WO2013123408A1 - Cellules déficientes en hypoxanthine-guanine phosphoribosyltransférase - Google Patents

Cellules déficientes en hypoxanthine-guanine phosphoribosyltransférase Download PDF

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WO2013123408A1
WO2013123408A1 PCT/US2013/026454 US2013026454W WO2013123408A1 WO 2013123408 A1 WO2013123408 A1 WO 2013123408A1 US 2013026454 W US2013026454 W US 2013026454W WO 2013123408 A1 WO2013123408 A1 WO 2013123408A1
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cell
hprt
cells
sequence encoding
chromosomal sequence
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PCT/US2013/026454
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English (en)
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Nan Lin
Trissa Borgschulte
Natalie SEALOVER
Henry George
Kevin Kayser
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Sigma-Aldrich Co. Llc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)

Definitions

  • the present invention generally relates to cells that are useful for recombinant protein production.
  • the invention relates to cells that are deficient in hypoxanthine-guanine phosphoribosyltransferase (HPRT) such that hypoxanthine-guanine phosphoribosyltransferase may be used as an amplifiable metabolic selectable marker.
  • HPRT hypoxanthine-guanine phosphoribosyltransferase
  • HPRT is a key enzyme in the purine salvage pathway in eukaryotic cells, and it is widely used as a selectable marker during the production of monoclonal antibodies in hybridoma technology. Although the mechanisms are not well understood, metabolic selection and gene amplification are the cornerstones of pharmaceutical recombinant protein production. These technologies have enabled the generation of highly productive mammalian cells lines, and in particular, Chinese hamster ovary (CHO) cell lines.
  • DHFR dihydrofolate reductase
  • GS glutamine synthase
  • HPRT as a third amplifiable selectable marker in addition to DHFR and GS would provide additional tools, and hence versatility, for developing high producing cell lines and/or reducing the time for developing high producing cell lines.
  • one aspect of the present disclosure provides an isolated cell deficient in hypoxanthine-guanine phosphoribosyltransferase (HPRT) and glutamine synthase (GS).
  • the cell comprises an edited
  • the targeting endonuclease is a meganuclease, a transcription activator-like effector (TALE) nuclease, a site-specific nuclease, or a zinc finger nuclease.
  • TALE transcription activator-like effector
  • the targeting endonuclease is a zinc finger nuclease.
  • the edited chromosomal sequence comprises a deletion of sequence encoding HPRT.
  • a zinc finger nuclease is used to delete all or part of the chromosomal sequence encoding HPRT.
  • all copies of chromosomal sequence encoding HPRT are inactivated, and the cell produces no HPRT.
  • the cell is a Chinese hamster ovary (CHO) cell line cell or a derivative thereof, and all copies of chromosomal sequence encoding HPRT are inactivated.
  • all copies of chromosomal sequence encoding HPRT are inactivated by deletion of all or part of the coding sequence.
  • the cell further comprises an edited chromosomal sequence encoding GS.
  • all copies of chromosomal sequence encoding GS are inactivated and the cell produces essentially no GS.
  • the cell is a Chinese hamster ovary (CHO) cell line cell or a derivative thereof, and all copies of chromosomal sequence encoding HPRT and all copies of chromosomal sequence encoding GS are inactivated such that the cell produces essentially no HPRT and no GS.
  • CHO Chinese hamster ovary
  • a further aspect of the disclosure encompasses a method for preparing a cell deficient in hypoxanthine-guanine phosphoribosyltransferase (HPRT) by editing a chromosomal sequence encoding HPRT.
  • HPRT hypoxanthine-guanine phosphoribosyltransferase
  • the method comprises introducing into the cell (i) at least one targeting endonuclease or at least one nucleic acid encoding a targeting endonuclease, each targeting endonuclease being able to introduce a double-stranded break at a targeted cleavage site in the chromosomal sequence encoding HPRT and, optionally, (ii) at least one polynucleotide comprising a sequence having substantial sequence identity to a sequence on at least one side of the targeted cleavage site in the chromosomal sequence encoding HPRT.
  • the method further comprises maintaining the cell under conditions such that the double-stranded break introduced by the targeting endonuclease is repaired by (i) a non-homologous end-joining repair process such that the chromosomal sequence encoding HPRT is edited, or (ii) a homology-directed repair process such that the chromosomal sequence is exchanged with the sequence of the polynucleotide such that the chromosomal sequence encoding HPRT is edited.
  • the targeting endonuclease is a zinc finger nuclease.
  • the optional polynucleotide is a single- stranded nucleic acid.
  • one targeting endonuclease is introduced into the cell.
  • one targeting endonuclease is introduced into the cell along with the optional polynucleotide, which comprises a first sequence having substantial sequence identity to a sequence adjacent to the targeted cleavage site and a second sequence having substantial sequence identity to a sequence located distally to the targeted cleavage site.
  • two targeting endonucleases are introduced into the cell.
  • the cell is a CHO cell line cell or a derivative thereof.
  • the cell is also deficient in GS.
  • the cell is a CHO cell and all copies of chromosomal sequence encoding GS are inactivated.
  • Still another aspect of the disclosure provides a method for identifying cells that produce high levels of at least one recombinant protein.
  • the method comprises expressing at least one nucleic acid encoding a recombinant protein in a population of cells deficient in hypoxanthine-guanine phosphoribosyltransferase (HPRT), wherein the nucleic acid also comprises sequence encoding HPRT.
  • HPRT hypoxanthine-guanine phosphoribosyltransferase
  • the method further comprises culturing the population of cells in the presence of
  • hypoxanthine, aminopterin, and thymidine selecting for cells that produces high levels of the recombinant protein.
  • all copies of chromosomal sequence encoding HPRT are inactivated in the population of cell.
  • the cells that are selected comprise additional copies of the nucleic acid encoding the recombinant protein and HPRT due to amplification of the nucleic acid.
  • the population of cells is also deficient in GS and the culturing step (b) further comprises methionine sulfoxide.
  • all copies of chromosomal sequence encoding GS are inactivated.
  • the method comprises expressing a first nucleic acid encoding a first recombinant protein and HPRT and a second nucleic acid encoding a second recombinant protein and GS.
  • the cells that are selected comprise additional copies of the first and second nucleic acids due to amplification of the nucleic acids.
  • the first recombinant protein is a therapeutic protein and the second recombinant protein is a therapeutic protein or a protein that provides an improved property to the population of cells.
  • the cell is a CHO cell line cell or a derivative thereof.
  • FIG. 1 illustrates ZFN-mediated cleavage of the hprt locus in CHO- K1 cells. Shown are the results of a Cel-1 nuclease assay performed using mock- transfected cells (i.e., negative control) or cells transfected with DNA encoding a ZFN targeting exon 3 of the hprt coding region. Sizes of cleavage products in the ZFN targeted cells are indicated at the right of the image. All intron-exon structure
  • FIG. 2 presents a schematic diagram of a targeted genomic deletion protocol utilizing a ZFN and a single-stranded oligonucleotide.
  • FIG. 3 shows a schematic diagram of a targeted genomic deletion protocol utilizing two ZFNs.
  • FIG. 4 illustrates ZFN-mediated cleavage of the hprt locus in CHO- K1 cells. Shown are the results of a Cel-1 nuclease assay using mock-transfected cells (i.e., negative control) or cells transfected with DNA encoding a ZFN targeting intron 2 of the hprt coding region. Sizes of cleavage products in the ZFN targeted cells are indicated at the right of the image.
  • the present disclosure provides compositions and methods for more efficient and/or increased production of recombinant protein pharmaceuticals.
  • the present disclosure provides cells deficient in HPRT.
  • the genome of the HPRT deficient cell is edited such that the chromosomal sequence encoding HPRT is inactivated.
  • the inactivated chromosomal sequence comprises a deletion of at least one nucleotide, an insertion of at least one nucleotide, and/or a substitution of at least one nucleotide.
  • the HPRT deficient cells comprise a deletion of all or most of the chromosomal sequence encoding HPRT.
  • an exogenous sequence encoding HPRT can be used as a metabolic selectable marker and a tool for gene amplification during recombinant protein production.
  • the HPRT deficient cells are also deficient in glutamine synthase (GS).
  • the present disclosure also provides methods for preparing the HPRT deficient cells, wherein the methods comprise using targeting endonucleases to inactivate the chromosomal sequence encoding HPRT. Also provided are methods for using the HPRT deficient cells for the production of high levels of recombinant proteins.
  • One aspect of the present disclosure encompasses an isolated cell that is deficient in HPRT.
  • the HPRT deficient cell produces reduced levels of HPRT.
  • the HPRT deficient cell produces essentially no HPRT.
  • the phrase "essentially no HPRT” means that HPRT mRNA and/or protein cannot be detected in the cell or lysates thereof using procedures well known in the art.
  • suitable procedures for determining the level of mRNA or protein include PCR, qPCR, Western blotting, and ELISA assay.
  • the cell deficient in HPRT comprises an inactivated chromosomal sequence encoding HPRT.
  • the genome of the cell can be edited using targeting endonuclease mediated genome editing to inactivate the chromosomal sequence encoding HPRT.
  • the term "inactivated chromosomal sequence” refers to a chromosomal sequence that is unable to generate a protein product.
  • the inactivated chromosomal sequence encoding HPRT is stable and the HPRT deficient cell does not revert to wild type.
  • the inactivated chromosomal sequence encoding HPRT can be monoallelic such that the cell produces reduced levels of HPRT. In other embodiments in which the cell is euploid, the inactivated chromosomal sequence encoding HPRT can be biallelic such that the cell produces no HPRT. In other embodiments in which the cell is aneuploid, one or more copies of the chromosomal sequence(s) encoding HPRT can be inactivated such that the cell produces a reduced amount of HPRT. In still other embodiments in which the cell is aneuploid, all copies of the chromosomal sequence(s) encoding HPRT are inactivated such that the cell line produces no HPRT. In an exemplary embodiment, the cell is haploid for the chromosomal sequence encoding HPRT, and inactivation of the chromosomal sequence encoding HPRT results in a complete loss of HPRT expression.
  • the chromosomal sequence encoding HPRT can be inactivated by a deletion of at least one base pair (bp), an insertion of at least one bp, a substitution of at least one bp, or combinations thereof. As a consequence of the deletion(s) insertion(s), and/or substitution(s), the HPRT coding sequence undergoes a shift in the reading frame, thereby preventing production of a protein product.
  • the chromosomal sequence encoding HPRT can be inactivated using targeting endonuclease-mediated genome editing technology as detailed below in section (II).
  • the chromosomal sequence encoding HPRT can be inactivated by deletion or disruption of all or part of the exonic coding region, deletion or disruption of all or part of a control region, deletion or disruption of all or part of an intron, deletion or disruption of a splice site such that expression of HPRT is abolished, or combinations thereof.
  • the chromosomal sequence encoding HPRT can be inactivated via deletions, insertions, and/or nucleotide substitutions to introduce a premature stop codon, new splice sites, and/or SNPs into the chromosomal sequence such that the cell is unable to produce HPRT.
  • the HPRT deficient cell comprises a deletion ranging from about 1 bp to the entire coding region of the chromosomal sequence encoding HPRT.
  • the deletion can encompass all or part of an exon and/or an intron in the HPRT chromosomal sequence.
  • the cell can comprise a deletion of all or part of exon 1 , intron 1 , exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, and/or exon 9 of the HPRT chromosomal sequence.
  • the cell can comprise a deletion of exon 3, exon 3-exon 9, exon 3-exon 8, exon 3-exon 7, exon 3-exon 6, exon 3-exon 5, exon 3-exon 4, intron 2-exon 9, intron 2-exon 8, intron 2-exon 7, intron 2-exon 6, intron 2-exon 5, intron 2-exon 4, exon 2-exon 9, exon 2-exon 8, exon 2-exon 7, exon 2-exon 6 exon 2-exon 5, or exon 2-exon 4 of the HPRT chromosomal sequence.
  • the deletion can be a partial or complete deletion of the exon(s) and/or intron(s).
  • the cell comprises a deletion of exon 3 of the HPRT coding sequence.
  • the cell deficient in HPRT is also deficient in GS.
  • a GS deficient cell produces essentially no GS protein.
  • the HPRT deficient cell also comprises inactivated GS chromosomal sequence(s), in which case the cell is a GS knockout, in addition to a HPRT knockout.
  • the chromosomal sequence encoding GS can be inactivated by targeting endonuclease-mediated genome editing (detailed below in section (II)). That is, the chromosomal sequence encoding GS can be edited to comprise a deletion of at least one nucleotide, an insertion of at least one nucleotide, or a substitution of at least one nucleotide, or a combination thereof such that the chromosomal sequence is inactivated.
  • the chromosomal sequence encoding GS can be inactivated via random mutagenesis (e.g., exposure to a mutagenic chemical, and/or ionizing radiation).
  • the chromosomal sequence encoding GS can be inactivated via random mutagenesis (e.g., exposure to a mutagenic chemical, and/or ionizing radiation).
  • the chromosomal sequence encoding GS can be inactivated via random mutagenesis (e.g., exposure to
  • chromosomal sequence encoding GS can be inactivated by contact with a transposon- mediated system or a site-specific recombination system.
  • the inactivated chromosomal sequence encoding GS can be a naturally occurring mutation.
  • the inactivated chromosomal sequence encoding GS can be monoallelic or biallelic. In embodiments in which the cell is aneuploid, at least one or all copies of the chromosomal sequence encoding GS can be inactivated. In exemplary embodiments, all copies of the chromosomal sequence encoding GS are inactivated such that the cell produced no GS.
  • the type of cell that is deficient in HPRT can and will vary.
  • the cell can be a human cell, a mammalian cell, a vertebrate cell, or an invertebrate cell.
  • suitable invertebrate cells include without limit insect cells, such as Sf9 cells from Spodoptera frugiperda or S2 cells from Drosophila melanogaster.
  • suitable vertebrate cells include those derived from frogs such as Xenopus, fish such as zebrafish, and fowl such as chickens.
  • suitable mammalian cells include without limit those derived from mice, rats, hamsters, rabbits, cats, dogs, horses, pigs, cattle, and non-human primates.
  • the cells can be primary cells. Examples of suitable primary cells include but are not limited to
  • fibroblasts fibroblasts, myoblasts, T or B cells, macrophages, epithelial cells, hepatocytes, and so forth.
  • the cell is a cell line cell.
  • the cell line can be any mammalian cell line, including, for example, any established cell line or a cell line derived from a primary cell or other cell in culture.
  • Suitable mammalian cell lines include Chinese hamster ovary (CHO) cells, monkey kidney CVI line transformed by SV40 (COS7); human embryonic kidney line 293; baby hamster kidney cells (BHK); monkey kidney cells (CVI-76); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); rat hepatoma cells (HTC); HIH/3T3 cells, the human U2-OS osteosarcoma cell line, the human A549 cell line, the human A-431 cell line, the human K562 cell line, the human HEK293 cell line, the human HEK293T cell line, and TRI cells.
  • CHO Chinese hamster ovary
  • COS7 monkey kidney CVI line transformed by SV40
  • BHK baby hamster kidney cells
  • CVI-76 monkey kidney cells
  • African green monkey kidney cells VERO
  • the cell is a stem cell.
  • Suitable stem cells include without limit embryonic stem cells, ES-like stem cells, fetal stem cells, adult stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, oligopotent stem cells, and unipotent stem cells.
  • the cell is of a type that is widely used for the production of recombinant proteins, such as antibodies, glycoproteins, and the like.
  • the cell is a CHO cell line cell. Numerous CHO cell lines are available from American Type Culture Collection (ATCC). Suitable CHO cell lines include, but are not limited to, CHO K1 cells and derivatives thereof.
  • the CHO cells further comprise a deletion of one or both alleles of glutamine synthase (GS).
  • GS glutamine synthase
  • Non-limiting examples of cells deficient in GS include CHO-S cells, CHOK1 SV cells, and CHOZN ® GS-/- cells.
  • the cells disclosed herein further comprise at least one nucleic acid sequence encoding a recombinant protein.
  • the recombinant protein is heterologous, meaning that the protein is not native to the cell.
  • the recombinant protein may be a therapeutic protein.
  • An exemplary recombinant therapeutic protein includes, without limit, an antibody, a fragment of an antibody, 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 nutraceutical protein, a functional fragment or functional variant of any
  • the recombinant protein may be a protein that imparts improved properties to the cell or improved properties to a first recombinant protein.
  • improved properties include increased robustness, increased viability, increased survival, increased proliferation, increased cell cycle progression (i.e., increased progression form G1 to S phase), increased cell growth, increased cell size, increased production of endogenous proteins, increased production of heterologous proteins, increased stability of a recombinant protein, altered post- translational processing of a recombinant protein, and combinations of any of the above.
  • the protein that improves cell properties can be overexpressed.
  • Non-limiting examples of suitable proteins include serpin proteins (e.g., SerpinBI ), cell regulatory proteins, cell cycle control proteins, apoptotic inhibitors, metabolic pathway proteins, post-translation modification proteins, artificial transcription factors, transcriptional activators, transcriptional inhibitors, and enhancer proteins.
  • the nucleic acid encoding the recombinant protein is DNA.
  • the DNA can be linear or circular.
  • the nucleic acid encoding the recombinant protein generally is operably linked to at least one expression control sequence.
  • Suitable expression control sequences include promoter sequences, enhancer sequences, polyadenylation signals, termination signals, and translational regulatory sequences (e.g., ribosome binding sites, translation initiation sites, stop codons, etc.).
  • the operably-linked promoter sequence can be constitutive or regulated.
  • Suitable constitutive promoter sequences include the cytomegalovirus immediate early promoter (CMV), the SV40 promoter, the adenovirus major late promoter, Rous sarcoma virus long terminal repeat promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, fragments thereof, or combinations of any of the foregoing.
  • the promoter sequence can be wild type or it can be modified for more efficient or efficacious expression.
  • the nucleic acid encoding the recombinant protein can be linked to a nucleic acid encoding HPRT, such that HPRT may be used as an amplifiable selectable marker.
  • HPRT may be used as an amplifiable selectable marker.
  • the amplifiable selectable marker not only allows for identification of stably transfected cells but also amplification of the nucleic acid sequence encoding the recombinant protein.
  • the sequence encoding HPRT can be operably linked to the sequence encoding the recombinant protein, such that both sequences are under the control of the same promoter and can be co-expressed.
  • sequence encoding HPRT can be operably linked to a separate promoter sequence such that it is expressed separately from the sequence encoding the recombinant protein.
  • Cells comprising a nucleic acid sequence encoding the recombinant protein and HPRT can be grown in the presence of increasing
  • hypoxanthine-aminopterin-thymidine HAT
  • cells can be selected for survival due to amplification of the nucleic acid sequence(s) encoding the
  • the nucleic acid encoding the recombinant protein can be linked to a nucleic acid encoding GS such that GS can also be used as an amplifiable selectable marker.
  • the cells are grown in the presence of methionine sulfoximide (MSX).
  • MSX methionine sulfoximide
  • the sequence encoding GS can be operably linked to the same promoter sequence controlling the expression of the recombinant protein, or the sequence encoding GS can be operably linked to a separate promoter sequence.
  • the cell comprises a first sequence encoding a first recombinant protein and HPRT and a second sequence encoding a second recombinant protein and GS
  • the cell can be selected (simultaneously or sequentially) for growth in the presence of HAT and MSX.
  • the cell can contain from 1 -10 additional copies, from 10-100 additional copies, from 100-1000, or many thousands of additional copies the nucleic acid sequence encoding the
  • the one or more copies of the nucleic acid sequence encoding the recombinant protein and the amplifiable selectable marker can be located episomally so as to be stably maintained in the cell or cell line. In other embodiments, the one or more copies of the nucleic acid sequence encoding the recombinant protein and the amplifiable selectable marker can be integrated into the nuclear genome of the cell line.
  • the HPRT deficient cell is a CHO cell line cell.
  • CHO cell line cells have a single intact X chromosome (Campbell et al., 1977, Cytogenetics and Cell Genetics 19, 303-319) and only one copy of the X-linked hprt gene (Chu et al., 1968, Proc. Natl. Acad. Sci. USA 61 , 1306-1312).
  • the HPRT deficient CHO cell comprises a deletion of all or part of the single copy of the chromosomal sequence encoding HPRT. The genotype of such cell is HPRT " 0 , and the cell produces no HPRT protein.
  • the HPRT deficient CHO cell is also deficient in GS. That is, the HPRT deficient cell comprises a biallelic inactivation of the chromosomal sequence encoding GS. The genotype of such cell is HPRT " 0 and GS " ' " , and the cell produced no HPRT or GS protein.
  • Yet another aspect of the present disclosure provides a method for preparing a cell deficient in HPRT by editing a chromosomal sequence encoding HPRT.
  • the method comprises introducing into a cell (i) at least one targeting endonuclease or at least one nucleic acid encoding a targeting endonuclease, wherein each targeting endonuclease is able to introduce a double-stranded break at a targeted cleavage site in the chromosomal sequence encoding HPRT, and optionally, (ii) at least one polynucleotide comprising a sequence having substantial sequence identity to a region on at least one side of the targeted cleavage site in the chromosomal sequence encoding HPRT.
  • the method further comprises maintaining the cell under conditions such that the double-stranded break introduced by the targeting endonuclease(s) is repaired by (i) a non-homologous end-joining repair process such that a mutation is introduced into the chromosomal sequence or, optionally, (ii) a homology-directed repair process such that the chromosomal sequence is exchanged with the sequence of the polynucleotide, thereby editing the chromosomal sequence encoding HPRT such that the cell is deficient in HPRT.
  • the type of targeting endonuclease used in the method disclosed herein can and will vary.
  • the targeting endonuclease can be a naturally-occurring protein or an engineered protein.
  • the targeting endonuclease is a meganuclease.
  • Meganucleases are endodeoxyribonucleases characterized by a large recognition site, i.e., the recognition site generally ranges from about 12 base pairs to about 40 base pairs. As a consequence of this requirement, the recognition site generally occurs only once in any given genome.
  • the family of homing endonudeases named LAGLIDADG has become a valuable tool for the study of genomes and genome engineering.
  • Meganucleases can be targeted to specific chromosomal sequence by modifying their recognition sequence using techniques well known to those skilled in the art.
  • the targeting endonuclease is a
  • TALE transcription activator-like effector nuclease.
  • TALEs are transcription factors from the plant pathogen Xanthomonas that can be readily engineered to bind new DNA targets.
  • TALEs or truncated versions thereof may be linked to the catalytic domain of endonudeases such as Fokl to create targeting endonuclease called TALE nucleases or TALENs.
  • the targeting endonuclease is a site- specific nuclease.
  • the site-specific nuclease can be a "rare-cutter" endonuclease whose recognition sequence occurs rarely in a genome.
  • the recognition sequence of the site-specific nuclease occurs only once in a genome.
  • the targeting nuclease can be an artificial targeted DNA double strand break inducing agent.
  • the targeting endonuclease is a zinc finger nuclease (ZFN).
  • ZFN zinc finger nuclease
  • a zinc finger nuclease comprises a DNA binding domain (i.e., zinc finger) and a cleavage domain (i.e., nuclease), both of which are described below.
  • Zinc finger binding domain i.e., zinc finger
  • cleavage domain i.e., nuclease
  • .Zinc finger binding domains may be engineered to recognize and bind to any nucleic acid sequence of choice. 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. 10:41 1 -416; Zhang et al. (2000) J. Biol. Chem.
  • An engineered zinc finger binding domain can have a novel binding specificity compared to a naturally- occurring zinc finger protein.
  • Engineering methods include, but are not limited to, rational design and various types of selection.
  • Rational design includes, for example, using databases comprising doublet, triplet, and/or quadruplet nucleotide sequences and individual zinc finger amino acid sequences, in which each doublet, triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence.
  • databases comprising doublet, triplet, and/or quadruplet nucleotide sequences and individual zinc finger amino acid sequences, in which each doublet, triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence.
  • a zinc finger binding domain may be designed to recognize and bind a DNA sequence ranging from about 3 nucleotides to about 21 nucleotides in length, for example, from about 9 to about 18 nucleotides in length.
  • Each zinc finger recognition region i.e., zinc finger
  • the zinc finger binding domains of the zinc finger nucleases disclosed herein comprise at least three zinc finger recognition regions (i.e., zinc fingers).
  • the zinc finger binding domain comprises four zinc finger recognition regions.
  • the zinc finger binding domain comprises five zinc finger recognition regions.
  • the 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. Pat. Nos. 6,607,882; 6,534,261 and 6,453,242, the disclosures of which are incorporated by reference herein in their entireties.
  • Exemplary methods of selecting a zinc finger recognition region include phage display and two-hybrid systems, and are disclosed 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; as well as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB 2,338,237, each of which is incorporated by reference herein in its entirety.
  • enhancement of binding specificity for zinc finger binding domains has been described, for example, in WO 02/077227, the disclosure of which is incorporated herein by reference.
  • Zinc finger recognition regions and/or multi-fingered zinc finger proteins may be linked together using suitable linker sequences, including for example, linkers of five or more amino acids in length. See, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949, the disclosures of which are incorporated by reference herein in their entireties, for non- limiting examples of linker sequences of six or more amino acids in length.
  • the zinc finger binding domain described herein may include a combination of suitable linkers between the individual zinc fingers (and additional domains) of the protein.
  • a zinc finger nuclease also includes a cleavage domain.
  • the cleavage domain portion of the zinc finger nuclease can be obtained from any endonuclease or exonuclease.
  • Non-limiting examples of endonucleases from which a cleavage domain can be derived include, but are not limited to, restriction
  • a cleavage domain also can be derived from an enzyme or portion thereof, as described above, that requires dimerization for cleavage activity.
  • Two zinc finger nucleases can be required for cleavage, as each nuclease comprises a monomer of the active enzyme dimer.
  • a single zinc finger nuclease can comprise both monomers to create an active enzyme dimer.
  • an "active enzyme dimer” is an enzyme dimer capable of cleaving a nucleic acid molecule.
  • the two cleavage monomers can be derived from the same endonuclease (or functional fragments thereof), or each monomer can be derived from a different endonuclease (or functional fragments thereof).
  • the recognition sites for the two zinc finger nucleases are preferably disposed such that binding of the two zinc finger nucleases to their respective recognition sites places the cleavage monomers in a spatial orientation to each other that allows the cleavage monomers to form an active enzyme dimer, e.g., by dimerizing.
  • the near edges of the recognition sites can be separated by about 5 to about 18 nucleotides. For instance, the near edges can be separated by about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17 or 18 nucleotides.
  • any integral number of nucleotides or nucleotide pairs can intervene between two recognition sites (e.g., from about 2 to about 50 nucleotide pairs or more).
  • the near edges of the recognition sites of the zinc finger nucleases can be separated by 6 nucleotides.
  • the site of cleavage lies between the recognition sites.
  • Restriction endonucleases are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding.
  • Certain restriction enzymes e.g., Type IIS
  • the Type IIS enzyme Fokl 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. See, for example, U.S. Pat. Nos.
  • a zinc finger nuclease can comprise the cleavage domain from at least one Type IIS restriction enzyme 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 WO 20140060600A1
  • WO 20140060600A1 International Publication WO 20140060600A1
  • An exemplary Type IIS restriction enzyme whose cleavage domain is separable from the binding domain, is Fokl. This particular enzyme is active as a dimer (Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10, 570-10, 575).
  • the portion of the Fokl enzyme used in a zinc finger nuclease is considered a cleavage monomer.
  • two zinc finger nucleases each comprising a Fokl cleavage monomer, can be used to reconstitute an active enzyme dimer.
  • a single polypeptide molecule containing a zinc finger binding domain and two Fokl cleavage monomers can also be used.
  • the cleavage domain comprises one or more engineered cleavage monomers that minimize or prevent homodimerization, as described, for example, in U.S. Patent Publication Nos. 20050064474, 20060188987, and 20080131962, each of which is incorporated by reference herein in its entirety.
  • amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491 , 496, 498, 499, 500, 531 , 534, 537, and 538 of Fokl are all targets for influencing dimerization of the Fokl cleavage half-domains.
  • Exemplary engineered cleavage monomers of Fokl that form obligate heterodimers include a pair in which a first cleavage monomer includes mutations at amino acid residue positions 490 and 538 of Fokl and a second cleavage monomer that includes mutations at amino-acid residue positions 486 and 499 (Miller et al., 2007, Nat. Biotechnol, 25:778-785; Szczpek et al., 2007, Nat. Biotechnol, 25:786-793).
  • modified Fokl cleavage domains can include three amino acid changes (Doyon et al. 201 1 , Nat. Methods, 8:74-81 ).
  • one modified Fokl domain (which is termed ELD) can comprise Q486E, I499L, N496D mutations and the other modified Fokl domain (which is termed KKR) can comprise E490K, I538K, H537R mutations.
  • the zinc finger nuclease further comprises at least one nuclear localization signal or sequence (NLS).
  • NLS nuclear localization signal or sequence
  • a NLS is an amino acid sequence which facilitates targeting the zinc finger nuclease protein into the nucleus to introduce a double stranded break at the target sequence in the chromosome.
  • Nuclear localization signals are known in the art. See, for example, Makkerh et al. (1996) Current Biology 6:1025-1027.
  • the NLS can be located at the N-terminus, the C- terminal, or in an internal location of the zinc finger nuclease.
  • the zinc finger nuclease can also comprise at least one cell-penetrating domain.
  • the cell-penetrating domain can be a cell-penetrating peptide sequence derived from the HIV-1 TAT protein, a cell- penetrating peptide sequence derived from the human hepatitis B virus, a cell penetrating peptide from Herpes simplex virus, MPG peptide, Pep-1 peptide, or a polyarginine peptide sequence.
  • the cell-penetrating domain can be located at the N- terminus, the C-terminal, or in an internal location of the zinc finger nuclease.
  • the targeting endonuclease is a zinc finger nuclease.
  • the zinc finger nuclease is engineered to recognize and cleave a target sequence in intron 2 of the chromosomal sequence encoding HPRT.
  • the zinc finger nuclease is engineered to recognize and cleave a target sequence in exon 3 of the chromosomal sequence encoding HPRT.
  • the method for targeted genome editing can further comprise introducing into the cell at least one polynucleotide comprising a sequence having substantial sequence identity to a sequence on at least one side of the targeted cleavage site.
  • the polynucleotide comprises a first sequence having substantial sequence identity to sequence on one side of the targeted cleavage site and a second sequence having substantial sequence identity to sequence on the other side of the targeted cleavage site.
  • the polynucleotide comprises a first sequence having substantial sequence identity to sequence on one side of the targeted cleavage site and a second sequence having substantial sequence identity to a sequence located away from the targeted cleavage site.
  • the sequence located away from the targeted cleavage site may be tens, hundreds, or thousands of nucleotides upstream or downstream of the targeted cleavage site.
  • the lengths of the first and second sequences in the polynucleotide that have substantial sequence identity to sequences in the chromosomal sequence encoding HPRT can and will vary. In general, each of the first and second sequences in the polynucleotide is at least about 10 nucleotides in length.
  • the polynucleotide sequences having substantial sequence identity with chromosomal sequences 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.
  • substantially sequence identity means that the sequences in the polynucleotide have at least about 75% sequence identity with the chromosomal sequences of interest. In some embodiments, the sequences in the polynucleotide 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 with the chromosomal sequences of interest.
  • the length of the polynucleotide can and will vary.
  • the polynucleotide can range from about 20 nucleotides in length up to about 200,000 nucleotides in length.
  • the polynucleotide ranges 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.
  • the polynucleotide is DNA.
  • the DNA can be single- stranded or double-stranded.
  • the polynucleotide can be a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PCR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
  • the polynucleotide is single-stranded.
  • the polynucleotide is a single-stranded oligonucleotide comprising less than about 200 nucleotides.
  • the polynucleotide further comprises a marker.
  • a marker may enable screening for targeted integrations.
  • the marker is a restriction endonuclease site.
  • the marker is a fluorescent protein, a purification tag, or an epitope tag.
  • the marker can be a fluorescent protein.
  • suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreenI ), yellow fluorescent proteins (e.g. YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl ,), blue fluorescent proteins (e.g. EBFP, EBFP2, Azurite, mKalamal , GFPuv, Sapphire, T-sapphire,), cyan fluorescent proteins (e.g.
  • ECFP Cerulean, CyPet, AmCyanl , Midoriishi-Cyan
  • red fluorescent proteins mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1 , DsRed-Express, DsRed2, DsRed-Monomer, HcRed- Tandem, HcRedl , AsRed2, eqFP61 1 , mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein.
  • red fluorescent proteins mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1 , DsRed-Express, DsRed2, DsRed-Monomer, HcRed- Tandem
  • the marker can be a purification tag and/or an epitope tag.
  • tags include, but are not limited to, glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1 , AU5, E, ECS, E2, FLAG, HA, nus, Softag 1 , Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1 , T7, V5, VSV-G, 6xHis, biotin carboxyl carrier protein (BCCP), and calmodulin.
  • GST glutathione-S-transferase
  • CBP chitin binding protein
  • TRX thioredoxin
  • poly(NANP) tandem affinity purification
  • TAP tandem affinity purification
  • the method further comprises introducing the targeting
  • the targeting endonuclease can be introduced into the cell as a purified isolated protein or as a nucleic acid encoding the targeting endonuclease.
  • the nucleic acid may be DNA or RNA.
  • the encoding nucleic acid is mRNA
  • the mRNA may be 5' capped and/or 3' polyadenylated.
  • the encoding nucleic acid is DNA
  • the DNA may be linear or circular.
  • the DNA may be part of a vector, wherein the encoding DNA may be operably linked to a suitable promoter.
  • the targeting endonudease molecule(s) and the optional polynucleotide(s) described above can be introduced into the cell by a variety of means. Suitable delivery means include microinjection, electroporation, sonoporation, biolistics, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection,
  • the targeting endonudease molecule(s) and polynucleotides(s) are introduced into the cell by nucleofection.
  • the molecules can be introduced simultaneously or sequentially.
  • targeting endonudease molecules each specific for a targeted cleavage site (and optional polynucleotides) can be introduced at the same time.
  • each targeting endonudease molecule, as well as the optional polynucleotides(s) can be introduced sequentially.
  • the ratio of the targeting endonudease molecule(s) to the optional polynucleotide(s) can and will vary. In general, the ratio of targeting endonudease molecule(s) to polynucleotide(s) ranges from about 1 : 10 to about 10:1 .
  • the ratio of the targeting endonudease molecule(s) to polynucleotide(s) may 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 .
  • the method further comprises maintaining the cell under
  • the method comprises maintaining the cell under appropriate conditions such that the cell expresses the targeting endonuclease(s).
  • the cell is maintained under conditions appropriate 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 of skill in the art appreciate that methods for culturing cells are known in the art and can and will vary depending on the cell type. Routine optimization may be used, in all cases, to determine the best techniques for a particular cell type.
  • the targeting endonuclease(s) recognizes, binds, and creates a double-stranded break(s) at the targeted cleavage site(s) in the chromosomal sequence, and during repair of the double-stranded break(s) a deletion, insertion, and/or substitution of at least one nucleotide is introduced into the chromosomal sequence encoding HPRT.
  • the chromosomal sequence encoding HPRT is edited such that the sequence is inactivated.
  • the frequency of the targeted editing of the chromosomal sequence can and will vary depending upon a variety of factors. In some embodiments, the frequency of editing is greater than about 0.01 %, 0.03%, 0.1 %, 0.3%, 1 %, 3%, 10%, 20%, or 30%.
  • Single cell clones comprising the edited chromosomal sequence can be isolated using techniques well known in the art.
  • one targeting endonuclease (and no)
  • polynucleotide is introduced into the cell and the double-stranded break introduced by the targeting endonuclease is repaired by a non-homologous end-joining repair process such that the chromosomal sequence encoding HRPT is inactivated.
  • the inactivation can be due to a deletion of at least one nucleotide, an insertion of at least one
  • nucleotide a substation of at least one nucleotide, or a combination thereof.
  • two targeting endonucleases are introduced into the cell (with no polynucleotide).
  • the first endonuclease targets and cleaves a sequence in intron 2 or exon 3 of the HPRT sequence and the second endonuclease targets and cleaves a sequence in any of exons 4-9 of the HPRT sequence.
  • the two double-stranded breaks are repaired by a non-homologous end- joining repair process such that the sequence between the two breaks is deleted, thereby inactivating the chromosomal sequence encoding HPRT.
  • one targeting endonuclease and one single-stranded polynucleotide are introduced into the cell, wherein the polynucleotide comprises a first sequence having substantial sequence identity to a sequence adjacent to one side of the targeted cleavage site and a second sequence having substantial sequence identity to a sequence located distally to the targeted cleavage site.
  • the endonuclease targets and cleaves intron 2 or exon 3 of the HPRT sequence
  • the first sequence of the polynucleotide has substantial sequence identity to a sequence in intron 2 or exon 3 of the HPRT sequence
  • the second sequence of the polynucleotide has substantial sequence identity to a sequence in any of exons 4-9 of the HPRT sequence.
  • the double-stranded break introduced by the targeting endonuclease is repaired by a homology-directed repair process such that the chromosomal sequence between the two sequences having substantial sequence identity with the first and second sequences in the single-stranded polynucleotide is deleted, thereby inactivating the chromosomal sequence encoding HPRT.
  • the HPRT deficient cell is also deficient in GS.
  • the cell is a double knock-out. That is, all copies of the chromosomal sequences encoding HPRT and GS are inactivated, and the cell produces not HPRT or GS.
  • Such a double knock-out cell can be prepared by using a GS deficient cell as the starting cell in the targeting endonuclease mediated genome editing method detailed above in sections (ll)(a)-(e).
  • Non-limiting examples of GS deficient cells are listed above in section (l)(b).
  • the starting cell is a CHOZN ® GS-/- cell.
  • the targeting endonuclease mediated genome editing method detailed above in sections (ll)(a)-(e) can be used to inactivate the chromosomal sequence encoding GS.
  • the chromosomal sequence encoding GS can be edited before, after, or concurrently with the editing of the chromosomal sequence encoding HPRT.
  • a targeting endonuclease such as a zinc finger nuclease, can be engineered to target a sequence in exon 1 , intron 1 , exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 6, exon 6, intron 6, or exon 7 of the GS chromosomal sequence.
  • a zinc finger nuclease is engineered to recognize and cleave a target site in exon 6 of the GS sequence.
  • the double-stranded break in exon 6 is repaired by the error-prone non-homologous end-joining repair process such that a short region is deleted, thereby shifting the reading frame and inactivating the GS gene.
  • a further aspect of the present disclosure encompasses a method for identifying cells that produce high levels of at least one recombinant protein.
  • the method comprises (a) expressing at least one nucleic acid encoding a recombinant protein in a population of cells deficient in HPRT, wherein the nucleic acid further comprises sequence encoding HPRT; (b) culturing the population of cells in the presence of hypoxanthine, aminopterin, and thymidine; and (c) selecting for cells that produces high levels of the at least one recombinant protein.
  • the recombinant protein can be a therapeutic protein or a protein that imparts improved properties to the cell as detailed above in section (l)(c).
  • Nucleic acids encoding the recombinant protein and the amplifiable selectable marker are also described in section (l)(c). Suitable cells are detailed above in section (l)(b).
  • a protocol for selecting cells that produce high levels of a recombinant protein is detailed below in Example 5.
  • Cells that are selected for producing high levels of the recombinant protein comprise additional copies of the nucleic acid encoding the recombinant protein and HPRT due to amplification of the nucleic acid during the selection procedure (detailed above in section (l)(c)).
  • the population of HPRT deficient cells is also deficient in GS.
  • the cells are double knockouts of HPRT and GS.
  • the method comprises expressing in the cells a first nucleic acid encoding a first recombinant protein and HPRT and a second nucleic acid encoding a second recombinant protein and GS, and selecting for cells that produce high levels of both recombinant proteins.
  • the culturing and selecting steps further comprise growth in the presence of MSX (see Example 6). The selection using GS can occur simultaneously with the selection using HPRT.
  • the two selection methods can be performed concurrently (and in either order).
  • the first recombinant protein can be a first subunit of a multi-subunit protein (e.g., heavy chain of IgG) and the second recombinant protein can be a second subunit of the multi-subunit protein (e.g., light chain of IgG).
  • the first recombinant protein can be a therapeutic protein and the second recombinant protein can be a therapeutic protein that is different from the first recombinant protein.
  • the first recombinant protein can be a therapeutic protein and the second recombinant protein can be a protein that provides an improved property to the population of cells (see examples provided above in section (l)(c)).
  • the amount of recombinant protein(s) produced by the selected cells may be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40- fold, 50-fold, 60-fold, 80-fold, 100-fold, or more than 100-fold of the amount of recombinant protein(s) produced by a comparable wild-type cell not deficient in HPRT (or deficient in both HPRT and GS) and not subjected to selection process.
  • endogenous sequence refers to a chromosomal sequence that is native to the cell.
  • exogenous sequence refers to a chromosomal sequence that is not native to the cell, or a chromosomal sequence that is located in a different chromosomal location.
  • the terms "editing,” “genome editing,” or “chromosomal editing” refer to a process by which a specific chromosomal sequence is changed.
  • the edited chromosomal sequence may comprise an insertion of at least one nucleotide, a deletion of at least one nucleotide, and/or a substitution of at least one nucleotide.
  • a "gene,” as used herein, refers to a DNA region (including exons and introns) encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, 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, replication origins, matrix attachment sites, and locus control regions.
  • nucleic acid and “polynucleotide” refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation. For the purposes of the present disclosure, these terms are not to be construed as limiting with respect to the length of a polymer.
  • the terms can encompass known analogs of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties.
  • 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, phosphoramidite, phosphorodiamidate bonds, or combinations thereof. .
  • nucleotide refers to deoxyribonucleotides
  • nucleotides may be standard nucleotides (i.e., adenosine, guanosine, cytidine, thymidine, and uridine) or nucleotide analogs.
  • a nucleotide analog refers to a nucleotide having a modified purine or pyrimidine base or a modified ribose moiety.
  • a nucleotide analog may be a naturally occurring nucleotide (e.g., inosine) or a non-naturally occurring nucleotide.
  • Non-limiting examples of modifications on the sugar or base moieties of a nucleotide include the addition (or removal) of acetyl groups, amino groups, carboxyl groups, carboxymethyl groups, hydroxyl groups, methyl groups, phosphoryl groups, and thiol groups, as well as the substitution of the carbon and nitrogen atoms of the bases with other atoms (e.g., 7-deaza purines).
  • Nucleotide analogs also include dideoxy nucleotides, 2'-O-methyl nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholinos.
  • polypeptide and “protein” are used interchangeably to refer to a polymer of amino acid residues.
  • target site or “target sequence” refer to a nucleic acid sequence that defines a portion of a chromosomal sequence to be edited and to which a targeting endonuclease is engineered to recognize and bind, provided sufficient conditions for binding exist.
  • upstream and downstream refer to locations in a nucleic acid sequence relative to a fixed position. Upstream refers to the region that is 5' (i.e., near the 5' end of the strand) to the position and downstream refers to the region that is 3' (i.e., near the 3' end of the strand) to the position.
  • nucleic acid and amino acid sequence identity are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this fashion. In general, identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides 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 is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100.
  • An approximate alignment for 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 by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation,
  • sequences described herein the range of desired degrees of sequence identity is approximately 80% to 100% and any integer value therebetween.
  • percent identities between sequences are 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.
  • a ZFN was designed to target and cleave exon 3 of the hprt locus of CHO cells (the exon/intron annotation is based on mousse homology).
  • the ZFN binds to the following sequence in the hprt locus:
  • CHO-K1 cells were transfected by electroporation with plasmid DNA encoding the ZFN. After a suitable period of incubation, the cells were harvested and subjected to a Cel-1 assay. The Cel-1 assay detects alleles of the target locus that deviate from wild type as a result of nonhomologous end-joining-mediated imperfect repair of ZFN-induced DNA double strand breaks. PCR amplification of the targeted region from a pool of ZFN-treated cells generates a mixture of WT and mutant amplicons.
  • Cells with an inactivated hprt gene can be selected for by growth in the presence of 6-thioguanine (i.e.., cells deficient in HPRT are resistant to 6- thioguanine).
  • the phenotype of the cells can be confirmed by the requirement for hypoxanthine and thymidine for survival (i.e., the cells die in the absence of hypoxanthine and thymidine).
  • a ZFN was designed to target and cleave intron 2 of the hprt locus of CHO cells.
  • the ZFN binds to the following sequence in the hprt locus:
  • CHO-K1 cells were transfected by nucleofection with DNA encoding the ZFN. After a suitable period of incubation, the cells were harvested and subjected to a Cel-1 assay to determine whether the ZFN cleaved the target sequence. As shown in FIG. 4, cleavage products of 401 , 246 and 155 bp were detected in CHO cells transfected with DNA encoding the ZFN (10.1 % modified).
  • ZFNs described above in Examples 1 and 2 can be used to target the hprt gene in GS-/- knock-out cells.
  • CHOZN ® GS-/- cells Sigma- Aldrich
  • the cells can be screened for ZFN-mediated deletions as detailed above.
  • the hprt locus in CHO cells can be inactivated via large deletions by using either of the following approaches.
  • CHO cells can be transfected with DNA or mRNA encoding the ZFN targeting intron 2 (described above in Example 2) and a single-stranded oligonucleotide comprising a first sequence having sequence identity with a sequence adjacent to the targeted cleavage site in intron 2 and a second sequence having sequence identity with a sequence located in any of exons 4-9.
  • the chromosomal sequence can recombine with the single-stranded oligonucleotide such that sequence between the two targeted sequences is deleted.
  • FIG. 2 presents a schematic diagram of this approach.
  • the single-stranded oligonucleotide comprises a first sequence having sequence identity with a sequence adjacent to the targeted cleavage site in exon 3 and a second sequence having sequence identity with a sequence located in any of exons 4-9.
  • CHO cells can be transfected with DNA or mRNA encoding a first ZFN and a second ZFN.
  • the first ZFN targets intron 2 and the second ZFN targets a sequence in any of exons 4-9.
  • the second ZFN targets a sequence in any of exons 4-9.
  • CHO cells can be transfected with DNA or mRNA encoding a first ZFN targeting exon 3 and a second ZFN targeting a sequence in any of exons 4-9, such that the sequence located between the targeted cleavage sites is deleted during repair of the two double-stranded DNA breaks.
  • HPRT deficient in HPRT can be used to select for cells that produce large amounts of a recombinant protein.
  • the HPRT deficient cells can be transfected using electroporation with DNA encoding the recombinant protein of interest and sequence encoding Chinese hamster HPRT. Twenty-four hours following transfection, the transfected cells are transferred to 6-well plates containing selection media (Ham's F-12 nutrient mix supplemented with 10% fetal bovine serum, 0.1 -4 ⁇ aminopterin, 100 ⁇ hypoxanthine, and 16 ⁇ thymidine). Aminopterin inhibits the enzyme dihydrofolate reductase which is responsible for the synthesis of tetrahydrofolate.
  • Tetrahydrofolate is used to produce nucleotides de novo. Only cells that can utilize hypoxanthine and thymidine in the salvage pathways for nucleotide biosynthesis survive in the presence of aminopterin. Cells that are deficient in HPRT cannot use
  • cells that grow in the presence of aminopterin constitute a selected pool that expresses HPRT and can be screened for the production of the recombinant protein of interest. Specifically, media is changed every 5-7 days during selection until viable cells start to form visible colonies (viewable by microscope). All viable cells are collected when the wells are >80% confluent and the cell cultures are expanded into T-75 and TPP bioreactor tubes. Expression levels of the recombinant protein of interest are evaluated in these stably transfected pools.
  • Individual producing clones can be isolated from these pools for recombinant protein production.
  • Cells deficient in both HPRT and GS can be used for the amplified production of more than one recombinant protein.
  • the cells can be transfected with a first DNA encoding a first recombinant protein and sequence encoding Chinese hamster HPRT and a second DNA encoding a second recombinant protein and sequence encoding Chinese hamster GS.
  • the first recombinant protein can be a therapeutic protein (or other protein of interest) and the second recombinant protein can be a different therapeutic protein (or a second protein of interest).
  • the first recombinant protein can be a therapeutic protein (or other protein of interest) and the second recombinant protein can be a protein that imparts improved properties to the cell, such as the exemplary recombinant proteins described elsewhere herein.
  • the second recombinant protein that imparts improved cellular properties may further enhance the production of the first recombinant protein or, alternatively or in addition, may enhance the growth and/or viability of the production cell itself.
  • the first recombinant protein can be a therapeutic protein (or other protein of interest) and the second recombinant protein can be a protein that modifies or affects the modification of the first recombinant protein (e.g., sialylation, glycosylation, or other post-translational modification proteins).
  • cells deficient in both HPRT and GS can be used for the production of a multi-subunit therapeutic protein (e.g., IgG protein).
  • a multi-subunit therapeutic protein e.g., IgG protein
  • the first recombinant protein can be the heavy chain of a desired IgG protein and the second recombinant protein can be the light chain of the IgG protein.
  • the cells can be transfected with a first DNA encoding a first recombinant protein and sequence encoding Chinese hamster HPRT and a second DNA encoding a second recombinant protein and sequence encoding Chinese hamster GS.
  • the two DNAs can be part of one vector or they can be separate.
  • the cells can be grown as detailed above in Example 5, except the selection media contains 25 ⁇ MSX with or without L-glutamine.

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Abstract

L'invention concerne des compositions et des procédés pour la fabrication de protéine améliorée qui fournissent des cellules déficientes en hypoxanthine-guanine phosphoribosyltransférase. L'invention concerne des procédés pour la préparation des cellules déficientes et des procédés pour l'utilisation des cellules déficientes pour produire de niveaux élevés de protéines recombinantes.
PCT/US2013/026454 2012-02-17 2013-02-15 Cellules déficientes en hypoxanthine-guanine phosphoribosyltransférase WO2013123408A1 (fr)

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EP2771457A4 (fr) * 2011-10-27 2015-04-08 Sangamo Biosciences Inc Procédés et compositions pour la modification du locus hprt

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