Metabolic selection through the serine biosynthetic pathway

Abstract

The present disclosure provides isolated mammalian cells comprising reduced or ablated expression of phosphoserine phosphatase (PSPH). Also provided are methods for preparing such cells and for using such cells in the production of recombinant proteins.

Description

[Technical Field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 377,876, filed September 30, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to mammalian cell lines for use in biological production systems, wherein the mammalian cell lines have been engineered to have reduced or eliminated expression of components of the serine biosynthetic pathway to create cell lines that cannot grow in the absence of exogenously supplied serine or heterologously expressed coding sequences required for serine biosynthesis. [Background technology]

[0003] The development of highly productive 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). While the GS system has become standard in the industry, there is a need for cell lines that allow for multiple selection methods 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

[0004] Various aspects of the present disclosure provide mammalian cell lines for use in biological production systems, wherein the mammalian cell lines have been engineered to have reduced or eliminated expression of the endogenous phosphoserine phosphatase (PSPH) gene. In the absence of endogenously expressed functional PSPH protein, cells require an exogenous source of the amino acid serine. The chromosomal PSPH sequence can be inactivated using genomic modification via targeted endonucleases, such as CRISPR ribonucleoprotein (RNP) complexes or zinc finger nucleases. Another aspect of the present disclosure provides mammalian cell lines, wherein the mammalian cell lines have been engineered to have reduced or eliminated expression of the endogenous PSPH gene and reduced or eliminated expression of the endogenous glutamine synthetase (GS) gene.

[0005] Another aspect of the present disclosure involves a process for selecting cell lines that enhance the productivity of expressed biotherapeutic proteins. Another aspect of the present disclosure is to provide a bioproduction system for the expression of bispecific antibodies or biotherapeutic proteins that require the expression of effector proteins, more conveniently by utilizing multiple selection systems. The process involves expressing at least one recombinant protein in any of the mammalian cell lines.

[0006] Other aspects and iterations of the present disclosure are described in more detail below. [Brief explanation of the drawings]

[0007] [Figure 1] Figure 1 shows that phosphoserine phosphatase (PSPH) catalyzes the final step in the de novo serine biosynthetic pathway. (1) Yang, M., Vousden, KH, Serine and one-carbon metabolism in cancer. Nature. 16, 650-660 (2016)

[0008] [Figure 2]Figure 2 shows the PSPH cDNA sequence in CHO. The gRNA target site is bold and underlined.

[0009] [Figure 3] FIG. 3 shows copy number analysis of the endogenous phosphoserine phosphatase gene via ddPCR.

[0010] [Figure 4] Figure 4 shows that Cas9 cleavage activity was assessed by adding Ser to the culture medium. The percentage of PSPH edited in the RNA transfected pool was determined via next-generation sequencing. Adding 10 mM Ser resulted in optimal cleavage efficiency.

[0011] [Figure 5] Figure 5 shows the cloned KO alleles generated by CRISPR / Cas9 targeting. All indels generate early stop codons in the coding sequence. The PSPH KO genotype of the clones was confirmed via NGS. KO indels and frequencies are highlighted in bold.

[0012] [Figure 6-1] Figure 6 shows how vectors were designed to allow for selection of GFP-positive cells using a glutamine-based selection system, selection of BFP-positive cells using a serine-based selection system, and expression of secreted recombinant proteins through the development of two similar vectors containing the mAb heavy chain, light chain, and either the PSPH or GS coding sequence. [Figure 6-2] Same as above [Figure 6-3] Same as above

[0013] [Figure 7] FIG. 7. PSPH KO clones show increased sensitivity to serine starvation in growth and a lower maximum VCD than parental PSPH+ / + controls.

[0014] [Figure 8] Figure 8 shows the growth and viability of pools co-expressing GFP+BFP using GS+PSPH selection.

[0015] [Figure 9] Figure 9 shows that CHO cells with genetically disrupted GS and PSPH genes were co-transfected with PSPH+BFP and GS+GFP, selected in medium lacking both glutamine and serine, and expressed both GFP and BFP.

[0016] [Figure 10] Figure 10 shows that CHO cells with genetically disrupted GS and PSPH genes were transfected with either GS-IgG or PSPH-IgG, or co-transfected with GS-IgG and PSPH IgG, and selected in the respective selective media. The titers of the bulk selection pool are shown. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present disclosure provides mammalian cell lines engineered to have reduced or eliminated expression of the endogenous PSPH gene. Also provided are mammalian cell lines engineered to have reduced or eliminated expression of the endogenous GS gene and reduced or eliminated expression of the endogenous PSPH gene. Methods for producing the engineered cell lines and methods for selecting and using the engineered cell lines to produce recombinant proteins are provided.

[0018] (I) Engineered cell lines Certain embodiments of the present disclosure include mammalian cell lines engineered to have reduced or eliminated expression of the endogenous PSPH gene, or alternatively, mammalian cell lines engineered to have reduced or eliminated expression of both the endogenous PSPH gene and the endogenous GS gene.

[0019] The cell lines disclosed herein with reduced or eliminated expression of PSPH or reduced expression of PSPH and GS are genetically engineered to modify the chromosomal sequence encoding the PSPH or GS protein. The chromosomal sequence can be modified using targeted endonuclease-mediated genome editing techniques, which are described in more detail in section (III) below. For example, the chromosomal sequence can be modified to include at least one nucleotide deletion, at least one nucleotide insertion, at least one nucleotide substitution, or a combination thereof, such that the reading frame is shifted and the protein product is not produced (i.e., the chromosomal sequence is inactivated). Inactivating one allele of the chromosomal sequence encoding either PSPH or GS reduces protein expression (i.e., knockdown). Inactivating both alleles of the chromosomal sequence encoding either PSPH or GS eliminates protein expression (i.e., knockout).

[0020] In some embodiments, the expression level of PSPH may 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, the expression level of PSPH may be reduced to a level that is undetectable using techniques standard in the art (e.g., Western immunoblotting assays, ELISA enzyme assays, SDS-polyacrylamide gel electrophoresis, etc.).

[0021] Generally, the cell viability, viable cell density, titer, proliferation rate, growth response, cell morphology, levels of apoptosis and autophagy, and / or overall cell health of the engineered cell lines disclosed herein are comparable to those of the unengineered parental cells when supplemented with serine and / or exogenous PSPH coding sequences.

[0022] (a) Cell typeThe 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 carcinoma cells (HELA); human embryonic retinal cells (PER.C6); human kidney cells (HKB-11); human hepatocytes (Huh-7); human lung cells (W138); human liver cells (Hep G2); human U2-OS osteosarcoma cells, human A549 lung cells, human A-431 epithelial cells, CACO-2 human colorectal adenocarcinoma cells, human pluripotent stem cells, Jurkat human T lymphocyte cells, or human K562 bone marrow cells. In other embodiments, the engineered cell lines can be derived from non-human cell lines. Suitable cell lines include Chinese hamster ovary (CHO) cells; baby hamster kidney (BHK) cells; mouse myeloma NS0 cells; mouse myeloma Sp2 / 0 cells; mouse mammary 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 carcinoma CT26 cells; and mouse prostate DuCuP cells. These include: mouse mammary EMT6 cells; mouse hepatoma Hepa1c1c7 cells; mouse myeloma J5582 cells; mouse epithelial MTD-1A cells; mouse cardiac MyEnd cells; mouse kidney RenCa cells; mouse pancreatic RIN-5F cells; mouse melanoma X64 cells; mouse lymphoma YAC-1 cells; rat glioblastoma 9L cells; rat B lymphoma RBL cells; rat neuroblastoma B35 cells; rat hepatocytes (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 fibroblast (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 American Type Culture Collection catalog (ATCC, Manassas, VA). In some embodiments, the cell lines disclosed herein are other than murine cell lines.In a particular embodiment, the engineered cell line is a CHO cell line. Suitable CHO cell lines are CHO-K1, CHO-K1SV, CHO GS. - / - , CHO S, DG44, DuxB11, and cell lines derived therefrom.

[0023] In various embodiments, the parent cell line may be deficient in glutamine synthetase (GS), dihydrofolate reductase (DHFR), hypoxanthine-guanine phosphoribosyltransferase (HPRT), asparagine synthetase (ASNS), or a combination thereof. For example, chromosomal sequences encoding GS, DHFR, HPRT, and / or ASNS may be inactivated. In certain embodiments, all chromosomal sequences encoding GS, DHFR, HPRT, and / or ASNS are inactivated in the parent cell line.

[0024] (b) an optional nucleic acid encoding a recombinant protein; In some embodiments, the engineered cell lines disclosed herein may further comprise at least one nucleic acid encoding a recombinant protein. Generally, the recombinant protein is heterologous, meaning that the protein is not native to the cell. The recombinant protein may be a therapeutic protein selected from, but not limited to, an antibody, an antibody fragment, a monoclonal antibody, a humanized antibody, a humanized monoclonal antibody, a chimeric antibody, an IgG molecule, an IgG heavy chain, an IgG light chain, an IgA molecule, an IgD molecule, an IgE molecule, an IgM molecule, a vaccine, a growth factor, a cytokine, an interferon, an interleukin, a hormone, a clotting (or coagulation) factor, a blood component, an enzyme, a therapeutic protein, a nutraceutical protein, a functional fragment or functional variant of any of the above, or a fusion protein comprising any of the above proteins and / or a functional fragment or variant thereof. In certain embodiments, the recombinant protein is a bispecific or multispecific antibody or a protein that requires an effector protein for expression.

[0025] In some embodiments, a nucleic acid encoding a recombinant protein may be linked to a sequence encoding phosphoserine phosphatase (PSPH), hypoxanthine-guanine phosphoribosyltransferase (HPRT), dihydrofolate reductase (DHFR), and / or glutamine synthetase (GS), such that PSPH, ASNS, HPRT, DHFR, and / or GS can be used as selectable markers. The nucleic acid encoding a recombinant protein may 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 a recombinant protein may be part of an expression construct. The expression construct or vector may include additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), selectable marker sequences, origins of replication, 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" Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd edition, 2001.

[0026] In some embodiments, the nucleic acid encoding the recombinant protein can be located extrachromosomally. That is, the nucleic acid encoding the recombinant protein can be transiently expressed from a plasmid, cosmid, artificial chromosome, minichromosome, or another extrachromosomal construct. In other embodiments, the nucleic acid encoding the recombinant protein can be chromosomally integrated into the genome of the cell. Integration can be random or targeted. Thus, the recombinant protein can be stably expressed. In some iterations of this embodiment, the nucleic acid sequence encoding the recombinant protein can be operably linked to an appropriate heterologous expression control sequence (i.e., promoter). In other iterations, the nucleic acid sequence encoding the recombinant protein can be placed under the control of an endogenous expression control sequence. The nucleic acid sequence encoding the recombinant protein can be integrated into the genome of the cell line using homologous recombination, targeting endonuclease-mediated genome editing, viral vectors, transposons, recombinase-mediated cassette exchange systems, plasmids, and other well-known means. Additional guidance can be found in Ausubel et al. 2003, supra, and Sambrook & Russell, 2001.

[0027] (II) Kit A further aspect of the present disclosure provides kits for recombinant protein production, wherein the kits include any of the engineered cell lines detailed in section (I) above. The kits may further include cell growth medium, transfection reagents, plasmid vectors, selective media, recombinant protein purification means, buffers, etc. The kits provided herein generally include instructions for growing the cell line and using it to produce the recombinant protein. The instructions included in the kit may be affixed to packaging material or may be included as a package insert. The instructions are typically, but are not limited to, written or printed material. Any medium capable of recording such instructions and transmitting them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROMs), etc. As used herein, the term "instructions" may include the address of an internet site providing the instructions.

[0028] (III) Methods for preparing engineered cell lines Yet another aspect of the present disclosure provides a method for preparing or engineering a cell line with reduced or eliminated expression of PSPH and / or GS, as described in section (I) above. The chromosomal sequences encoding PSPH and / or GS can be knocked down or knocked out using various techniques. Typically, engineered cell lines are prepared using a targeted endonuclease-mediated genome modification process. Those skilled in the art will understand that the engineered cell line can also be prepared using a site-specific recombination system, random mutagenesis, or other methods known in the art.

[0029] Generally, engineered cell lines are prepared by a method comprising introducing at least one targeting endonuclease or a nucleic acid encoding the targeting endonuclease into a parent cell line of interest, wherein the targeting endonuclease is targeted to a chromosomal sequence encoding PSPH and / or GS. The targeting endonuclease recognizes and binds to a specific chromosomal sequence, introducing a double-strand break. In some embodiments, the double-strand break is repaired by the non-homologous end joining (NHEJ) repair process. Because NHEJ is error-prone, deletion, insertion, and / or substitution of at least one nucleotide may occur, disrupting the reading frame of the chromosomal sequence and resulting in the production of a non-functional protein, for example, through disruption of the protein's enzymatic active site. In other embodiments, targeting endonucleases can also be used to alter chromosomal sequences via homologous recombination reactions by co-introducing a polynucleotide with substantial sequence identity to a portion of the targeted chromosomal sequence. In such a situation, the double-strand break introduced by the targeted endonuclease is repaired by a homology-directed repair process in which the chromosomal sequence is replaced with the polynucleotide in such a way that the chromosomal sequence is altered or altered (e.g., integration of the exogenous sequence).

[0030] (a) Targeting endonuclease Various targeting endonucleases can be used to modify the chromosomal sequence encoding PSPH and / or GS.Targeting endonucleases can be naturally occurring proteins or engineered proteins.Suitable targeting 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 targeting DNA double-strand break inducers.

[0031] (i) Zinc finger nuclease In certain embodiments, the targeting endonuclease can be a pair of zinc finger nucleases (ZFNs). ZFNs bind to specific target sequences and introduce double-strand breaks at the targeted cleavage site. Typically, ZFNs comprise a DNA binding domain (i.e., zinc finger) and a cleavage domain (i.e., nuclease), each of which is described below.

[0032] DNA-binding domain DNA binding domains or zinc fingers can be engineered to recognize and bind to any nucleic acid sequence. 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. 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, for example, involves using a database containing duplicate, triplet, and / or quadruplet nucleotide sequences and individual zinc finger amino acid sequences, where each duplicate, triplet, or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers that bind to a particular triplet or quadruplet sequence. See, for example, U.S. Patent Nos. 6,453,242 and 6,534,261, the disclosures of which are incorporated herein by reference in their entireties. As an example, the algorithm described in U.S. Patent No. 6,453,242 can be used to design zinc finger binding domains that target preselected sequences.Alternative methods, such as rational design using nondegenerate recognition code tables, may be used to design zinc finger binding domains that 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 for 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. 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).

[0033] A zinc finger binding domain can be designed to recognize and bind to a DNA sequence ranging from about 3 to about 21 nucleotides in length. In certain embodiments, a zinc finger binding domain can be designed to recognize and bind to a DNA sequence ranging from about 9 to about 18 nucleotides in length. Generally, the zinc finger binding domain of a zinc finger nuclease used herein comprises at least three zinc finger recognition regions or zinc fingers, where each zinc finger binds to three nucleotides. In certain embodiments, a zinc finger binding domain comprises four zinc finger recognition regions. In another embodiment, a zinc finger binding domain comprises five zinc finger recognition regions. In yet another embodiment, a zinc finger binding domain comprises six zinc finger recognition regions. A zinc finger binding domain can be designed to bind to any suitable target DNA sequence. See, for example, U.S. Patent Nos. 6,607,882; 6,534,261; and 6,453,242, the disclosures of which are incorporated herein by reference in their entireties.

[0034] Exemplary methods for selecting zinc finger recognition regions include phage display and two-hybrid systems, which are described in U.S. Patent 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, as well as International Publication Nos. WO 98 / 37186, WO 98 / 53057, WO 00 / 27878, WO 01 / 88197, and British Patent No. 2,338,237, each of which is incorporated herein by reference in its entirety. In addition, enhancement of the binding specificity of zinc finger binding domains is described, for example, in International Publication No. WO 02 / 077227, the entire disclosure of which is incorporated herein by reference.

[0035] Methods for designing and constructing zinc finger binding domains and fusion proteins (and polynucleotides encoding them) are known to those skilled in the art and are described in detail, for example, in U.S. Patent No. 7,888,121, the entire contents of which are incorporated herein by reference. Zinc finger recognition regions and / or multi-finger zinc finger proteins can be linked using appropriate linker sequences, including, for example, linkers of 5 or more amino acids in length. For non-limiting examples of linker sequences of 6 or more amino acids in length, see U.S. Patent Nos. 6,479,626, 6,903,185, and 7,153,949, the disclosures of which are incorporated herein by reference in their entireties. The zinc finger binding domains described herein may include a combination of appropriate linkers between the individual zinc fingers of the protein.

[0036] Cleavage domain Zinc finger nucleases also contain a cleavage domain. The cleavage domain portion of zinc finger nucleases 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. For example, see New England Biolabs Catalog or Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388. Other enzymes that cleave DNA are known (e.g., S1 nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease). 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 the source of the cleavage domain.

[0037] The cleavage domain can also be derived from an enzyme or portion thereof that requires dimerization for cleavage activity, as described above. Two zinc finger nucleases may be required for cleavage, with each nuclease containing a monomer of the active enzyme dimer. Alternatively, a single zinc finger nuclease may contain both monomers to form an active enzyme dimer. As used herein, an "active enzyme dimer" is an enzyme dimer that can cleave a nucleic acid molecule. The two cleavage monomers may be derived from the same endonuclease (or functional fragments thereof), or each monomer may be derived from a different endonuclease (or functional fragments thereof).

[0038] When two cleavage monomers are used to form an active enzyme dimer, the recognition sites of the two zinc fingers are preferably positioned such that the cleavage monomers are spatially oriented relative to one another such that binding of the two zinc fingers to their respective recognition sites allows the cleavage monomers to form an active enzyme dimer (e.g., by dimerization). Consequently, the near edges of the recognition sites can be about 5 to about 18 nucleotides apart. For example, the near edges can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides apart. However, it will be understood that any integer number of nucleotides or nucleotide pairs can be interposed between the two recognition sites (e.g., about 2 to about 50 nucleotide pairs or more). For example, the near edges of the recognition sites of zinc finger nucleases, such as those described in detail herein, can be 6 nucleotides apart. Typically, the cleavage site is located between the recognition sites.

[0039] Restriction endonucleases (restriction enzymes) exist in many species and can bind to DNA (at recognition sites) in a sequence-specific manner and cleave the DNA at or near the binding site. Some restriction enzymes (e.g., Type IIS) cleave DNA at sites distant from the recognition site and have separable binding and cleavage domains. For example, the Type IIS enzyme FokI catalyzes a double-stranded cleavage of DNA 9 nucleotides from the recognition site on one strand and 13 nucleotides from the recognition site on the other strand. See, e.g., U.S. Patent 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 91:883-887; Kim et al. (1994b) J. Biol. Chem. 269:31978-31982. Thus, a zinc finger nuclease can comprise at least one Type IIS restriction enzyme cleavage domain and one or more zinc finger binding domains, which may or may not be engineered. Exemplary Type IIS restriction enzymes are described, for example, in International Publication No. 07 / 014,275, the disclosure of which is incorporated herein by reference in its entirety. Additional restriction enzymes also contain separable binding and cleavage domains and are also contemplated by the present disclosure. See, e.g., Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

[0040] An exemplary Type IIS restriction enzyme in which the cleavage domain is separable from the binding domain is FokI. This particular enzyme is active as a dimer (Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10, 570-10, 575). Therefore, in the present disclosure, the portion of the FokI enzyme used in a zinc finger nuclease is considered a cleavage monomer. Thus, for targeted double-strand cleavage using a Fok cleavage domain, two zinc finger nucleases, each containing a FokI cleavage monomer, can be used to reconstitute an active enzyme dimer. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two FokI cleavage monomers can be used.

[0041] In certain embodiments, the cleavage domain contains one or more engineered cleavage monomers that minimize or inhibit homodimerization. As a non-limiting example, the 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 all targets for affecting dimerization of the Fok cleavage half-domain. Exemplary engineered cleavage monomers of FokI that form obligate heterodimers include a pair in which the first cleavage monomer contains a mutation at amino acid residue positions 490 and 538 of FokI, and the second cleavage monomer contains a mutation at amino acid residue positions 486 and 499.

[0042] Thus, in one embodiment of an engineered cleavage monomer, a mutation at amino acid position 490 replaces Glu(E) with Lys(K); a mutation at amino acid residue 538 replaces Iso(I) with Lys(K); a mutation at amino acid residue 486 replaces Gln(Q) with Glu(E); and a mutation at position 499 replaces Iso(I) with Lys(K). Specifically, engineered cleavage monomers can be prepared by mutating position 490 from E to K and position 538 from I to K in one cleavage monomer to create an engineered cleavage monomer designated "E490K:I538K," and mutating position 486 from Q to E and position 499 from I to K in another cleavage monomer to create an engineered cleavage monomer designated "Q486E:I499K." The engineered cleavage monomers are obligate heterodimer mutants in which aberrant cleavage is minimized or eliminated. The engineered cleavage monomers can be prepared using suitable methods, for example, by site-directed mutagenesis of the wild-type cleavage monomer (FokI), as described in U.S. Patent No. 7,888,121, the entire contents of which are incorporated herein.

[0043] Additional Domains In some embodiments, the zinc finger nuclease further comprises at least one nuclear localization sequence (NLS). An NLS is an amino acid sequence that promotes targeting of the zinc finger nuclease protein into the nucleus and introduction of a double-strand break at the target sequence in the chromosome. Nuclear localization signals are known in the art (see, for example, 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), SALIKKKKKKMAP (SEQ ID NO: 11), NLSs include 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 RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 18). The NLS can be located at the N-terminus, C-terminus, or internally of the zinc finger nuclease.

[0044] In further embodiments, the zinc finger nuclease may also comprise at least one cell membrane-permeable domain. Examples of suitable cell membrane-permeable domains include, but are not limited to, GRKKRRQRRRPPQPKKKRKV (SEQ ID NO: 19), PLSSIFSRIGDPPKKKRKV (SEQ ID NO: 20), GALFLGWLGAAGSTMGAPKKKRKV (SEQ ID NO: 21), GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 22), KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 23), YARAAARQARA (SEQ ID NO: 24), THLPRRRRR (SEQ ID NO: 25), GGRRRRR (SEQ ID NO: 26), RRQRRRTSKLMKR (SEQ ID NO: 27), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 28), KALAWEAKLAKALAKHLAKALAKALKCEA (SEQ ID NO: 29), and RQIKIWFQNRRMKWKK (SEQ ID NO: 30). The cell membrane permeation domain can be located at the N-terminus, C-terminus, or internally of the zinc finger nuclease.

[0045] In still other embodiments, the zinc finger nuclease may further comprise at least one marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, and epitope tags. In certain embodiments, the marker 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, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric In another embodiment, the marker domain may be a purification tag and / or an epitope tag.Suitable tags include, but are not limited to, poly(His) tag, FLAG (or DDK) tag, Halo tag, AcV5 tag, AU1 tag, AU5 tag, biotin carboxyl carrier protein (BCCP), calmodulin-binding protein (CBP), chitin-binding domain (CBD), E tag, E2 tag, ECS tag, eXact tag, Glu-Glu tag, glutathione-S-transferase (GST), HA tag, HSV tag, KT3 tag, maltose-binding protein (MBP), MAP tag, Myc tag, NE tag, NusA tag, PDZ tag, S tag, S1 tag, SBP tag, Softag 1 tag, Softag 3 tag, Spot tag, Strep tag, SUMO tag, T7 tag, tandem affinity purification (TAP) tag, thioredoxin (TRX), V5 tag, VSV-G tag, and Xa tag. The marker domain can be located at the N-terminus, C-terminus, or internally of the zinc finger nuclease.

[0046] At least one nuclear localization signal, at least one cell membrane-permeable domain, and / or at least one marker domain can be directly linked 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 membrane-permeable domain, and / or at least one marker domain can be indirectly linked to the zinc finger nuclease via one or more linkers. Suitable linkers include amino acids, peptides, nucleotides, nucleic acids, organic linker molecules (e.g., maleimide derivatives, N-ethoxybenzylimidazole, biphenyl-3,4',5-tricarboxylic acid, p-aminobenzyloxycarbonyl, etc.), disulfide linkers, and polymer linkers (e.g., PEG). The linker can include one or more spacer groups, including, but not limited to, alkylene, alkenylene, alkynylene, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl, etc. The linker may be neutral or may have a positive or negative charge. In addition, the linker may be cleavable such that the covalent bond of the linker connecting the linker to another chemical group can be broken or cleaved under certain conditions, including pH, temperature, salt concentration, light, catalyst, or enzyme. In some embodiments, the linker may be a peptide linker. The peptide linker may be a flexible amino acid linker or a rigid amino acid linker. Further examples of suitable linkers are well known in the art, and programs for designing linkers are readily available (Crasto et al., Protein Eng., 2000, 13(5):309-312).

[0047] (ii) CRISPR ribonucleoproteins (RNPs) In other embodiments, targeting endonuclease can be clustered regularly interspaced short palindromic repeats (CRISPR) nuclease.CRISPR nuclease is the RNA-guided nuclease derived from bacterial or archaeal CRISPR / CRIPSR-associated (Cas) system.CRISPR RNP system comprises CRISPR nuclease and guide RNA.

[0048] 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 present in a variety of bacteria and archaea. For example, CRISPR nucleases can be used to inhibit the growth of Streptococcus species (e.g., S. pyogenes, S. thermophilus, S. pasteurianus), Campylobacter species (e.g., Campylobacter jejuni), Francisella species (e.g., Francisella novicida), Acaryochloris species, Acetohalobium species, Acidaminococcus species, Acidithiobacillus species, sp., Alicyclobacillus sp., Allochromatium sp., Ammonifex sp., Anabaena sp., Arthrospira sp., Bacillus sp., Burkholderiales sp., Caldicelulosiruptor sp., Candidatus sp., Clostridium sp., Crocosphaera sp., Cyanothece sp., Exiguobacterium sp., Finegoldia sp. 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. sp., Pelotomaculum sp., Pseudoalteromonas sp., Petrotoga sp., Prevotella sp., Staphylococcus sp., Streptomyces sp., Streptosporangium sp., Synechococcus sp., Thermosipho sp., or Verrucomicrobia sp. In other embodiments, the CRISPR nuclease can 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).

[0049] In some embodiments, the CRISPR nuclease can be derived from a type II CRISPR nuclease. For example, the type II CRISPR nuclease can be a Cas9 protein. Suitable Cas9 nucleases include Streptococcus pyogenes Cas9 (SpCas9), Francisella novicida Cas9 (FnCas9), Staphylococcus aureus (SaCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (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 Cpf1 nuclease. Suitable Cpf1 nucleases include Francisella novicida Cpf1 (FnCpf1), Acidaminococcus sp. Cpf1 (AsCpf1), or Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1). In yet another embodiment, the CRISPR nuclease can be derived from a Type VI CRISPR nuclease, such as Leptotrichia wadei Cas13a (LwaCas13a) or Leptotrichia shahii Cas13a (LshCas13a).

[0050] CRISPR nuclease can be wild-type CRISPR nuclease, modified CRISPR nuclease, or a fragment of wild-type or modified CRISPR nuclease.CRISPR nuclease can be modified to increase nucleic acid binding affinity and / or specificity, change enzymatic activity, and / or change other properties of protein.For example, the nuclease (i.e., DNase, RNase) domain of CRISPR nuclease can be modified, deleted, or inactivated.CRISPR nuclease can be truncated to remove domains that are not essential for nuclease function.

[0051] CRISPR nuclease comprises two nuclease domains.For example, Cas9 nuclease comprises HNH domain, which cuts the complementary strand of guide RNA, and RuvC domain, which cuts non-complementary strand; Cpf1 nuclease comprises RuvC domain and NUC domain, and Cas13a nuclease comprises two HNEPN domains.When both nuclease domains function, CRISPR nuclease introduces double-strand break.Either nuclease domain can be inactivated by one or more mutations and / or deletions, thereby creating a variant that introduces single-strand breaks in one strand of double-stranded sequence. For example, one or more mutations in the RuvC domain of 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 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. Equivalent mutations can convert Cpfl and Cas13a nucleases into nickases. Two CRISPR nickases that target opposite strands of a chromosomal sequence (via a pair of offset guide RNAs) can be used in combination to generate double-strand breaks in the chromosomal sequence. Dual CRISPR nickase RNPs can improve target specificity and reduce off-target effects.

[0052] Additional Domains CRISPR nuclease may further comprise at least one nuclear localization sequence (NLS).NLS is an amino acid sequence that targets zinc finger nuclease protein into the nucleus and promotes the introduction of double-strand breaks in the target sequence in chromosome.Nuclear localization signals are known in the art (see, for example, 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), SALIKKKKKKMAP (SEQ ID NO: 11), NLSs include 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 RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 18). The NLS can be located at the N-terminus, C-terminus, or internally of the CRISPR nuclease.

[0053] In further embodiments, CRISPR nucleases can also comprise at least one cell membrane permeation domain. Examples of suitable cell membrane permeation domains include, but are not limited to, GRKKRRQRRRPPQPKKKRKV (SEQ ID NO: 19), PLSSIFSRIGDPPKKKRKV (SEQ ID NO: 20), GALFLGWLGAAGSTMGAPKKKRKV (SEQ ID NO: 21), GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 22), KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 23), YARAAARQARA (SEQ ID NO: 24), THLPRRRRR (SEQ ID NO: 25), GGRRRRR (SEQ ID NO: 26), RRQRRRTSKLMKR (SEQ ID NO: 27), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 28), KALAWEAKLAKALAKHLAKALAKALKCEA (SEQ ID NO: 29) and RQIKIWFQNRRMKWKK (SEQ ID NO: 30). The cell membrane permeation domain can be located at the N-terminus, C-terminus, or internally of the CRISPR protein.

[0054] In yet other embodiments, the CRISPR nuclease may further comprise at least one marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, and epitope tags. In certain embodiments, the marker 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, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric In another embodiment, the marker domain may be a purification tag and / or an epitope tag.Suitable tags include, but are not limited to, poly(His) tag, FLAG (or DDK) tag, Halo tag, AcV5 tag, AU1 tag, AU5 tag, biotin carboxyl carrier protein (BCCP), calmodulin-binding protein (CBP), chitin-binding domain (CBD), E tag, E2 tag, ECS tag, eXact tag, Glu-Glu tag, glutathione-S-transferase (GST), HA tag, HSV tag, KT3 tag, maltose-binding protein (MBP), MAP tag, Myc tag, NE tag, NusA tag, PDZ tag, S tag, S1 tag, SBP tag, Softag 1 tag, Softag 3 tag, Spot tag, Strep tag, SUMO tag, T7 tag, tandem affinity purification (TAP) tag, thioredoxin (TRX), V5 tag, VSV-G tag, and Xa tag. The marker domain can be located at the N-terminus, C-terminus, or internally of the CRISPR nuclease.

[0055] At least one nuclear localization signal, at least one cell membrane permeation domain, and / or at least one marker domain can be directly linked 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 membrane permeation domain, and / or at least one marker domain can be indirectly linked to the CRISPR nuclease via one or more linkers. Suitable linkers include amino acids, peptides, nucleotides, nucleic acids, organic linker molecules (e.g., maleimide derivatives, N-ethoxybenzylimidazole, biphenyl-3,4',5-tricarboxylic acid, p-aminobenzyloxycarbonyl, etc.), disulfide linkers, and polymer linkers (e.g., PEG). The linker can include one or more spacer groups, including, but not limited to, alkylene, alkenylene, alkynylene, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl, etc. The linker may be neutral or may have a positive or negative charge. In addition, the linker may be cleavable such that the covalent bond of the linker connecting the linker to another chemical group can be broken or cleaved under certain conditions, including pH, temperature, salt concentration, light, catalyst, or enzyme. In some embodiments, the linker may be a peptide linker. The peptide linker may be a flexible amino acid linker or a rigid amino acid linker. Further examples of suitable linkers are well known in the art, and programs for designing linkers are readily available in the art.

[0056] guide RNA CRISPR nucleases are guided to their target sites by guide RNAs. The guide RNA hybridizes to the target site, interacts with the CRISPR nuclease, and directs the CRISPR nuclease to the target site in the chromosomal sequence. The target site contains protospacer adjacent motifs ( p rotospacer a djacent mThere are no sequence limitations other than the presence of a PAM (protif). CRISPR proteins in 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 either G or A, W is defined as either A or T, Y is defined as either C or T, and V is defined as A, C, or G. The Cas9 PAM is located 3' of the target site, and the cpf1 PAM is located 5' of the target site.

[0057] A guide RNA contains 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 essentially 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 called scaffold regions) of each guide RNA can be the same for all guide RNAs.

[0058] The first region of the guide RNA is complementary to the sequence of the target site (i.e., the protospacer sequence) so that the first region of the guide RNA can base pair with the sequence of the target site. The complementarity between the first region of the guide RNA (i.e., the crRNA) and the target sequence can be at least 80%, at least 85%, at least 90%, at least 95%, or more. Generally, there is no mismatch between the first region of the guide RNA and the sequence of the target site (i.e., complete complementarity). In various embodiments, the first region of the guide RNA can comprise from about 10 nucleotides to more than about 25 nucleotides. For example, the region of base pairing between the first region of the guide RNA and the target site of the chromosomal sequence can 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 exemplary embodiments, the first region of the guide RNA is about 19, 20, or 21 nucleotides in length.

[0059] The guide RNA also comprises a second region that forms a secondary structure. In some embodiments, the secondary structure comprises a stem (or hairpin) and a loop. The lengths of the loop and stem can vary. For example, the loop can range from about 3 to about 10 nucleotides in length, and the stem can range from about 6 to about 20 base pairs in length. The stem can include one or more bulges of 1 to about 10 nucleotides in length. That is, the entire length of the second region can range from about 16 to 60 nucleotides in length. In an exemplary embodiment, the loop is about 4 nucleotides in length, and the stem comprises about 12 base pairs.

[0060] Guide RNA also comprises a third region at the 3' end, which remains essentially single-stranded.Therefore, the third region has no complementarity to any chromosomal sequence in target cell, and also has no complementarity to the rest of guide RNA.The length of the third region can vary.Generally, the third region is more than about 4 nucleotides in length.For example, the length of the third region can be in the range of about 5 to about 60 nucleotides in length.

[0061] The combined length of the second and third regions (or scaffold) of the guide RNA can range from about 30 to about 120 nucleotides in length, hi some embodiments, the combined length of the second and third regions of the guide RNA ranges from about 70 to 100 nucleotides in length.

[0062] In some embodiments, the guide RNA comprises a single molecule comprising all three regions. In other embodiments, the guide RNA may comprise two separate molecules. The first RNA molecule may comprise the first (5') region of the guide RNA and half of the "stem" of the second region of the guide RNA. The second RNA molecule may comprise the other half of the "stem" of the second region of the guide RNA and the third region of the guide RNA. Thus, in this embodiment, the first and second RNA molecules each contain a sequence of nucleotides complementary to the other. For example, in certain embodiments, the first and second RNA molecules each comprise a sequence (of about 6 to about 20 nucleotides) that base-pairs with the other sequence to form a functional guide RNA.

[0063] (iii) Other targeting endonucleases In a further embodiment, the targeting endonuclease can be a meganuclease. Meganucleases are endodeoxyribonucleases characterized by long recognition sequences, i.e., the recognition sequence generally ranges from about 12 base pairs to about 40 base pairs. Because of this requirement, the recognition sequence generally occurs only once in any genome. Among meganucleases, a family of homing endonucleases named LAGLIDADG has become a valuable tool for genome research and genome engineering (see, for example, Arnould et al., 2011, Protein Eng Des Sel, 24(1-2):27-31). Other suitable meganucleases include I-CreI and I-Dmol. Meganucleases can be targeted to specific chromosomal sequences by modifying their recognition sequences using techniques well known to those skilled in the art.

[0064] In a further embodiment, the targeting endonuclease can be a transcription activator-like effector (TALE) nuclease. TALEs are transcription factors from the plant pathogen Xanthomonas that can be easily engineered to bind to new DNA targets. TALEs, or truncated versions thereof, can be linked to the catalytic domain of an endonuclease such as FokI to create targeting endonucleases called TALE nucleases or TALENs (Sanjana et al., 2012, Nat Protoc, 7(1):171-192) and Arnould et al., 2011, Protein Engineering, Design & Selection, 24(1-2):27-31).

[0065] In another embodiment, 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 means for generating such chimeric nuclease fusions.

[0066] In yet another embodiment, the targeting endonuclease can be a site-specific endonuclease. In particular, the site-specific endonuclease can be a "rare-cutter" endonuclease, whose recognition sequence occurs rarely in the genome. Alternatively, the site-specific endonuclease can be engineered to cleave a site of interest (Friedhoff et al., 2007, Methods Mol Biol 352:1110123). Generally, the recognition sequence of the site-specific endonuclease occurs only once in the genome. In another further embodiment, the targeting endonuclease can be an artificial targeted DNA double-strand break inducer.

[0067] (b) Delivery of the targeted endonuclease to cells The method includes introducing a targeting endonuclease into a parent cell line of interest. The targeting endonuclease can be introduced into cells as a purified and isolated protein or as a nucleic acid encoding the targeting endonuclease. The nucleic acid can be DNA or RNA. In embodiments where the encoding nucleic acid is mRNA, the mRNA can 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 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 cells of interest. In embodiments where the targeting endonuclease is a CRISPR nuclease, the CRISPR nuclease system can be introduced into cells as a gRNA-protein complex.

[0068] The targeting endonuclease molecule can be introduced into cells by various means. Suitable delivery methods include microinjection, electroporation, sonoporation, particle bombardment, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impalefection, optical transfection, nucleic acid uptake enhancement by proprietary agents, and delivery via liposomes, immunoliposomes, virosomes, or artificial virions. In certain embodiments, the targeting endonuclease molecule is introduced into cells by nucleofection.

[0069] Optional Donor Polynucleotide The method for targeted genome modification or manipulation further comprises introducing at least one donor polynucleotide into cells, the donor polynucleotide comprising a sequence having at least one nucleotide change compared to the target chromosomal sequence.The donor polynucleotide has substantial sequence identity with the sequence at or near the target site in the chromosomal sequence, so that the double-strand break introduced by the targeting endonuclease can be repaired by homology-directed repair process, and the sequence of the donor polynucleotide can be inserted into or exchanged with the chromosomal sequence, thereby modifying the chromosomal sequence.For example, the donor polynucleotide can comprise a first sequence that has substantial sequence identity with the sequence on one side of the target site and a second sequence that has substantial sequence identity with the sequence on the other side of the target site.The donor polynucleotide can further comprise a donor sequence for integration into the target chromosomal sequence.For example, the donor sequence can be an exogenous sequence (such as a marker sequence), so that the integration of the exogenous sequence disrupts the reading frame and inactivates the target chromosomal sequence.

[0070] The length of the first and second sequences of donor polynucleotide that have substantial sequence identity with the sequence of target site or nearby in chromosome sequence can and will be various.Generally, each of the first and second sequences of donor polynucleotide is at least about 10 nucleotides in length.In various embodiments, the donor polynucleotide sequence that has substantial sequence identity with 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 in length.

[0071] The phrase "substantial sequence identity" means that the sequence of a polynucleotide has at least about 75% sequence identity to a chromosomal sequence of interest. In some embodiments, the sequence of a 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%, or 99% sequence identity to a chromosomal sequence of interest.

[0072] The length of the donor polynucleotide can and will vary. For example, the donor polynucleotide can range from about 20 nucleotides to about 200,000 nucleotides in length. In various embodiments, the donor polynucleotide can range from about 20 nucleotides to about 100 nucleotides in length, from about 100 nucleotides to about 1000 nucleotides in length, from about 1000 nucleotides to about 10,000 nucleotides in length, from about 10,000 nucleotides to about 100,000 nucleotides in length, or from about 100,000 nucleotides to about 200,000 nucleotides in length.

[0073] Typically, the donor polynucleotide is DNA. The DNA can be single-stranded or double-stranded. The DNA can be linear or circular. In some embodiments, the donor polynucleotide can be a single-stranded, linear oligonucleotide containing less than about 200 nucleotides. In other embodiments, the donor polynucleotide can be part of a vector. Suitable vectors include DNA plasmids, viral vectors, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs). In still other embodiments, the donor polynucleotide can be a PCR fragment or nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.

[0074] The donor polynucleotide and the targeting endonuclease molecule can be introduced into the cell simultaneously. Alternatively, the donor polynucleotide and the targeting endonuclease molecule can be introduced into the cell sequentially. The ratio of the targeting endonuclease molecule to the donor polynucleotide can and will vary. Generally, the ratio of the targeting endonuclease molecule to the donor polynucleotide ranges from 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 certain embodiments, the ratio is about 1:1.

[0075] (c) Cell culture The method further includes maintaining the cells under appropriate conditions so that the double-strand break introduced by the targeting endonuclease can be repaired (i) by 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) by a homology-directed repair process, such that the chromosomal sequence is exchanged with the sequence of the polynucleotide such that the chromosomal sequence is modified. In embodiments in which a nucleic acid encoding the targeting endonuclease is introduced into the cells, the method includes maintaining the cells under appropriate conditions so that the cells express the targeting endonuclease.

[0076] Generally, 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 understand that cell culture methods are known in the art and can and will vary depending on cell type.In all cases, routine optimization may be used to determine the optimal method for a particular cell type.

[0077] During this step of the process, the targeting endonuclease recognizes and binds to the target cleavage site in the chromosomal sequence, and generates a double-strand break, and in the process of repairing the double-strand break, at least one nucleotide deletion, insertion, and / or substitution is introduced into the target chromosomal sequence. In certain embodiments, the target chromosomal sequence is inactivated.

[0078] Upon confirmation that the chromosomal sequence of interest has been modified, a single clone can be isolated and genotyped (via DNA sequencing and / or protein analysis). Cells containing one modified chromosomal sequence can undergo one or more additional rounds of targeted genome modification to modify additional chromosomal sequences, thereby creating double knockouts, triple knockouts, etc.

[0079] (IV) Production of recombinant proteins Another aspect of the present disclosure encompasses a method for producing a recombinant protein in a biological production system. Suitable recombinant proteins are described in Section (I)(c). The method includes expressing a recombinant protein of interest in any of the engineered cell lines described in Section (I) above, and purifying the expressed recombinant protein. Means for producing or manufacturing recombinant proteins are well known in the art (see, e.g., "Biopharmaceutical Production Technology," Subramanian (ed), 2012, Wiley-VCH; ISBN: 978-3-527-33029-4).

[0080] Recombinant proteins can be purified via a process that includes a clarification step, such as filtration, and one or more chromatography steps, such as affinity chromatography, Protein A (or G) chromatography, ion exchange (i.e., cation and / or anion) chromatography, etc.

[0081] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention pertains. The following references provide those skilled in the art with general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The HarperCollins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless otherwise specified.

[0082] When referring to elements of the disclosure or preferred embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0083] As used herein, the term "endogenous sequence" refers to a chromosomal sequence that is native to the cell.

[0084] The term "exogenous sequence" refers to a chromosomal sequence that is not native to the cell or that has been moved to a different chromosomal location.

[0085] An "engineered" or "genetically modified" cell refers to a cell whose genome has been altered or manipulated, i.e., the cell contains at least one chromosomal sequence that has been engineered to contain an insertion of at least one nucleotide, a deletion of at least one nucleotide, and / or a substitution of at least one nucleotide.

[0086] The terms "genome modification" and "genome editing" refer to a process in which a specific endogenous chromosomal sequence is altered such that the chromosomal sequence is modified. The chromosomal sequence may be modified to include the insertion of at least one nucleotide, the deletion of at least one nucleotide, and / or the substitution of at least one nucleotide. The modified chromosomal sequence is inactivated so that no product is produced. Alternatively, the chromosomal sequence may be modified so that an altered product is produced.

[0087] As used herein, "gene" refers to a DNA region (including exons and introns) that encodes a gene product, as well as a DNA region that regulates the production of the gene product, regardless of whether such regulatory sequences are adjacent to the coding sequence and / or the transcribed sequence. Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0088] The term "heterologous" refers to material that is not native to the cell or species of interest.

[0089] The terms "nucleic acid" and "polynucleotide" refer to a polymer of deoxyribonucleotides or ribonucleotides in a linear or cyclic conformation. In this disclosure, these terms should not be construed as limiting the length of the polymer. These terms can encompass known analogs of natural nucleotides as well as nucleotides modified in the base, sugar, and / or phosphate moieties. Generally, an analog of a particular nucleotide has the same base-pairing specificity; i.e., an analog of A will base pair with T. The nucleotides of a nucleic acid or polynucleotide may be linked by phosphodiester, phosphothioate, phosphoramidate, or phosphorodiamidate linkages, or a combination thereof.

[0090] The term "nucleotide" refers to deoxyribonucleotides or ribonucleotides. Nucleotides may 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 may be naturally occurring nucleotides (e.g., inosine) or non-naturally occurring nucleotides. Non-limiting examples of modifications to the sugar or base moiety of a nucleotide include the addition (or removal) of acetyl, amino, carboxyl, carboxymethyl, hydroxy, methyl, phosphoryl, and thiol groups, as well as the substitution of carbon and nitrogen atoms of the base with other atoms (e.g., 7-deazapurines). Nucleotide analogs also include dideoxynucleotides, 2'-O-methyl nucleotides, locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholinos.

[0091] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues.

[0092] As used herein, the term "target site" or "target sequence" refers to a nucleic acid sequence that defines a portion of a chromosomal sequence to be modified or edited and that a targeting endonuclease is engineered to recognize and bind to, provided sufficient conditions for binding exist.

[0093] The terms "upstream" and "downstream" refer to positions relative to a fixed position in a nucleic acid sequence, with upstream referring to the region that is 5' to that position (i.e., closer to the 5' end of the strand) and downstream referring to the region that is 3' to that position (i.e., closer to the 3' end of the strand).

[0094] Techniques for determining the identity of nucleic acid and amino acid sequences are well known in the art. Typically, such techniques involve determining the nucleotide sequence of a gene's mRNA and / or the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this manner. Generally, identity refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity. The percent identity of two sequences, whether nucleic acid or amino acid, is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence, multiplied by 100. Approximate alignment of nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences 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 can be normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of this algorithm for determining the percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the utility application "BestFit". Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art; for example, another alignment program is BLAST, used with default parameters.For example, BLASTN and BLASTP can be used with the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found on the GenBank website. For the sequences described herein, desirable degrees of sequence identity range from approximately 80% to 100% and any integer value therebetween. Typically, the percent identity between sequences is at least 70-75%, preferably 80-82%, more preferably 85-90%, even more preferably 92%, even more preferably 95%, and most preferably 98% sequence identity.

[0095] It is intended that all matter contained in the above description and the following examples be interpreted as illustrative and not limiting, as various changes can be made in the cells and methods described above without departing from the scope of the invention. [Example]

[0096] The following examples illustrate certain aspects of the present invention.

[0097] Example 1: Design of a serine-mediated selection system To develop a serine-mediated metabolic selection system, we first developed a CHO cell line sensitive to the deficiency of the non-essential amino acid serine (Ser). A comprehensive search was performed on the Reactome and KEGG databases to identify all genes related to the serine synthesis pathway. The endogenous phosphoserine phosphatase gene (PSPH) was identified as the only non-redundant gene responsible for de novo Ser synthesis (Figure 1). To generate the Ser-sensitive CHO cell line, we used the glutamine (Gln) auxotrophic CHOZN gene from MilliporeSigma. (登録商標) GS - / - Cell line (CHOZN (登録商標) ) was used. (登録商標) Through whole genome sequencing (WGS) of the cell line, we revealed the endogenous PSPH coding sequence (Figure 2), which, as determined by digital droplet PCR (ddPCR) analysis, is CHOZN (登録商標) It was found to be present in two copies of the genome of CHOZN (Figure 3). CRISPR / Cas9 gene editing reagents were designed to disrupt the second exon of the PSPH gene. The CRISPR / Cas9 target sequence is underlined and bolded in Figure 2. (登録商標) Cells were cultured in EX-CELL medium supplemented with 6 mM L-glutamine (MilliporeSigma G7513). (登録商標)Cells were cultured in CD CHO Fusion medium (MilliporeSigma 14365C) ​​(Fusion+Gln) at 37°C with shaking and 5% CO2. Cells were seeded at 0.5e6 cells per well the day before transfection, and cultures were maintained in logarithmic growth phase. Cas9 RNP was complexed by mixing 50 pmol of Cas9 (Sigma CAS9PROT-250UG) with 150 pmol of sgRNA (Sigma) for 15 minutes at room temperature. 4e5 cells were transfected with a total of 200 pmol of complexed RNP using Lonza's 4DX nucleofector system with programs DT-133 and SF nucleofector solution. Cas9 cleavage activity was assessed via next-generation sequencing (NGS), and it was determined that an additional 10 mM serine was required in the medium to allow optimal cell survival during PSPH gene modification (Figure 4). Transfected cells were cultured in EX-CELL medium supplemented with 6 mM L-glutamine (MilliporeSigma G7513) and 10 mM serine (MilliporeSigma S4311-100G). (登録商標) The cells were transferred to a 6-well culture flask containing 3 mL of CD CHO Fusion medium (MilliporeSigma 14365C) ​​(Fusion + Gln + Ser). The cells were incubated in a static environment at 37°C / 5% CO for 96 hours after transfection. (登録商標) Cells were scaled up to T-25 flasks. Single-cell clones were isolated from the Cas9-modified pool into 96-well culture plates via a fluorescence-activated cell sorter (FACS). To identify clones that successfully disrupted both copies of PSPH, single-cell clones were evaluated via next-generation sequencing (NGS) (Figure 5).

[0098] To demonstrate the effectiveness of the serine-mediated selection mechanism both as a stand-alone system and as part of a dual metabolic selection system, we generated stable selected cell populations expressing multiple molecules. The molecules used to validate this system included cyan fluorescent protein (BFP), Dasher green fluorescent protein (GFP), and human IgG1. (登録商標) GS - / - PSPH - / - Cells were cultured in Fusion+Gln+Ser medium. The expression vectors used in this study contain either a phosphoserine phosphatase (PSPH) or glutamine synthetase (GS) selectable marker, as specified by the experimental design. Expression of Chinese hamster PSPH (protein: phosphoserine phosphatase; gene: PSPH; UniProtKB ID: G3I2M2) or mouse GS (protein: glutamine synthetase {glutamate ammonia ligase}; gene: Glul; UniProtKB ID: P15105) was driven by a 5' SV40 promoter with an SV40 polyadenylation sequence at the 3' end of the gene (Figure 6).

[0099] CHOZN (登録商標) GS - / - PSPH - / -Cells were cultured in Fusion+Gln+Ser under shaking conditions at 37°C and 5% CO2. 1.0e6 cells per condition were transfected with 7.5 μg of plasmid DNA using electroporation. Transfected cells were transferred to 6-well plates containing 3 mL of Fusion+Gln+Ser. After cells recovered to >98% viability, they were pelleted, the medium was aspirated, and then resuspended in 10 mL of the appropriate selection medium at 5e5 viable cells / mL and transferred to T-75 flasks. Glutamine-based selection was performed using Fusion-Gln, serine-based selection was performed using Fusion-Ser, and glutamine / serine double selection was performed using Fusion-Gln and -Ser. Cell viability and viable cell density of the various selection cultures were monitored over time. Upon recovery from selection, stable selected cultures were transferred to TPP for scale-up and adaptation to shaking conditions. (登録商標) TubeSpin Bioreactor Tubes (TPP (登録商標) ) was moved.

[0100] EX-CELL (登録商標) Advanced CHO Fed-batch medium (MilliporeSigma 14366C), EX-CELL (登録商標) Advanced CHO Feed (MilliporeSigma 24367C), and Cellvento (登録商標)Custom Serine (Ser)-deficient formulations of 4 Feed (MilliporeSigma 1.03796.0005) were developed (Advanced-Ser, Feed-Ser, and 4 Feed-Ser, respectively). Stable selected cultures transfected with an IgG1 expression vector were pelleted, the selection medium aspirated, and then resuspended in Advanced-Ser at 3e5 viable cells / mL for productivity analysis in fed-batch conditions. Viable cell density and viability data for each culture were collected every other day starting on day 3 post-seeding. Starting on day 3 post-seeding, 1.5 mL of a 50:50 mixture of Advanced Feed-Ser and 4 Feed-Ser was added to each culture. Glucose measurements were obtained from each culture every other day starting on day 5, and D-glucose (MilliporeSigma G8769) was added to maintain appropriate glucose levels. Productivity was monitored over time and fed batch titers were recorded every other day from day 9 until the culture viability fell below 70%. Titers were determined using interferometry on a ForteBio Octet and subsequently confirmed via HPLC Protein A affinity chromatography.

[0101] Example 2: Based on EX-CELL (登録商標) A custom formulation of CD CHO Fusion medium that does not contain serine (Ser) was developed (Fusion-Gln-Ser). (登録商標) GS - / - PSPH - / - Clones were cultured in Fusion+Gln+Ser or Fusion+Gln-Ser for at least 7 days. Viability and viable cell density measurements were performed twice a week. Figure 7 shows the CHOZN (登録商標) GS - / - PSPH - / - Cells cannot grow in the absence of serine, but when serine is added to the medium, CHOZN (登録商標) GS - / - PSPH - / - It is shown that cell proliferation is rescued.

[0102] Example 3: To demonstrate that stable selected cell populations can produce proteins of interest using the Ser-mediated selection system described in Example 1, a vector was developed containing coding sequences for IgG heavy chain, IgG light chain, and phosphoserine phosphatase (PSPH). (登録商標) GS - / - PSPH - / - The cell lines were transfected with the Advanced Feed-Ser medium. Mock transfections without DNA were used as controls. Populations were subcultured under selection pressure in Fusion+Gln-Ser. The conditions used for selection were also applied during recovery, scale-up, and productivity assays. 3e5 viable cells / mL in Advanced+Gln-Ser medium were inoculated into the Fed-Batch productivity assay. The viable cell density and viability of each culture were collected every other day starting on day 3 after seeding. Starting on day 3 after seeding, 1.5 mL of a 50:50 mixture of Advanced Feed-Ser and 4 Feed-Ser was added to each culture. Glucose measurements were obtained every other day starting on day 5, and D-glucose (MilliporeSigma G8769) was added to maintain appropriate glucose levels. The resulting IgG titers are shown in Figure 10.

[0103] Example 4: To test whether stable cell populations could produce two independent intracellular fluorescent proteins under glutamine- and serine-selective conditions, two vectors were developed: one containing sequences encoding GFP and GS, and the second containing sequences encoding BFP and PSPH (Figure 5). These two plasmids were transfected into CHOZN (登録商標) GS - / - PSPH - / -Cells were co-transfected with both vectors (GFP + BFP). They were then subcultured under dual metabolic selection conditions (Fusion-Gln-Ser). The conditions used for selection were also applied during recovery, scale-up, and all other assays. Figure 8 shows growth and viability data from the selection assay, demonstrating that cells co-transfected with both vectors (GFP + BFP) survive and grow in -Gln-Ser medium. Figures 8 and 9 suggest that cells co-transfected with both vectors (GFP + BFP) that survive and grow in -Gln-Ser medium are positive for both GFP and BFP. This data suggests that the GS + PSPH dual metabolic selection system provides an opportunity to select for cells transfected with multiple independent vectors encoding intracellular proteins without the need to add any selective substances, such as antibiotics, to the medium.

[0104] Example 5: To test whether stable cell populations could produce secreted proteins under glutamine- and serine-free conditions, two vectors expressing IgG1 were developed: one containing sequences encoding the IgG heavy chain, the IgG light chain, and GS, and the second containing sequences encoding the same IgG heavy chain, the IgG light chain, and PSPH. These two independent vectors were then cloned into CHOZN. (登録商標) GS - / - PSPH - / - As a control, each vector was co-transfected into CHOZN cells (GS+ASNS). (登録商標) GS - / - PSPH - / -The cells were separately transfected with either GS or PSPH (GS only and PSPH only). The cells were then subcultured under selection in medium supplemented with Fusion-Gln (GS-only transfected cells), Fusion-Ser (PSPH-only transfected cells), or Fusion-Gln-Ser (GS + PSPH transfected cells). The conditions used for selection were also applied during recovery, scale-up, and productivity assays. The GS-only selected culture fully recovered after 14 days, whereas the PSPH-only and GS + PSPH double-selected cultures required 21 days to recover. In fed-batch assays, the GS-only, PSPH-only, and GS + PSPH-selected pools were all capable of driving IgG production (Figure 10). This suggests that upon expression of exogenous GS and / or PSPH coding sequences, the GS and PSPH produced by the cells are sufficient for sustained production of secreted proteins. This provides the possibility for running large-scale production bioreactors under dual-selection conditions. Doing this using antibiotic selection methods would be difficult due to either the need to separate or purify the antibiotic from the desired secreted protein or the cost of adding antibiotics to large-scale bioreactors. Furthermore, this dual metabolic selection system (GS+PSPH) offers the opportunity to more efficiently select cells transfected with multiple large vectors (e.g., those expressing bispecific antibodies or other large and / or complex proteins).

Claims

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

2. 2. The method of claim 1, wherein the mammalian cell line of (a) further comprises reduced or abolished expression of endogenous glutamine synthetase (GS) and / or asparagine synthetase (ASNS) activity.

3. 2. The method of claim 1, wherein endogenous PSPH expression is reduced or eliminated by inactivation of the endogenous PSPH gene in the mammalian cell line.

4. 2. The method of claim 1, wherein the endogenous PSPH gene is inactivated using a targeted endonuclease-mediated genome modification approach.

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

6. 2. 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 NS0 mouse myeloma cell line, an HEK293 cell line, or a Vero African green monkey kidney cell line.

7. The method of any one of claims 1 to 6, wherein the cell line is a CHO cell line.

8. The method of any one of claims 1 to 7, wherein the recombinant protein product is selected from an antibody, an antibody fragment, a vaccine, a growth factor, a cytokine, a hormone, or a clotting 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 biological production system that has been engineered to have reduced or abolished expression of endogenous PSPH.

11. 11. The mammalian cell line of claim 10, wherein expression of PSPH is reduced or eliminated through inactivation of at least one allele of the chromosomal sequence encoding PSPH.

12. 12. The mammalian cell line of claim 11, wherein one or more alleles of the chromosomal sequence encoding PSPH are inactivated.

13. 11. The mammalian cell line of claim 10, which has been engineered to have reduced or eliminated expression of endogenous glutamine synthetase (GS) and / or asparagine synthetase (ASNS).

14. The mammalian cell line of claim 12, wherein the chromosomal sequence has been inactivated using a targeted endonuclease-mediated genome modification approach.

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

16. 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 NS0 mouse myeloma cell line, a HEK293 cell line, or a Vero African green monkey kidney cell line.

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

18. 18. The mammalian cell line of claim 17, wherein the cell viability, viable cell density, titer, growth rate, growth response, cell morphology, and / or overall cell health is comparable to that of the non-manipulated parent mammalian cell line.

19. 19. The mammalian cell line of any of claims 10 to 18, 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 clotting 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 PSPH and at least one recombinant protein of interest.

22. a) a nucleic acid sequence encoding a functional PSPH; b) a nucleic acid sequence encoding a functional GS and / or ASNS; and c) nucleic acid sequences encoding mutations in the ASNS and / or PSPH coding sequences that attenuate the activity of either one or both of said enzymes. d) a nucleic acid sequence encoding the recombinant protein of interest A polynucleotide comprising:

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