Metabolic selection via asparagine biosynthetic pathway

Abstract

The present disclosure provides an isolated mammalian cell comprising reduced or eliminated asparagine synthetase (ASNS) expression. Methods of making such cells and methods of using such cells to produce recombinant proteins are also provided.

Description

Technical Field

[0001] The present disclosure relates to mammalian cell lines for use in biologic production systems, wherein the mammalian cell lines are engineered to have reduced or eliminated expression of components of the asparagine biosynthetic pathway to create asparagine auxotrophic cell lines. Background Art

[0002] The development of high-yield clonal cell lines for biomanufacturing typically utilizes one or more well-known selection methods, such as glutamine synthetase (GS for glutamine selection), dihydrofolate receptor (DHFR for hypoxanthine and thymidine selection), antibiotic selection (puromycin, hygromycin, blasticidin, etc.), or P5C synthetase (P5CS-proline selection). The GS system has become the industry standard, but cell lines that allow for multiple selection methods are needed so that more than one vector can be introduced into the cell line to facilitate the production of molecules, such as bispecific antibodies, multispecific antibodies, and other multi-chain enzymes / proteins or proteins / enzymes that require effector proteins for expression. Summary of the invention

[0003] Various aspects of the present disclosure provide mammalian cell lines for bioproduction systems, wherein the mammalian cell lines are engineered to have reduced or eliminated endogenous asparagine synthetase (ASNS) gene expression. In the absence of endogenously expressed functional ASNS proteins, cells require exogenous amino acid asparagine. Targeted endonuclease-mediated genome modification, such as CRISPR ribonucleoprotein (RNP) complexes or zinc finger nucleases, can be used to inactivate chromosomal ASNS sequences. Other aspects of the present disclosure provide mammalian cell lines, wherein the mammalian cell lines are engineered to have reduced or eliminated endogenous ASNS gene expression, and reduced or eliminated endogenous glutamine synthetase (GS) gene expression.

[0004] Another aspect of the present disclosure includes a process for selecting a cell line with enhanced productivity of expressed biotherapeutic proteins. In other aspects of the present disclosure, a bioproduction system is provided for more conveniently expressing bispecific antibodies or biotherapeutic proteins requiring expression of effector proteins by utilizing a variety of selection systems. These processes include expressing at least one recombinant protein in any mammalian cell line.

[0005] Other aspects and iterations of the disclosure are described in greater detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 The role of asparagine synthetase in the final step of asparagine biosynthesis is shown.

[0007] Figure 2 Shown GS - / - The cDNA sequence of ASNS in CHO cells, including the preferred ZFN binding site (underlined).

[0008] Figure 3 Shows the CHO GS - / - Data showing that there are two copies of the ASNS gene in the cell line genome.

[0009] Figure 4 Shows targeted Figure 2 Cleavage activity of ZFNs at the binding sites identified in .

[0010] Figure 5 Alterations in the ASNS coding sequence resulting from ZFN cleavage are shown.

[0011] Figure 6 Vectors for use with glutamine and / or asparagine based selection systems are shown.

[0012] Figure 7 Endogenous expression control sequences (promoter) of the ASNS gene are shown, including the sequences used in the transgenic vector (underlined).

[0013] Figure 8 Efficient asparagine selection of ASNS knockout clones is shown.

[0014] Fig. 9 Efficient IgG expression driven by an asparagine-based selection system was demonstrated.

[0015] Fig.10 Shown are growth and survival data for pools expressing GFP, RFP, or GFP+RFP selected using GS, ASNS, or double GS+ASNS, respectively.

[0016] Fig.11 Efficient selection using CHO cells in which both GS and ASNS were knocked out and plasmids containing the GS coding sequence, the ASNS coding sequence, or both GS and ASNS coding sequences was demonstrated.

[0017] Fig.12 Fluorescence expression data of pools expressing GFP, RFP, or GFP+RFP selected using GS, ASNS, or double GS+ASNS, respectively, are shown.

[0018] Fig.13 IgG expression data are shown for pools expressing IgG heavy chains and IgG light chains selected using GS, ASNS, or double GS+ASNS, respectively. DETAILED DESCRIPTION

[0019] The present disclosure provides mammalian cell lines that are engineered to have reduced or eliminated endogenous ASNS gene expression. Further provided are mammalian cell lines that are engineered to have reduced or eliminated endogenous GS gene expression and reduced or eliminated endogenous ASNS gene expression. Methods of producing the engineered cell lines are provided, as well as methods of selecting and using the engineered cell lines to produce recombinant proteins.

[0020] (I) Engineered cell lines

[0021] One aspect of the present disclosure includes a mammalian cell line engineered to have reduced or eliminated expression of an endogenous ASNS gene. Alternatively, the mammalian cell line is engineered to have reduced or eliminated expression of an endogenous ASNS gene and an endogenous GS gene.

[0022] The cell lines disclosed herein with reduced or eliminated ASNS expression or reduced ASNS and GS expression are genetically engineered to modify the chromosomal sequence encoding the ASNS or GS protein. The chromosomal sequence can be modified using the targeted endonuclease-mediated genome editing technology described in detail in Section (III) below. For example, the chromosomal sequence can be modified to include a deletion of at least one nucleotide, an insertion of at least one nucleotide, a replacement of at least one nucleotide, or a combination thereof, so that the reading frame is shifted without producing a protein product (ie, the chromosomal sequence is inactivated). Inactivating one allele of the chromosomal sequence encoding ASNS or GS results in reduced protein expression (ie, knockdown). Inactivating two alleles of the chromosomal sequence encoding ASNS or GS results in no protein expression (ie, knockout).

[0023] In some embodiments, ASNS expression levels can be reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, 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 more than about 99%. In other embodiments, ASNS expression levels can be reduced to a level that is undetectable using standard techniques in the art (e.g., Western immunoblot analysis, ELISA enzyme analysis, SDS polyacrylamide gel electrophoresis, etc.).

[0024] Generally, the engineered cell lines disclosed herein have cell viability, viable cell density, titer, growth rate, proliferative response, cell morphology, apoptosis and autophagy levels, and / or general cellular health similar to their non-engineered parental cells when supplemented with asparagine and / or exogenous ASNS coding sequences.

[0025] (a) Cell type

[0026] The engineered cell lines disclosed herein are mammalian cell lines. In some embodiments, the engineered cell lines can be derived from human cell lines. Non-limiting examples of suitable human cell lines include human embryonic kidney cells (HEK293, HEK293T); human connective tissue cells (HT-1080); human cervical cancer cells (HELA); human embryonic retinal cells (PER.C6); human kidney cells (HKB-11); human liver cells (Huh-7); human lung cells (W138); human liver cells (Hep G2); human U2-OS osteosarcoma cells, human A549 lung cells, human A-431 epidermal cells, CACO-2 human colon adenocarcinoma cells, human pluripotent stem cells, Jurkat human T lymphocytes, or human K562 bone marrow cells. In other embodiments, the engineered cell lines can be derived from non-human cell lines. Suitable cell lines also include Chinese hamster ovary (CHO) cells; hamster kidney (BHK) cells; mouse myeloma NS0 cells; mouse myeloma Sp2 / 0 cells; mouse mammary adenocarcinoma C127 cells; mouse embryonic fibroblast 3T3 cells (NIH3T3); mouse B lymphoma A20 cells; mouse melanoma B16 cells; mouse myoblast C2C12 cells; mouse embryonic mesenchymal C3H-10T1 / 2 cells; mouse cancer CT26 cells, mouse prostate DuCuP cells; Mouse mammary gland EMT6 cells; mouse hepatoma Hepa1c1c7 cells; mouse myeloma J5582 cells; mouse epithelial MTD-1A cells; mouse myocardial MyEnd cells; mouse kidney Renca cells; mouse pancreatic RIN-5F cells; mouse melanoma X64 cells; mouse lymphoma YAC-1 cells; rat glioma 9L cells; rat B lymphoma RBL cells; rat neuroblastoma B35 cells; rat hepatoma cells (HTC); Buffalo rat liver BRL 3A cells; canine kidney cells (MDCK); canine mammary gland (CMT) cells; rat osteosarcoma D17 cells; rat monocyte / macrophage DH82 cells; monkey kidney SV-40 transformed fibroblasts (COS7) cells; monkey kidney CVI-76 cells; or African green monkey kidney (VERO, VERO-76) cells. An extensive list of mammalian cell lines can be found in the catalog of the American Type Culture Collection (ATCC, Manassas, VA). In some embodiments, the cell lines disclosed herein are not mouse cell lines. In certain embodiments, the engineered cell lines are CHO cell lines. Suitable CHO cell lines include, but are not limited to, CHO-K1, CHO-K1SV, CHO GS- / -, CHO S, DG44, DuxB11 and derivatives thereof.

[0027] In various embodiments, the parental cell line can lack glutamine synthetase (GS), dihydrofolate reductase (DHFR), hypoxanthine-guanine phosphoribosyl transferase (HPRT), or a combination thereof. For example, the chromosomal sequences encoding GS, DHFR, and / or HPRT can be inactivated. In specific embodiments, all chromosomal sequences encoding GS, DHFR, and / or HPRT are inactivated in the parental cell line.

[0028] (b) Optional nucleic acid encoding a recombinant protein

[0029] In some embodiments, the engineered cell lines disclosed herein may further include at least one nucleic acid encoding a recombinant protein. Typically, a recombinant protein is heterologous, meaning that the protein is not natural to the cell. Recombinant proteins can be therapeutic proteins without limitation, selected from antibodies, antibody fragments, monoclonal antibodies, humanized antibodies, humanized monoclonal antibodies, chimeric antibodies, IgG molecules, IgG heavy chains, IgG light chains, IgA molecules, IgD molecules, IgE molecules, IgM molecules, vaccines, growth factors, cytokines, interferons, interleukins, hormones, coagulation (clotting) (or coagulation (coagulation)) factors, blood components, enzymes, therapeutic proteins, nutritional proteins, any of the above functional fragments or functional variants or fusion proteins comprising the above proteins and / or their functional fragments or variants. In a specific embodiment, the recombinant protein is a bispecific antibody or a multispecific antibody, or a protein that requires an effector protein for expression.

[0030] In some embodiments, the nucleic acid encoding the recombinant protein can be linked to a sequence encoding asparagine synthetase (ASNS), hypoxanthine-guanine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR) and / or glutamine synthetase (GS), so that ASNS, HPRT, DHFR and / or GS can be used as a selectable marker. The nucleic acid encoding the recombinant protein can also be linked to a sequence encoding at least one antibiotic resistance gene and / or a sequence encoding a marker protein such as a fluorescent protein. In some embodiments, the nucleic acid encoding the recombinant protein can be part of an expression construct. The expression construct or vector can include additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), selectable marker sequences, replication origins, etc. Additional information can be found in "Current Protocols in Molecular Biology" Ausubel et al., John Wiley & Sons, New York, 2003 or "Molecular Cloning: A Laboratory Manual"

[0031] Found in Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd edition, 2001.

[0032] In some embodiments, the nucleic acid encoding the recombinant protein can be located outside the chromosome. That is, the nucleic acid encoding the recombinant protein can be transiently expressed from a plasmid, a cosmid, an artificial chromosome, a minichromosome, or another extrachromosomal construct. In other embodiments, the nucleic acid encoding the recombinant protein can be integrated into the genome of the cell on the chromosome. Integration can be random or targeted. Therefore, the recombinant protein can be stably expressed. In some iterations of this embodiment, the nucleic acid sequence encoding the recombinant protein can be operably connected to an appropriate heterologous expression control sequence (i.e., a promoter). In other iterations, the nucleic acid sequence encoding the recombinant protein can be placed under the control of an endogenous expression control sequence. Homologous recombination, targeted endonuclease-mediated genome editing, viral vectors, transposons, recombinase-mediated cassette exchange systems, plasmids, and other well-known means can be used to integrate the nucleic acid sequence encoding the recombinant protein into the genome of the cell line. Other guidelines can be found in Ausubel et al. 2003, the same and Sambrook & Russell, 2001, the same.

[0033] (II) Kit

[0034] Another aspect of the present disclosure provides a kit for producing a recombinant protein, wherein the kit includes any engineered cell line described in detail in section (I) above. The kit may also include a cell growth medium, a transfection reagent, a plasmid vector, a selective medium, a recombinant protein purification means, a buffer, etc. The kit provided herein generally includes a growth cell line and instructions for using them to produce a recombinant protein. The instructions contained in the kit may be fixed on a packaging material or included as a packaging insert. Although the instructions are generally written or printed materials, they are not limited thereto. The present disclosure contemplates any medium capable of storing such instructions and communicating them to an end user. Such media include, but are not limited to, electronic storage media (e.g., disks, tapes, cassettes, chips), optical media (e.g., CD ROMs), etc. As described above, the term "instructions" may include the address of an Internet site providing the instructions.

[0035] (III) Method for preparing engineered cell lines

[0036] Yet another aspect of the present disclosure provides methods for preparing or engineering a cell line having reduced or eliminated ASNS and / or GS expression as described in Section (I) above. Various techniques can be used to knock down or knock out chromosomal sequences encoding ASNS and / or GS. Typically, engineered cell lines are prepared using a targeted endonuclease-mediated genome modification process. Those skilled in the art will appreciate that the engineered cell line can also be prepared using a site-specific recombination system, random mutagenesis, or other methods known in the art.

[0037] Typically, engineered cell lines are prepared by the following method, including introducing at least one targeted endonuclease or a nucleic acid encoding the targeted endonuclease into a target parental cell line, wherein the targeted endonuclease targets a chromosomal sequence encoding ASNS and / or GS. The targeted endonuclease recognizes and binds to a specific chromosomal sequence and introduces a double-strand break. In some embodiments, the double-strand break is repaired by a non-homologous end joining (NHEJ) repair process. Because NHEJ is prone to errors, deletion, insertion and / or substitution of at least one nucleotide may occur, thereby destroying the reading frame of the chromosomal sequence, so that no protein product is produced or non-functional protein is produced via destruction (e.g., destruction of the enzyme active site of the protein). In other embodiments, the targeted endonuclease can also be used to change the chromosomal sequence via a homologous recombination reaction, by co-introducing a polynucleotide having substantial sequence identity with a portion of the targeted chromosomal sequence. In such cases, the double-strand break introduced by the targeted endonuclease is repaired by a homology-directed repair process, so that the chromosomal sequence is exchanged with the polynucleotide in a manner that causes the chromosomal sequence to change or change (e.g., by the integration of an exogenous sequence).

[0038] (a) Targeted endonucleases

[0039] A variety of targeted endonucleases can be used to modify the chromosomal sequence encoding ASNS and / or GS. The targeted endonuclease can be a naturally occurring protein or an engineered protein. Suitable targeted endonucleases include, but are not limited to, zinc finger nucleases (ZFNs), CRISPR nucleases, transcription activator-like effector (TALE) nucleases (TALENs), meganucleases, chimeric nucleases, site-specific endonucleases, and artificial targeted DNA double-strand break inducers.

[0040] (i) Zinc finger nuclease

[0041] In certain embodiments, the targeting endonuclease can be a pair of zinc finger nucleases (ZFNs). ZFNs bind to a specific targeting sequence and introduce a double-strand break into the targeted cleavage site. Typically, ZFNs comprise a DNA binding domain (i.e., zinc finger) and a cleavage domain (i.e., nuclease), which are described separately below.

[0042] DNA binding domain. DNA binding domains or zinc fingers can be engineered to recognize and bind to any selected nucleic acid sequence. See, for example, Beerli et al. (2002) Nat. Biotechnol. 20: 135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70: 313-340; Isalan et al. (2001) Nat. Biotechnol. 19: 656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12: 632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10: 411-416; Zhang et al. (2000) J. Biol. Chem.

[0043] 275(43):33850-33860; Doyon et al. (2008) Nat. Biotechnol. 26:702-708; and Santiago et al. (2008) Proc. Natl. Acad. Sci. USA 105:5809-5814. Engineered zinc finger binding domains may have novel binding specificities compared to naturally occurring zinc finger proteins. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases containing doublet, triplet and / or quadruple nucleotide sequences and individual zinc finger amino acid sequences, in which each doublet, triplet or quadruple nucleotide sequence is associated with one or more amino acid sequences of zinc fingers that bind to a particular triplet or quadruple nucleotide sequence. See, for example, U.S. Pat. Nos. 6,453,242 and 6,534,261, the disclosures of which are incorporated herein by reference in their entirety. For example, the algorithm described in U.S. Pat. No. 6,453,242 can be used to design zinc finger binding domains that target a preselected sequence. Alternative approaches can also be used, such as the rational design of a non-degenerate recognition code table to design zinc finger binding domains to target specific sequences (Sera et al. (2002) Biochemistry 41:7074-7081). Publicly available web-based tools for identifying potential target sites in DNA sequences and designing zinc finger binding domains are known in the art. For example, tools for identifying potential target sites in DNA sequences can be found at zincfingertools.org.

[0044] Tools for designing zinc finger binding domains can be found at zifit.partners.org / ZiFiT. (See also Mandell et al. (2006) Nuc. Acid Res. 34:W516-W523; Sander et al. (2007) Nuc. Acid Res. 35:W599-W605).

[0045] Zinc finger binding domains can be designed to recognize and bind DNA sequences with a length ranging from about 3 nucleotides to about 21 nucleotides. In one embodiment, the zinc finger binding domain can be designed to recognize and bind DNA sequences with a length ranging from about 9 to about 18 nucleotides. Typically, the zinc finger binding domain in the zinc finger nuclease used herein includes at least three zinc finger recognition domains or zinc fingers, wherein each zinc finger binds 3 nucleotides. In one embodiment, the zinc finger binding domain includes four zinc finger recognition domains. In another embodiment, the zinc finger binding domain includes five zinc finger recognition domains. In still another embodiment, the zinc finger binding domain includes six zinc finger recognition domains. Zinc finger binding domains can be designed to bind any appropriate target DNA sequence. See, for example, U.S. Patent Nos. 6,607,882, 6,534,261 and 6,453,242, the entire contents of which are incorporated herein by reference.

[0046] Exemplary methods for selecting zinc finger recognition domains include phage display and two-hybrid systems, which are described in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; and WO 98 / 37186; WO 98 / 53057; WO 00 / 27878; WO 01 / 88197 and GB 2,338,237, the entire contents of each of which are incorporated herein by reference. In addition, enhancement of the binding specificity of zinc finger binding domains has been described (e.g., in WO

[0047] 02 / 077227, the entire contents of which are incorporated herein by reference).

[0048] Zinc finger binding domains and methods of designing and constructing fusion proteins (and polynucleotides encoding them) are known to those skilled in the art and are described in detail, for example, in U.S. Pat. No. 7,888,121, the entire contents of which are incorporated herein by reference. Zinc finger recognition domains and / or multi-finger zinc finger proteins can be linked together using suitable linker sequences, including, for example, linker sequences of five or more amino acids in length. For non-limiting examples of linker sequences of six or more amino acids in length, see U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949, the entire contents of which are incorporated herein by reference. The zinc finger binding domains described herein may include a combination of suitable linker sequences between the individual zinc fingers of the protein.

[0049] Cleavage domain. Zinc finger nucleases also include a cleavage domain. The cleavage domain portion of a zinc finger nuclease can be obtained from any endonuclease or exonuclease. Non-limiting examples of endonucleases from which the cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, New England Biolabs Catalog or Belfort et al. (1997) Nucleic Acids Res. 25: 3379-3388. Other enzymes known to cut DNA (e.g., S1 nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO nuclease). See also Linn et al. (eds.), Nucleases, Cold Spring Harbor Laboratory Press, 1993. One or more of these enzymes (or their functional fragments) can be used as a source of a cleavage domain.

[0050] As described above, the cleavage domain can also be derived from an enzyme or a portion thereof, which requires dimerization to obtain cleavage activity. Two zinc finger nucleases may be required for cleavage because each nuclease includes a monomer of an active enzyme dimer. Alternatively, a zinc finger nuclease can include two monomers to create an active enzyme dimer. As used herein, an "active enzyme dimer" is an enzyme dimer that can cut a nucleic acid molecule. Two cleavage monomers can be derived from the same endonuclease (or its functional fragment), or each monomer can be derived from a different endonuclease (or its functional fragment).

[0051] When two cleavage monomers are used to form an active enzyme dimer, the recognition sites of the two zinc fingers are preferably arranged so that the combination of the two zinc fingers with their respective recognition sites positions the cleavage monomers to allow the cleavage monomers to form an active enzyme dimer (e.g., by dimerization) in a spatial orientation relative to each other. Therefore, the proximal end of the recognition site can be separated by about 5 to about 18 nucleotides. For example, the proximal end can be separated by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides. However, it will be understood that any integer number of nucleotides or nucleotide pairs can be located between the two recognition sites (e.g., about 2 to about 50 nucleotide pairs or more). The proximal end of the recognition site of the zinc finger nuclease, such as those described in detail herein, can be separated by 6 nucleotides. Typically, the cleavage site is located between the recognition sites.

[0052] Restriction endonucleases (restriction enzymes) are present in many species and are able to bind specifically to a DNA sequence (at a recognition site) and cleave the DNA at or near the binding site. Certain restriction endonucleases (e.g., Type IIS) cleave DNA at sites away from the recognition site and have separable binding and cleavage domains. For example, the Type IIS enzyme FokI catalyzes double-stranded cleavage of DNA at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand. See, e.g., U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; and Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA

[0053] 91:883-887; Kim et al. (1994b) J. Biol. Chem. 269:31978-31982. Thus, a zinc finger nuclease may include at least one IIS type restriction enzyme cleavage domain and one or more zinc finger binding domains, which may be engineered or non-engineered. Exemplary IIS type restriction enzymes are disclosed, for example, in International Publication WO 07 / 014,275, the entire disclosure of which is incorporated herein by reference. Additional restriction enzymes also contain separable binding and cleavage domains, and the present disclosure also contemplates these. See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

[0054] An exemplary type IIS restriction enzyme is FokI, whose cleavage domain can be separated from the binding domain. This particular enzyme is active as a dimer (Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575). Therefore, for the purposes of this disclosure, the portion of the FokI enzyme used for the zinc finger nuclease is considered to be a cleavage monomer. Therefore, for targeted double-stranded cleavage using the FokI cleavage domain, two zinc finger nucleases each containing a FokI cleavage monomer can be used to reconstitute the active enzyme dimer. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two FokI cleavage monomers can also be used.

[0055] In certain embodiments, the cleavage domain comprises one or more engineered cleavage monomers that minimize or prevent homodimerization. As non-limiting examples, amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of FokI are targets that affect dimerization of the FokI cleavage half-domain. Exemplary engineered cleavage monomers of FokI that form obligate heterodimers include a pair in which the first cleavage monomer includes mutations at amino acid residue positions 490 and 538 of FokI, and the second cleavage monomer includes mutations at amino acid residue positions 486 and 499.

[0056] Thus, in one embodiment of the engineered cleavage monomer, the mutation at amino acid position 490 replaces Glu (E) with Lys (K); the mutation at amino acid residue 538 replaces Iso (I) with Lys (K); the mutation at amino acid residue 486 replaces Gln (Q) with Glu (E); and the mutation at position 499 replaces Iso (I) with Lys (K). Specifically, the engineered cleavage monomer can be prepared by mutating position 490 from E to K and position 538 from I to K in one cleavage monomer to produce an engineered cleavage monomer designated as "E490K:I538K", and by mutating position 486 from Q to E and position 499 from I to K in another cleavage monomer to produce an engineered cleavage monomer designated as "Q486E:I499K". The above-mentioned engineered cleavage monomers are obligate heterodimer mutants in which abnormal cleavage is minimized or eliminated. Engineered cleavage monomers can be prepared using suitable methods, such as by site-directed mutagenesis of the wild-type cleavage monomer (FokI), as described in US Pat. No. 7,888,121, which is incorporated herein in its entirety.

[0057] Additional domains.In some embodiments, the zinc finger nuclease further comprises at least one nuclear localization signal (NLS). NLS is an amino acid sequence that promotes targeting of the zinc finger nuclease protein to the nucleus to introduce double-strand breaks at the target sequence in the chromosome. Nuclear localization signals are known in the art (see, e.g., Lange et al., J. Biol. Chem., 2007, 282: 5101-5105). Non-limiting examples of nuclear localization signals include PKKKRKV (SEQ ID NO: 1), PKKKRRV (SEQ ID NO: 2), KRPAATKKAGQAKKKK (SEQ ID NO: 3), YGRKKRRQRRR (SEQ ID NO: 4), RKKRRQRRR (SEQ ID NO: 5), PAAKRVKLD (SEQ ID NO: 6), RQRRNELKRSP (SEQ ID NO: 7), VSRKRPRP (SEQ ID NO: 8), PPKKARED (SEQ ID NO: 9), PQPKKKPL (SEQ ID NO: 10), SALIKKKKKMAP (SEQ ID NO: 11), PKQKKRK (SEQ ID NO: 12), RKLKKKIKKL (SEQ ID NO: 13), REKKKFLKRR (SEQ ID NO: 14), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 15), RKCLQAGMNLEARKTKK (SEQ ID NO: 16), ID NO:16), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:17) and

[0058] RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 18). The NLS can be located at the N-terminus, C-terminus or an internal position of the zinc finger nuclease.

[0059] In other embodiments, the zinc finger nuclease may further include at least one cell penetrating domain. Examples of suitable cell penetrating domains include, but are not limited to

[0060] GRKKRRQRRRPPQPKKKRKV(SEQ ID NO:19), PLSSIFSRIGDPPKKKRKV(SEQ ID NO:20), GALFLGWLGAAGSTMGAPKKKRKV(SEQ ID NO:21), GALFLGFLGAAGSTMGAWSQPKKKRKV(SEQ ID NO:22), KETWWETWWTEWSQPKKKRKV(SEQ ID NO:22) NO:23), YARAAARQARA (SEQ ID NO:24), THRLPRRRRRR (SEQ ID NO:25), GGRRARRRRRR (SEQ ID NO:26), RRQRRTSKLMKR (SEQ ID NO:27), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:28), KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:28) NO:29) and RQIKIWFQNRRMKWKK (SEQ ID NO:30). The cell penetrating domain can be located at the N-terminus, C-terminus, or an internal position of the zinc finger nuclease.

[0061] In still other embodiments, the zinc finger nuclease may further include at least one tag domain. Non-limiting examples of tag domains include fluorescent proteins, purification tags, and epitope tags. In one embodiment, the tag domain may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (mKate, m In another embodiment, the marker domain can be a purification tag and / or an epitope tag. Suitable tags include, but are not limited to, poly (His) tags, FLAG (or DDK) tags, Halo tags, AcV5 tags, AU1 tags, AU5 tags, biotin carboxyl carrier protein (BCCP), calmodulin binding protein (CBP), chitin binding domain (CBD), E tags, E2 tags, ECS tags, eXact tags, Glu-Glu tags, glutathione-S-transferase (GST), HA tags, HSV tags, KT3 tags, maltose binding protein (MBP), MAP tags, Myc tags, NE tags, NusA tags, PDZ tags, S tags, S1 tags, SBP tags, Softag 1 tags, Softag 3 tags, Spot tags, Strep tags, SUMO tags, T7 tags, tandem affinity purification (TAP) tags, thioredoxin (TRX), V5 tags, VSV-G tags, and Xa tags. The tag domain can be located at the N-terminus, C-terminus, or internal position of the zinc finger nuclease.

[0062] At least one nuclear localization signal, at least one cell penetration domain and / or at least one tag domain can be directly connected to the zinc finger nuclease via one or more chemical bonds (e.g., covalent bonds). Alternatively, at least one nuclear localization signal, at least one cell penetration domain and / or at least one tag domain can be indirectly connected to the zinc finger nuclease via one or more joints. Suitable joints include amino acids, peptides, nucleotides, nucleic acids, organic joint molecules (e.g., maleimide derivatives, N-ethoxybenzylimidazole, diphenyl-3,4′,5-tricarboxylic acid, p-aminobenzyloxycarboxyl, etc.), disulfide joints and polymer joints (e.g., PEG). The joint may include one or more spacer groups, including but not limited to alkylene, alkenylene, alkynylene, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, aralkyl, aralkenyl and aralkyne. The joint may be neutral, or positively or negatively charged. In addition, the joint may be cleavable so that the covalent bond connecting the joint to another chemical group can be broken or cut under certain conditions, including pH, temperature, salt concentration, light, catalyst or enzyme. In some embodiments, the linker can be a peptide linker. The peptide linker can be a flexible amino acid linker or a rigid amino acid linker. Examples of other suitable linkers are well known in the art, and the procedure for designing linkers is easy in the art (Crasto et al., Protein Eng., 2000, 13 (5): 309-312).

[0063] (ii) CRISPR ribonucleoprotein (RNP)

[0064] In other embodiments, the targeting endonuclease can be a clustered regularly interspaced short palindromic repeats (CRISPR) nuclease. CRISPR nucleases are RNA-guided nucleases derived from bacterial or archaeal CRISPR / CRISPR-associated (Cas) systems. The CRISPR RNP system includes a CRISPR nuclease and a guide RNA.

[0065] Nuclease.CRISPR nucleases can be derived from type I (i.e., IA, IB, IC, ID, IE, or IF), type II (i.e., IIA, IIB, or IIC), type III (i.e., IIIA or IIIB), type V, or type VI CRISPR systems, which are found in various bacteria and archaea. For example, the CRISPR nuclease can be from a Streptococcus sp. (e.g., S. pyogenes, S. thermophilus, S. pasteurianus), a Campylobacter sp. (e.g., Campylobacter jejuni), a Francisella sp. (e.g., Francisella novicida), an Acaryochloris sp., an Acetohalobium sp., an Acidaminococcus sp., an Acidithiobacillus sp., an Alicyclobacillus sp., an Allochromatium sp., an Ammonifex sp., an Anabaena sp. sp.), Arthrospira sp., Bacillus sp., Burkholderiales sp., Caldicelulosiruptor sp., Candidatus sp., Clostridium sp., Crocosphaera sp., Cyanothece sp., Exiguobacterium sp., Finegoldia sp., Ktedonobacter sp., Lachnospiraceae sp., Lactobacillus sp., Lyngbya sp., Marinobacter sp., Methanohalobium sp., Microscilla sp.), Microcoleus sp., Microcystis sp., Natranaerobius sp., Neisseria sp., Nitrosococcus sp., Nocardiopsis sp., Nodularia sp., Nostoc sp., Oscillatoria sp., Polaromonas sp., Pelotomaculum sp., Pseudoalteromonas sp., Petrotoga sp., Prevotella sp., Staphylococcus sp., Streptomyces sp., Streptosporangium sp. sp.), Synechococcus sp., Thermosipho sp., or Verrucomicrobia sp. In other embodiments, the CRISPR nuclease may be derived from an archaeal CRISPR system, a CRISPR / CasX system, or a CRISPR / CasY system (Burstein et al., Nature, 2017, 542(7640):237-241). .

[0066] In some embodiments, the CRISPR nuclease can be derived from a type II CRISPR nuclease. For example, a type II CRISPR nuclease can be a Cas9 protein. Suitable Cas9 nucleases include Streptococcus pyogenes Cas9 (SpCas9), Francisella novo Cas9 (FnCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus Cas9 (SpaCas9), Campylobacter jejuni Cas9 (CjCas9), Neisseria meningitis Cas9 (NmCas9) or Neisseria cinerea Cas9 (NcCas9). In other embodiments, the CRISPR nuclease can be derived from a type V CRISPR nuclease, such as a Cpf1 nuclease. Suitable Cpf1 nucleases include Francisella novicida Cpf1 (FnCpf1), Acidaminococcus sp. Cpf1 (AsCpf1), or Lachnospiraceae ND2006 Cpf1.

[0067] (LbCpf1). In yet another embodiment, the CRISPR nuclease may be derived from a Type VI CRISPR nuclease, such as Leptotrichia wadei Cas13a (LwaCas13a) or Leptotrichia shahii Cas13a (LshCas13a).

[0068] The CRISPR nuclease can be a wild-type CRISPR nuclease, a modified CRISPR nuclease, or a fragment of a wild-type or modified CRISPR nuclease. The CRISPR nuclease can be modified to increase nucleic acid binding affinity and / or specificity, change enzyme activity, and / or change other properties of the protein. For example, the nuclease (i.e., DNA enzyme, RNA enzyme) domain of the CRISPR nuclease can be modified, deleted, or inactivated. The CRISPR nuclease can be truncated to remove domains that are not essential for nuclease function.

[0069] CRISPR nucleases include two nuclease domains. For example, the Cas9 nuclease includes an HNH domain, which cuts the guide RNA complementary strand, and a RuvC domain, which cuts the non-complementary strand; the Cpf1 nuclease includes a RuvC domain and a NUC domain; and the Cas13a nuclease includes two HNEPN domains. When both nuclease domains are functional, the CRISPR nuclease introduces double-strand breaks. Both nuclease domains can be inactivated by one or more mutations and / or deletions, thereby creating variants that introduce single-strand breaks in one strand of the double-stranded sequence. For example, one or more mutations in the RuvC domain of the Cas9 nuclease (e.g., D10A, D8A, E762A, and / or D986A) result in an HNH nickase that nicks the complementary strand of the guide RNA; and one or more mutations in the HNH domain of the Cas9 nuclease (e.g., H840A, H559A, N854A, N856A, and / or N863A) result in a RuvC nickase that nicks the non-complementary strand of the guide RNA. Similar mutations can convert Cpf1 and Cas13a nucleases into nickases. Two CRISPR nickases targeting chains opposite to the chromosome sequence (via a pair of complementary guide RNAs) can be used in combination to establish double-strand breaks in the chromosome sequence. Double CRISPR nickase RNPs can improve target specificity and reduce off-target effects.

[0070] Additional domains. The CRISPR nuclease may further include at least one nuclear localization signal (NLS). NLS is an amino acid sequence that promotes the targeting of zinc finger nuclease proteins into the nucleus to introduce double-strand breaks at the target sequence in the chromosome. Nuclear localization signals are known in the art (see, e.g., Lange et al., J. Biol. Chem., 2007, 282: 5101-5105). Non-limiting examples of nuclear localization signals include PKKKRKV (SEQ ID NO: 1), PKKKRRV (SEQ ID NO: 2), KRPAATKKAGQAKKKK (SEQ ID NO: 3), YGRKKRRQRRR (SEQ ID NO: 4), RKKRRQRRR (SEQ ID NO: 5), PAAKRVKLD (SEQ ID NO: 6), RQRRNELKRSP (SEQ ID NO: 7).

[0071] NO:7), VSRKRPRP (SEQ ID NO:8), PPKKARED (SEQ ID NO:9), PQPKKKPL (SEQ ID NO:10), SALIKKKKKMAP (SEQ ID NO:11), PKQKKRK (SEQ ID NO:12), RKLKKKIKKL (SEQ ID NO:13), REKKKFLKRR (SEQ ID NO:14), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:15), RKCLQAGMNLEARKTKK (SEQ ID NO:16), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:17) and

[0072] RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 18). The NLS can be located at the N-terminus, C-terminus or an internal position of the CRISPR nuclease.

[0073] In further embodiments, the CRISPR nuclease may further include at least one cell penetrating domain. Examples of suitable cell penetrating domains include, but are not limited to

[0074] GRKKRRQRRRPPQPKKKRKV(SEQ ID NO:19), PLSSIFSRIGDPPKKKRKV(SEQ ID NO:20), GALFLGWLGAAGSTMGAPKKKRKV(SEQ ID NO:21), GALFLGFLGAAGSTMGAWSQPKKKRKV(SEQ ID NO:22), KETWWETWWTEWSQPKKKRKV(SEQ ID NO:22) NO:23), YARAAARQARA (SEQ ID NO:24), THRLPRRRRRR (SEQ ID NO:25), GGRRARRRRRR (SEQ ID NO:26), RRQRRTSKLMKR (SEQ ID NO:27), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:28), KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:28) NO:29) and RQIKIWFQNRRMKWKK (SEQ ID NO:30). The cell-penetrating domain can be located at the N-terminus, C-terminus, or an internal location of the CRISPR protein.

[0075] In still other embodiments, the CRISPR nuclease may further include at least one marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, and epitope tags. In one embodiment, the marker domain can be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (mKate, m In another embodiment, the marker domain can be a purification tag and / or an epitope tag. Suitable tags include, but are not limited to, poly (His) tags, FLAG (or DDK) tags, Halo tags, AcV5 tags, AU1 tags, AU5 tags, biotin carboxyl carrier protein (BCCP), calmodulin binding protein (CBP), chitin binding domain (CBD), E tags, E2 tags, ECS tags, eXact tags, Glu-Glu tags, glutathione-S-transferase (GST), HA tags, HSV tags, KT3 tags, maltose binding protein (MBP), MAP tags, Myc tags, NE tags, NusA tags, PDZ tags, S tags, S1 tags, SBP tags, Softag 1 tags, Softag 3 tags, Spot tags, Strep tags, SUMO tags, T7 tags, tandem affinity purification (TAP) tags, thioredoxin (TRX), V5 tags, VSV-G tags, and Xa tags. The tag domain can be located at the N-terminus, C-terminus, or internal position of the CRISPR nuclease.

[0076] At least one nuclear localization signal, at least one cell penetration domain and / or at least one tag domain can be directly connected to the CRISPR nuclease via one or more chemical bonds (e.g., covalent bonds). Alternatively, at least one nuclear localization signal, at least one cell penetration domain and / or at least one tag domain can be indirectly connected to the CRISPR nuclease via one or more joints. Suitable joints include amino acids, peptides, nucleotides, nucleic acids, organic joint molecules (e.g., maleimide derivatives, N-ethoxybenzylimidazole, diphenyl-3,4′,5-tricarboxylic acid, p-aminobenzyloxycarboxyl, etc.), disulfide joints and polymer joints (e.g., PEG). The joint may include one or more spacer groups, including but not limited to alkylene, alkenylene, alkynylene, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, aralkyl, aralkenyl and aralkyne, etc. The joint may be neutral, or positively or negatively charged. In addition, the joint may be cleavable so that the covalent bond connecting the joint to another chemical group can be broken or cut under certain conditions, including pH, temperature, salt concentration, light, catalyst or enzyme. In some embodiments, the linker can be a peptide linker. The peptide linker can be a flexible amino acid linker or a rigid amino acid linker. Additional examples of suitable linkers are well known in the art, and procedures for designing linkers are readily available in the art.

[0077] Guide RNA. The CRISPR nuclease is guided to its target site by a guide RNA. The guide RNA hybridizes to the target site and interacts with the CRISPR nuclease to direct the CRISPR nuclease to the target site in the chromosomal sequence. The target site is surrounded by a preceding spacer adjacent motif ( p rotospacer a djacent m There are no sequence restrictions outside the restriction boundaries of the target site (otif, PAM). CRISPR proteins from different bacterial species recognize different PAM sequences. For example, PAM sequences include 5'-NGG (SpCas9, FnCas9), 5'-NGRRT (SaCas9), 5'-NNAGAAW (StCas9), 5'-NNNNGATT (NmCas9), 5'-NNNNRYAC (CjCas9), and 5'-TTTV (Cpf1), where N is defined as any nucleotide, R is defined as G or A, W is defined as A or T, Y is defined as C or T, and V is defined as A, C, or G. The Cas9 PAM is located at the 3' end of the target site, while the cpf1 PAM is located at the 5' end of the target site.

[0078] The guide RNA includes three regions: a first region at the 5' end that is complementary to the sequence of the target site, a second internal region that forms a stem-loop structure, and a third 3' region that remains substantially single-stranded. The first region of each guide RNA is different so that each guide RNA guides the CRISPR nuclease to a specific target site. The second and third regions (also referred to as scaffold regions) of each guide RNA can be the same in all guide RNAs.

[0079] The first region of the guide RNA is complementary to the sequence of the target site (i.e., the pre-spacer sequence) so that the first region of the guide RNA can be base-paired with the sequence of the target site. The complementarity between the first region of the guide RNA (i.e., crRNA) and the target site sequence can be at least 80%, at least 85%, at least 90%, at least 95% or more. Typically, there is no mismatch between the first region sequence of the guide RNA and the target site sequence (i.e., the complementarity is complete). In various embodiments, the first region of the guide RNA may include about 10 nucleotides to more than about 25 nucleotides. For example, the first region of the guide RNA and the base pairing region of the target site in the chromosome sequence may be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25 or more than 25 nucleotides in length. In an exemplary embodiment, the first region of the guide RNA is about 19, 20 or 21 nucleotides in length.

[0080] The guide RNA also includes a second region forming a secondary structure. In some embodiments, the secondary structure includes a stem (or hairpin) and a loop. The length of the loop and the stem can vary. For example, the length of the loop can be in the range of about 3 nucleotides to about 10 nucleotides, and the length of the stem can be in the range of about 6 to about 20 base pairs. The stem can include one or more protrusions of 1 to about 10 nucleotides. Therefore, the total length of the second region can be in the range of about 16 nucleotides to about 60 nucleotides. In an exemplary embodiment, the loop is about 4 nucleotides long, and the stem includes about 12 base pairs.

[0081] The guide RNA also includes a third region at the 3' end, which remains substantially single-stranded. Therefore, the third region has no complementarity with any chromosomal sequence in the target cell, nor with the rest of the guide RNA. The length of the third region can vary. Typically, the length of the third region is more than about 4 nucleotides. For example, the length of the third region can be in the range of about 5 nucleotides to about 60 nucleotides long.

[0082] The combined length of the second and third regions (or scaffolds) of the guide RNA can be in the range of about 30 nucleotides to about 120 nucleotides in length. In one aspect, the combined length of the second and third regions of the guide RNA is in the range of about 70 nucleotides to about 100 nucleotides in length.

[0083] In some embodiments, the guide RNA includes one molecule that includes all three regions. In other embodiments, the guide RNA may include two separate molecules. The first RNA molecule may include the first (5') region of the guide RNA and half of the second region "stem" of the guide RNA. The second RNA molecule may include the other half of the second region "stem" of the guide RNA and the third region of the guide RNA. Therefore, in this embodiment, the first and second RNA molecules each include nucleotide sequences that are complementary to each other. For example, in one embodiment, the first and second RNA molecules each include base pairing with each other to form a sequence of (about 6 to about 20 nucleotides) of a functional guide RNA.

[0084] (iii) Other targeted endonucleases

[0085] In a further embodiment, the targeting endonuclease can be a meganuclease. A meganuclease is an endodeoxyribonuclease characterized by a long recognition sequence, i.e., a recognition sequence generally within the range of about 12 base pairs to about 40 base pairs. As a result of this requirement, the recognition sequence generally only occurs once in any given genome. In meganucleases, the homing endonuclease family named LAGLIDADG has become a valuable tool for studying genomes and genome engineering (see, e.g., Arnould et al., 2011, Protein Eng Des Sel, 24 (1-2): 27-31). Other suitable meganucleases include I-CreI and I-Dmol. A meganuclease can target a specific chromosome sequence by modifying its recognition sequence using techniques well known to those skilled in the art.

[0086] In another embodiment, the targeting endonuclease can be a transcription activator-like effector (TALE) nuclease. TALE is a transcription factor from the plant pathogen Xanthomonas, which can be easily engineered to bind new DNA targets. TALE or its truncated version can be connected to the catalytic domain of endonucleases such as FokI to establish a targeting endonuclease called TALE nuclease or TALEN (Sanjana et al., 2012, Nat Protoc, 7 (1): 171-192) and Arnould et al., 2011, Protein Engineering, Design & Selection, 24 (1-2): 27-31).

[0087] In alternative embodiments, the targeting endonuclease can be a chimeric nuclease. Non-limiting examples of chimeric nucleases include ZF-meganucleases, TAL-meganucleases, Cas9-FokI fusions, ZF-Cas9 fusions, TAL-Cas9 fusions, etc. Those skilled in the art are familiar with the means of generating such chimeric nuclease fusions.

[0088] In still other embodiments, the targeting endonuclease can be a site-specific endonuclease. In particular, the site-specific endonuclease can be a "rare cutting" endonuclease that recognizes sequences that rarely occur in the genome. Alternatively, the site-specific endonuclease can be engineered to cut the target site (Friedhoff et al., 2007, Methods Mol Biol 352: 1110123). Typically, the recognition sequence of the site-specific endonuclease only occurs once in the genome. In alternative further embodiments, the targeting endonuclease can be an artificially targeted DNA double-strand break inducing agent.

[0089] (b) Delivery of targeted endonucleases to cells

[0090] The method includes introducing the targeted endonuclease into the target parent cell line. The targeted endonuclease can be introduced into the cell as a purified isolated protein or as a nucleic acid encoding the targeted endonuclease. The nucleic acid can be DNA or RNA. In embodiments where the encoding nucleic acid is mRNA, the mRNA may be 5' capped and / or 3' polyadenylated. In embodiments where the encoding nucleic acid is DNA, the DNA can be linear or circular. The nucleic acid can be part of a plasmid or a viral vector, where the encoding DNA can be operably linked to a suitable promoter. Those skilled in the art are familiar with suitable vectors, promoters, other control elements, and means for introducing vectors into target cells. In embodiments where the targeted endonuclease is a CRISPR nuclease, the CRISPR nuclease system can be introduced into the cell as a gRNA-protein complex.

[0091] The targeting endonuclease molecule can be introduced into the cell by various means. Suitable delivery means include microinjection, electroporation, sonoporation, biotrajectory, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nuclear transfection, magnetofection, lipofection, puncture transfection, optical transfection, nucleic acid uptake enhanced by proprietary agents and delivery via liposomes, immunoliposomes, virions or artificial virions. In a specific embodiment, the targeting endonuclease molecule is introduced into the cell by nuclear transfection.

[0092] Optional donor polynucleotide. The method for targeted genome modification or engineering modification can further include introducing at least one donor polynucleotide into the cell, and the donor polynucleotide includes the sequence with at least one nucleotide change compared with the target chromosome sequence. The donor polynucleotide has basic sequence identity with the sequence at or near the chromosome sequence targeting site, so that the double-strand break introduced by the targeting endonuclease can be repaired by the homology directed repair process, and the sequence of the donor polynucleotide can be inserted in the chromosome sequence or exchanged with the chromosome sequence, thereby modifying the chromosome sequence. For example, the donor polynucleotide can include a first sequence with basic sequence identity with the sequence on one side of the target site, and a second sequence with basic sequence identity with the sequence on the other side of the target site. The donor polynucleotide can further include a donor sequence for integration into the targeted chromosome sequence. For example, the donor sequence can be an exogenous sequence (such as a marker sequence) so that the integration of the exogenous sequence destroys the reading frame and inactivates the targeted chromosome sequence.

[0093] The length of the first and second sequences in the donor polynucleotides having a substantial sequence identity to the sequence at or near the chromosome sequence targeting site can vary. Typically, the length of each of the first and second sequences in the donor polynucleotides is at least about 10 nucleotides. In various embodiments, the length of the donor polynucleotide sequence having a substantial sequence identity to the chromosome sequence can be about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 100 nucleotides, or more than 100 nucleotides.

[0094] The phrase "substantial sequence identity" means that the sequence in the polynucleotide has at least about 75% sequence identity to the target chromosome sequence. In some embodiments, the sequence in the polynucleotide has about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121

[0095] 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0096] The length of the donor polynucleotide can and will vary. For example, the length of the donor polynucleotide can be in the range of about 20 nucleotides to about 200,000 nucleotides at most. In various embodiments, the length of the donor polynucleotide can be in the range of about 20 nucleotides to about 100 nucleotides, about 100 nucleotides to about 1000 nucleotides, about 1000 nucleotides to about 10,000 nucleotides, about 10,000 nucleotides to about 100,000 nucleotides, or about 100,000 nucleotides to about 200,000 nucleotides.

[0097] In some embodiments, the donor polynucleotide can be a single-stranded or double-stranded DNA. The DNA can be linear or circular. In some embodiments, the donor polynucleotide can be a single-stranded, linear oligonucleotide, comprising less than about 200 nucleotides. In other embodiments, the donor polynucleotide can be a part of a vector. Suitable vectors include DNA plasmids, viral vectors, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC). In still other embodiments, the donor polynucleotide can be a PCR fragment or a nucleic acid compounded with a delivery vehicle such as a liposome or poloxamer.

[0098] The donor polynucleotide can be introduced while the targeting endonuclease molecule is introduced into the cell. Alternatively, the donor polynucleotide and the targeting endonuclease molecule can be sequentially introduced into the cell. The ratio of the targeting endonuclease molecule to the donor polynucleotide can and will change. Typically, the ratio of the targeting endonuclease molecule to the donor polynucleotide is within the range of about 1:10 to about 10:1. In various embodiments, the ratio of the targeting endonuclease molecule to the polynucleotide can be about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1. In one embodiment, the ratio is about 1:1.

[0099] (c) Cultured cells

[0100] The method further includes culturing the cell under appropriate conditions such that the double-strand break introduced by the targeted endonuclease can be repaired by: (i) a non-homologous end joining repair process such that the chromosomal sequence is modified by deletion, insertion and / or substitution of at least one nucleotide, or optionally (ii) a homology directed repair process such that the chromosomal sequence is exchanged with a polynucleotide sequence such that the chromosomal sequence is modified. In embodiments where a nucleic acid encoding a targeted endonuclease is introduced into the cell, the method includes maintaining the cell under appropriate conditions such that the cell expresses the targeted endonuclease.

[0101] Typically, cells are maintained under conditions suitable for cell growth and / or maintenance. Suitable cell culture conditions are well known in the art and are described, for example, in Santiago et al. (2008) PNAS 105:5809-5814; Moehle et al. (2007) PNAS 104:3055-3060; Urnov et al. (2005) Nature 435:646-651; and Lombardo et al. (2007) Nat. Biotechnology 25:1298-1306. Those skilled in the art will appreciate that methods for culturing cells are known in the art and can and will vary depending on the cell type. Routine optimization can be used in all cases to determine the best technique for a particular cell type.

[0102] During this step, the targeted endonuclease recognizes, binds and produces a double-strand break at the targeted cleavage site of the chromosome sequence, and during the repair of the double-strand break, at least one nucleotide deletion, insertion and / or substitution is introduced into the targeted chromosome sequence. In a specific embodiment, the targeted chromosome sequence is inactivated.

[0103] After confirming that the target chromosome sequence has been modified, single cell clones can be isolated and genotyped (via DNA sequencing and / or protein analysis). Cells containing a modified chromosome sequence can undergo one or more rounds of additional targeted genome modification to modify additional chromosome sequences, thereby establishing double knockouts, triple knockouts, etc.

[0104] (IV) Production of recombinant proteins

[0105] Another aspect of the present disclosure includes a method for producing a recombinant protein in a biopharmaceutical production system. Suitable recombinant proteins are described in section (I) (c). The method includes expressing the target recombinant protein in any engineered cell line described in section (I) above, and purifying the expressed recombinant protein. The means for producing or manufacturing recombinant proteins are well known in the art (see, for example, "Biopharmaceutical Production Technology", Subramanian (ed.), 2012, Wiley-VCH; ISBN: 978-3-527-33029-4).

[0106] The recombinant protein can be purified via a process comprising a clarification step (eg, filtration) and one or more chromatography steps (eg, affinity chromatography, protein A (or G) chromatography, ion exchange (ie, cation and / or anion) chromatography).

[0107] definition

[0108] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the invention belongs. The following references provide general definitions of many of the terms used in the present invention for technicians: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd Edition. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Edition, R. Rieger et al. (ed.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise indicated, the following terms have the meanings assigned to them.

[0109] When introducing elements of the present disclosure or the preferred embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements in addition to the listed elements.

[0110] As used herein, the term "endogenous sequence" refers to a chromosomal sequence that is native to a cell.

[0111] The term "exogenous sequence" refers to a chromosomal sequence that is not native to the cell, or a chromosomal sequence that has been moved to a different chromosomal location.

[0112] An "engineered" or "genetically modified" cell refers to a cell whose genome has been modified or engineered, i.e., the cell contains at least one chromosomal sequence that has been engineered to contain at least one nucleotide insertion, at least one nucleotide deletion and / or at least one nucleotide substitution.

[0113] The terms "genomic modification" and "genomic editing" refer to a process by which a specific endogenous chromosome sequence is changed so that the chromosome sequence is modified. The chromosome sequence can be modified to include the insertion of at least one nucleotide, the deletion of at least one nucleotide, and / or the replacement of at least one nucleotide. The modified chromosome sequence can be inactivated so that no product is produced. Alternatively, the chromosome sequence can be modified so that a changed product is produced.

[0114] As used herein, "gene" refers to a DNA region encoding a gene product (including exons and introns), as well as all DNA regions that regulate the production of a gene product, whether or not these regulatory sequences are adjacent to coding and / or transcribed sequences. Thus, a gene includes, but is not limited to, promoter sequences, terminators, translation regulatory sequences, such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and site control regions.

[0115] The term "heterologous" refers to an entity that is not native to the cell or species of interest.

[0116] The terms "nucleic acid" and "polynucleotide" refer to a deoxyribonucleotide or ribonucleotide polymer, in a linear or circular conformation. For the purposes of this disclosure, these terms are not to be construed as limiting the length of the polymer. The terms may encompass analogs of known natural nucleotides, as well as nucleotides modified at the base, sugar and / or phosphate moieties. Typically, analogs of a particular nucleotide have the same base pairing specificity; i.e., an analog of A will pair with T. The nucleotides of a nucleic acid or polynucleotide may be linked by phosphodiester, phosphorothioate, phosphoramidite, or phosphorodiamidite bonds, or a combination thereof.

[0117] The term "nucleotide" refers to a deoxyribonucleotide or a ribonucleotide. Nucleotides can be standard nucleotides (i.e., adenosine, guanosine, cytidine, thymidine and uridine) or nucleotide analogs. Nucleotide analogs refer to nucleotides with modified purine or pyrimidine bases or modified ribose moieties. Nucleotide analogs can be naturally occurring nucleotides (e.g., inosine) or non-naturally occurring nucleotides. Non-limiting examples of modifications of nucleotide sugar or base moieties include the addition (or removal) of acetyl, amino, carboxyl, carboxymethyl, hydroxyl, methyl, phosphate and sulfhydryl groups, and the replacement of the carbon and nitrogen atoms of the bases by other atoms (e.g., 7-deazapurine). Nucleotide analogs also include dideoxynucleotides, 2'-O-methyl nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA) and morpholinos.

[0118] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues.

[0119] As used herein, the term "target site" or "target sequence" refers to a nucleic acid sequence that defines the portion of the chromosomal sequence to be modified or edited, and the targeting endonuclease is engineered to recognize and bind to the nucleic acid sequence, provided that sufficient binding conditions exist.

[0120] The terms "upstream" and "downstream" refer to positions in a nucleic acid sequence relative to a fixed position. Upstream refers to the region 5' (i.e., near the 5' end of the chain) from the position, and downstream refers to the region 3' (i.e., near the 3' end of the chain) from the position.

[0121] The technology of determining nucleic acid and amino acid sequence identity is known in the art. Generally, such technology includes determining the nucleotide sequence of the mRNA of the gene and / or determining the encoded amino acid sequence thereby, and comparing these sequences with a second nucleotide or amino acid sequence. It is also possible to determine and compare genomic sequences in this way. Generally, identity refers to the exact nucleotide to nucleotide or amino acid to amino acid correspondence between two polynucleotides or polypeptide sequences respectively. Two or more sequences (polynucleotides or amino acids) can be compared by determining their percentage identity. The percentage identity of two sequences (whether nucleic acid or amino acid sequence) is the numerical value of dividing the exact matching number between two aligned sequences by the length of the shorter sequence and multiplying by 100. For the approximate alignment of nucleic acid sequences, the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489,1981 is provided. The algorithm can be applied to amino acid sequences by using a scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, MO Dayhoff ed., 5 suppl. 3: 353-358, National Biomedical Research Foundation, Washington, DC, USA, and normalized by Gribskov, Nucl. Acids Res. 14 (6): 6745-6763 (1986). An exemplary embodiment of this algorithm for determining the percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the "BestFit" utility application. Other programs suitable for calculating percent identity or similarity between sequences are known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; chain = both; cutoff value

[0122] =60; Expectation = 10; Matrix = BLOSUM62; Description = 50 sequences; Sort by = Sort by high score; Database = non-redundant,

[0123] GenBank+EMBL+DDBJ+PDB+GenBank CDS Translation+Swiss Protein

[0124] +Spupdate+PIR. The detailed information of these programs can be found on the GenBank website. With respect to the sequences described herein, the desired degree of sequence identity is approximately in the range of 80% to 100%, and any integer value therebetween. Typically, the percentage identity between the sequences is at least 70-75%, preferably 80-82%, more preferably 85-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity.

[0125] As various changes could be made in the above cells and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and the following examples shall be interpreted as illustrative and not in a limiting sense.

[0126] Example

[0127] The following examples illustrate certain aspects of the invention.

[0128] Example 1: Design of asparagine-mediated selection system

[0129] To develop an asparagine-mediated metabolic selection system, a CHO cell line auxotrophic for the non-essential amino acid asparagine (Asn) was first developed. A comprehensive search against the Reactome and KEGG databases was performed to identify all genes associated with the asparagine biosynthesis pathway. The endogenous glutamine hydrolyzing asparagine synthetase gene (ASNS) was identified as the only non-redundant gene responsible for Asn biosynthesis ( Figure 1 To generate Asn auxotrophic CHO cell lines, glutamine (Gln) auxotrophic cells from MilliporeSigma were used. GS - / - Cell lines via Whole genome sequencing (WGS) of the cell lines elucidated the endogenous ASNS coding sequence ( Figure 2 ) and found it in The gene is present in two copies in the genome, as determined by digital droplet PCR (ddPCR) analysis ( Figure 3 ). Zinc finger nucleases (ZFNs) were designed to disrupt the tenth exon of the ASNS gene encoding the active subunit of the ASNS protein. The ZFN target sequence is Figure 2 is underlined. Cells were grown in medium supplemented with 6 mM L-glutamine

[0130] (MilliporeSigma G7513) CD CHO fusion medium (MilliporeSigma 14365C) ​​(Fusion + Gln) was incubated at 37°C and 5% CO 2 The cells were cultured under shaking conditions of 5% RT. Cells were seeded at a density of 0.5e6 one day before transfection to maintain the culture in logarithmic growth phase. 1.0e6 cells were transfected with 6 μg mRNA of each ZFN arm using electroporation. ZFN cleavage activity was assessed via the Surveyor nuclease S mutation detection assay ( Figure 4 The transfected cells were transferred to 3 mL of medium supplemented with 6 mM L-glutamine (MilliporeSigma G7513) and 2 mM L-anhydrous asparagine (MilliporeSigma 1043502). CD CHO fusion medium (MilliporeSigma 14365C) ​​(fusion + Gln + Asn) was added to 6-well culture dishes. The cells were kept at 30°C and 5% CO 2 Incubate in a static environment for 48 hours and then transfer to 37°C / 5% CO 2 96 hours after transfection, ZFN-modified Cells were scaled up into T-75 flasks. Monoclonal cells from the ZFN-modified pools were isolated into 96-well culture plates via fluorescence activated cell sorting (FACS). Monoclonal cells were evaluated via next generation sequencing (NGS) to identify clones containing two copies of the successful genetic disruption of ASNS ( Figure 5 ).

[0131] To demonstrate the effectiveness of the asparagine-mediated selection mechanism as a stand-alone system and as part of a dual metabolic selection system, stably selected cell populations were generated expressing a variety of molecules. Molecules used in validating this system included paprika red fluorescent protein (RFP), Dasher green fluorescent protein (GFP), and human IgG1. GS - / - ASNS - / - Cells were cultured in Fusion+Gln+Asn medium. The expression vectors used in the current work contained either asparagine synthetase (ASNS) or glutamine synthetase (GS) selection markers, as indicated by the experimental design. Expression of murine ASNS (protein: asparagine synthetase {glutamine hydrolyzes}; gene: ASNS; UniProtKB ID: Q61024) or murine GS (protein: glutamine synthetase {glutamine ammonia ligase}; gene: Glul; UniProtKB ID: P15105) was driven by either the 5' endogenous expression control sequence (promoter) or the 5' SV40 promoter with the SV40 polyadenylation sequence at the 3' end of the gene ( Figure 6 and 7).

[0132] GS - / - ASNS - / - Cells were maintained in confluence + Gln + Asn at 37°C and 5% CO 2 Cells were grown under shaking conditions at 4 °C. Cells were seeded at a density of 0.5e6 the day before transfection to maintain the culture in logarithmic growth phase. 1.0e6 cells of each condition were transfected with 12 μg of plasmid DNA using electroporation. Transfected cells were transferred to T-25 dishes containing Fusion+Gln+Asn and an additional 2 mL of media was added 48 hours after transfection. 96 hours after transfection, cells were pelleted and media aspirated, then 4e5 viable cells / mL were resuspended in 12 mL of the appropriate selection media and transferred to T-75 flasks. Glutamine-based selection was performed using Fusion-Gln, asparagine-based selection was performed using Fusion-Asn, and glutamine / asparagine dual selection was performed using Fusion with low concentrations of glutamine and -Asn. Cell viability and viable cell density of the various selection cultures were monitored over time. After recovery from selection, stable selection cultures were transferred to TubeSpin Bioreactor Tubes Used to amplify and adapt to oscillating conditions.

[0133] Developed a product that does not contain L-anhydrous asparagine (Asn) Advanced CHO fed-batch medium (MilliporeSigma 14366C), Advanced CHO feed (MilliporeSigma 24367C) and 4 Feed (MilliporeSigma

[0134] 1.03796.0005) (Advanced-Asn, Feed-Asn and 4-Feed-Asn, respectively). Stably selected cultures transfected with the IgG1 expression vector were pelleted, the selection medium was aspirated, and then resuspended in Advanced-Asn at 3e5 viable cells / mL for productivity analysis under fed-batch conditions. Starting from the 3rd day after inoculation, the viable cell density and viability of each culture were collected every other day. Starting from the 3rd day after inoculation, 1.5mL of a 50 / 50 mixture of Advanced Feed-Asn and 4-Feed-Asn was added to each culture medium. Glucose and glutamine readings were taken from each culture every other day starting from the 5th day, with D-+-glucose (MilliporeSigma G8769) and L-glutamine (MilliporeSigma G7513) added to maintain appropriate glucose and glutamine levels. Productivity was monitored over time, and the fed batch titer was recorded every other day starting from the 9th day until the viability of the culture dropped below 70%. Titers were determined using interferometry with the ForteBioOctet and then confirmed via HPLC protein A affinity chromatography.

[0135] Embodiment 2:

[0136] Developed a product that does not contain L-anhydrous asparagine (Asn) Custom formulation of CD CHO Fusion Medium (Fusion-Gln-Asn). GS - / - ASNS - / - Clones were cultured in Fusion+Gln+Asn or Fusion+Gln-Asn for at least ten days. Viability and viable cell density were measured twice a week. Figure 8 It was shown that in the absence of asparagine, GS - / - ASNS - / - The cells were unable to grow, but when asparagine was supplemented into the medium, GS - / - ASNS - / - Cell growth was restored.

[0137] Embodiment 3:

[0138] To demonstrate that the stably selected cell population is capable of producing the protein of interest using the asparagine-mediated selection system described in Example 1, cells were transfected and passaged in fusion-Asn under selection pressure. GS - / - ASNS - / -Cells were transfected with ASNS / RFP plasmids and grown in Fusion+Gln+Asn (non-selective medium) or Fusion+Gln-Asn (Asn selective medium) to recover from the selection pressure. The surviving cell population was analyzed by FACS, and the mean fluorescence intensity (MFI) and the percentage of RFP+ cells were measured. Compared with cells selected for asparagine in Fusion+Gln-Asn, cells grown in Fusion+Gln+Asn showed a small percentage of RFP-positive cells and low MFI. The results are summarized in Table 1.

[0139] Table 1

[0140]

[0141] Embodiment 4:

[0142] To demonstrate that the stably selected cell population is capable of producing the protein of interest using the asparagine-mediated selection system described in Example 1, we developed a vector with an IgG heavy chain, an IgG light chain, and an asparagine synthetase (ASNS) coding sequence driven by either a 5' endogenous expression control sequence or a 5' SV40 promoter. Figure 6 and 7 ). This vector was transfected into GS - / - ASNS - / - In cell line. As a control, a simulated transfection without DNA was used. These colonies have been passaged under the selection pressure in fusion+Gln-Asn. The conditions for selection are also applied during recovery, amplification and productivity determination. The fed-batch productivity determination is inoculated in advanced+Gln-Asn culture medium with 3e5 viable cells / mL. From the 3rd day after inoculation, the viable cell density and viability of each culture medium are collected every other day. From the 3rd day after inoculation, a 50 / 50 mixture of 1.5mL of advanced feed-Asn and 4 feed-Asn is added to each culture medium. From the 5th day, glucose and glutamine readings are read every other day, wherein D-+-glucose (MilliporeSigma G8769) and L-glutamine (MilliporeSigma G7513) are added, to maintain appropriate glucose and glutamine levels. Cells selected with the ASNS coding sequence driven by the endogenous expression control sequence (ASNS promoter) were shown to exhibit IgG expression levels approximately 3-fold higher than cells expressing the same IgG when selected with the ASNS coding sequence driven by the SV40 promoter ( Fig. 9 ).

[0143] Embodiment 5:

[0144] To test whether stable cell populations could produce two independent intracellular fluorescent proteins under dual selection conditions, we developed two vectors, one containing the GFP and GS coding sequences and the second containing the RFP and ASNS coding sequences ( Figure 6 These two plasmids were co-transfected into GS - / - ASNS - / - As a control, each plasmid was also independently transfected into GS - / - ASNS - / - Cells were then passaged under GS selective conditions (fused-Gln), ASNS selective conditions (fused-Asn) and dual metabolic selective conditions (fused-Gln-Asn, supplemented with amino acids if necessary). The conditions used for selection were also applied during recovery, amplification and all other assays. Fig.10 Growth and viability data from selection assays are shown, indicating that cells transfected with GFP vector can survive and grow under -Gln conditions, but require Asn supplementation in the medium. On the other hand, cells transfected with RFP vector can survive and grow under -Asn conditions, but require Gln supplementation in the medium. Cells co-transfected with both plasmids (GFP+RFP) can survive and grow in -Gln medium, -Asn medium, and -Gln-Asn medium (supplemented with amino acids as necessary). Fig.11 and 12 It is shown that cells transfected with GFP vectors that survive and grow in -Gln medium are GFP positive; cells transfected with RFP vectors that survive and grow in -Asn medium are RFP positive; and cells co-transfected with two plasmids (GFP+RFP) that survive and grow in -Gln-Asn medium or in -Asn medium supplemented with different levels of Gln and / or Asn are positive for both GFP and RFP. These data show that the GS+ASNS dual metabolic selection system provides a unique opportunity to select cells into which multiple independent vectors encoding intracellular proteins have been introduced without adding any selection agent (e.g., antibiotics) to the culture medium.

[0145] Embodiment 6:

[0146] To test whether the stable cell populations were able to produce secreted proteins under dual selection conditions, we developed two vectors expressing IgG1, one containing the IgG heavy chain, IgG light chain, and GS coding sequences, and the second containing the same IgG heavy chain, IgG light chain, and ASNS coding sequences ( Figure 6 These two independent vectors were co-transfected into GS - / -ASNS - / - As a control, each vector was also independently transfected into GS - / - ASNS - / - cells (GS only and ASNS only). The cells were then passaged under selection pressure in fusion-Gln (cells transfected with GS only), in fusion-Asn (cells transfected with ASNS only), or in fusion-Gln–Asn (cells transfected with GS+ASNS) or in medium supplemented with trace amounts of Gln and / or Asn. The conditions used for selection were also applied during recovery, scale-up, and productivity assays. The GS only selection culture fully recovered after 14 days, while the ASNS only and GS+ASNS dual selection cultures had similar recovery profiles and required 21 days for full recovery. In the fed-batch assay, GS only cells, ASNS only cells produced IgG ( Fig.13 ). This suggests that exogenous GS and ASNS produced by the cells, plus supplementation with Gln and Asn when necessary, are sufficient to maintain production of secretory proteins without slowing cell growth and proliferation due to increased metabolic burden. This offers the potential to run large-scale production bioreactors under dual metabolic selection conditions, which may be difficult to perform using antibiotic selection methods, either because of the need to later separate or purify the antibiotics from the desired secreted proteins, or because of the cost of adding antibiotics to large-scale bioreactors. In addition, this dual metabolic selection system (GS+ASNS) provides the opportunity to more effectively select for cells in which multiple large vectors have been introduced into the cells, such as when expressing bispecific antibodies or other large and / or complex proteins.

Claims

1. A method for producing a recombinant protein product, comprising: (a) providing a mammalian cell line engineered to have reduced or eliminated expression of endogenous asparagine synthetase (ASNS); (b) introducing a polynucleotide into the mammalian cell line, wherein the polynucleotide encodes a functional ASNS gene and a recombinant protein; (c) culturing the cell line; and (d) purifying the recombinant protein to form the recombinant protein product.

2. The method of claim 1, wherein the mammalian cell line of (a) further comprises reduced or eliminated endogenous glutamine synthetase (GS) expression.

3. The method of claim 1, wherein endogenous ASNS expression is reduced or eliminated by inactivating an endogenous ASNS gene of the mammalian cell line.

4. The method of claim 1, wherein the endogenous ASNS gene is inactivated using targeted endonuclease-mediated genome modification technology.

5. The method of claim 4, wherein the targeting endonuclease is a CRISPR ribonucleoprotein complex or a pair of zinc finger nucleases.

6. The method of claim 1, wherein the mammalian cell line is a Chinese hamster ovary (CHO) cell line, a baby hamster kidney (BHK) cell line, an NSO mouse myeloma cell line, a HEK293 cell line, or a Vero African green monkey kidney cell line.

7. The method of claim 1, wherein the cell line is a CHO cell line.

8. The method of claim 1, wherein the recombinant protein product is selected from an antibody, an antibody fragment, a vaccine, a growth factor, a cytokine, a hormone or a coagulation factor.

9. The method of claim 8, wherein the antibody is a bispecific or multispecific antibody.

10. A genetically engineered mammalian cell line for use in a biologic production system, wherein the mammalian cell line is engineered to have reduced or eliminated endogenous ASNS expression.

11. The mammalian cell line of claim 10, wherein ASNS expression is reduced or eliminated by inactivating at least one allele of a chromosomal sequence encoding ASNS.

12. The mammalian cell line of claim 11, wherein both alleles of the chromosomal sequence encoding ASNS are inactivated.

13. The mammalian cell line of claim 10, wherein the cell line is engineered to have reduced or eliminated expression of endogenous glutamine synthetase (GS).

14. The mammalian cell line of claim 12, wherein the chromosomal sequence is inactivated using targeted endonuclease-mediated genome modification technology.

15. The mammalian cell line of claim 14, wherein the targeting endonuclease is a ribonucleoprotein complex or a pair of zinc finger nucleases.

16. The mammalian cell line of claim 15, wherein the non-human cell line is a Chinese hamster ovary (CHO) cell line, a baby hamster kidney (BHK) cell line, an NSO mouse myeloma cell line, a HEK293 cell line, or a Vero African green monkey kidney cell line.

17. The mammalian cell line of claim 16, wherein the cell line is a CHO cell line.

18. The mammalian cell line of claim 17, wherein cell viability, viable cell density, titer, growth rate, proliferative response, cell morphology and / or general cell health is comparable to that of a non-engineered parental mammalian cell line.

19. The mammalian cell line of claim 10, further comprising at least one nucleic acid encoding a recombinant protein selected from an antibody, an antibody fragment, a vaccine, a growth factor, a cytokine, a hormone or a coagulation factor.

20. The mammalian cell line of claim 19, wherein the antibody is a bispecific or multispecific antibody.

21. A polynucleotide comprising a nucleic acid sequence encoding a functional ASNS and at least one recombinant protein of interest.

22. A polynucleotide comprising a) a nucleic acid sequence encoding a functional ASNS; b) a nucleic acid sequence encoding a functional GS; and c) a polynucleotide encoding a nucleic acid sequence of a target recombinant protein. The method according to claim 1, wherein the functional ASNS gene comprises a heterologous promoter sequence.

23. The method of claim 1, wherein the functional ASNS gene comprises an endogenous promoter sequence.

24. The method of claim 24, wherein the endogenous promoter is an endogenous ASNS promoter.

25. The polynucleotide of claim 22, wherein the functional ASNS comprises an endogenous ASNS promoter.

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