and Cas9 molecules are administered to the host cell in a single vector. Alternatively, each gRNA and Cas9 molecule can be administered via a separate vector. In some embodiments, the CRISPR / Cas9 system can be delivered to the host cell as a ribonucleoprotein complex (RNP) comprising the Cas9 protein complexed with one or more gRNAs, for example, via electroporation (see, for example, DeWitt et al., Methods 121-122:9-15 (2017) on other methods of delivering RNPs to cells). In some embodiments, administration of the CRISPR / Cas9 system to host cells causes a reduction or elimination of the expression of endogenous products (e.g., BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK peptides).

[0255] CRISPR / Cas9 can be used to target specific target genes at one, two, three, or more different sites. For example, but not limited to, multiplex ribonucleoprotein delivery can be used to simultaneously target three different sites within a coding sequence using three different gRNAs. In some embodiments, multiplex ribonucleoprotein delivery exhibits higher gene editing efficiency and specificity compared to conventional plasmid-based CRISPR / Cas9 editing. In some embodiments, double-strand breaks at the gene target site induce insertion / deletion formation. In some embodiments, for example, when multiple sites are targeted due to the use of multiple gRNAs, deletions of sequences (e.g., inserted exons) between target sites result in frameshifts of the target protein's CDS.

[0256] In some embodiments, sequencing of PCR-amplified loci in the modified cell pool will reveal a break in the sequencing reaction at the first gRNA site, indicating successful targeting of the gene. In some embodiments, the cell pool will contain modifications at all target genes in at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the cells in the pool. In some embodiments, the cell pool will contain modifications at "n-1" of "n" target genes out of "n" target genes (where "n" is the number of target genes) in at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the cells in the pool. In some embodiments, the cell pool will contain modifications at “n-2” target genes out of “n” target genes in at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the cells in the pool. In some embodiments, the cell pool will contain modifications at “n-3” target genes out of “n” target genes in at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the cells in the pool.In some embodiments, the cell pool will contain modifications at “n-4” target genes out of “n” target genes in at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the cells in the pool. In some embodiments, the cell pool will contain modifications at one to n target genes in at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the cells in the pool.

[0257] In some embodiments, the genetically engineered system is a ZFN system used to reduce or eliminate the expression of one or more specific endogenous proteins (e.g., BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK peptides) in CHO cells. ZFNs can be used as restriction endonucleases, generated by combining a zinc finger DNA-binding domain with a DNA-cutting domain. The zinc finger domains can be engineered to target specific DNA sequences, thereby enabling zinc finger nucleases to target desired sequences within the genome. The DNA-binding domain of each ZFN typically comprises multiple individual zinc finger repeat sequences, and each zinc finger repeat sequence can recognize multiple base pairs. The most common method for generating new zinc finger domains is to combine smaller zinc finger “modules” with known specificity. The most common cleavage domain in ZFNs is the non-specific cleavage domain derived from the type IIs restriction endonuclease FokI. ZFNs regulate protein expression by inducing double-strand breaks (DSBs) in the target DNA sequence. In the absence of a homologous template, these DSBs are repaired via non-homologous end joining (NHEJ). This repair can result in the deletion or insertion of base pairs, creating a frameshift and preventing the production of harmful proteins (Durai et al., Nucleic Acids Res.; 33(18): 5978–90 (2005)). Multiple ZFN pairs can also be used to completely remove large segments of the genomic sequence (Lee et al., Genome Res.; 20(1): 81–9 (2010)).

[0258] In some embodiments, the genetically engineered system is a TALEN system used to reduce or eliminate the expression of one or more specific endogenous products (e.g., BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK peptides) in CHO cells. TALENs are restriction enzymes that can be engineered to cleave specific DNA sequences. The TALEN system operates similarly to ZFNs. TALENs are generated by combining a transcription activator-like effector DNA-binding domain with a DNA-cleaving domain. A transcription activator-like effector (TALE) consists of a 33- to 34-amino acid repeat motif with two variable positions and a strong ability to recognize specific nucleotides. By assembling arrays of these TALEs, the TALE DNA-binding domains can be engineered to bind desired DNA sequences, thereby guiding nucleases to cleave at specific locations in the genome (Boch et al., Nature Biotechnology; 29(2):135-6 (2011)). In some embodiments, the target genes encode BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK.

[0259] In some embodiments, the expression of one or more specific endogenous products (e.g., BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK peptides) can be reduced or eliminated using oligonucleotides having sequences complementary to the corresponding nucleic acids (e.g., mRNA). Non-limiting examples of such oligonucleotides include small interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA). In some embodiments, such oligonucleotides may be homologous to at least a portion of the nucleic acid sequences of BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LIPA; LPLA2; BCKDHA; BCKDHB; PPT1; and / or PERK, wherein the homology of the portion with respect to the corresponding nucleic acid sequence is at least about 75% or at least about 80% or at least about 85% or at least about 90% or at least about 95% or at least about 98%. In some non-limiting embodiments, the complementary portion may constitute at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, or at least 30 nucleotides, and the length of the antisense nucleic acid, shRNA, mRNA, or siRNA molecule may be up to 15, 20, 30, 40, 50, 75, or 100 nucleotides. The antisense nucleic acid, shRNA, mRNA, or siRNA molecule may contain DNA or atypical or non-naturally occurring residues, such as, but not limited to, phosphate thioester residues.

[0260] The genetically engineered systems disclosed herein can be delivered into CHO cells using viral vectors, such as retroviral vectors like gamma-retroviral vectors and lentiviral vectors. A combination of retroviral vectors and appropriate packaging lines is suitable, wherein the capsid protein will have the function of infecting human cells. Various cell lines producing amphiphilic viruses are known, including but not limited to PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphobic particles are also suitable, for example, particles encapsulated with VSVG, RD114, or GALV and any other particle pseudomorphs known in the art. Possible transduction methods also include direct co-culture of cells with producing cells, for example, by Bregni et al. (1992) Blood 80:1418-1422, or culturing with a single viral supernatant or a concentrated vector stock with or without appropriate growth factors and polycations, for example, by Xu et al. (1994) Exp. Hemat. 22:223-230; and Hughes et al. (1992) J. Clin. Invest. 89:1817.

[0261] Other transduction viral vectors can be used to modify the CHO cells disclosed herein. In some embodiments, the selected vectors have exhibited efficient infection as well as stable integration and expression (see, for example, Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. USA94:10319, 1997). Other viral vectors that can be used include, for example, adenovirus, lentivirus and adeno-associated virus vectors, vaccinia virus, bovine papillomavirus or herpesvirus, such as Epstein-Barr virus (see also, for example, Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; LeGal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995 (vectors). Retroviral vectors have been particularly well-developed and used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., US Patent No. 5,399,346).

[0262] Non-viral methods can also be used for genetic engineering of CHO cells disclosed in this paper. For example, nucleic acid molecules can be introduced into CHO cells in the presence of lipid transfection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), desialylated glycomucoid-polylysine binding (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or via microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990). Other non-viral methods for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may also be advantageous for delivering nucleic acid molecules into CHO cells. Transplantation of normal genes into the affected tissue of a subject can also be accomplished by transferring normal nucleic acids ex vivo into a cultured cell type (e.g., autologous or allogeneic primary cells or their progeny), followed by injection of those cells (or their progeny) into the target tissue or systemic injection.

[0263] 4. Integration of exogenous nucleic acids

[0264] As used herein, an exogenous nucleotide sequence is a nucleotide sequence that is not derived from a host cell but can be introduced into a host cell by conventional DNA delivery methods (e.g., by transfection, electroporation, transformation). In some embodiments, the exogenous nucleotide sequence is a target sequence (SOI), such as a nucleotide sequence encoding a target polypeptide. However, in some embodiments, the exogenous nucleotide sequence used in the context of this disclosure includes elements that facilitate the introduction of additional nucleic acid sequences (e.g., SOIs), such as one or more recombination recognition sequences (RRs) and one or more selection markers. In some embodiments, the exogenous nucleotide sequence that facilitates the introduction of additional nucleic acid sequences is referred to herein as a “landing pad.” Thus, in some embodiments, a TI host cell may comprise: (1) an exogenous nucleotide sequence comprising one or more SOIs, such as an SOI targeted and integrated into a specific locus in the host cell genome via an exogenous site-specific nuclease-mediated (e.g., CRISPR / Cas9-mediated) approach; (2) an exogenous nucleotide sequence comprising one or more landing pads; or (3) an exogenous nucleotide sequence comprising one or more landing pads in which one or more SOIs have been integrated.

[0265] In some embodiments, the TI host cell includes at least one exogenous nucleotide sequence at one or more integration sites integrated into the genome of the TI host cell. In some embodiments, the exogenous nucleotide sequence is integrated into one or more integration sites within a locus of the genome of a particular TI host cell.

[0266] 4.1 Landing mat

[0267] In some embodiments, the integrated foreign nucleotide sequence comprises one or more recombinant recognition sequences (RRSs), wherein the RRSs are recognizable by a recombinase. In some embodiments, the integrated foreign nucleotide sequence comprises at least two RRSs. In some embodiments, the integrated foreign nucleotide sequence comprises two identical RRSs. In some embodiments, the integrated foreign nucleotide sequence comprises two species-specific RRSs, i.e., not recognized by the same recombinase. In some embodiments, the integrated foreign nucleotide sequence comprises three RRSs, wherein the third RRS is located between the first and second RRSs. In some embodiments, the first and second RRSs are identical, and the third RRS is different from either the first or second RRS. In some embodiments, all three RRSs are species-specific. In some embodiments, the integrated foreign nucleotide sequence comprises four, five, six, seven, or eight RRSs. In some embodiments, the integrated foreign nucleotide sequence comprises multiple RRSs. In some embodiments, the multiple (two or more) RRSs are identical. In some embodiments, the two or more RRSs are species-specific. In some embodiments, each RRS may be recognized by a different recombinase. In some embodiments, subsets of all RRS have homospecificity, i.e., are recognized by the same recombinase, and subsets of all RRS have heterospecificity, i.e., are not recognized by the same recombinase. In some embodiments, one or more RRS may 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, φC31attP sequence, and φC31 attB sequence.

[0268] In some embodiments, the integrated exogenous nucleotide sequence includes at least one selection marker. In some embodiments, the integrated exogenous nucleotide sequence includes an RRS and at least one selection marker. In some embodiments, the integrated exogenous nucleotide sequence includes a first RRS and a second RRS, and at least one selection marker. In some embodiments, the selection marker is located between the first RRS and the second RRS. In some embodiments, the two RRSs are side-attached to at least one selection marker, i.e., the first RRS is located upstream of the 5' end of the selection marker and the second RRS is located downstream of the 3' end of the selection marker. In some embodiments, the first RRS is adjacent to the 5' end of the selection marker, and the second RRS is adjacent to the 3' end of the selection marker.

[0269] In some embodiments, the selector is located between the first RRS and the second RRS, and these two flanking RRSs are identical. In some embodiments, both RRS flanking the selector are LoxP sequences. In some embodiments, both RRS flanking the selector are FRT sequences. In some embodiments, the selector is located between the first RRS and the second RRS, and these two flanking RRSs are xenogeneic specific. In some embodiments, the first flanking RRS is a LoxP L3 sequence, and the second flanking RRS is a LoxP 2L sequence. In some embodiments, the LoxP L3 sequence is located at the 5' end of the selector, and the LoxP 2L sequence is located at the 3' end of the selector. In some embodiments, the first flanking RRS is a wild-type FRT sequence, and the second flanking RRS is a mutant FRT sequence. In some embodiments, the first flanking RRS is a Bxb1 attP sequence, and the second flanking RRS is a Bxb1 attB sequence. In some embodiments, the first side-connected RRS is a φC31 attP sequence, and the second side-connected RRS is a φC31 attB sequence. In some embodiments, the two RRSs are positioned in the same orientation. In some embodiments, both RRSs are in either a forward or reverse orientation. In some embodiments, the two RRSs are positioned in opposite orientations.

[0270] In some embodiments, the selection markers may be aminoglycoside phosphotransferases (APHs) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthase (GS), asparagine synthase, tryptophan synthase (indole), histamine dehydrogenase (histamine D), and genes encoding resistance to puromycin, blastomycin, bleomycin, humicin, chloramphenicol, zeocin, or mycophenolic acid. In some embodiments, the selection markers may be GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomers, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, or T-Sapphire markers. In some embodiments, the selection marker may be a fusion construct comprising at least two selection markers. In some embodiments, a gene encoding a selection marker or a fragment of a selection marker may be fused to a gene encoding a different selection marker or a fragment thereof.

[0271] In some embodiments, the integrated exogenous nucleotide sequence includes two selectable markers flanked by two RRSs, wherein the first selectable marker differs from the second selectable marker. In some embodiments, both selectable markers are selected from the group consisting of: glutamine synthetase selectable markers, thymidine kinase selectable markers, HYG selectable markers, and puromycin resistance selectable markers. In some embodiments, the integrated exogenous nucleotide sequence includes both thymidine kinase selectable markers and HYG selectable markers. In some embodiments, the first selectable marker is selected from the group consisting of: aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthase (GS), asparagine synthase, tryptophan synthase (indole), histamine dehydrogenase (histamine D), and genes encoding resistance to puromycin, blastomycin, bleomycin, cyprodinil, chloramphenicol, zeocin, or mycophenolic acid; and the second selectable marker is selected from the group consisting of: GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire markers. In some embodiments, the first selection marker is a glutamine synthase selection marker, and the second selection marker is a GFP marker. In some embodiments, the two RRSs of the two selection markers are identical. In some embodiments, the two RRSs of the two selection markers are different.

[0272] In some embodiments, the selection marker is operatively linked to a promoter sequence. In some embodiments, the selection marker is operatively linked to the SV40 promoter. In some embodiments, the selection marker is operatively linked to a cytomegalovirus (CMV) promoter.

[0273] In some embodiments, the integrated exogenous nucleotide sequence comprises at least one selectable marker and an IRES, wherein the IRES is operatively linked to the selectable marker. In some embodiments, the selectable marker operatively linked to the IRES is selected from the group consisting of: GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire markers. In some embodiments, the selectable marker operatively linked to the IRES is a GFP marker. In some embodiments, the integrated exogenous nucleotide sequence comprises an IRES flanked by two RRSs and two selectable markers, wherein the IRES is operatively linked to the second selectable marker. In some embodiments, the integrated exogenous nucleotide sequence comprises an IRES flanked by two RRSs and three selectable markers, wherein the IRES is operatively linked to a third selectable marker. In some embodiments, the integrated exogenous nucleotide sequence comprises an IRES flanked by two RRSs and three selectable markers, wherein the IRES is operatively linked to a third selectable marker. In some embodiments, the third selectable marker is different from either the first or second selectable marker. In some embodiments, the integrated exogenous nucleotide sequence comprises a first selectable marker operatively linked to a promoter and a second selectable marker operatively linked to an IRES. In some embodiments, the integrated exogenous nucleotide sequence comprises a glutamine synthetase selectable marker operatively linked to an SV40 promoter and a GFP selectable marker operatively linked to an IRES. In some embodiments, the integrated exogenous nucleotide sequence comprises a thymidine kinase selectable marker and a HYG selectable marker operatively linked to a CMV promoter and a GFP selectable marker operatively linked to an IRES.

[0274] In some embodiments, the integrated exogenous nucleotide sequence comprises three RRSs. In some embodiments, the third RRS is located between the first and second RRSs. In some embodiments, all three RRSs are identical. In some embodiments, the first and second RRSs are identical, and the third RRS is different from either the first or second RRS. In some embodiments, all three RRSs are xenogeneic specific.

[0275] 4.2 Target Sequence (SOI)

[0276] In some embodiments, the integrated exogenous nucleotide sequence comprises at least one exogenous SOI. In some embodiments, the integrated exogenous nucleotide sequence comprises at least one selection marker and at least one exogenous SOI. In some embodiments, the integrated exogenous nucleotide sequence comprises at least one selection marker, at least one exogenous SOI, and at least one RRS. In some embodiments, the integrated exogenous nucleotide sequence comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or more SOIs. In some embodiments, the SOIs are the same. In some embodiments, the SOIs are different.

[0277] In some embodiments, the SOI encodes a single-chain antibody or a fragment thereof. In some embodiments, the SOI encodes an antibody heavy chain sequence or a fragment thereof. In some embodiments, the SOI encodes an antibody light chain sequence or a fragment thereof. In some embodiments, the integrated exogenous nucleotide sequence comprises an SOI encoding an antibody heavy chain sequence or a fragment thereof and an SOI encoding an antibody light chain sequence or a fragment thereof. In some embodiments, the integrated exogenous nucleotide sequence comprises an SOI encoding a first antibody heavy chain sequence or a fragment thereof, an SOI encoding a second antibody heavy chain sequence or a fragment thereof, and an SOI encoding an antibody light chain sequence or a fragment thereof. In some embodiments, the number of SOIs encoding the heavy chain and light chain sequences may be selected to achieve desired expression levels of the heavy chain and light chain peptides, for example, to achieve desired bispecific antibody yield. In some embodiments, the individual SOI encoding heavy and light chain sequences may be integrated, for example, into a single exogenous nucleic acid sequence at an integration site, into multiple exogenous nucleic acid sequences at a single integration site, or into multiple exogenous nucleic acid sequences at different integration sites within the TI host cell.

[0278] In some embodiments, the integrated exogenous nucleotide sequence comprises at least one selectable marker, at least one exogenous SOI, and one RRS. In some embodiments, the RRS is adjacent to at least one selectable marker or at least one exogenous SOI. In some embodiments, the integrated exogenous nucleotide sequence comprises at least one selectable marker, at least one exogenous SOI, and two RRSs. In some embodiments, the integrated exogenous nucleotide sequence comprises at least one selectable marker and at least one exogenous SOI located between a first RRS and a second RRS. In some embodiments, the two RRS flanking the selectable marker and the exogenous SOI are identical. In some embodiments, the two RRS flanking the selectable marker and the exogenous SOI are different. In some embodiments, the first flanking RRS is a LoxP L3 sequence, and the second flanking RRS is a LoxP 2L sequence. In some embodiments, the L3 LoxP sequence is located at the 5' end of the selectable marker and the exogenous SOI, and the LoxP 2L sequence is located at the 3' end of the selectable marker and the exogenous SOI.

[0279] In some embodiments, the integrated exogenous nucleotide sequence comprises three RRSs and two exogenous SOIs, with the third RRS located between the first and second RRSs. In some embodiments, the first SOI is located between the first and third RRSs, and the second SOI is located between the third and second RRSs. In some embodiments, the first and second SOIs are different. In some embodiments, the first and second RRSs are the same, and the third RRS is different from either the first or second RRS. In some embodiments, all three RRSs are xenogeneic specific. In some embodiments, the first RRS is a LoxP L3 site, the second RRS is a LoxP 2L site, and the third RRS is a LoxFas site. In some embodiments, the integrated exogenous nucleotide sequence comprises three RRSs, one exogenous SOI, and a selection marker. In some embodiments, the SOI is located between the first and third RRSs, and the selection marker is located between the third and second RRSs. In some embodiments, the integrated exogenous nucleotide sequence comprises three RRSs, two exogenous SOIs, and a selection marker. In some embodiments, the first SOI and the selection marker are located between the first and third RRSs, and the second SOI is located between the third and second RRSs.

[0280] In some embodiments, the exogenous SOI encodes a target polypeptide. Such target polypeptides may be selected from the group consisting of, but not limited to, antibodies, enzymes, cytokines, growth factors, hormones, viral proteins, bacterial proteins, vaccine proteins, or proteins with therapeutic functions. In some embodiments, the exogenous SOI encodes an antibody or an antigen-binding fragment thereof. In some embodiments, the exogenous 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. In some embodiments, the exogenous SOI (or SOI) encodes a standard antibody. In some embodiments, the exogenous SOI (or SOI) encodes a hapten, such as, but not limited to, antibodies B, Q, T, and mAb I of this disclosure. In some embodiments, the exogenous SOI (or SOI) encodes a complex antibody. In some embodiments, the complex antibody may be a bispecific antibody, such as, but not limited to, bispecific molecules A, B, C, or D of this disclosure. In some embodiments, the external SOI is operatively connected to at least one cis-acting element, such as a promoter or enhancer. In some embodiments, the external SOI is operatively connected to a CMV promoter.

[0281] In some embodiments, the integrated exogenous nucleotide sequence comprises two RRSs and at least two exogenous SOIs located between the two RRSs. In some embodiments, an SOI encoding one heavy chain and one light chain of the antibody is located between the two RRSs. In some embodiments, an SOI encoding one heavy chain and two light chains of the antibody is located between the two RRSs. In some embodiments, SOIs encoding different combinations of copies of the heavy chain and light chain of the antibody are located between the two RRSs.

[0282] In some embodiments, the integrated exogenous nucleotide sequence comprises three RRSs and at least two exogenous SOIs, with the third RRS located between the first and second RRSs. In some embodiments, at least one SOI is located between the first and third RRSs, and at least one SOI is located between the third and second RRSs. In some embodiments, the first and second RRSs are identical, and the third RRS is different from either the first or second RRS. In some embodiments, all three RRSs are xenogeneic specific. In some embodiments, an SOI encoding one heavy chain and one light chain of a first antibody is located between the first and third RRSs, and an SOI encoding one heavy chain and one light chain of a second antibody is located between the third and second RRSs. In some embodiments, an SOI encoding one heavy chain and two light chains of a first antibody is located between the first and third RRSs, and an SOI encoding one heavy chain and one light chain of a second antibody is located between the third and second RRSs. In some embodiments, an SOI encoding one heavy chain and three light chains of a first antibody is located between the first RRS and the third RRS, and an SOI encoding one light chain of the first antibody and one heavy chain and one light chain of the second antibody is located between the third RRS and the second RRS. In some embodiments, an SOI encoding one heavy chain and one light chain of the first antibody is located between the first RRS and the third RRS, and an SOI encoding two light chains of the first antibody and one heavy chain and one light chain of the second antibody is located between the third RRS and the second RRS. In some embodiments, SOIs encoding different combinations of copies of heavy chains and light chains of multiple antibodies are located between the first RRS and the third RRS and between the third RRS and the second RRS.

[0283] In some embodiments, the number of SOIs is selected to increase the expression titer and / or the specific productivity of host cells expressing the SOI. For example, but not as a limitation, combining two, three, four, five, six, seven, eight or more SOIs may yield higher titers and / or specific productivity.

[0284] In the context of antibody expression, including additional heavy or light chain-encoding SOIs can result in higher titers and / or specific productivity. For example, but not as a limitation, increasing the copy number from one heavy chain and one light chain (HL) to one heavy chain and two light chain-encoding sequences (HLL) can achieve higher titers and / or specific productivity. Similarly, as outlined in the following examples, increasing the HLL (three SOIs) to HLL-HL (five SOIs) or HLL-HLL (six) can provide higher titers and / or specific productivity. Furthermore, increasing the copy number to HLL-HL (five SOIs) or HLL-HLHL (seven SOIs) can provide higher titers and / or specific productivity. Further options for heavy and light chain SOI copy numbers include, but are not limited to: HHL; HHL-H; HLL-H; HHL-HH; HHL-HL; HHL-LL; HLL-HH; HLL-HL; HLL-LL; HHL-HHL; HHL-HHH; HHL-HLL; HHL-LLL; HLL-HHL; HLL-HHH; HLL-LLL; HHL-HHHL; HHL-HHHH; HHL-HHLL; HHL-HLLL; HHL-LLLL; HLL-HHHL; HLL-HHHH; HLL-HLLL; and HLL-LLLL. In some embodiments, the inclusion of additional copies occurs at a single genomic locus, while in other embodiments, SOI copies may be integrated at two or more loci; for example, multiple copies may be integrated at a single locus and one or more copies may be integrated at one or more additional loci.

[0285] In some embodiments, the location of the SOIs (e.g., whether one SOI is located at the 3' end or the 5' end relative to another SOI) is chosen to improve titers and / or the specific productivity of host cells expressing the SOIs. For example, but not as a limitation, the integration location of the heavy and light chain SOIs in the context of antibody production can lead to higher titers and / or specific productivity. In some embodiments, the relative positions of the heavy and light chain SOIs can affect titers and specific productivity even though the SOI copy number remains unchanged.

[0286] In some embodiments, targeted integration may be combined with transposon-mediated genome integration. In some embodiments, targeted integration may occur prior to transposon-mediated genome integration. In some embodiments, targeted integration may occur simultaneously with transposon-mediated genome integration. In some embodiments, targeted integration may occur prior to transposon-mediated genome integration.

[0287] 4.3. Targeted integration of recombinase-mediated recombination

[0288] A "recombination recognition sequence" (RRS) is a nucleotide sequence that is recognized by recombinases and is both necessary and sufficient for recombinase-mediated recombination events. RRS can be used to define the location in a nucleotide sequence where a recombination event will occur.

[0289] In some embodiments, the RRS is selected from the group consisting of: LoxP sequence, LoxP L3 sequence, LoxP2L 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.

[0290] In some embodiments, the RRS may be recognized by the Cre recombinase. In some embodiments, the RRS may be recognized by the FLP recombinase. In some embodiments, the RRS may be recognized by the Bxb1 integrase. In some embodiments, the RRS may be recognized by the φC31 integrase.

[0291] In some embodiments, when the RRS is a LoxP site, the host cell requires Cre recombinase to perform recombination. In some embodiments, when the RRS is an FRT site, the host cell requires FLP recombinase to perform recombination. In some embodiments, when the RRS is a Bxb1 attP or Bxb1 attB site, the host cell requires Bxb1 integrase to perform recombination. In some embodiments, when the RRS is a φC31 attP or φC31 attB site, the host cell requires φC31 integrase to perform recombination. The recombinase can be introduced into the host cell using an expression vector containing the coding sequence of the enzyme.

[0292] The Cre-LoxP site-specific recombination system has been widely used in many biological experimental systems. Cre is a 38kDa site-specific DNA recombinase that recognizes a 34 bp LoxP sequence. Cre originates from bacteriophage P1 and belongs to the tyrosine family of site-specific recombinases. Cre recombinase mediates intramolecular and intermolecular recombination between LoxP sequences. The LoxP sequence consists of an 8 bp non-palindromic core region flanked by two 13 bp inverted repeat sequences. Cre recombinase binds to the 13 bp repeat sequences, thereby mediating recombination within the 8 bp core region. Cre-LoxP-mediated recombination occurs with high efficiency and requires no additional host factors. If two LoxP sequences are placed in the same nucleotide sequence with the same orientation, Cre-mediated recombination will excise the DNA sequence located between the two LoxP sequences, resulting in a covalently closed loop. If two LoxP sequences are placed in opposite positions in the same nucleotide sequence, Cre-mediated recombination will reverse the orientation of the DNA sequence located between the two sequences. LoxP sequences can also be placed on different chromosomes to promote recombination between different chromosomes. If two LoxP sequences are on two different DNA molecules, and if one of the DNA molecules is circular, Cre-mediated recombination will result in the integration of the circular DNA sequence.

[0293] In some embodiments, the LoxP sequence is the wild-type LoxP sequence. In some embodiments, the LoxP sequence is a mutant LoxP sequence. Mutant LoxP sequences have been developed to improve the efficiency of Cre-mediated integration or substitution. In some embodiments, the mutant LoxP sequence is selected from the group consisting of: LoxP L3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, and Lox66 sequence. For example, the Lox71 sequence has a 5 bp mutation in the left 13 bp repeat sequence. The Lox66 sequence has a 5 bp mutation in the right 13 bp repeat sequence. Both wild-type and mutant LoxP sequences can mediate Cre-dependent recombination.

[0294] The FLP-FRT site-specific recombination system is similar to the Cre-Lox system. It involves the flippant enzyme (FLP), a recombinase derived from a 2 µm plasmid of *Saccharomyces cerevisiae*. FLP also belongs to the tyrosine family of site-specific recombinases. The FRT sequence is a 34 bp sequence consisting of two 13 bp palindromic sequences, each flanked by an 8 bp spacer. FLP binds to the 13 bp palindromic sequence and mediates DNA breaks, exchanges, and ligations within the 8 bp spacer. Similar to the Cre recombinase, the position and orientation of the two FRT sequences determine the FLP-mediated recombination outcome. In some embodiments, the FRT sequence is a wild-type FRT sequence. In some embodiments, the FRT sequence is a mutant FRT sequence. Both wild-type and mutant FRT sequences can mediate FLP-dependent recombination. In some embodiments, the FRT sequence is fused to a reactive receptor domain sequence, such as, but not limited to, a tamoxifen reactive receptor domain sequence.

[0295] Bxb1 and φC31 belong to the serine recombinase family. They are both derived from bacteriophages and are used by these phages to establish lysogenicity, facilitating site-specific integration of the phage genome into the bacterial genome. These integrases catalyze site-specific recombination events between short (40-60 bp) DNA substrates called attP and attB sequences, which are originally attachment sites on the phage DNA and bacterial DNA, respectively. Recombination results in two new sequences, called attL and attR, each containing half a sequence from attP and attB, respectively. Recombination can also occur between attL and attR sequences, cleaving the integrated phage from the bacterial DNA. Both integrases can catalyze recombination without the aid of any additional host factors. In the absence of any cofactors, these integrases mediate unidirectional recombination between attP and attB with an efficiency greater than 80%. Because of the short DNA sequences that can be recognized by these integrases and the one-way recombination, these recombination systems have been developed as a complement to the widely used Cre-LoxP and FRT-FLP systems for genetic engineering purposes.

[0296] The terms "matching RRS" and "homologous RRS" indicate that recombination has occurred between two RRS. In some embodiments, the two matching RRS are identical. In some embodiments, both RRS are wild-type LoxP sequences. In some embodiments, both RRS are mutant LoxP sequences. In some embodiments, both RRS are wild-type FRT sequences. In some embodiments, both RRS are mutant FRT sequences. In some embodiments, the two matching RRS are different sequences, but can be recognized by the same recombinase. In some embodiments, the first matching RRS is a Bxb1 attP sequence, and the second matching RRS is a Bxb1 attB sequence. In some embodiments, the first matching RRS is a φC31 attB sequence, and the second matching RRS is a φC31 attB sequence.

[0297] In some embodiments, the integrated foreign nucleotide sequence contains two RRSs, and the vector contains two RRSs that match the two RRSs on the integrated foreign nucleotide sequence; that is, the first RRS on the integrated foreign nucleotide sequence matches the first RRS on the vector, and the second RRS on the integrated foreign nucleotide sequence matches the second RRS on the vector. In some embodiments, the first RRS on the integrated foreign nucleotide sequence and the first RRS on the vector are identical to the second RRS on the integrated foreign nucleotide sequence and the second RRS on the vector. A non-limiting example of such a “single-vector RMCE” strategy is provided in Figure 2A of PCT application PCT / US2018 / 067070 (publication number WO2019126634). In some embodiments, the first RRS on the integrated foreign nucleotide sequence and the first RRS on the vector are different from the second RRS on the integrated foreign nucleotide sequence and the second RRS on the vector. In some embodiments, the first RRS on the integrated exogenous nucleotide sequence and the first RRS on the vector are both LoxP L3 sequences, and the second RRS on the integrated exogenous nucleotide sequence and the second RRS on the vector are both LoxP 2L sequences.

[0298] In some embodiments, a “dual-vector RMCE” strategy is employed. For example, but not as a limitation, the integrated exogenous nucleotide sequence may contain three RRSs, such as in the following arrangement: a third RRS (“RRS3”) is present between the first RRS (“RRS1”) and the second RRS (“RRS2”), and the first vector contains two RRSs that match the first and third RRSs on the integrated exogenous nucleotide sequence, and the second vector contains two RRSs that match the third and second RRSs on the integrated exogenous nucleotide sequence. An example of a dual-vector RMCE strategy is illustrated in Figure 4 of PCT application PCT / US2018 / 067070 (publication number WO2019126634). In such examples, RRS1, RRS2, and RRS3 are heterospecific, i.e., they do not cross-react with each other. In some embodiments, one vector (front) contains RRS1, a first SOI, and a promoter, followed by a start codon and RRS3 (in that order). Another vector (later) contains RRS3, fused to a marker coding sequence excluding the start codon (ATG), and SOI 2 and RRS2 (in that order). Additional nucleotides may be inserted between the RRS3 site and the selected marker sequence to ensure frame translation of the fusion protein. In some embodiments, the first SOI encodes an antibody. In some embodiments, the antibody is a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv), or an Fc fusion protein. In some embodiments, the second SOI encodes an antibody. In some embodiments, the antibody is a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv), or an Fc fusion protein. In some embodiments, the first SOI and the second SOI pair encode antibodies to form multispecific (e.g., bispecific) antibodies.

[0299] This type of dual-carrier RMCE strategy allows for the introduction of eight or more SOIs by combining an appropriate number of SOIs between each pair of RRSs.

[0300] Both single-vector and dual-vector RMCEs allow for the unidirectional integration of one or more donor DNA molecules into a predetermined site in the host cell genome, precisely exchanging a DNA cassette present on the donor DNA with a DNA cassette on the host genome where the integration site is located. The DNA cassette is characterized by two xenogeneic specific RRSs (although in some dual-vector RMCE instances, “separate selection markers” as outlined herein) and / or at least one exogenous SOI. RMCE involves a dual recombination exchange event between two xenogeneic specific RRSs within the target genome locus and the donor DNA molecule, catalyzed by recombinases. RMCEs are designed to introduce a copy of the SOI or selection marker into a predetermined locus in the host cell genome. Unlike recombination, which involves only one crossover event, RMCEs can be implemented such that prokaryotic vector sequences are not introduced into the host cell genome, thereby reducing and / or preventing unwanted triggering of host immune or defense mechanisms. The RMCE method can be repeated with multiple DNA cassettes.

[0301] In some embodiments, targeted integration is achieved through a single recombination event, wherein a foreign nucleotide sequence is integrated into a predetermined site in the host cell genome, the foreign nucleotide sequence comprising an RRS adjacent to at least one foreign SOI or at least one selection marker. In some embodiments, targeted integration is achieved through a single RMCE, wherein a DNA cassette is integrated into a predetermined site in the host cell genome, the DNA cassette comprising at least one foreign SOI or at least one selection marker flanked by two xenogeneic specific RRSs. In some embodiments, targeted integration is achieved through two RMCEs, wherein two different DNA cassettes are integrated into predetermined sites in the host cell genome, each of the two DNA cassettes comprising at least one foreign SOI or at least one selection marker flanked by two xenogeneic specific RRSs. In some embodiments, targeted integration is achieved through multiple RMCEs, wherein DNA cassettes from multiple vectors are all integrated into predetermined sites in the host cell genome, each of these DNA cassettes comprising at least one foreign SOI or at least one selection marker flanked by two xenogeneic specific RRSs. In some embodiments, the selection marker may be encoded partially on a first carrier and partially on a second carrier, such that the integration of the two RMCEs allows the expression of the selection marker. An example of such a system is presented in Figure 4 of PCT application PCT / US2018 / 067070 (publication number WO2019126634).

[0302] In some embodiments, targeted integration of recombinase-mediated recombination results in the integration of a selection marker or one or more exogenous SOIs into one or more predetermined integration sites in a host cell genome containing a sequence from a prokaryotic vector. In some embodiments, targeted integration of recombinase-mediated recombination results in the integration of a selection marker or one or more exogenous SOIs into one or more predetermined integration sites in a host cell genome that does not contain a sequence from a prokaryotic vector.

[0303] 4.4 Targeted integration via homologous recombination, HDR, or NHEJ

[0304] The subject matter of this disclosure also relates to targeted integration mediated by homologous recombination or by exogenous site-specific nucleases followed by HDR or NHEJ.

[0305] Homologous recombination is the recombination between DNA molecules that share extensive sequence homology. It can be used to guide error-free repair of double-stranded DNA breaks and to produce sequence changes in gametes during meiosis. Because homologous recombination involves the exchange of genetic information between two homologous DNA molecules, it does not alter the overall arrangement of genes on a chromosome. During homologous recombination, a gap or break forms in double-stranded DNA (dsDNA), and a homologous dsDNA molecule is subsequently introduced through the ends of single-stranded DNA. Homologous sequence pairing and branching occur to form a Holliday linker, which is eventually separated.

[0306] Double-strand breaks (DSBs) are the most serious form of DNA damage, and their repair is crucial for maintaining the genome integrity of all organisms. There are two main DSB repair pathways. The first is homology-mediated repair (HDR), with homologous recombination being the most common form. Because HDR requires the presence of homologous DNA in the cell, this pathway is typically active during the S and G2 phases of the cell cycle, where newly replicated sister chromatids can serve as homologous templates. HDR is also the primary repair pathway for repairing folded replication forks during DNA replication. HDR is considered a relatively error-free repair pathway. The second DSB repair pathway is non-homologous end joining (NHEJ). NHEJ is a repair pathway that joins the ends of broken DNA together without requiring a homologous DNA template.

[0307] Exogenous site-specific nucleases and subsequent HDR can promote targeted integration. This is because introducing DSBs at specific target genomic sites can increase the frequency of homologous recombination. In some embodiments, the exogenous nuclease may be selected from the group consisting of: zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly spaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases.

[0308] CRISPR / Cas and TALEN systems are two genome editing tools that offer optimal ease of construction and high efficiency. CRISPR / Cas has been identified as an immune defense mechanism in bacteria against invading bacteriophages. Cas is a nuclease that, when guided by synthetic guide RNA (gRNA), can associate with a specific nucleotide sequence in a cell and edit the DNA in or around that sequence, for example, by forming one or more of single-strand breaks, DSBs, and / or point mutations. TALEN is an engineered site-specific nuclease consisting of the DNA-binding domain of TALE (a transcription activator-like effector nuclease) and the catalytic domain of the restriction endonuclease FokI. Artificial TALENs targeting various nucleotide sequences can be created by altering the amino acids in a highly variable residue region of the monomer present in the DNA-binding domain. The DNA-binding domain then guides the nuclease to the target sequence and forms a DSB.

[0309] Targeted integration via homologous recombination or HDR involves the presence of a homologous sequence at the integration site. In some embodiments, the homologous sequence is present on the vector. In some embodiments, the homologous sequence is present on a polynucleotide.

[0310] In some embodiments, the vector for targeted integration of exogenous nucleotide sequences into host cells comprises a nucleotide sequence homologous to at least one selection marker, consisting of: an endogenous sequence comprising a portion of a contiguous group sequence of one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1; or a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2; or a sequence selected from SEQ ID No. 1 to 7. In some embodiments, the vector for targeted integration of exogenous nucleotide sequences into host cells comprises a nucleotide sequence homologous to at least one selection marker and at least one exogenous SOI, consisting of: an endogenous sequence comprising a portion of a contiguous group sequence comprising one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1; or a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2; or a sequence selected from SEQ ID No. 1 to 7. In some embodiments, the vector for targeted integration of exogenous nucleotide sequences into host cells comprises a nucleotide sequence with a side-attached DNA cassette that is at least 50% homologous to a sequence selected from the group consisting of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, and SEQ ID Nos. 1 to 7, wherein the DNA cassette comprises at least one selection marker with two RRSs and at least one exogenous SOI.In some embodiments, the vector for targeted integration of exogenous nucleotide sequences into host cells comprises a nucleotide sequence with a side-attached DNA cassette that is at least 50% homologous to the following: an endogenous sequence that is a portion of a contiguous group sequence of one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, wherein the DNA cassette comprises two side-attached RRSs. At least one selected marker and at least one exogenous SOI. In some embodiments, the vector nucleotide sequence is homologous to at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% of the following contiguous groups: NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or an endogenous sequence selected from LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2. The gene constituting the group, or the sequence selected from SEQ ID No. 1 to 7. In some embodiments, the vector is selected from the group consisting of: adenovirus vector, adeno-associated virus vector, lentiviral vector, retroviral vector, integrative phage vector, nonviral vector, transposon and / or transposase vector, integrase substrate and plasmid.

[0311] In some embodiments, the polynucleotide for targeted integration of exogenous nucleotide sequences into host cells comprises a nucleotide sequence homologous to at least one selection marker, consisting of: an endogenous sequence being a portion of a contiguous group sequence of one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1; or a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2; or a sequence selected from SEQ ID No. 1 to 7. In some embodiments, the polynucleotide for targeted integration of exogenous nucleotide sequences into host cells comprises a nucleotide sequence homologous to at least one selection marker and at least one exogenous SOI, consisting of: an endogenous sequence that is part of a contiguous group sequence of one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1; or a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2; or a sequence selected from SEQ ID No. 1 to 7. In some embodiments, the polynucleotide used to target and integrate a foreign nucleotide sequence into a host cell comprises a nucleotide sequence that is at least 50% homologous to a sequence selected from SEQ ID Nos. 1 to 7 of a side-attached DNA cassette, wherein the DNA cassette comprises at least one selection marker with two RRSs attached and at least one foreign SOI.In some embodiments, the polynucleotide for targeted integration of exogenous nucleotide sequences into host cells comprises a nucleotide sequence with at least 50% homology to the following contiguous DNA cassette: an endogenous sequence comprising a portion of a contiguous sequence of one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, wherein the DNA cassette comprises two RRS contiguous DNA cassettes. At least one selected marker and at least one exogenous SOI. In some embodiments, the flanking nucleotide sequences are homologous to at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% of the following: contiguous groups NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or to endogenous nucleic acids selected from LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2. The genes that make up the group, or sequences selected from SEQ ID No. 1 to 7.

[0312] In some embodiments, homologous recombination occurs without any auxiliary factors. In some embodiments, there are vectors capable of integration to facilitate homologous recombination. In some embodiments, the integration vector is selected from the group consisting of adeno-associated virus vectors, lentiviral vectors, retroviral vectors, and integration phage vectors.

[0313] 4.5 Transposon-mediated genome integration

[0314] In some embodiments, the subject matter of this disclosure also covers transposon-mediated genome integration of one or more exogenous nucleic acids into host cells. As outlined herein, in some embodiments, targeted integration may occur concurrently with transposon-mediated genome integration, which may occur simultaneously with targeted integration. In some embodiments, targeted integration may follow transposon-mediated genome integration. In some embodiments, targeted integration may precede transposon-mediated genome integration.

[0315] Available transposons associated with the methods described herein are known in the art, and include, but are not limited to, the following:

[0316] ●piggyBac transposon (see, for example, Wilson et al., Molecular Therapy, 15(1):139-145 (2007));

[0317] ● Sleeping Beauty Transposition (see, for example, Ivics et al., Cell 91:501-510 (1997)); and

[0318] ●Tol2 transposons (see, for example, Balciunas et al., PLoS Genent. Nov 10;2(11):e169. doi: 10.1371 / journal.pgen.0020169.Epub 28 August 2006).

[0319] Generally, transposons available in connection with the methods of this disclosure are translocated via a non-replicative "cut-and-paste" mechanism. For example, without being bound by theory, transposition catalyzed by available transposons in connection with the methods of this disclosure can be carried out by a DNA transposase recognizing two inverted terminal repeat (TIR) ​​sequences, which cleaves its target and thus releases the DNA transposon from its donor sequence (e.g., a donor plasmid). After excision, the transposon can be integrated into the host cell genome cleaved by the same transposase at the corresponding sequence within the genome.

[0320] 4.6 Tunable Expression of SOI

[0321] In many cases, protein expression levels are not optimal, primarily because the encoding protein is difficult to express. Low expression levels of difficult-to-express proteins can have a variety of difficult-to-identify causes. One possibility is the toxicity of the protein expressed in the host cell. In such cases, tunable expression systems can be used to express toxic proteins, where the target sequence encoding the protein is under the control of an inducible promoter. In these systems, the expression of the difficult-to-express protein is promoted only when a regulatory factor (e.g., a small molecule, such as, but not limited to, tetracycline or its analogues, doxycycline (DOX)) is added to the culture medium. Regulating the expression of toxic proteins can mitigate toxicity, allowing the culture to achieve the desired cell growth prior to production. In some embodiments, the tunable expression system comprises at least one SOI transcribed under a tunable promoter operably linked thereto. In some embodiments, the tunable expression system can be used to determine the root cause of low protein expression levels of difficult-to-express molecules (such as, but not limited to, antibodies). In some embodiments, the ability to selectively shut down SOI expression in the tunable expression system can be used to correlate SOI expression with observed adverse effects.

[0322] In some embodiments, tunable expression systems may be used to minimize the effects of transcriptional and cell line variability during the root cause analysis of molecules that are difficult to express. For example, but not limited to, SOI expression can be triggered by adding a regulatory factor (e.g., doxycycline) to the culture. In some embodiments, tunable expression vectors utilize a tetracycline-regulated promoter to express SOI, thereby allowing tunable expression of SOI.

[0323] In some embodiments, the tunable expression system described herein, compared to a control cell line, can be used to successfully determine the potential cause of low protein expression levels of SOIs (e.g., therapeutic antibodies). In some embodiments, once lower relative expression of SOIs (e.g., therapeutic antibodies) is confirmed in the tunable expression cell line, treatment with protein translation inhibitors (e.g., Dox and actinomycin) can be used to assess intracellular accumulation and secretion levels of SOIs.

[0324] As detailed herein, tunable expression can be based on gene switches to block or activate mRNA synthesis through tunable coupling of transcriptional repressors or activators with constitutive or minimal promoters. In some non-limiting embodiments, repression can be achieved by binding to repressor proteins, for example, wherein the protein spatially blocks transcription initiation or actively represses transcription through a transcriptional silencer. In some non-limiting embodiments, activation of a minimal promoter of a mammalian or virus-free enhancer can be achieved through tunable coupling with an activation domain.

[0325] In some embodiments, conditional coupling of transcriptional repressors or activators can be achieved by using allosteric proteins that bind promoters in response to external stimuli. In some embodiments, conditional coupling of transcriptional repressors or activators can be achieved by using intracellular receptors released from chelating proteins, thereby binding to target promoters. In some embodiments, conditional coupling of transcriptional repressors or activators can be achieved by using chemically induced dimers.

[0326] In some embodiments, the allosteric protein used in the tunable expression system of this disclosure may be a protein that regulates transcriptional activity in response to antibiotics, bacterial quorum sensing messengers, catabolites, or culture parameters such as temperature, e.g., cold or hot. In some embodiments, such tunable expression systems may be based on catabolites, for example, where a bacterial repressor controlling a catabolite gene for an alternative carbon source has been transferred to mammalian cells. In some embodiments, repression of a target promoter may be achieved via a cumate reaction binding of the repressor CymR. In some embodiments, catabolite-based systems may rely on a 6-hydroxynicotinic reaction binding of the prokaryotic repressor HdnoR fused to the transactivation domain of herpes simplex VP16 to activate a chimeric promoter.

[0327] In some embodiments, the tunable expression system of this disclosure may employ a quorum-sensing-based expression system derived from prokaryotes, which manages intra- and inter-population communication through quorum-sensing molecules. These quorum-sensing molecules bind to receptors in target cells, modulating the affinity of the receptors for homologous promoters, thereby activating specific regulator switches. In some embodiments, the quorum-sensing molecule may be N-(3-oxo-octanoyl)-homoserine lactone, in which the TraR-p65 fusion protein activates expression of the minimal promoter fused to the TraR-specific operator sequence in its presence. In some embodiments, the quorum-sensing molecule may be butyrolactone SCB1 (racemic 2-(1'-hydroxy-6-methylheptyl)-3-(hydroxymethyl)-butyrolactone) in a system based on the Streptomyces coelicolor A3(2) ScbR repressor, which binds to its homologous operator OScbR in the absence of SCB1. In some embodiments, the quorum sensing molecule may be a homoserine-derived inducer used in the RTI system, wherein the Pseudomonas aeruginosa quorum sensing inhibitors RhlR and LasR are fused to the SV40 T antigen nuclear localization sequence and the herpes simplex VP16 domain, and can activate a promoter containing a specific operator gene sequence (las box).

[0328] In some embodiments, the inducing molecules for regulating the allosteric proteins used in the tunable expression system of this disclosure may be, but are not limited to, cumate, isopropyl-β-D-galactopyranoside (IPTG), macrolides, 6-hydroxynicotinic acid, doxycycline, streptozotocin, NADH, and tetracycline.

[0329] In some embodiments, the intracellular receptor used in the tunable expression system of this disclosure may be a cytoplasmic or nuclear receptor. In some embodiments, the tunable expression system of this disclosure may use small molecules to release transcription factors from chelating and repressing proteins. In some embodiments, the tunable expression system of this disclosure relies on steroid regulation, wherein the hormone receptor is fused to a natural or artificial transcription factor that can be released from HSP90 in the cytosol, migrate to the nucleus, and activate a selected promoter. In some embodiments, mutant receptors regulated by synthetic steroid analogs may be used to avoid crosstalk of endogenous steroid hormones. In some embodiments, the receptor may be an estrogen receptor variant responsive to 4-hydroxytamoxifen or a progesterone receptor mutant induced by RU486. In some embodiments, a rosiglitazone-reactive transcriptional switch derived from the nuclear receptor of human nuclear peroxisome proliferator-activated receptor γ (PPARγ) may be used in the tunable expression system of this disclosure. In some embodiments, a variant of the steroid response receptor may be RheoSwitch, which is based on a modified spruce leafroller (Choristoneura fumiferana) ecdysone receptor and mouse retinoic acid X receptor (RXR) fused to the Gal4 DNA-binding domain and VP16 trans-activator. In the presence of synthetic ecdysone, the RheoSwitch variant can bind to and activate a minimal promoter fused to several repetitive sequences of the Gal4 response element.

[0330] In some embodiments, the tunable expression system disclosed herein utilizes chemically induced dimerization of DNA-binding proteins and transcription activators to activate a minimal core promoter fused to a homologous operator. In some embodiments, the tunable expression system disclosed herein utilizes dimerization of FKBP and FRB regulated by rapamycin. In this system, FRB is fused to a p65 trans-activator, and FKBP is fused to a zinc finger domain targeting specific homologous operator sites upstream of an engineered minimal interleukin-12 promoter. In some embodiments, FKBP may be mutated. In some embodiments, the tunable expression system disclosed herein utilizes the bacterial gyrase B subunit (GyrB), wherein GyrB dimerizes in the presence of the antibiotic coumarin and dissociates from neomycin.

[0331] In some embodiments, the tunable expression system of this disclosure can be used for tunable siRNA expression. In some embodiments, the tunable siRNA expression system may be a tetracycline, a macrolide, or a turn-off and turn-on QuoRex system. In some embodiments, the RTI system may utilize the Xenopus terminal oligopyrimidine element (TOP), which blocks translation initiation by forming a hairpin structure in the 5' untranslated region.

[0332] In some embodiments, the tunable expression systems described in this disclosure may utilize gas-phase controlled expression, such as acetaldehyde-induced regulation (AIR) systems. AIR systems may employ the Aspergillus nidulans AlcR transcription factor, which, in the presence of non-toxic concentrations of gaseous or liquid acetaldehyde, specifically activates the PAIR promoter, a genome-specifically manipulated AlcR fused to the microcytomegalovirus promoter.

[0333] In some embodiments, the tunable expression system of this disclosure may utilize a Tet-On or Tet-Off system. In such systems, the expression of one or more SOIs may be regulated by tetracycline or its analogue doxycycline.

[0334] In some embodiments, the tunable expression system of this disclosure may utilize a PIP-on or PIP-off system. In such systems, SOI expression may be regulated by, for example, primordial mycin, tetracycline, and / or erythromycin.

[0335] 5. Preparation and use of RVLP-reduced host cells

[0336] This disclosure relates to methods for targeted integration of exogenous nucleotide sequences into host cells. In some embodiments, these methods involve integrating exogenous nucleotide sequences into host cells to generate host cells suitable for subsequent targeted integration of SOIs. In some embodiments, the methods include recombinase-mediated recombination. In some embodiments, the methods involve homologous recombination, HDR, and / or NHEJ.

[0337] In some embodiments, the subject matter of this disclosure relates to methods for targeted integration of exogenous nucleotide sequences into host cells and to transposon-mediated integration of exogenous nucleotide sequences into host cells. In some embodiments, these methods involve targeted integration of exogenous nucleotide sequences into host cells to produce host cells suitable for subsequent targeted integration of SOIs, and to integration of the same or different SOIs with transposon-mediated genome integration. In some embodiments, the methods include recombinase-mediated recombination. In some embodiments, the methods involve homologous recombination, HDR, and / or NHEJ.

[0338] In some embodiments, the target polypeptide is generated and secreted into a cell culture medium. In some embodiments, the target polypeptide is expressed and retained within the host cell. In some embodiments, the target polypeptide is expressed, inserted into, and retained in the host cell membrane.

[0339] Desired exogenous nucleotides or vectors can be introduced into host cells using conventional cell biology methods, including but not limited to transfection, transduction, electroporation, or injection. In some embodiments, chemical-based transfection methods are used to introduce the desired exogenous nucleotides or vectors into host cells, including lipid-based, calcium phosphate-based, cationic polymer-based, or nanoparticle-based transfection methods. In some embodiments, virus-mediated transduction is used to introduce the desired exogenous nucleotides or vectors into host cells, including but not limited to lentivirus, retrovirus, adenovirus, or adeno-associated virus-mediated transduction. In some embodiments, the desired exogenous nucleotides or vectors are introduced into host cells via gene gun-mediated injection. In some embodiments, both DNA and RNA molecules are introduced into host cells using the methods described herein.

[0340] 5.1 Preparation of TI host cells using recombinase-mediated recombinant synthesis

[0341] In some embodiments, this disclosure provides methods for preparing TI host cells to express a target peptide, the methods comprising: a) providing TI host cells containing a foreign nucleotide sequence at a locus integrated into the genome of the host cell, wherein the locus is at least about 90% homologous to SEQ ID No. 1 to 7, wherein the foreign nucleotide sequence contains two RRSs side-gated with at least one first selectable marker; b) introducing a vector into the cells provided in a) the vector containing two RRSs that match two RRSs on the integrated foreign nucleotide sequence and side-gated 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, thereby isolating TI host cells expressing the target peptide.

[0342] In some embodiments, this disclosure provides methods for preparing TI host cells to express a target polypeptide, the methods comprising: a) providing TI host cells containing a foreign nucleotide sequence integrated into a site of an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2 and sequences at least about 90% homologous thereto, wherein the foreign nucleotide sequence contains two RRSs side-joined with at least one first selectable marker; b) introducing a vector into the cells provided in a) the vector containing two RRSs that match two RRSs on the integrated foreign nucleotide sequence and side-joined 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, thereby isolating TI host cells expressing the target polypeptide.

[0343] In some embodiments, this disclosure provides methods for preparing TI host cells to express a target peptide, the methods comprising: a) providing TI host cells containing a foreign nucleotide sequence at a locus integrated into the genome of the TI host cell, wherein the locus is at least about 90% homologous to SEQ ID No. 1 to 7, wherein the foreign nucleotide sequence contains a first DNA cassette containing two xenogeneic specific RRSs side-gated with at least one first selectable marker; b) introducing a vector containing a second DNA cassette into the cells provided in a) the second DNA cassette containing two xenogeneic specific RRSs that match two RRSs on the integrated foreign nucleotide sequence and side-gated with at least one foreign SOI and at least one second selectable marker; c) introducing a recombinase, wherein the recombinase recognizes the RRSs and performs one RMCE; and d) selecting TI cells expressing the second selectable marker, thereby isolating TI host cells expressing the target peptide.

[0344] In some embodiments, this disclosure provides methods for preparing TI host cells to express a target polypeptide, the methods comprising: a) providing TI host cells containing exogenous nucleotide sequences integrated into sites within endogenous sequences of all or part of a contiguous group consisting of one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and at least about 90% thereof. The method involves: a) introducing a homologous sequence, wherein the exogenous nucleotide sequence contains a first DNA cassette containing two xenogeneic specific RRSs side-mounted with at least one first selectable marker; b) introducing a vector containing a second DNA cassette into the cells provided in a) the second DNA cassette containing two xenogeneic specific RRSs that match two RRSs on the integrated exogenous nucleotide sequence and side-mounted with at least one exogenous SOI and at least one second selectable marker; c) introducing a recombinase, wherein the recombinase recognizes the RRSs and performs one RMCE; and d) selecting TI cells expressing the second selectable marker to isolate TI host cells expressing the target peptide.

[0345] In some embodiments, this disclosure provides methods for preparing TI host cells to express a first target polypeptide and a second target polypeptide (wherein the first and second polypeptides may be the same or different), the methods comprising: a) providing TI host cells comprising a foreign nucleotide sequence at a locus integrated into the genome of the host cell, wherein the locus comprises all or a portion of a contig sequence of one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1 and NW_003615411.1 or a sequence at least about 90% homologous to SEQ ID No. 1 to 7, wherein the foreign nucleotide sequence comprises a first RRS and a second RRS side-mounted with at least one first selection marker and located between the first RRS and the second RRS. a) Introducing a first vector into the cells provided in a), the first vector comprising two RRSs that match the first and third RRSs on the integrated exogenous nucleotide sequence and are side-mounted with at least one first exogenous SOI and at least one second selectable marker; c) Introducing a second vector into the cells provided in a), the second vector comprising two RRSs that match the second and third RRSs on the integrated exogenous nucleotide sequence and are side-mounted with at least one second exogenous SOI; d) Introducing one or more recombinases that recognize the RRSs; and e) Selecting TI cells expressing the second selectable marker, thereby isolating TI host cells expressing the first and second target peptides. In some embodiments, instead of having the entire selection marker on the first vector, the first vector contains a promoter sequence operatively linked to the ATG codon with a first SOI upstream and an RRS downstream; and the second vector contains a selection marker lacking the ATG transcription start codon with an RRS upstream and a second SOI downstream.

[0346] In some embodiments, this disclosure provides methods for preparing TI host cells to express a first target polypeptide and a second target polypeptide (wherein the first and second polypeptides may be the same or different), the methods comprising: a) providing TI host cells containing exogenous nucleotide sequences at sites integrated within the following endogenous sequences: contigs including NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1 a) Introducing a first vector into the cells provided in a), the first vector containing two RRSs that match the first and third RRSs on the integrated foreign nucleotide sequence and are side-linked with at least one first foreign SOI and at least one second selection marker; c) Introducing a second vector into the cells provided in a), the second vector containing two RRSs that match the first and third RRSs on the integrated foreign nucleotide sequence and are side-linked with at least one first foreign SOI and at least one second selection marker. The process involves: d) matching and side-attaching at least one second exogenous SOI; d) introducing one or more recombinases that recognize RRS; and e) selecting TI cells expressing a second selection marker to isolate TI host cells expressing a first target polypeptide and a second target polypeptide. In some embodiments, instead of having the entire selection marker on the first vector, the first vector contains a promoter sequence operatively linked to the ATG codon, which is upstream side-attached to the first SOI and downstream side-attached to the RRS; and the second vector contains a selection marker lacking the ATG transcription start codon, which is upstream side-attached to the RRS and downstream side-attached to the second SOI.

[0347] In some embodiments, this disclosure provides methods for preparing TI host cells to express a first target polypeptide and a second target polypeptide (wherein the first and second polypeptides may be the same or different), the methods comprising: a) providing TI host cells comprising a foreign nucleotide sequence at a locus integrated into the genome of the host cell, wherein the locus is homologous to all or part of a sequence comprising one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1 and NW_003615411.1 or to at least about 90% homologous to SEQ ID Nos. 1 to 7, wherein the foreign nucleotide sequence comprises a first DNA cassette comprising a first RRS and a second RRS side-mounted with at least one first selection marker. a) Introducing a first vector containing a second DNA cassette into the cells provided in a), wherein the second DNA cassette contains two xenogeneic specific RRSs that match the first and third RRSs of the first DNA cassette and are side-attached with at least one first exogenous SOI and at least one second selection marker; c) Introducing a second vector containing a third DNA cassette into the cells provided in a), wherein the third DNA cassette contains two xenogeneic specific RRSs that match the second and third RRSs of the first DNA cassette and are side-attached with at least one second exogenous SOI; d) Introducing one or more recombinases that recognize the RRSs and perform RMCE twice; and e) Selecting TI cells expressing the second selection marker to isolate TI host cells expressing the first and second target peptides. In some embodiments, instead of having the entire selection marker on the first vector, the first vector contains a promoter sequence operatively linked to the ATG codon with a first SOI upstream and an RRS downstream; and the second vector contains a selection marker lacking the ATG transcription start codon with an RRS upstream and a second SOI downstream.

[0348] In some embodiments, this disclosure provides methods for preparing TI host cells to express a first target polypeptide and a second target polypeptide (wherein the first and second polypeptides may be the same or different), the methods comprising: a) providing TI host cells containing exogenous nucleotide sequences at sites integrated within the following endogenous sequences: contigs including NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1 a) Introducing into the cells provided in a) a first vector containing a second DNA cassette, wherein the second DNA cassette contains two xenogeneic specific RRSs that match the first and third RRSs of the first DNA cassette and are side-linked with at least one first exogenous SOI and at least one second selection marker; b) Introducing into the cells provided in a) a first vector containing a second DNA cassette, wherein the second DNA cassette contains two xenogeneic specific RRSs that match the first and third RRSs of the first DNA cassette and are side-linked with at least one first exogenous SOI and at least one second selection marker; c) Introducing into the cells provided in a) a first vector containing a third DNA cassette. The third DNA cassette comprises a second vector containing two xenogeneic specific RRSs that match the second and third RRSs of the first DNA cassette and are side-joined with at least one second exogenous SOI; d) introducing one or more recombinases that recognize the RRSs and perform RMCE twice; and e) selecting TI cells expressing a second selection marker to isolate TI host cells expressing the first and second target peptides. In some embodiments, instead of having the entire selection marker on the first vector, the first vector contains a promoter sequence operatively linked to the ATG codon, which is side-joined upstream with the first SOI and downstream with the RRS; and the second vector contains a selection marker lacking the ATG transcription start codon, which is side-joined upstream with the RRS and downstream with the second SOI.

[0349] In some embodiments, this disclosure provides methods for preparing TI host cells to express a target polypeptide, the methods comprising: a) providing TI host cells containing a foreign nucleotide sequence at a locus integrated into the genome of the host cell, wherein the locus is homologous to all or part of a sequence selected from one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1 or to at least about 90% homologous to SEQ ID No. 1 to 7, wherein the foreign nucleotide sequence contains an RRS adjacent to at least one first selective marker; b) introducing a vector into the cells provided in a) the vector containing an RRS, the RRS being The process involves: a) matching the RRS on the integrated exogenous nucleotide sequence and side-attaching at least one exogenous SOI and at least one second selectable marker; b) introducing a recombinase that recognizes the RRS; and c) selecting TI cells expressing the second selectable marker to isolate TI host cells expressing the target peptide.

[0350] In some embodiments, this disclosure provides methods for preparing TI host cells to express a target polypeptide, the methods comprising: a) providing TI host cells containing exogenous nucleotide sequences integrated into sites within endogenous sequences of all or part of a contiguous group consisting of one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and at least about 90% thereof. a) A homologous sequence, wherein the exogenous nucleotide sequence contains an RRS adjacent to at least one first selectable marker; b) Introducing a vector into the cells provided in a) the vector contains an RRS that matches an RRS on an integrated exogenous nucleotide sequence and is side-attached to at least one exogenous SOI and at least one second selectable marker; c) Introducing a recombinase, wherein the recombinase recognizes the RRS; and d) Selecting TI cells expressing the second selectable marker to isolate TI host cells expressing the target polypeptide.

[0351] The subject matter of this disclosure also relates to methods for generating a target peptide, the methods comprising: a) providing TI host cells as described herein; b) culturing the TI host cells of a) under conditions suitable for SOI expression, and recovering the target peptide therefrom.

[0352] In some embodiments, this disclosure provides methods for preparing TI host cells suitable for subsequent targeted integration, the methods comprising: a) providing TI host cells comprising a foreign nucleotide sequence at a locus within a host cell genome, wherein the locus is homologous to all or a portion of a sequence comprising one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1 or to a sequence selected from SEQ ID No. 1 to 7, wherein the foreign nucleotide sequence comprises two RRS sidebanded with at least one foreign SOI and at least one first selectable marker; b) to a) The method involves introducing a vector into cells containing two RRSs that match two RRSs on an integrated exogenous nucleotide sequence and side-attached at least one second selectable marker; c) introducing a recombinase that recognizes the RRSs; and d) selecting TI cells expressing the second selectable marker to isolate TI host cells suitable for subsequent targeted integration.

[0353] In some embodiments, this disclosure provides methods for preparing TI host cells suitable for subsequent targeted integration, the methods comprising: a) providing TI host cells containing exogenous nucleotide sequences at sites integrated within endogenous sequences: all or a portion of a contiguous sequence comprising one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and at least about 90% thereof. The method involves: a) introducing a homologous sequence, wherein the exogenous nucleotide sequence comprises two RRSs side-joined with at least one exogenous SOI and at least one first selectable marker; b) introducing a vector into the cells provided in a) the vector comprising two RRSs that match two RRSs on the integrated exogenous nucleotide sequence and side-joined with 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 isolate TI host cells suitable for subsequent targeted integration.

[0354] In some embodiments, this disclosure provides methods for preparing TI host cells suitable for subsequent targeted integration, the methods comprising: a) providing TI host cells comprising a foreign nucleotide sequence at a locus within a host cell genome, wherein the locus is homologous to all or a portion of a sequence comprising one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1 or to a sequence selected from SEQ ID No. 1 to 7, wherein the foreign nucleotide sequence comprises a first RRS and a second RRS sidebanded with at least one foreign SOI and at least one first selection marker; b) to a) The method involves introducing a vector into cells provided in the study, the vector containing three RRSs, wherein the first RRS of the vector matches a first RRS on the integrated foreign nucleotide sequence, the second RRS of the vector matches a second RRS on the integrated foreign nucleotide sequence, and at least one second selection marker is located between the first RRS and the second RRS; c) introducing a recombinase, wherein the recombinase recognizes the first RRS and the second RRS on both the vector and the integrated foreign nucleotide sequence; and d) selecting TI host cells expressing the second selection marker, thereby isolating TI host cells suitable for subsequent targeted integration.

[0355] In some embodiments, this disclosure provides methods for preparing TI host cells suitable for subsequent targeted integration, the methods comprising: a) providing TI host cells containing exogenous nucleotide sequences at sites integrated within endogenous sequences: all or a portion of a contiguous sequence comprising one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and at least about 90% thereof. The method involves: a) introducing a homologous sequence, wherein the exogenous nucleotide sequence comprises a first RRS and a second RRS side-mounted with at least one exogenous SOI and at least one first selectable marker; b) introducing a vector into the cells provided in a) the vector comprising three RRSs, wherein the first RRS of the vector matches the first RRS on the integrated exogenous nucleotide sequence, the second RRS of the vector matches the second RRS on the integrated exogenous nucleotide sequence, and at least one second selectable marker is located between the first RRS and the second RRS; c) introducing a recombinase, wherein the recombinase recognizes the first RRS and the second RRS on both the vector and the integrated exogenous nucleotide sequence; and d) selecting TI host cells expressing the second selectable marker to isolate TI host cells suitable for subsequent targeted integration.

[0356] 5.2 Methods for targeting and modifying host cells using homologous recombination, HDR, or NHEJ

[0357] In some embodiments, this disclosure provides methods for preparing TI host cells to express a target polypeptide, the methods comprising: a) providing TI host cells containing a locus of the host cell's genome, wherein the locus is at least about 90% homologous to SEQ ID Nos. 1 to 7; b) introducing a vector into the TI host cells, wherein the vector contains a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID Nos. 1 to 7 with a side-attached DNA cassette, wherein the DNA cassette contains at least one selection marker and at least one exogenous SOI; and c) selecting for the selection marker to isolate TI host cells having an SOI integrated into a locus of the genome and expressing the target polypeptide. In some embodiments, the DNA cassette of the vector further comprises at least one selection marker with two side-attached RRSs and at least one exogenous SOI.

[0358] In some embodiments, this disclosure provides methods for preparing TI host cells to express a target polypeptide, the methods comprising: a) providing TI host cells containing a locus of the host cell's genome, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to 7; b) introducing a polynucleotide into the TI host cell, wherein the polynucleotide contains a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID No. 1 to 7 with a side-attached DNA cassette, wherein the DNA cassette contains at least one selection marker and at least one exogenous SOI; and c) selecting for the selection marker to isolate TI host cells having an SOI integrated into a locus of the genome and expressing the target polypeptide. In some embodiments, the DNA cassette of the vector further comprises at least one selection marker with two side-attached RRSs and at least one exogenous SOI.

[0359] In some embodiments, homologous recombination is facilitated by an integration vector. In some embodiments, the vector is selected from the group consisting of: adenoviral vectors, adeno-associated virus vectors, lentiviral vectors, retroviral vectors, integration phage vectors, nonviral vectors, transposon and / or transposase vectors, integrase substrates, and plasmids. In some embodiments, the transposon may be a piggyBac (PB) transposon system.

[0360] In some embodiments, integration is facilitated by exogenous nucleases. In some embodiments, the exogenous nuclease is selected from the group consisting of: zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly spaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases.

[0361] In some embodiments, this disclosure provides methods for preparing TI host cells suitable for subsequent targeted integration, the methods comprising: a) providing TI host cells containing a locus of the host cell's genome, wherein the locus is homologous to all or a portion of a sequence containing one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1 or to a sequence selected from SEQ ID No. 1 to 7 at least about 90% homologous; b) introducing a vector into the TI host cells, wherein the vector contains side-attached DNA. The DNA cassette contains all or part of the contig sequence of one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1 and NW_003615411.1, or a nucleotide sequence that is at least 50% homologous to a sequence selected from SEQ ID No. 1 to 7, wherein the DNA cassette contains at least one selection marker with two RRSs attached to its sides; c) Selecting against the selection marker to isolate host cells suitable for subsequent targeted integration of TI.

[0362] In some embodiments, this disclosure provides methods for preparing TI host cells suitable for subsequent targeted integration, the methods comprising: a) providing TI host cells containing a locus of the host cell's genome, wherein the locus is homologous to all or a portion of a sequence containing one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1 or to a sequence selected from SEQ ID No. 1 to 7 by at least about 90%; b) introducing a polynucleotide into the TI host cell, wherein the polynucleotide contains side-attached DNA. The DNA cassette contains all or part of the contig sequence of one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1 and NW_003615411.1, or a nucleotide sequence that is at least 50% homologous to a sequence selected from SEQ ID No. 1 to 7, wherein the DNA cassette contains at least one selection marker with two RRSs attached to its sides; c) Selecting against the selection marker to isolate host cells suitable for subsequent targeted integration of TI.

[0363] In some embodiments, this disclosure provides methods for preparing TI host cells suitable for subsequent targeted integration, the methods comprising: a) providing TI host cells containing a locus of the host cell's genome, wherein the locus is homologous to all or a portion of a sequence containing one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1 or to a sequence selected from SEQ ID No. 1 to 7 at least about 90% homologous; b) introducing a vector into the host cell, wherein the vector contains side-attached DNA. The DNA cassette comprises all or a portion of the contig sequence of one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1 and NW_003615411.1, or a nucleotide sequence that is at least 50% homologous to a sequence selected from SEQ ID No. 1 to 7, wherein the DNA cassette contains three RRSs, wherein a third RRS and at least one selection marker are located between the first RRS and the second RRS; and c) selecting for the selection marker to isolate TI host cells suitable for subsequent targeted integration.

[0364] In some embodiments, this disclosure provides methods for preparing TI host cells suitable for subsequent targeted integration, the methods comprising: a) providing TI host cells containing a locus of the host cell's genome, wherein the locus is homologous to all or a portion of a sequence containing one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1 or to a sequence selected from SEQ ID No. 1 to 7 by at least about 90%; b) introducing a polynucleotide into the host cell, wherein the polynucleotide contains side-attached DNA. The DNA cassette comprises all or a portion of the contig sequence of one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1 and NW_003615411.1, or a nucleotide sequence that is at least 50% homologous to a sequence selected from SEQ ID No. 1 to 7, wherein the DNA cassette contains three RRSs, wherein a third RRS and at least one selection marker are located between the first RRS and the second RRS; and c) selecting for the selection marker to isolate TI host cells suitable for subsequent targeted integration.

[0365] In some embodiments, this disclosure provides methods for preparing TI host cells expressing at least one target polypeptide, the methods comprising: a) providing TI host cells comprising at least one exogenous nucleotide sequence at a site integrated into one or more loci in the genome of the TI host cell, wherein the one or more loci are homologous to all or part of a sequence comprising a contiguous group sequence comprising one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1 and NW_003615411.1 or to a sequence selected from SEQ ID No. 1 to 7 at least 90% homologous, wherein the at least one exogenous nucleotide sequence comprises two RRS side-joined with at least one first selectable marker; b) Introduce a vector into the cells provided in a), the vector containing two RRS that match two RRS on an integrated exogenous nucleotide sequence and side-attached to at least one exogenous SOI and at least one second selectable marker; c) introduce a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRS; and select TI cells expressing the second selectable marker to isolate TI host cells expressing the at least one target polypeptide.

[0366] In some embodiments, this disclosure provides methods for preparing TI host cells expressing at least one first target polypeptide and a second target polypeptide (wherein the first and second polypeptides may be the same or different), the methods comprising: a) providing TI host cells comprising at least one exogenous nucleotide sequence at a site integrated into one or more loci of the host cell's genome, wherein the one or more loci are at least 90% homologous to a sequence comprising all or a portion of a contiguous group sequence comprising one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1 or to a sequence selected from SEQ ID No. 1 to 7, wherein the exogenous nucleotide sequence comprises a first RRS and a second RRS side-joined with at least one first selection marker. a) and a third RRS located between the first RRS and the second RRS, and all RRS are heterospecific; b) introducing a first vector into the cells provided in a), the first vector containing two RRSs that match the first and third RRSs on at least one integrated exogenous nucleotide sequence and are side-attached to at least one first exogenous SOI and at least one second selection marker; c) introducing a second vector into the cells provided in a), the second vector containing two RRSs that match the second and third RRSs on at least one integrated exogenous nucleotide sequence and are side-attached to 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 RRSs; and e) selecting TI cells expressing the second selection marker, thereby isolating TI host cells expressing at least one first target polypeptide and a second target polypeptide. In some embodiments, instead of having the entire selection marker on the first vector, the first vector contains a promoter sequence operatively linked to the ATG codon with a first SOI upstream and an RRS downstream; and the second vector contains a selection marker lacking the ATG transcription start codon with an RRS upstream and a second SOI downstream.

[0367] In some embodiments, this disclosure provides methods for preparing TI host cells expressing a target polypeptide, the methods comprising: a) providing TI host cells comprising at least one exogenous nucleotide sequence at a site integrated into one or more loci of the genome of the TI host cell, wherein the one or more loci are at least about 90% homologous to a sequence of a contiguous group of one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1 and NW_003615411.1 or to a sequence selected from SEQ ID No. 1 to 7, wherein the exogenous nucleotide sequence comprises one or more RRS; b) introducing a vector into the cells provided in a) the vector comprising one or more RRS, the one or more RRS being a) matching one or more RRSs on the integrated exogenous nucleotide sequence and operatively tethered to a tunable promoter at least one exogenous SOI; b) introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRS; and c) selecting TI cells that express the exogenous SOI in the presence of an inducer, thereby isolating TI host cells expressing the target polypeptide.

[0368] In some embodiments, this disclosure provides methods for expressing a target polypeptide, the methods comprising: a) providing a host cell containing at least one exogenous SOI with two RRSs side-mounted at a locus integrated into the host cell's genome and a tunable promoter, wherein the locus is homologous to all or a portion of a sequence comprising one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1 and NW_003615411.1 or to a sequence selected from SEQ ID No. 1 to 7 at least about 90% homologous; and b) culturing the cell under conditions suitable for SOI expression and recovering the target polypeptide therefrom.

[0369] In some embodiments, this disclosure provides methods for preparing TI host cells expressing a first target polypeptide and a second target polypeptide (wherein the first and second polypeptides may be the same or different), the methods comprising: a) providing TI host cells comprising a foreign nucleotide sequence at a locus integrated into the genome of the host cell, wherein the locus is homologous to all or part of a sequence comprising one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1 and NW_003615411.1 or to a sequence selected from SEQ ID No. 1 to 7, wherein the foreign nucleotide sequence comprises a first RRS, a second RRS, and a sequence located between the first RRS and the second RRS. a) Introducing a first vector into the cells provided in a), the first vector comprising two RRSs that match the first and third RRSs on the integrated exogenous nucleotide sequence and are operatively tethered to at least one first exogenous SOI of a tunable promoter; c) Introducing a second vector into the cells provided in a), the second vector comprising two RRSs that match the second and third RRSs on the integrated exogenous nucleotide sequence and are operatively tethered to at least one second SOI of a tunable promoter; 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 cells expressing the first and second exogenous SOIs in the presence of an inducer, thereby isolating TI host cells expressing the target polypeptide. In some embodiments, instead of having the entire selection marker on the first vector, the first vector contains a promoter sequence operatively linked to the ATG codon with a first SOI upstream and an RRS downstream; and the second vector contains a selection marker lacking the ATG transcription start codon with an RRS upstream and a second SOI downstream.

[0370] 6. Products

[0371] The RVLP-reduced CHO host cells of this disclosure can be used to express any target molecule (e.g., a target peptide). In some embodiments, the host cells of this disclosure can be used to express peptides (e.g., mammalian peptides). Non-limiting examples of such peptides include hormones, receptors, fusion proteins, regulatory factors, growth factors, complement system factors, enzymes, coagulation factors, anticoagulation factors, kinases, cytokines, CD proteins, interleukins, therapeutic proteins, diagnostic proteins, and antibodies. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a therapeutic antibody. In some embodiments, the antibody is a diagnostic antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a humanized antibody.

[0372] In some embodiments, the target peptide is a bispecific antibody, a trispecific antibody, or a multispecific antibody, such as a bispecific antibody. Various molecular forms of multispecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol Immunol 67 (2015) 95-106). Also included herein is a particular type of multispecific antibody that is a bispecific antibody designed to simultaneously bind to a surface antigen on a target cell (e.g., tumor cells) and an activation-invariant component of the T cell receptor (TCR) complex (such as CD3) for retargeting T cells to kill the target cells. Other examples of bispecific antibody forms include, but are not limited to, so-called “BiTE” (bispecific T-cell conjugate) molecules, in which two scFv molecules are fused via a flexible linker (see, for example, WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261, and WO 2008 / 119567; Nagorsen and Bäuerle, Exp Cell Res 317, 1255-1260 (2011)); bispecific antibodies (Holliger et al., ProtEng 9, 299-305 (1996)) and their derivatives, such as tandem bispecific antibodies (“TandAb”; Kipriyanov et al., JMol Biol 293, 41-56 (1999)); and “DART” (dual affinity retargeting) molecules, which are based on bispecific antibody forms but are characterized by a C-terminal disulfide bridge for achieving additional stabilization (Johnson). (e.g., J Mol Biol 399, 436-449 (2010)), and so-called triomab, which is a fully hybridized mouse / rat IgG molecule (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). The specific T-cell bispecific antibody forms included in this article are described in the following references: WO 2013 / 026833; WO 2013 / 026839; WO2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

[0373] Therapeutic antibodies include, but are not limited to, anti-HER receptor family antibodies (such as anti-HER1 (EGFR), anti-HER2, anti-HER3, and anti-HER4); anti-CD protein antibodies (such as anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD20, anti-CD21, anti-CD22, anti-CD25, anti-CD33, anti-CD34, anti-CD38, and anti-CD52); anti-IL-8 antibodies; anti-VEGF antibodies; anti-CD40 antibodies, anti-CD11a antibodies; anti-CD18 antibodies; anti-IgE antibodies; anti-Apo-2 receptor antibodies; anti-tissue factor (TF) antibodies; and anti-cell adhesion molecules such as LFA-1, Mol, p150,95, VLA-4, ICAM-1, VCAM, anti-human α4β7 integrin antibodies, and anti-human α vβ8 integrin antibodies, anti-αvβ3 antibodies including their α or β or subunits (e.g., anti-CD11a, anti-CD18, or anti-CD11b antibodies); anti-EGFR antibodies; anti-Fc receptor antibodies; anti-carcinoembryonic antigen (CEA) antibodies; anti-human renal cell carcinoma antibodies; anti-human colorectal tumor antibodies; anti-human melanoma antibody R24 against GD3 gangliosides; anti-human squamous cell carcinoma; antibodies against mammary epithelial cells; antibodies binding to colon cancer cells; anti-EpCAM antibodies; anti-GpIIb / IIIa antibodies; anti-RSV antibodies; anti-CMV antibodies; anti-HIV antibodies; anti-hepatitis antibodies; anti-CA125 antibodies; anti-human 17-1A antibodies; and anti-human leukocyte antigen (HLA) antibodies and anti-HLA DR antibodies; anti-growth factors, such as vascular endothelial growth factor (anti-VEGF) or fragments; anti-IgE; anti-blood group antigens; anti-flk2 / flt3. Receptors; and anti-obesity (OB) receptors.Other exemplary proteins for designing therapeutic antibodies include anti-amyloid antibodies, anti-α-synuclein (e.g., prasinezumab), anti-amyloid β, anti-growth hormone (GH) (including human growth hormone (HGH) and bovine growth hormone (bGH)); growth hormone-releasing factor; parathyroid hormone; thyroid-stimulating hormone; lipoproteins; α-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle-stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VIIIC, tissue factor, or von Willebrands factor; anticoagulation factors such as protein C; atrial natriuretic factor; pulmonary surfactant; plasminogen activator such as urokinase or tissue-type plasminogen activator (t-PA); bombazine; thrombin; tumor necrosis factor-α and-β; enkephalin; and RANTES (regulators of normal T... Cellular expression and secretion of activating factors); human macrophage inflammatory protein (MIP-1-α); serum albumin, such as human serum albumin (HSA); Müllerian duct inhibitory substance; relaxin A chain; relaxin B chain; pro-relaxin; mouse gonadotropin-related peptide; DNase; inhibin; activin; hormone or growth factor receptor; protein A or D; rheumatoid factor; neurotrophic factors such as bone-derived neurotrophic factor (BDNF), neurotrophic factor-3,-4,-5 or-6 (NT-3, NT-4, NT-5 or NT-6), or nerve growth factors such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factors such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF), such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I); insulin-like growth factor binding protein (IGFBP); erythropoietin (EPO); thrombopoietin (TPO); bone-inducing factor; immunotoxin; bone morphogenetic protein (BMP); interferons such as interferon-α, -β and -γ; colony-stimulating factors (CSF), such as M-CSF, GM-CSF and G-CSF; interleukins (ILS), such as IL-1 to IL-10; superoxide dismutase; T cell receptor; surface membrane protein; decay accelerator factor (DAF); viral antigens, such as, for example, part of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; immunoadhesins; and biologically active fragments or variants of any of the polypeptides listed above.The use of many other antibodies and / or other proteins in this disclosure, as well as the list above, is not intended to be limiting.

[0374] Specific therapeutic antibodies include those in clinical practice or under development, such as commercially available AVASTIN® (bevacizumab), HERCEPTIN® (trastuzumab), LUCENTIS® (ranibizumab), RAPTIVA® (efalizumab), RITUXAN® (rituximab), and XOLAIR® (omalizumab), anti-β amyloid (Aβ), anti-CD4 (MTRX1011A), anti-EGFL7 (EGF-like domain 7), anti-IL13, Apomab (anti-DR5 targeting apoptosis receptor agonist (PARA)), anti-BR3 (CD268), anti-BLyS receptor 3, anti-BAFF-R (BAFF receptor), anti-β7 integrin subunit, and anti-α v β8 integrin antibody, dasi-1 antibody (anti-CD40), GA101 (anti-CD20 monoclonal antibody), MetMAb (anti-MET receptor tyrosine kinase), anti-neuropiliin-1 (NRP1), OCREVUS ® (Aureiz monoclonal antibody - anti-CD20 antibody), anti-OX40 ligand, antioxidant LDL (oxLDL), PERJETA ® (Pertuzumab - HER dimerization inhibitor (HDI)), TECENTRIQ ® (Anti-PD-L1 antibody), anti-CD79b antibody, LUNSUMIO ® Or COLUMVI TM (Anti-CD20 X anti-CD3 bispecific antibody), VABYSMO ® (Anti-VEGF-A X anti-angiogenic-2 bispecific antibody), rhuMAb IFNα, etc. Many other antibodies and / or other proteins used in this disclosure, as well as the list above, are not intended to be limiting.

[0375] The host cells disclosed herein can be used to produce target molecules on a production scale. "Manufacturing-scale" production of therapeutic proteins or other proteins utilizes cell cultures ranging from about 400 L to about 80,000 L, depending on the protein being produced and the demand. Typically, such manufacturing-scale production utilizes cell cultures ranging from about 400 L to about 25,000 L. Within this range, specific cell cultures such as 4,000 L, about 6,000 L, about 8,000 L, about 10,000 L, about 12,000 L, about 14,000 L, or about 16,000 L can be used.

[0376] The host cells of this disclosure can be used to produce large quantities of target molecules in a shorter time compared to non-TI cells used in current cell culture methods. In some embodiments, the host cells of this disclosure can be used to improve the quality of target molecules compared to non-TI cells used in current cell culture methods. In some embodiments, the host cells of this disclosure can enhance the stability of seed series by preventing chronic toxicity caused by products that may induce cellular stress and clonal instability over time. In some embodiments, the host cells of this disclosure can be used to optimize the expression of acutely toxic products.

[0377] In some embodiments, the host cell (TI system) disclosed herein can be used for cell culture process optimization and / or process development.

[0378] In some embodiments, the host cells of this disclosure can be used to accelerate the production of target molecules by about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, or about 10 weeks compared to non-TI cells used in conventional cell culture methods. In some embodiments, the host cells of this disclosure can be used to accelerate the harvest of target molecules by about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, or about 10 weeks compared to non-TI cells used in conventional cell culture methods.

[0379] In some embodiments, the host cells of this embodiment can be used to reduce the aggregation level of the target molecule compared to non-TI cells used in conventional cell culture methods.

[0380] In some embodiments, the host cells of this disclosure can be used to achieve higher levels of expression of the one or more target peptides relative to host cells in which exogenous sequences expressing one or more target peptides are randomly integrated. For example, but not as a limitation, the host cells of this disclosure can express standard antibodies and haptens at titers of at least 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L, 6.5 g / L, 7 g / L, 7.5 g / L, 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, 10 g / L, 10.5 g / L, 11 g / L or higher, and can express multispecific antibodies (e.g., bispecific antibodies) at titers of at least 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L or higher. In some embodiments, the host cells of this disclosure can achieve higher levels of bispecificity compared to host cells in which one or more exogenous sequences expressing bispecific content are randomly integrated. For example, but not as a limitation, the host cells disclosed herein can achieve a bispecificity content of at least 80%, 85%, 90%, 95%, 96%, 98%, 99% or higher.

[0381] In some embodiments, the host cells of this disclosure can be used as research tools. In some embodiments, the host cells of this disclosure can be used as diagnostic tools to identify the root cause of low protein expression levels of problem molecules in various cells. In some embodiments, the host cells of this disclosure can be used to directly link observed phenomena or cellular behaviors to transgene expression in cells. The host cells of this disclosure can also be used to demonstrate whether observed behaviors are reversible in cells. In some embodiments, the host cells of this disclosure can be used to identify and alleviate problems related to transgene transcription and expression in cells.

[0382] 7. Example

[0383] 7.1 Inactivation of the ERV gene: RVLP titer and integral viable cell concentration

[0384] CRISPR reagents. Guide RNAs targeting the Gag genes of CHERV-3g and CHERV-1b for insertion / deletion (ID) knockout were designed using CRISPOR software (Concordet JP, Haeussler M. CRISPOR:intuitive guide selection for CRISPR / Cas9 genome editing experiments and screens. Nucleic Acids Res. 2018 Jul 2;46(W1):W242-W245. doi: 10.1093 / nar / gky354). Guide RNAs targeting the Gag genes of CHERV-3g and CHERV-1b for base editing (BE) knockout were designed using BE-Designer software (Hwang, GH., Park, J., Lim, K. et al. Web-based design and analysis tools for CRISPR base editing. BMC Bioinformatics 19, 542 (2018). https: / / doi.org / 10.1186 / s12859-018-2585-4). All ID guide RNAs were chemically synthesized by Integrated DNA Technologies, Inc.

[0385] Guide RNAs targeting flanking regions of the CHERV-3g and CHERV-1b genomes for deletion (DL) knockout were designed using CRISPOR software (Concordet JP, Haeussler M. CRISPOR: intuitive guide selection for CRISPR / Cas9 genome editing experiments and screens. Nucleic Acids Res. 2018 July 2;46(W1):W242-W245. doi: 10.1093 / nar / gky354). All DL guide RNAs were chemically synthesized by Integrated DNA Technologies, Inc.

[0386] The Cas9 nuclease was purchased from Integrated DNA Technologies, Inc., and the plasmid used to express AncBE4max (Koblan, L., Doman, J., Wilson, C. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat Biotechnol 36, 843–846 (2018). https: / / doi.org / 10.1038 / nbt.4172) was chemically synthesized by Genscript Biotech.

[0387] Transfection. For the results depicted in Figures 1A-1D, knockout was performed using a CHO-K1-derived targeted integration host cell line (lineage A) with a known deletion at the ETC109F locus. For insertion-deletion knockout, the Cas9 protein, along with CHERV-3g Gag sgRNA and CHERV-1b Gag sgRNA, was conjugated into an RNP and electroporated into cells using a Neon transfection system (ThermoFisher). Cells were transfected again under the same conditions four days later. For base editing knockout, the AncBE4max plasmid, along with CHERV-3g Gag sgRNA and CHERV-1b Gag sgRNA, was electroporated into cells using a Neon transfection system (ThermoFisher).

[0388] Regarding the results depicted in Figures 2B-2C, knockout was performed using a CHO-K1-derived targeted integration host cell line, a “host control,” with a known deletion at the ETC109F locus. For deletion knockout, the Cas9 protein, along with CHERV-3g 5' flanking sgRNA, CHERV-3g 3' flanking sgRNA, CHERV-1b 5' flanking sgRNA, and CHERV-1b 3' flanking sgRNA, were complexed into RNPs and electroporated into cells using a Neon transfection system (ThermoFisher). Two days later, cells were transfected again under the same conditions.

[0389] Single-cell cloning. For the results depicted in Figures 1A-1D, knockout pool single-cell clones were cloned into 384-well plates using limiting dilution (0.4 cells / well). Day 14 confluence wells were scaled up to 96-well plates. Culture supernatants were treated with TurboDNase (ThermoFisher), and CHERV-3g, CHERV-1b, and Actb titers were quantified using a one-step RT-ddPCR Advanced Kit for probes (Bio-Rad). Clones with the lowest CHERV-3g + CHERV-1b titer (relative to Actb) were amplified, along with control clones without CHERV-3g KO, CHERV-1b KO, or both CHERV-3g and CHERV-1b KO.

[0390] For the results depicted in Figures 2B-2C, knockout pool single-cell clones were cloned into 384-well plates using an UP.SIGHT single-cell dispenser (Cytena). Day 14 confluence wells were scaled up to 96-well plates. Clones were screened for large deletions at the CHERV-3g and CHERV-1b loci by genomic PCR. Furthermore, the culture supernatant was treated with TurboDNase (ThermoFisher), and the titers of CHERV-3g, CHERV-1b, and Actb were quantified using a one-step RT-ddPCR advanced probe kit (Bio-Rad). Clones with the target genotypes were amplified and evaluated through production culture.

[0391] Production Culture. RVLP KO clones were cultured in shake flasks using chemically defined media and feed in a 7-day fed batch culture. Cell counts were performed periodically to calculate the integrated viable cell concentration (IVCC). The supernatant was harvested on day 7 and treated with Turbo DNase (ThermoFisher). RNA was extracted using the MagNA Pure 96 system (Roche Diagnostics). The extracted RNA was evaluated for titers of CHERV-3g, CHERV-1b, and CHERV-2g using a one-step RT-ddPCR advanced probe kit (Bio-Rad). The total RVLP titer was calculated by summing the titers of CHERV-3g, CHERV-1b, and CHERV-2g.

[0392] TEM titration. Cell cultures were harvested by centrifugation at 250 x g for 10 min. The supernatant was submitted to Charles River Laboratories for RVLP quantification by TEM analysis. Briefly, 40 mL of the supernatant was ultracentrifuged and the precipitate was fixed in 2% glutaraldehyde and 0.1 M sodium dimethylarsinate. Thin sections of the precipitate were cut at 70–90 nm and mounted on a 200-mesh copper grid, stained with uranyl methyl acetate and lead renin-Citrate, and examined by TEM. Ten grid spaces were identified and calculated for particles with retrovirus-like morphology by trained technicians. RVLP titers were calculated based on grid cross-sectional volume, precipitate volume, and sample volume.

[0393] Results. Figure 1A depicts the expression distribution of the three ERV substances in the Gag knockout clone and the control, where ID corresponds to insertion / deletion knockout and BE corresponds to base editing as discussed above. Figure 1B depicts the total RVLP titer (the sum of the three ERVs) in the Gag knockout clone and the control. Figure 1C depicts the production culture integral viable cell concentration (IVCC) in the Gag knockout clone and the control. Figure 1D shows that, as assessed by TEM, the Gag knockout clone produces less RVLP. In fact, in two of the three knockout clones, the RVLP titer was below the detection limit.

[0394] Figure 2A depicts genomic loci identified by BLAST and / or RNA sequencing and verified by genomic PCR and sequencing to match the RVLP sequences of CHERV-3g and CHERV-1b. CHERV-3g was found to be present in the CHO genome assembly (GCF_003668045.3). Two alleles of CHERV-3g were found in CHO cells, with slight differences between the two alleles. CHERV-1b was found to exist as a copy and was not present in the CHO genome assembly. A portion of the 3'LTR of CHERV-1b was truncated, and long telomere-like repeat sequences were present, also on the suspected 3' flanking genomic sequences at the integration site. Guide RNAs targeting the CHO genome regions flanking these two loci were designed, and four sites were simultaneously targeted using CRISPR / Cas9 to stimulate massive deletions. Figures 2B and 2C show that DL-8 and DL-17 exhibited reduced RVLP titers by RT-ddPCR and TEM. RT-ddPCR showed that DL-11 and DL-19 exhibited decreased CHERV-3g titers but high CHERV-1b titers. Therefore, DL-19 showed a slight decrease in TEM titer. However, DL-11 showed a significant decrease in TEM titer.

Claims

1. A modified Chinese hamster ovary (CHO) cell, wherein prior to modification, the CHO cell contains two or more endogenous retroviral (ERV) loci selected from: (a) CHERV-1b (Genbank accession number MN527960, SEQ ID NO. 8); (b) CHERV-2g (Genbank accession number MN527961, SEQ ID NO. 9); (c) ETC109F (SEQ ID NO. 10, 30021-39247); (d) CHERV-3g (Genbank accession number MN527962, SEQ ID NO. 11); and (e) An ERV locus containing a sequence that has at least 90% identity with any of (a) to (d). And the modification described therein includes the inactivation of two or more of the ERV loci in (a) to (e).

2. The modified CHO cells according to claim 1, wherein the inactivation of the ERV locus comprises the knockout of the corresponding ERV GAG coding sequence.

3. The modified CHO cells of claim 2, wherein knockout of each corresponding ERV GAG coding sequence comprises introducing an insertion deletion into the ERV GAG coding sequence.

4. The modified CHO cells of claim 2, wherein knockout of each corresponding ERV GAG coding sequence comprises introducing base editing into the ERV GAG coding sequence.

5. The modified CHO cells of claim 2, wherein knockout of each corresponding ERV GAG coding sequence includes a deletion introduced flanking the ERV GAG coding sequence.

6. The modified CHO cells of claim 1, wherein the inactivation of one or more of the ERV loci comprises the deletion of at least one ERV locus.

7. The modified CHO cells of claim 6, wherein inactivation of one or more of the ERV loci comprises deletion of each ERV locus.

8. The modified CHO cells of claim 6, wherein inactivation of one or more of the ERV loci comprises deletion of the ERV locus and introduction of insertion / deletion or base editing into the ERVGAG coding sequence of the ERV locus.

9. The modified CHO cells according to any one of claims 1 to 8, wherein the modified cells express the target recombinant product.

10. The modified CHO cells according to any one of claims 1 to 8, wherein the modified cells are generated from recombinant cells expressing the desired recombinant product.

11. The modified CHO cells according to claim 9 or 10, wherein the desired recombinant product comprises a recombinant protein.

12. The modified CHO cells according to claim 11, wherein the recombinant protein is an antibody or an antigen-binding fragment thereof.

13. The modified CHO cells according to claim 12, wherein the antibody is a multispecific antibody or an antigen-binding fragment thereof.

14. The modified CHO cells of claim 13, wherein the antibody comprises a single heavy chain sequence and a single light chain sequence or an antigen-binding fragment thereof.

15. The modified CHO cells according to any one of claims 12 to 14, wherein the antibody is a chimeric antibody, a human antibody, or a humanized antibody.

16. The modified CHO cells according to any one of claims 12 to 15, wherein the antibody is a monoclonal antibody.

17. The modified CHO cells according to claim 12, wherein the antibody is selected from: bevacizumab; trastuzumab; ranibizumab; efalizumab; rituximab; omalizumab; anti-β amyloid (Aβ) antibody; anti-CD4 (MTRX1011A) antibody; anti-EGFL7 (EGF-like domain 7) antibody; anti-IL13 antibody; anti-Apomab antibody; anti-DR5-targeted apoptosis-promoting receptor agonist (PARA) antibody; anti-BR3 antibody; anti-CD268 antibody; anti-BLyS receptor 3 antibody; anti-BAFF-R (BAFF receptor) antibody; anti-β7 integrin subunit antibody; anti-α v β8 integrin antibody; Darcy's group monoclonal antibody (anti-CD40); GA101 (anti-CD20 monoclonal antibody); MetMAb (anti-MET receptor tyrosine kinase antibody); anti-neuropiliin-1 (NRP1) antibody; Oreogroup monoclonal antibody (anti-CD20 antibody); anti-OX40 ligand antibody; anti-oxidative LDL (oxLDL) antibody; pertuzumab (HER dimerization inhibitor (HDI)); anti-PD-L1 antibody; anti-CD79b antibody; anti-CD20 X anti-CD3 bispecific antibody; anti-VEGF-A X anti-angiogenic-2 bispecific antibody; and rhuMAb IFNα.

18. The modified CHO cell according to claim 10 or 11, wherein the target recombinant product is encoded by an exogenous nucleic acid sequence integrated into the cellular genome of the CHO cell at one or more target sites.

19. The modified CHO cells of claim 18, wherein the target site is a sequence that is at least about 90% homologous to a portion of the contiguous group sequence of one of the contiguous groups NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1 and NW_003615411.1 or to a sequence selected from SEQ ID No. 1 to 7.

20. The modified CHO cells of claim 18, wherein the target site is a sequence of 1000 nucleotides that is at least about 50% homologous to the following sequences: NW_006874047.1 containing position 45269; NW_006884592.1 containing position 207911; NW_006881296.1 containing position 491909; NW_003616412.1 containing position 79768; NW_003615063.1 containing position 315265; NW_006882936.1 containing position 2662054; or NW_003615411.1 containing position 97705.

21. The modified CHO cells according to claim 19 or 20, wherein the modified CHO cells comprise gene knockouts selected from: a) BAX; BAK; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; b) BAX; BAK; ICAM-1; SIRT-1; GGTA1; CMAH; LPL, LPLA2; and PPT1; c) BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; d) BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; e) BAX; BAK; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; f) BAX; BAK; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; g) BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; h) BAX; BAK; ICAM-1; LPL; LPLA2; and PPT1; i) BAX; BAK; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; j) BAX; BAK; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1; k) BAX; BAK; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA; l) BAX; BAK; ICAM-1; SIRT-1; and MYC; m) BAX; BAK; ICAM-1; PERK; SIRT-1; and MYC; n) BAX; BAK; ICAM-1; SIRT-1; PERK; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; o) BAX; BAK; LPL; LPLA2; GGTA1; and CMAH; p) BAX;BAK;PERK;LPL;LPLA2;GGTA1;and CMAH;q) BAX;BAK;MYC;LPL;LPLA2;GGTA1;and CMAH;r) BAX;BAK;MYC;PERK;LPL;LPLA2;GGTA1;and CMAH;s) BAX;BAK;LPL;LPLA2;GGTA1;CMAH;and PPT1;t) BAX; BAK; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; u) BAX; BAK; MYC; LPL;LPLA2; GGTA1; CMAH; and PPT1; v) BAX; BAK; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; w) BAX; BAK; ICAM-1; and SIRT-1; x) BAX; BAK; and ICAM-1; y) BAX; BAK; BCKDHA; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; z) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; aa) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; bb) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; cc) BAX; BAK; BCKDHA; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; dd) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ee) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ff) BAX; BAK; BCKDHA; ICAM-1; LPL; LPLA2; and PPT1; gg) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; hh) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1; ii) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA; jj) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; and MYC; kk) BAX; BAK; BCKDHA; ICAM-1; PERK; SIRT-1; and MYC; ll) BAX; BAK; BCKDHA; ICAM-1; PERK; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; mm) BAX; BAK; BCKDHA; LPL; LPLA2; GGTA1; and CMAH; nn) BAX; BAK; BCKDHA; PERK; LPL; LPLA2; GGTA1;and CMAH; oo) BAX; BAK; BCKDHA; MYC; LPL; LPLA2; GGTA1; and CMAH; pp) BAX; BAK; BCKDHA; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH; qq) BAX; BAK; BCKDHA; LPL; LPLA2; GGTA1; CMAH; and PPT1; rr) BAX; BAK; BCKDHA; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; ss) BAX; BAK; BCKDHA; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1; tt) BAX; BAK; BCKDHA; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; uu) BAX; BAK; BCKDHA; ICAM-1; and SIRT-1; vv) BAX; BAK; BCKDHA; and ICAM-1; ww) BAX; BAK; BCKDHB; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; xx) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; yy) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; zz) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; aaa) BAX; BAK; BCKDHB; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; bbb) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ccc) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ddd) BAX; BAK; BCKDHB; ICAM-1; LPL; LPLA2; and PPT1; eee) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; fff) BAX; BAK; BCKDHB; ICAM-IP; SIRT-1; MYC; LPL; LPLA2; and PPT1; ggg) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; LPL;LPLA2; PPT1; and LIPA; hhh) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; and MYC; iii) BAX; BAK; BCKDHB; ICAM-1; PERK; SIRT-1; and MYC; jjj) BAX; BAK; BCKDHB; ICAM-1; PERK; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; kkk) BAX; BAK; BCKDHB; LPL; LPLA2; GGTA1; and CMAH; lll) BAX; BAK; BCKDHB; PERK; LPL; LPLA2; GGTA1; and CMAH; mmm) BAX; BAK; BCKDHB; MYC; LPL; LPLA2; GGTA1; and CMAH; nnn) BAX; BAK; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH; ooo) BAX; BAK; BCKDHB; LPL; LPLA2; GGTA1; CMAH; and PPT1; ppp) BAX; BAK; BCKDHB; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; qqq) BAX; BAK; BCKDHB; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1; rrr) BAX; BAK; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; sss) BAX; BAK; BCKDHB; ICAM-1; and SIRT-1; ttt) BAX; BAK; BCKDHB; and ICAM-1; uuu) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; vvv) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; www) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; xxx) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; yyy) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; zzz) BAX; BAK;BCKDHA; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; aaaa) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; bbbb) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; LPL; LPLA2; and PPT1; cccc) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; dddd) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1; eeee) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA; ffff) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; and MYC; gggg) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; PERK; SIRT-1; and MYC; hhhh) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; PERK; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; iiiii) BAX; BAK; BCKDHA; BCKDHB; LPL; LPLA2; GGTA1; and CMAH; jjjj) BAX; BAK; BCKDHA; BCKDHB; PERK; LPL; LPLA2; GGTA1; and CMAH; kkkk) BAX; BAK; BCKDHA; BCKDHB; MYC; LPL; LPLA2; GGTA1; and CMAH; llll) BAX; BAK; BCKDHA; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH; mmmm) BAX; BAK; BCKDHA; BCKDHB; LPL; LPLA2; GGTA1; CMAH; and PPT1; nnnn) BAX; BAK; BCKDHA; BCKDHB; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; oooo) BAX; BAK; BCKDHA; BCKDHB; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1; pppp) BAX; BAK; BCKDHA; BCKDHB; MYC; PERK; LPL;LPLA2; GGTA1; CMAH; and PPT1; qqqq) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; and SIRT-1; or rrrr) BAX; BAK; BCKDHA; BCKDHB; and ICAM-1.

22. A method for producing modified CHO cells, the method comprising: (a) Contacting cells with a nuclease-assisted gene-targeting system and / or nucleic acids that target at least two endogenous ERVs, wherein the endogenous ERVs are selected from: (i) CHERV-1b (Genbank accession number MN527960, SEQ ID NO. 8); (ii) CHERV-2g (Genbank accession number MN527961, SEQ ID NO. 9); (iii) ETC109F (SEQ ID NO. 10, 30021-39247); (iv) CHERV-3g (Genbank accession number MN527962, SEQ ID NO. 11); and (v) An ERV locus containing a sequence that has at least 90% identity with any of (i) to (iv), and (b) Select modified CHO cells in which the expression of the ERV has been reduced or eliminated compared with unmodified CHO cells.

23. The method of claim 22, wherein, after the modification according to claim 21, the exogenous nucleic acid encoding the desired recombinant product is introduced into the CHO cells.

24. The method of claim 23, wherein prior to the modification of claim 21, the exogenous nucleic acid encoding the desired recombinant product is introduced into the CHO cells.

25. The method of claim 23 or claim 24, wherein the exogenous nucleic acid encoding the desired recombinant product is integrated into the cellular genome of the modified cell at one or more target sites.

26. The method of claim 25, wherein the target location is a sequence that is part of a contiguous group sequence of one of the contiguous groups NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1 and NW_003615411.1 or is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to 7.

27. The method of claim 25, wherein the target position is within a sequence that is at least about 50% homologous to the following: containing position 45269 of NW_006874047.1; containing position 207911 of NW_006884592.1; containing position 491909 of NW_006881296.1; containing position 79768 of NW_003616412.1; containing position 315265 of NW_003615063.1; containing position 2662054 of NW_006882936.1; or containing position 97705 of NW_003615411.1, comprising 1000 nucleotides.

28. The method of claim 23 or claim 24, wherein the exogenous nucleic acid encoding the desired recombinant product is randomly integrated into the cellular genome of the modified CHO cells.

29. The method according to any one of claims 22 to 28, wherein the desired recombinant product comprises a recombinant protein.

30. The method of claim 29, wherein the recombinant protein is an antibody or an antigen-binding fragment thereof.

31. The method of claim 30, wherein the antibody is a multispecific antibody or an antigen-binding fragment thereof.

32. The method of claim 31, wherein the antibody comprises a single heavy chain sequence and a single light chain sequence or an antigen-binding fragment thereof.

33. The method according to any one of claims 29 to 32, wherein the antibody is a chimeric antibody, a human antibody, or a humanized antibody.

34. The method according to any one of claims 29 to 33, wherein the antibody is a monoclonal antibody.

35. The method of claim 30, wherein the antibody is selected from: bevacizumab; trastuzumab; ranibizumab; efalizumab; rituximab; omalizumab; anti-β amyloid (Aβ) antibody; anti-CD4 (MTRX1011A) antibody; anti-EGFL7 (EGF-like domain 7) antibody; anti-IL13 antibody; anti-Apomab antibody; anti-DR5-targeted apoptosis-promoting receptor agonist (PARA) antibody; anti-BR3 antibody; anti-CD268 antibody; anti-BLyS receptor 3 antibody; anti-BAFF-R (BAFF receptor) antibody; anti-β7 integrin subunit antibody; anti-α v β8 integrin antibody; Darcy's group monoclonal antibody (anti-CD40); GA101 (anti-CD20 monoclonal antibody); MetMAb (anti-MET receptor tyrosine kinase antibody); anti-neuropiliin-1 (NRP1) antibody; Oreogroup monoclonal antibody (anti-CD20 antibody); anti-OX40 ligand antibody; anti-oxidative LDL (oxLDL) antibody; pertuzumab (HER dimerization inhibitor (HDI)); anti-PD-L1 antibody; anti-CD79b antibody; anti-CD20 X anti-CD3 bispecific antibody; anti-VEGF-A X anti-angiogenic-2 bispecific antibody; and rhuMAb IFNα.

36. The method of claim 23 or claim 24, wherein a transposase-mediated gene integration system is used to integrate the nucleic acid sequence encoding the desired recombinant product into the cellular genome of the CHO cell.

37. The method according to any one of claims 22 to 36, wherein the nuclease-assisted gene targeting system is selected from the group consisting of CRISPR / Cas9, CRISPR / Cpf1, zinc finger nucleases, TALEN, or meganucleases.

38. The method according to any one of claims 22 to 37, wherein the reduction in ERV expression is mediated by RNA silencing.

39. The method of claim 38, wherein RNA silencing is selected from the group consisting of siRNA gene targeting and knockdown, shRNA gene targeting and knockdown, and miRNA gene targeting and knockdown.

40. The method according to any one of claims 22 to 39, wherein the modified CHO cells comprise gene knockouts selected from: a) BAX; BAK; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; b) BAX; BAK; ICAM-1; SIRT-1; GGTA1; CMAH; LPL, LPLA2; and PPT1; c) BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; d) BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; e) BAX; BAK; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; f) BAX; BAK; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; g) BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; h) BAX; BAK; ICAM-1; LPL; LPLA2; and PPT1; i) BAX; BAK; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; j) BAX; BAK; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1; k) BAX; BAK; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA; l) BAX; BAK; ICAM-1; SIRT-1; and MYC; m) BAX; BAK; ICAM-1; PERK; SIRT-1; and MYC; n) BAX; BAK; ICAM-1; SIRT-1; PERK; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; o) BAX; BAK; LPL; LPLA2; GGTA1; and CMAH; p) BAX;BAK;PERK;LPL;LPLA2;GGTA1;and CMAH;q) BAX;BAK;MYC;LPL;LPLA2;GGTA1;and CMAH;r) BAX;BAK;MYC;PERK;LPL;LPLA2;GGTA1;and CMAH;s) BAX;BAK;LPL;LPLA2;GGTA1;CMAH;and PPT1;t) BAX; BAK; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; u) BAX; BAK; MYC; LPL;LPLA2; GGTA1; CMAH; and PPT1; v) BAX; BAK; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; w) BAX; BAK; ICAM-1; and SIRT-1; x) BAX; BAK; and ICAM-1; y) BAX; BAK; BCKDHA; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; z) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; aa) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; bb) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; cc) BAX; BAK; BCKDHA; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; dd) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ee) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ff) BAX; BAK; BCKDHA; ICAM-1; LPL; LPLA2; and PPT1; gg) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; hh) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1; ii) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA; jj) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; and MYC; kk) BAX; BAK; BCKDHA; ICAM-1; PERK; SIRT-1; and MYC; ll) BAX; BAK; BCKDHA; ICAM-1; PERK; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; mm) BAX; BAK; BCKDHA; LPL; LPLA2; GGTA1; and CMAH; nn) BAX; BAK; BCKDHA; PERK; LPL; LPLA2; GGTA1;and CMAH; oo) BAX; BAK; BCKDHA; MYC; LPL; LPLA2; GGTA1; and CMAH; pp) BAX; BAK; BCKDHA; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH; qq) BAX; BAK; BCKDHA; LPL; LPLA2; GGTA1; CMAH; and PPT1; rr) BAX; BAK; BCKDHA; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; ss) BAX; BAK; BCKDHA; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1; tt) BAX; BAK; BCKDHA; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; uu) BAX; BAK; BCKDHA; ICAM-1; and SIRT-1; vv) BAX; BAK; BCKDHA; and ICAM-1; ww) BAX; BAK; BCKDHB; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; xx) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; yy) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; zz) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; aaa) BAX; BAK; BCKDHB; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; bbb) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ccc) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ddd) BAX; BAK; BCKDHB; ICAM-1; LPL; LPLA2; and PPT1; eee) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; fff) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1; ggg) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; LPL;LPLA2; PPT1; and LIPA; hhh) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; and MYC; iii) BAX; BAK; BCKDHB; ICAM-1; PERK; SIRT-1; and MYC; jjj) BAX; BAK; BCKDHB; ICAM-1; PERK; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; kkk) BAX; BAK; BCKDHB; LPL; LPLA2; GGTA1; and CMAH; lll) BAX; BAK; BCKDHB; PERK; LPL; LPLA2; GGTA1; and CMAH; mmm) BAX; BAK; BCKDHB; MYC; LPL; LPLA2; GGTA1; and CMAH; nnn) BAX; BAK; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH; ooo) BAX; BAK; BCKDHB; LPL; LPLA2; GGTA1; CMAH; and PPT1; ppp) BAX; BAK; BCKDHB; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; qqq) BAX; BAK; BCKDHB; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1; rrr) BAX; BAK; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; sss) BAX; BAK; BCKDHB; ICAM-1; and SIRT-1; ttt) BAX; BAK; BCKDHB; and ICAM-1; uuu) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; vvv) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; www) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; xxx) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; yyy) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; zzz) BAX; BAK;BCKDHA; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; aaaa) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; bbbb) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; LPL; LPLA2; and PPT1; cccc) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; dddd) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1; eeee) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA; ffff) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; and MYC; gggg) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; PERK; SIRT-1; and MYC; hhhh) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; PERK; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; iiiii) BAX; BAK; BCKDHA; BCKDHB; LPL; LPLA2; GGTA1; and CMAH; jjjj) BAX; BAK; BCKDHA; BCKDHB; PERK; LPL; LPLA2; GGTA1; and CMAH; kkkk) BAX; BAK; BCKDHA; BCKDHB; MYC; LPL; LPLA2; GGTA1; and CMAH; llll) BAX; BAK; BCKDHA; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH; mmmm) BAX; BAK; BCKDHA; BCKDHB; LPL; LPLA2; GGTA1; CMAH; and PPT1; nnnn) BAX; BAK; BCKDHA; BCKDHB; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; oooo) BAX; BAK; BCKDHA; BCKDHB; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1; pppp) BAX; BAK; BCKDHA; BCKDHB; MYC; PERK; LPL;LPLA2; GGTA1; CMAH; and PPT1; qqqq) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; and SIRT-1; or rrrr) BAX; BAK; BCKDHA; BCKDHB; and ICAM-1.

41. A method for producing a recombinant product for a specific purpose, the method comprising: (a) Culturing CHO cells according to any one of claims 9 to 21 under conditions that induce the expression of the desired recombinant product, and (b) The target recombinant product is recovered from the culture medium or modified CHO cells.

42. The method of claim 41, wherein the exogenous nucleic acid encoding the desired recombinant product is integrated into the cellular genome of the modified cell at one or more target sites.

43. The method of claim 42, wherein the target location is a sequence that is part of a contiguous group sequence of one of the contiguous groups NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1 and NW_003615411.1 or is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to 7.

44. The method of claim 42, wherein the target position is within a sequence that is at least about 50% homologous to the following: containing position 45269 of NW_006874047.1; containing position 207911 of NW_006884592.1; containing position 491909 of NW_006881296.1; containing position 79768 of NW_003616412.1; containing position 315265 of NW_003615063.1; containing position 2662054 of NW_006882936.1; or containing position 97705 of NW_003615411.1, comprising 1000 nucleotides.

45. The method of claim 41, wherein the exogenous nucleic acid encoding the desired recombinant product is randomly integrated into the cellular genome of the modified CHO cells.

46. ​​The method according to any one of claims 41 to 45, wherein the desired recombinant product comprises a recombinant protein.

47. The method of claim 46, wherein the recombinant protein is an antibody or an antigen-binding fragment thereof.

48. The method of claim 47, wherein the antibody is a multispecific antibody or an antigen-binding fragment thereof.

49. The method of claim 47, wherein the antibody comprises a single heavy chain sequence and a single light chain sequence or an antigen-binding fragment thereof.

50. The method according to any one of claims 47 to 49, wherein the antibody is a chimeric antibody, a human antibody, or a humanized antibody.

51. The method according to any one of claims 47 to 50, wherein the antibody is a monoclonal antibody.

52. The method of claim 47, wherein the antibody is selected from: bevacizumab; trastuzumab; ranibizumab; efalizumab; rituximab; omalizumab; anti-β amyloid (Aβ) antibody; anti-CD4 (MTRX1011A) antibody; anti-EGFL7 (EGF-like domain 7) antibody; anti-IL13 antibody; anti-Apomab antibody; anti-DR5-targeted apoptosis-promoting receptor agonist (PARA) antibody; anti-BR3 antibody; anti-CD268 antibody; anti-BLyS receptor 3 antibody; anti-BAFF-R (BAFF receptor) antibody; anti-β7 integrin subunit antibody; anti-α v β8 integrin antibody; Darcy's group monoclonal antibody (anti-CD40); GA101 (anti-CD20 monoclonal antibody); MetMAb (anti-MET receptor tyrosine kinase antibody); anti-neuropiliin-1 (NRP1) antibody; Oreogroup monoclonal antibody (anti-CD20 antibody); anti-OX40 ligand antibody; anti-oxidative LDL (oxLDL) antibody; pertuzumab (HER dimerization inhibitor (HDI)); anti-PD-L1 antibody; anti-CD79b antibody; anti-CD20 X anti-CD3 bispecific antibody; anti-VEGF-A X anti-angiogenic-2 bispecific antibody; and rhuMAb IFNα.

53. The method of claim 41, wherein a transposase-mediated gene integration system is used to integrate the nucleic acid sequence encoding the desired recombinant product into the cellular genome of the CHO cell.

54. The method according to any one of claims 41 to 53, wherein the nuclease-assisted gene targeting system is selected from the group consisting of CRISPR / Cas9, CRISPR / Cpf1, zinc finger nucleases, TALEN, or meganucleases.

55. The method according to any one of claims 41 to 54, wherein the modified CHO cells comprise gene knockouts selected from: a) BAX; BAK; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; b) BAX; BAK; ICAM-1; SIRT-1; GGTA1; CMAH; LPL, LPLA2; and PPT1; c) BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; d) BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; e) BAX; BAK; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; f) BAX; BAK; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; g) BAX; BAK; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; h) BAX; BAK; ICAM-1; LPL; LPLA2; and PPT1; i) BAX; BAK; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; j) BAX; BAK; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1; k) BAX; BAK; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA; l) BAX; BAK; ICAM-1; SIRT-1; and MYC; m) BAX; BAK; ICAM-1; PERK; SIRT-1; and MYC; n) BAX; BAK; ICAM-1; SIRT-1; PERK; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; o) BAX; BAK; LPL; LPLA2; GGTA1; and CMAH; p) BAX;BAK;PERK;LPL;LPLA2;GGTA1;and CMAH;q) BAX;BAK;MYC;LPL;LPLA2;GGTA1;and CMAH;r) BAX;BAK;MYC;PERK;LPL;LPLA2;GGTA1;and CMAH;s) BAX;BAK;LPL;LPLA2;GGTA1;CMAH;and PPT1;t) BAX; BAK; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; u) BAX; BAK; MYC; LPL;LPLA2; GGTA1; CMAH; and PPT1; v) BAX; BAK; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; w) BAX; BAK; ICAM-1; and SIRT-1; x) BAX; BAK; and ICAM-1; y) BAX; BAK; BCKDHA; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; z) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; aa) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; bb) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; cc) BAX; BAK; BCKDHA; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; dd) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ee) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ff) BAX; BAK; BCKDHA; ICAM-1; LPL; LPLA2; and PPT1; gg) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; hh) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1; ii) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA; jj) BAX; BAK; BCKDHA; ICAM-1; SIRT-1; and MYC; kk) BAX; BAK; BCKDHA; ICAM-1; PERK; SIRT-1; and MYC; ll) BAX; BAK; BCKDHA; ICAM-1; PERK; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; mm) BAX; BAK; BCKDHA; LPL; LPLA2; GGTA1; and CMAH; nn) BAX; BAK; BCKDHA; PERK; LPL; LPLA2; GGTA1;and CMAH; oo) BAX; BAK; BCKDHA; MYC; LPL; LPLA2; GGTA1; and CMAH; pp) BAX; BAK; BCKDHA; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH; qq) BAX; BAK; BCKDHA; LPL; LPLA2; GGTA1; CMAH; and PPT1; rr) BAX; BAK; BCKDHA; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; ss) BAX; BAK; BCKDHA; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1; tt) BAX; BAK; BCKDHA; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; uu) BAX; BAK; BCKDHA; ICAM-1; and SIRT-1; vv) BAX; BAK; BCKDHA; and ICAM-1; ww) BAX; BAK; BCKDHB; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; xx) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; yy) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; zz) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; aaa) BAX; BAK; BCKDHB; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; bbb) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ccc) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; ddd) BAX; BAK; BCKDHB; ICAM-1; LPL; LPLA2; and PPT1; eee) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; fff) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1; ggg) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; MYC; LPL;LPLA2; PPT1; and LIPA; hhh) BAX; BAK; BCKDHB; ICAM-1; SIRT-1; and MYC; iii) BAX; BAK; BCKDHB; ICAM-1; PERK; SIRT-1; and MYC; jjj) BAX; BAK; BCKDHB; ICAM-1; PERK; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; kkk) BAX; BAK; BCKDHB; LPL; LPLA2; GGTA1; and CMAH; lll) BAX; BAK; BCKDHB; PERK; LPL; LPLA2; GGTA1; and CMAH; mmm) BAX; BAK; BCKDHB; MYC; LPL; LPLA2; GGTA1; and CMAH; nnn) BAX; BAK; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH; ooo) BAX; BAK; BCKDHB; LPL; LPLA2; GGTA1; CMAH; and PPT1; ppp) BAX; BAK; BCKDHB; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; qqq) BAX; BAK; BCKDHB; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1; rrr) BAX; BAK; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; sss) BAX; BAK; BCKDHB; ICAM-1; and SIRT-1; ttt) BAX; BAK; BCKDHB; and ICAM-1; uuu) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; vvv) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPL; LPLA2; and PPT1; www) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL; LPLA2; and PPT1; xxx) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; yyy) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; zzz) BAX; BAK;BCKDHA; BCKDHB; ICAM-1; SIRT-1; GGTA1; CMAH; LPLA2; PPT1; and LIPA; aaaa) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; GGTA1; CMAH; LPLA2; PPT1; and LIPA; bbbb) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; LPL; LPLA2; and PPT1; cccc) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; LPL, LPLA2; and PPT1; dddd) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2; and PPT1; eeee) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; MYC; LPL; LPLA2; PPT1; and LIPA; ffff) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; SIRT-1; and MYC; gggg) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; PERK; SIRT-1; and MYC; hhhh) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; PERK; SIRT-1; MYC; GGTA1; CMAH; LPL, LPLA2; PPT1; and LIPA; iiiii) BAX; BAK; BCKDHA; BCKDHB; LPL; LPLA2; GGTA1; and CMAH; jjjj) BAX; BAK; BCKDHA; BCKDHB; PERK; LPL; LPLA2; GGTA1; and CMAH; kkkk) BAX; BAK; BCKDHA; BCKDHB; MYC; LPL; LPLA2; GGTA1; and CMAH; llll) BAX; BAK; BCKDHA; BCKDHB; MYC; PERK; LPL; LPLA2; GGTA1; and CMAH; mmmm) BAX; BAK; BCKDHA; BCKDHB; LPL; LPLA2; GGTA1; CMAH; and PPT1; nnnn) BAX; BAK; BCKDHA; BCKDHB; PERK; LPL; LPLA2; GGTA1; CMAH; and PPT1; oooo) BAX; BAK; BCKDHA; BCKDHB; MYC; LPL; LPLA2; GGTA1; CMAH; and PPT1; pppp) BAX; BAK; BCKDHA; BCKDHB; MYC; PERK; LPL;LPLA2; GGTA1; CMAH; and PPT1; qqqq) BAX; BAK; BCKDHA; BCKDHB; ICAM-1; and SIRT-1; or rrrr) BAX; BAK; BCKDHA; BCKDHB; and ICAM-1.

56. The method according to any one of claims 41 to 55, comprising purifying the target recombinant product and / or formulating the target recombinant product.

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