US20070134796A1 - Targeted integration and expression of exogenous nucleic acid sequences - Google Patents

Targeted integration and expression of exogenous nucleic acid sequences Download PDF

Info

Publication number
US20070134796A1
US20070134796A1 US11/493,423 US49342306A US2007134796A1 US 20070134796 A1 US20070134796 A1 US 20070134796A1 US 49342306 A US49342306 A US 49342306A US 2007134796 A1 US2007134796 A1 US 2007134796A1
Authority
US
United States
Prior art keywords
sequence
cell
gene
cleavage
sequences
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US11/493,423
Other languages
English (en)
Inventor
Michael Holmes
Fyodor Urnov
Philip Gregory
Edward Rebar
Sean Brennan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sangamo Therapeutics Inc
Original Assignee
Sangamo Biosciences Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sangamo Biosciences Inc filed Critical Sangamo Biosciences Inc
Priority to US11/493,423 priority Critical patent/US20070134796A1/en
Assigned to SANGAMO BIOSCIENCES, INC. reassignment SANGAMO BIOSCIENCES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BRENNAN, SEAN M., GREGORY, PHILIP D., HOLMES, MICHAEL C., REBAR, EDWARD J., URNOV, FYODOR
Publication of US20070134796A1 publication Critical patent/US20070134796A1/en
Priority to US12/381,217 priority patent/US8313925B2/en
Priority to US13/134,766 priority patent/US9045763B2/en
Priority to US13/787,473 priority patent/US9260726B2/en
Priority to US14/699,908 priority patent/US9376685B2/en
Priority to US15/146,276 priority patent/US9765360B2/en
Assigned to SANGAMO THERAPEUTICS, INC. reassignment SANGAMO THERAPEUTICS, INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: SANGAMO BIOSCIENCES, INC.
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding

Definitions

  • the present disclosure is in the fields of genome engineering, gene targeting, targeted chromosomal integration and protein expression.
  • sickle cell anemia is caused by mutation of a single nucleotide pair in the human ⁇ -globin gene.
  • the ability to convert the endogenous genomic copy of this mutant nucleotide pair to the wild-type sequence in a stable fashion and produce normal ⁇ -globin would provide a cure for sickle cell anemia, as would introduction of a functional ⁇ -globin gene into a genome containing a mutant ⁇ -globin gene.
  • DNA cleavage in the desired genomic region was accomplished by inserting a recognition site for a meganuclease (i.e., an endonuclease whose recognition sequence is so large that it does not occur, or occurs only rarely, in the genome of interest) into the desired genomic region.
  • a recognition site for a meganuclease i.e., an endonuclease whose recognition sequence is so large that it does not occur, or occurs only rarely, in the genome of interest
  • meganuclease cleavage-stimulated homologous recombination relies on either the fortuitous presence of, or the directed insertion of, a suitable meganuclease recognition site in the vicinity of the genomic region to be altered. Since meganuclease recognition sites are rare (or nonexistent) in a typical mammalian genome, and insertion of a suitable meganuclease recognition site is plagued with the same difficulties as associated with other genomic alterations, these methods are not broadly applicable.
  • compositions and methods for targeted alteration of sequences in any genome and for compositions and methods for targeted introduction of exogenous sequences into a genome.
  • the present disclosure provides method and compositions for expressing the product of an exogenous nucleic acid sequence (i.e. a protein or a RNA molecule) in a cell.
  • the exogenous nucleic acid sequence can comprise, for example, one or more genes or cDNA molecules, or any type of coding or noncoding sequence, and is introduced into the cell such that it is integrated into the genome of the cell in a predetermined region of interest. Integration of the exogenous nucleic acid sequence is facilitated by targeted double-strand cleavage of the genome in the region of interest.
  • Cleavage is targeted to a particular site through the use of fusion proteins comprising a zinc finger binding domain, which can be engineered to bind any sequence of choice in the region of interest, and a cleavage domain or a cleavage half-domain.
  • cleavage stimulates integration of exogenous polynucleotide sequences at or near the cleavage site. Said integration of exogenous sequences can proceed through both homology-dependent and homology-independent mechanisms.
  • exogenous sequences can include, for example, transcriptional control sequences such as promoters and enhancers.
  • Modulation can include transcriptional activation (e.g., enhancement of transcription by, for example, insertion of a promoter and/or enhancer sequence) and transcriptional repression (e.g., functional “knock-out” by, for example, inserting an exogenous sequence into an endogenous transcriptional regulatory sequence, inserting a sequence facilitating transcriptional repression, or inserting a sequence that interrupts a coding region).
  • a recognition sequence for a sequence-specific DNA-cleaving enzyme can be introduced at a predetermined location in a genome so that targeted cleavage by the cleaving enzyme, at the predetermined location in the genome, can be accomplished.
  • Exemplary DNA-cleaving enzymes include, but are not limited to, restriction enzymes, meganucleases and homing endonucleases.
  • a method for expressing the product of an exogenous nucleic acid sequence in a cell comprising: (a) expressing a first fusion protein in the cell, the first fusion protein comprising a first zinc finger binding domain and a first cleavage half-domain, wherein the first zinc finger binding domain has been engineered to bind to a first target site in a region of interest in the genome of the cell; (b) expressing a second fusion protein in the cell, the second fusion protein comprising a second zinc finger binding domain and a second cleavage half domain, wherein the second zinc finger binding domain binds to a second target site in the region of interest in the genome of the cell, wherein the second target site is different from the first target site; and (c) contacting the cell with a polynucleotide comprising an exogenous nucleic acid sequence and a first nucleotide sequence that is homologous to a first sequence in the region of interest; wherein binding of the first
  • the exogenous nucleic acid sequence may comprise a cDNA and/or a promoter. In other embodiments, the exogenous nucleic acid sequence encodes a siRNA.
  • the first nucleotide sequence may be identical to the first sequence in the region of interest.
  • the polynucleotide further comprises a second nucleotide sequence that is homologous to a second sequence in the region of interest.
  • the second nucleotide sequence may be identical to the second sequence in the region of interest.
  • the first nucleotide sequence may identical to the first sequence in the region of interest and the second nucleotide sequence may be homologous but non-identical to a second sequence in the region of interest.
  • the first and second nucleotide sequences flank the exogenous sequence.
  • the polynucleotide is a plasmid. In other embodiments, the polynucleotide is a linear DNA molecule.
  • the region of interest is in an accessible region of cellular chromatin, a chromosome, and/or a gene (e.g., a gene comprising a mutation such as a point mutation, a substitution, a deletion, an insertion, a duplication, an inversion and/or a translocation).
  • the exogenous nucleic acid sequence comprises the wild-type sequence of the gene.
  • the exogenous nucleic acid sequence comprises a portion of the wild-type sequence of the gene.
  • the exogenous nucleic acid sequence comprises a cDNA copy of a transcription product of the gene.
  • the region of interest is in a region of the genome that is not essential for viability. In other embodiments, the region of interest is in a region of the genome that is transcriptionally active. The region of interest is in a region of the genome that is transcriptionally active and not essential for viability (e.g., the human Rosa26 genome, the human homologue of the murine Rosa26 gene, or a CCR5 gene).
  • a method for integrating an exogenous sequence into a region of interest in the genome of a cell comprising: (a) expressing a first fusion protein in the cell, the first fusion protein comprising a first zinc finger binding domain and a first cleavage half-domain, wherein the first zinc finger binding domain has been engineered to bind to a first target site in a region of interest in the genome of the cell; (b) expressing a second fusion protein in the cell, the second fusion protein comprising a second zinc finger binding domain and a second cleavage half domain, wherein the second zinc finger binding domain binds to a second target site in the region of interest in the genome of the cell, wherein the second target site is different from the first target site; and (c) contacting the cell with a polynucleotide comprising an exogenous nucleic acid sequence; wherein binding of the first fusion protein to the first target site, and binding of the second fusion protein to the second target site,
  • the integration inactivates gene expression in the region of interest.
  • the exogenous nucleic acid sequence may comprise, for example, a sequence of between 1 and 50 nucleotides in length. Furthermore, the exogenous nucleic acid sequence may encode a detectable amino acid sequence.
  • the region of interest may be in an accessible region of cellular chromatin.
  • the first and second cleavage half-domains are from a Type IIS restriction endonuclease, for example, FokI or StsI.
  • at least one of the fusion proteins may comprise an alteration in the amino acid sequence of the dimerization interface of the cleavage half-domain.
  • the cell can be a mammalian cell, for example, a human cell.
  • the cell may be arrested in the G2 phase of the cell cycle.
  • FIG. 1 shows the nucleotide sequence, in double-stranded form, of a portion of the human hSMC1L1 gene encoding the amino-terminal portion of the protein (SEQ ID NO: 1) and the encoded amino acid sequence (SEQ ID NO:2).
  • Target sequences for the hSMC1-specific ZFPs are underlined (one on each DNA strand).
  • FIG. 2 shows a schematic diagram of a plasmid encoding a ZFP-FokI fusion for targeted cleavage of the hSMC1 gene.
  • FIG. 3A -D show a schematic diagram of the hSMC1 gene.
  • FIG. 3A shows a schematic of a portion of the human X chromosome which includes the hSMC1 gene.
  • FIG. 3B shows a schematic of a portion of the hSMC1 gene including the upstream region (left of +1), the first exon (between +1 and the right end of the arrow labeled “SMC1 coding sequence”) and a portion of the first intron. Locations of sequences homologous to the initial amplification primers and to the chromosome-specific primer (see Table 3) are also provided.
  • FIG. 3A shows a schematic of a portion of the human X chromosome which includes the hSMC1 gene.
  • FIG. 3B shows a schematic of a portion of the hSMC1 gene including the upstream region (left of +1), the first exon (between +1 and the right end of the arrow labeled “SMC1 coding sequence”) and
  • FIG. 3C shows the nucleotide sequence of the human X chromosome in the region of the SMC1 initiation codon (SEQ ID NO: 3), the encoded amino acid sequence (SEQ ID NO: 4), and the target sites for the SMC1-specific zinc finger proteins.
  • FIG. 3D shows the sequence of the corresponding region of the donor molecule (SEQ ID NO: 5), with differences between donor and chromosomal sequences underlined. Sequences contained in the donor-specific amplification primer (Table 3) are indicated by double underlining.
  • FIG. 4 shows a schematic diagram of the hSMC1 donor construct.
  • FIG. 5 shows PCR analysis of DNA from transfected HEK293 cells. From left, the lanes show results from cells transfected with a plasmid encoding GFP (control plasmid), cells transfected with two plasmids, each of which encodes one of the two hSMC1-specific ZFP-FokI fusion proteins (ZFPs only), cells transfected with two concentrations of the hSMC1 donor plasmid (donor only), and cells transfected with the two ZFP-encoding plasmids and the donor plasmid (ZFPs+ donor). See Example 1 for details.
  • FIG. 6 shows the nucleotide sequence of an amplification product derived from a mutated hSMC1 gene (SEQ ID NO:6) generated by targeted homologous recombination. Sequences derived from the vector into which the amplification product was cloned are single-underlined, chromosomal sequences not present in the donor molecule are indicated by dashed underlining (nucleotides 32-97), sequences common to the donor and the chromosome are not underlined (nucleotides 98-394 and 402-417), and sequences unique to the donor are double-underlined (nucleotides 395-401). Lower-case letters represent sequences that differ between the chromosome and the donor.
  • FIG. 7 shows the nucleotide sequence of a portion of the human IL2R ⁇ gene comprising the 3′ end of the second intron and the 5′ end of third exon (SEQ ID NO:7) and the amino acid sequence encoded by the displayed portion of the third exon (SEQ ID NO:8).
  • Target sequences for the second pair of IL2R ⁇ -specific ZFPs are underlined. See Example 2 for details.
  • FIG. 8 shows a schematic diagram of a plasmid encoding a ZFP-FokI fusion for targeted cleavage of IL2R ⁇ gene.
  • FIG. 9A -D show a schematic diagram of the IL2R ⁇ gene.
  • FIG. 9A shows a schematic of a portion of the human X chromosome which includes the IL2R ⁇ gene.
  • FIG. 9B shows a schematic of a portion of the IL2R ⁇ gene including a portion of the second intron, the third exon and a portion of the third intron. Locations of sequences homologous to the initial amplification primers and to the chromosome-specific primer (see Table 5) are also provided.
  • FIG. 9A shows a schematic of a portion of the human X chromosome which includes the IL2R ⁇ gene.
  • FIG. 9B shows a schematic of a portion of the IL2R ⁇ gene including a portion of the second intron, the third exon and a portion of the third intron. Locations of sequences homologous to the initial amplification primers and to the chromosome-specific primer (see Table 5) are also provided.
  • FIG. 9C shows the nucleotide sequence of the human X chromosome in the region of the third exon of the IL2R ⁇ gene (SEQ ID NO: 9), the encoded amino acid sequence (SEQ ID NO: 10), and the target sites for the first pair of IL2R ⁇ -specific zinc finger proteins.
  • FIG. 9D shows the sequence of the corresponding region of the donor molecule (SEQ ID NO: 11), with differences between donor and chromosomal sequences underlined. Sequences contained in the donor-specific amplification primer (Table 5) are indicated by double overlining.
  • FIG. 10 shows a schematic diagram of the IL2R ⁇ donor construct.
  • FIG. 11 shows PCR analysis of DNA from transfected K652 cells. From left, the lanes show results from cells transfected with two plasmids, each of which encodes one of a pair of IL2R ⁇ -specific ZFP-FokI fusion proteins (ZFPs only, lane 1), cells transfected with two concentrations of the IL2R ⁇ donor plasmid (donor only, lanes 2 and 3), and cells transfected with the two ZFP-encoding plasmids and the donor plasmid (ZFPs+ donor, lanes 4-7).
  • FIG. 12 shows the nucleotide sequence of an amplification product derived from a mutated IL2R ⁇ gene (SEQ ID NO: 12) generated by targeted homologous recombination. Sequences derived from the vector into which the amplification product was cloned are single-underlined, chromosomal sequences not present in the donor molecule are indicated by dashed underlining (nucleotides 460-552), sequences common to the donor and the chromosome are not underlined (nucleotides 32-42 and 59-459), and a stretch of sequence containing nucleotides which distinguish donor sequences from chromosomal sequences is double-underlined (nucleotides 44-58). Lower-case letters represent nucleotides whose sequence differs between the chromosome and the donor.
  • FIG. 13 shows the nucleotide sequence of a portion of the human beta-globin gene encoding segments of the core promoter, the first two exons and the first intron (SEQ ID NO: 13).
  • a missense mutation changing an A (in boldface and underlined) at position 5212541 on Chromosome 11 (BLAT, UCSC Genome Bioinformatics site) to a T results in sickle cell anemia.
  • a first zinc finger/FokI fusion protein was designed such that the primary contacts were with the underlined 12-nucleotide sequence AAGGTGAACGTG (nucleotides 305-316 of SEQ ID NO: 13), and a second zinc finger/FokI fusion protein was designed such that the primary contacts were with the complement of the underlined 12-nucleotide sequence CCGTTACTGCCC (nucleotides 325-336 of SEQ ID NO:13).
  • FIG. 14 is a schematic diagram of a plasmid encoding ZFP-FokI fusion for targeted cleavage of the human beta globin gene.
  • FIG. 15 is a schematic diagram of the cloned human beta globin gene showing the upstream region, first and second exons, first intron and primer binding sites.
  • FIG. 16 is a schematic diagram of the beta globin donor construct, pCR4-TOPO-HBBdonor.
  • FIG. 17 shows PCR analysis of DNA from cells transfected with two pairs of ⁇ -globin-specific ZFP nucleases and a beta globin donor plasmid.
  • the panel on the left is a loading control in which the initial amp 1 and initial amp 2 primers (Table 7) were used for amplification.
  • the “chromosome-specific and “donor-specific” primers (Table 7) were used for amplification.
  • the leftmost lane in each panel contains molecular weight markers and the next lane shows amplification products obtained from mock-transfected cells.
  • Remaining lanes, from left to right, show amplification product from cells transfected with: a GFP-encoding plasmid, 100 ng of each ZFP/FokI-encoding plasmid, 200 ng of each ZFP/FokI-encoding plasmid, 200 ng donor plasmid, 600 ng donor plasmid, 200 ng donor plasmid +100 ng of each ZFP/FokI-encoding plasmid, and 600 ng donor plasmid +200 ng of each ZFP/FokI-encoding plasmid.
  • FIG. 18 shows the nucleotide sequence of an amplification product derived from a mutated beta-globin gene (SEQ ID NO: 14) generated by targeted homologous recombination.
  • Chromosomal sequences not present in the donor molecule are indicated by dashed underlining (nucleotides 1-72), sequences common to the donor and the chromosome are not underlined (nucleotides 73-376), and a stretch of sequence containing nucleotides which distinguish donor sequences from chromosomal sequences is double-underlined (nucleotides 377-408).
  • Lower-case letters represent nucleotides whose sequence differs between the chromosome and the donor.
  • FIG. 19 shows the nucleotide sequence of a portion of the fifth exon of the Interleukin-2 receptor gamma chain (IL-2R ⁇ ) gene (SEQ ID NO: 15). Also shown (underlined) are the target sequences for the 5-8 and 5-10 ZFP/FokI fusion proteins. See Example 5 for details.
  • IL-2R ⁇ Interleukin-2 receptor gamma chain
  • FIG. 20 shows the amino acid sequence of the 5-8 ZFP/FokI fusion targeted to exon 5 of the human IL-2R ⁇ gene (SEQ ID NO: 16).
  • Amino acid residues 1-17 contain a nuclear localization sequence (NLS, underlined); residues 18-130 contain the ZFP portion, with the recognition regions of the component zinc fingers shown in boldface; the ZFP-FokI linker (ZC linker, underlined) extends from residues 131 to 140 and the FokI cleavage half-domain begins at residue 141 and extends to the end of the protein at residue 336.
  • the residue that was altered to generate the Q486E mutation is shown underlined and in boldface.
  • FIG. 21 shows the amino acid sequence of the 5-10 ZFP/FokI fusion targeted to exon 5 of the human IL-2R ⁇ gene (SEQ ID NO: 17).
  • Amino acid residues 1-17 contain a nuclear localization sequence (NLS, underlined); residues 18-133 contain the ZFP portion, with the recognition regions of the component zinc fingers shown in boldface; the ZFP-FokI linker (ZC linker, underlined) extends from residues 134 to 143 and the FokI cleavage half-domain begins at residue 144 and extends to the end of the protein at residue 339.
  • the residue that was altered to generate the E490K mutation is shown underlined and in boldface.
  • FIG. 22 shows the nucleotide sequence of the enhanced Green Fluorescent Protein gene (SEQ ID NO: 18) derived from the Aequorea victoria GFP gene (Tsien (1998) Ann. Rev. Biochem. 67:509-544). The ATG initiation codon, as well as the region which was mutagenized, are underlined.
  • FIG. 23 shows the nucleotide sequence of a mutant defective eGFP gene (SEQ ID NO: 19). Binding sites for ZFP-nucleases are underlined and the region between the binding sites corresponds to the region that was modified.
  • FIG. 24 shows the structures of plasmids encoding Zinc Finger Nucleases targeted to the eGFP gene.
  • FIG. 25 shows an autoradiogram of a 10% acrylamide gel used to analyze targeted DNA cleavage of a mutant eGFP gene by zinc finger endonucleases. See Example 8 for details.
  • FIG. 26 shows the structure of plasmid pcDNA4/TO/GFPmut (see Example 9).
  • FIG. 27 shows levels of eGFPmut mRNA, normalized to GAPDH mRNA, in various cell lines obtained from transfection of human HEK293 cells. Light bars show levels in untreated cells; dark bars show levels in cell that had been treated with 2 ng/ml doxycycline. See Example 9 for details.
  • FIG. 28 shows the structure of plasmid pCR(R)4-TOPO-GFPdonor5. See Example 10 for details.
  • FIG. 29 shows the nucleotide sequence of the eGFP insert in pCR(R)4-TOPO-GFPdonor5 (SEQ ID NO:20).
  • the insert contains sequences encoding a portion of a non-modified enhanced Green Fluorescent Protein, lacking an initiation codon. See Example 10 for details.
  • FIG. 30 shows a FACS trace of T18 cells transfected with plasmids encoding two ZFP nucleases and a plasmid encoding a donor sequence, that were arrested in the G2 phase of the cell cycle 24 hours post-transfection with 100 ng/ml nocodazole for 48 hours. The medium was replaced and the cells were allowed to recover for an additional 48 hours, and gene correction was measured by FACS analysis. See Example 11 for details.
  • FIG. 31 shows a FACS trace of T18 cells transfected with plasmids encoding two ZFP nucleases and a plasmid encoding a donor sequence, that were arrested in the G2 phase of the cell cycle 24 hours post-transfection with 0.2 ⁇ M vinblastine for 48 hours. The medium was replaced and the cells were allowed to recover for an additional 48 hours, and gene correction was measured by FACS analysis. See Example 11 for details.
  • FIG. 32 shows the nucleotide sequence of a 1,527 nucleotide eGFP insert in pCR(R)4-TOPO (SEQ ID NO:21). The sequence encodes a non-modified enhanced Green Fluorescent Protein lacking an initiation codon. See, Example 13 for details.
  • FIG. 33 shows a schematic diagram of an assay used to measure the frequency of editing of the endogenous human IL-2R ⁇ gene. See, Example 14 for details.
  • FIG. 34 shows autoradiograms of acrylamide gels used in an assay to measure the frequency of editing of an endogenous cellular gene by targeted cleavage and homologous recombination.
  • the lane labeled “GFP” shows assay results from a control in which cells were transfected with an eGFP-encoding vector; the lane labeled “ZFPs only” shows results from another control experiment in which cells were transfected with the two ZFP/nuclease-encoding plasmids (50 ng of each) but not with a donor sequence.
  • Lanes labeled “donor only” show results from a control experiment in which cells were transfected with 1 ⁇ g of donor plasmid but not with the ZFP/nuclease-encoding plasmids.
  • 50Z refers to cells transfected with 50 ng of each ZFP/nuclease expression plasmid
  • 100Z refers to cells transfected with 100 ng of each ZFP/nuclease expression plasmid
  • 0.5D refers to cells transfected with 0.5 ⁇ g of the donor plasmid
  • 1D refers to cells transfected with 1.0 ⁇ g of the donor plasmid.
  • ⁇ refers to cells that were not exposed to vinblastine.
  • wt refers to the fragment obtained after BsrBI digestion of amplification products obtained from chromosomes containing the wild-type chromosomal IL-2R ⁇ gene; “rflp” refers to the two fragments (of approximately equal molecular weight) obtained after BsrBI digestion of amplification products obtained from chromosomes containing sequences from the donor plasmid which had integrated by homologous recombination.
  • FIG. 35 shows an autoradiographic image of a four-hour exposure of a gel used in an assay to measure targeted recombination at the human IL-2R ⁇ locus in K562 cells.
  • wt identifies a band that is diagnostic for chromosomal DNA containing the native K562 IL-2R ⁇ sequence
  • rflp identifies a doublet diagnostic for chromosomal DNA containing the altered IL-2R ⁇ sequence present in the donor DNA molecule.
  • the symbol “+” above a lane indicates that cells were treated with 0.2 ⁇ M vinblastine; the symbol “ ⁇ ” indicates that cells were not treated with vinblastine.
  • the numbers in the “ZFP+ donor” lanes indicate the percentage of total chromosomal DNA containing sequence originally present in the donor DNA molecule, calculated using the “peak finder, automatic baseline” function of Molecular Dynamics' ImageQuant v. 5.1 software as described in Ch. 8 of the manufacturer's manual (Molecular Dynamics ImageQuant User's Guide; part 218-415). “Untr” indicates untransfected cells. See Example 15 for additional details.
  • FIG. 36 shows an autoradiographic image of a four-hour exposure of a gel used in an assay to measure targeted recombination at the human IL-2R ⁇ locus in K562 cells.
  • “wt” identifies a band that is diagnostic for chromosomal DNA containing the native K562 IL-2R ⁇ sequence
  • “rflp” identifies a band that is diagnostic for chromosomal DNA containing the altered IL-2R ⁇ sequence present in the donor DNA molecule.
  • the symbol “+” above a lane indicates that cells were treated with 0.2 ⁇ M vinblastine; the symbol “ ⁇ ” indicates that cells were not treated with vinblastine.
  • the numbers beneath the “ZFP+ donor” lanes indicate the percentage of total chromosomal DNA containing sequence originally present in the donor DNA molecule, calculated as described in Example 35. See Example 15 for additional details.
  • FIG. 37 shows an autoradiogram of a four-hour exposure of a DNA blot probed with a fragment specific to the human IL-2R ⁇ gene.
  • the arrow to the right of the image indicates the position of a band corresponding to genomic DNA whose sequence has been altered by homologous recombination.
  • the symbol “+” above a lane indicates that cells were treated with 0.2 ⁇ M vinblastine; the symbol “ ⁇ ” indicates that cells were not treated with vinblastine.
  • the numbers beneath the “ZFP+ donor” lanes indicate the percentage of total chromosomal DNA containing sequence originally present in the donor DNA molecule, calculated as described in Example 35. See Example 15 for additional details.
  • FIG. 38 shows autoradiographic images of gels used in an assay to measure targeted recombination at the human IL-2R ⁇ locus in CD34 + human bone marrow cells.
  • the left panel shows a reference standard in which the stated percentage of normal human genomic DNA (containing a MaeII site) was added to genomic DNA from Jurkat cells (lacking a MaeII site), the mixture was amplified by PCR to generate a radiolabelled amplification product, and the amplification product was digested with MaeII. “wt” identifies a band representing undigested DNA, and “rflp” identifies a band resulting from MaeII digestion.
  • the right panel shows results of an experiment in which CD34 + cells were transfected with donor DNA containing a BsrBI site and plasmids encoding zinc finger-FokI fusion endonucleases.
  • the relevant genomic region was then amplified and labeled, and the labeled amplification product was digested with BsrBI.
  • GFP indicates control cells that were transfected with a GFP-encoding plasmid
  • Donor only indicates control cells that were transfected only with donor DNA
  • ZFP+ Donor indicates cells that were transfected with donor DNA and with plasmids encoding the zinc finger/FokI nucleases.
  • “wt” identifies a band that is diagnostic for chromosomal DNA containing the native IL-2R ⁇ sequence
  • “rflp” identifies a band that is diagnostic for chromosomal DNA containing the altered IL-2R ⁇ sequence present in the donor DNA molecule.
  • the rightmost lane contains DNA size markers. See Example 16 for additional details.
  • FIG. 39 shows an image of an immunoblot used to test for Ku70 protein levels in cells transfected with Ku70-targeted siRNA.
  • the T7 cell line (Example 9, FIG. 27 ) was transfected with two concentrations each of siRNA from two different siRNA pools (see Example 18).
  • Lane 1 70 ng of siRNA pool D
  • Lane 2 140 ng of siRNA pool D
  • Lane 3 70 ng of siRNA pool E
  • Lane 4 140 ng of siRNA pool E.
  • “Ku70” indicates the band representing the Ku70 protein
  • “TFIIB” indicates a band representing the TFIIB transcription factor, used as a control.
  • FIG. 40 shows the amino acid sequences of four zinc finger domains targeted to the human ⁇ -globin gene: sca-29b (SEQ ID NO:22); sca-36a (SEQ ID NO:23); sca-36b (SEQ ID NO:24) and sca-36c (SEQ ID NO:25).
  • the target site for the sca-29b domain is on one DNA strand, and the target sites for the sca-36a, sca-36b and sca-36c domains are on the opposite strand. See Example 20.
  • FIG. 41 shows results of an in vitro assay, in which different combinations of zinc finger/FokI fusion nucleases (ZFNs) were tested for sequence-specific DNA cleavage.
  • ZFNs zinc finger/FokI fusion nucleases
  • the lane labeled “U” shows a sample of the DNA template.
  • the next four lanes show results of incubation of the DNA template with each of four ⁇ -globin-targeted ZFNs (see Example 20 for characterization of these ZFNs).
  • the rightmost three lanes show results of incubation of template DNA with the sca-29b ZFN and one of the sca-36a, sca-36b or sca-36c ZFNs (all of which are targeted to the strand opposite that to which sca-29b is targeted).
  • FIG. 42 shows levels of eGFP mRNA in T18 cells (bars) as a function of doxycycline concentration (provided on the abscissa). The number above each bar represents the percentage correction of the eGFP mutation, in cells transfected with donor DNA and plasmids encoding eGFP-targeted zinc finger nucleases, as a function of doxycycline concentration.
  • FIG. 43A -C show schematic diagrams of different fusion protein configurations.
  • FIG. 43A shows two fusion proteins, in which the zinc finger domain is nearest the N-terminus and the FokI cleavage half-domain is nearest the C-terminus, binding to DNA target sites on opposite strands whose 5′ ends are proximal to each other.
  • FIG. 43B shows two fusion proteins, in which the FokI cleavage half-domain is nearest the N-terminus and the zinc finger domain is nearest the C-terminus, binding to DNA target sites on opposite strands whose 3′ ends are proximal to each other.
  • FIG. 43A shows two fusion proteins, in which the zinc finger domain is nearest the N-terminus and the FokI cleavage half-domain is nearest the C-terminus, binding to DNA target sites on opposite strands whose 3′ ends are proximal to each other.
  • 43C shows a first protein in which the FokI cleavage half-domain is nearest the N-terminus and the zinc finger domain is nearest the C-terminus and a second protein in which the zinc finger domain is nearest the N-terminus and the FokI cleavage half-domain is nearest the C-terminus, binding to DNA target sites on the same strand, in which the target site for the first protein is upstream (i.e. to the 5′ side) of the binding site for the second protein.
  • three-finger proteins are shown binding to nine-nucleotide target sites. 5′ and 3′ polarity of the DNA strands is shown, and the N-termini of the fusion proteins are identified.
  • FIG. 44 is an autoradiogram of an acrylamide gel in which cleavage of a model substrate by zinc finger endonucleases was assayed.
  • Lane 1 shows the migration of uncleaved substrate.
  • Lane 2 shows substrate after incubation with the IL2-1R zinc finger/FokI fusion protein.
  • Lane 3 shows substrate after incubation with the5-9DR zinc finger/FokI fusion protein.
  • Lane 4 shows substrate after incubation with both proteins. Approximate sizes (in base pairs) of the substrate and its cleavage products are shown to the right of the image. Below the image, the nucleotide sequence (SEQ ID NO:211) of the portion of the substrate containing the binding sites for the 5-9D and IL2-1 zinc finger binding domains is shown. The binding sites are identified and indicated by underlining.
  • FIG. 45 is an autoradiogram of an acrylamide gel in which cleavage of a model substrate by zinc finger endonucleases was assayed.
  • Lane 1 shows the migration of uncleaved substrate.
  • Lane 2 shows substrate after incubation with the IL2-1C zinc finger/FokI fusion protein.
  • Lane 3 shows substrate after incubation with the IL2-1R zinc finger/FokI fusion protein.
  • Lane 4 shows substrate after incubation with the5-9DR zinc finger/FokI fusion protein.
  • Lane 5 shows substrate after incubation with both the IL2-1R and 5-9DR fusion proteins.
  • Lane 6 shows substrate after incubation with both the IL2-1C and 5-9DR proteins.
  • FIG. 46 is a schematic diagram of a plasmid containing mutant eGFP coding sequences containing an insertion of sequences from exon 5 of the IL-2R ⁇ gene. See Example 29 for details.
  • FIG. 47 shows an autoradiographic image of a gel in which amplification products of DNA from transfected K562 cells were incubated with the restriction enzyme Stu I.
  • Headings above the lane indicate DNA from cells transfected with a GFP-encoding plasmid (GFP); DNA from cells transfected with a vector encoding the 5-8G and 5-9D ZFP/FokI fusion proteins (ZFNs); DNA from cells transfected with a plasmid containing a 12-nucleotide pair exogenous sequence (including a StuI recognition site) flanked on either side by 750 nucleotide pairs of sequence homologous to exon 5 of the IL-2R ⁇ gene, wherein the two exon 5-homologous sequences are adjacent to one another in the wild-type IL-2R ⁇ gene (donor); and DNA from cells transfected with a vector encoding the 5-8G and 5-9D ZFP/FokI fusion proteins and a plasmid containing
  • FIG. 48 shows images of gels in which amplification products of DNA from transfected K562 cells were analyzed. Headings above the lane indicate DNA from cells transfected with a vector encoding the 5-8G and 5-9D ZFP/FokI fusion proteins (ZFNs); DNA from cells transfected with a plasmid containing a 720 nucleotide pair open reading frame encoding eGFP (donor 1); DNA from cells transfected with a plasmid containing a 924 nucleotide pair sequence that included an eGFP open reading frame and a downstream polyadenylation signal (donor 2); DNA from cells transfected with a vector encoding the 5-8G and 5-9D ZFP/FokI fusion proteins and with a plasmid containing a 720 nucleotide pair open reading frame encoding eGFP (ZFNs +donor 1) and DNA from cells transfected with a vector encoding the 5-8G and 5-9D ZFP
  • the leftmost and rightmost lanes of the top panel contains molecular weight markers.
  • the top panel is a photograph of an ethidium bromide-stained gel; the bottom panel shows an autoradiogram of a gel from a separate experiment in which labeled amplification products were analyzed. See also Example 34.
  • FIG. 49 is a schematic diagram describing an experiment in which a “therapeutic half-gene” was introduced into the endogenous human IL-2R ⁇ gene.
  • the top line represents chromosomal IL-2R ⁇ sequences
  • the middle line represents donor sequences, with exons indicated by boxes and introns by horizontal lines. Numbers inside the boxes identify the exons of the IL-2R ⁇ gene, with “5” representing the fifth exon of the chromosomal IL-2R gene, “5u” representing the upstream portion of the fifth exon, “5d” representing the downstream portion of the fifth exon and “5d(m)” representing the downstream portion of the fifth exon containing several silent sequence changes (i.e., changes that do not alter the encoded amino acid sequence).
  • Diagonal lines demarcate regions of homology between donor and chromosomal sequences.
  • the bottom line shows the expected product of homologous recombination, in which exons 5d(m), 6, 7, and 8 are inserted within the fifth exon of the chromosomal gene. See also Example 35.
  • FIG. 50 shows an autoradiogram of a gel in which amplification products of DNA from transfected K562 cells were analyzed. Headings above the lane indicate DNA from control cells transfected with a vector encoding green fluorescent protein (“GFP”) and from experimental cells transfected with a vector encoding the 5-8G and 5-9D ZFP/FokI fusion proteins and a plasmid containing a 720-nucleotide cDNA construct containing part of exon 5 and exons 6, 7 and 8 of the IL-2R ⁇ gene, flanked on either side by 750 nucleotide pairs of sequence homologous to exon 5 and surrounding regions of the IL-2R ⁇ gene, wherein the two exon 5-homologous sequences are adjacent to one another in the wild-type IL-2R ⁇ gene (ZFNs+ donor). Bands arising from chromosomes containing wild-type IL-2R sequences (“WT”) and chromosomes into which exogenous sequences have been integrated (“
  • FIG. 51 shows the design and results of an experiment in which a 7.7 kbp antibody expression construct was inserted into the endogenous chromosomal IL-2R ⁇ gene.
  • the upper portion of the figure is a schematic diagram showing the result of homology-dependent targeted integration of a 7.7 kilobase pair expression construct (shaded) into exon 5 of the endogenous chromosomal IL-2R ⁇ gene.
  • Arrows indicate the locations and polarities of amplification primers used to detect the junctions between exogenous and endogenous sequences that result from targeted integration.
  • the lower portion of the figure shows photographs of ethidium bromide-stained gels in which amplification products were analyzed.
  • the left panel shows products of cellular DNA that was amplified using primers that detect the upstream junction (Primer set A) and the right panel shows products of cellular DNA that was amplified using primers that detect the downstream junction (Primer set B).
  • DNA samples used as templates for amplification are identified below the gel, as follows: DNA from cells transfected with a vector encoding green fluorescent protein (GFP); DNA from cells transfected only with the donor DNA molecule containing the 7.7 kbp expression construct (don.); DNA from cells transfected with a vector encoding the 5-8G and 5-9D ZFP/FokI fusion proteins and with the donor DNA molecule (ZFNs+ donor).
  • GFP green fluorescent protein
  • Don. DNA from cells transfected with a vector encoding the 5-8G and 5-9D ZFP/FokI fusion proteins and with the donor DNA molecule (ZFNs+ donor).
  • the topology of the donor DNA (circular or linear) is also indicated. See also Example 36.
  • FIG. 52 is an autoradiogram of a gel in which amplification products from the CHO DHFR gene were analyzed for mismatches using a Cel-1 assay. Amplification products were obtained from wild-type CHO cell DNA (W) or DNA from CHO cells that had been treated with zinc finger nucleases (Mu), and were then exposed to Cel-1 nuclease (+) or not ( ⁇ ), as indicated above the gel. To the right of the gel, bands indicative of wild-type DHFR sequences (WT) and mutant DHFR sequences containing a 157-nucleotide insertion (Mutant) are indicated. See Example 37 for details.
  • FIG. 53 shows a portion of the nucleotide sequence of the CHO dihydrofolate reductase (DHFR) gene (upper lines) and a portion of the nucleotide sequence of a mutant DHFR gene generated by targeted homology-independent integration of exogenous sequences (lower lines).
  • Target sequences for the zinc finger nucleases described in Table 28 are boxed; changes from wild-type sequence are underlined. See also Example 37.
  • FIG. 54 shows the amino acid sequences of the wild-type FokI cleavage half-domain and of several mutant cleavage half-domains containing alterations in the amino acid sequence of the dimerization interface. Positions at which the sequence was altered (amino acids 486, 490 and 538) are underlined.
  • compositions and methods useful for targeted cleavage of cellular chromatin and for targeted alteration of a cellular nucleotide sequence e.g., by targeted cleavage followed by non-homologous end joining (with or without an exogenous sequence inserted therebetween) or by targeted cleavage followed by homologous recombination between an exogenous polynucleotide (comprising one or more regions of homology with the cellular nucleotide sequence) and a genomic sequence.
  • Genomic sequences include those present in chromosomes, episomes, organellar genomes (e.g., mitochondria, chloroplasts), artificial chromosomes and any other type of nucleic acid present in a cell such as, for example, amplified sequences, double minute chromosomes and the genomes of endogenous or infecting bacteria and viruses.
  • Genomic sequences can be normal (i.e., wild-type) or mutant; mutant sequences can comprise, for example, insertions, deletions, translocations, rearrangements, and/or point mutations.
  • a genomic sequence can also comprise one of a number of different alleles.
  • compositions useful for targeted cleavage and recombination include fusion proteins comprising a cleavage domain (or a cleavage half-domain) and a zinc finger binding domain, polynucleotides encoding these proteins and combinations of polypeptides and polypeptide-encoding polynucleotides.
  • a zinc finger binding domain can comprise one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers), and can be engineered to bind to any genomic sequence.
  • a fusion protein or proteins
  • the presence of such a fusion protein (or proteins) in a cell will result in binding of the fusion protein(s) to its (their) binding site(s) and cleavage within or near said genomic region.
  • an exogenous polynucleotide homologous to the genomic region is also present in such a cell, homologous recombination occurs at a high rate between the genomic region and the exogenous polynucleotide.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, “Chromatin” (P.M. Wassarman and A. P.
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form.
  • polynucleotide refers to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form.
  • these terms are not to be construed as limiting with respect to the length of a polymer.
  • the terms can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones).
  • an analogue of a particular nucleotide has the same base-pairing specificity; i.e., an analogue of A will base-pair with T.
  • polypeptide “peptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues.
  • the term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of a corresponding naturally-occurring amino acids.
  • Binding refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific. Such interactions are generally characterized by a dissociation constant (K d ) of 10 ⁇ 6 M ⁇ 1 or lower. “Affinity” refers to the strength of binding: increased binding affinity being correlated with a lower K d .
  • a “binding protein” is a protein that is able to bind non-covalently to another molecule.
  • a binding protein can bind to, for example, a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein) and/or a protein molecule (a protein-binding protein).
  • a DNA-binding protein a DNA-binding protein
  • an RNA-binding protein an RNA-binding protein
  • a protein-binding protein it can bind to itself (to form homodimers, homotrimers, etc.) and/or it can bind to one or more molecules of a different protein or proteins.
  • a binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding and protein-binding activity.
  • a “zinc finger DNA binding protein” (or binding domain) is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion.
  • the term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.
  • Zinc finger binding domains can be “engineered” to bind to a predetermined nucleotide sequence.
  • methods for engineering zinc finger proteins are design and selection.
  • a designed zinc finger protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.
  • a “selected” zinc finger protein is a protein not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. See e.g., US 5,789,538; US 5,925,523; US 6,007,988; US 6,013,453; US 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197 and WO 02/099084.
  • sequence refers to a nucleotide sequence of any length, which can be DNA or RNA; can be linear, circular or branched and can be either single-stranded or double stranded.
  • donor sequence refers to a nucleotide sequence that is inserted into a genome.
  • a donor sequence can be of any length, for example between 2 and 10,000 nucleotides in length (or any integer value therebetween or thereabove), preferably between about 100 and 1,000 nucleotides in length (or any integer therebetween), more preferably between about 200 and 500 nucleotides in length.
  • a “homologous, non-identical sequence” refers to a first sequence which shares a degree of sequence identity with a second sequence, but whose sequence is not identical to that of the second sequence.
  • a polynucleotide comprising the wild-type sequence of a mutant gene is homologous and non-identical to the sequence of the mutant gene.
  • the degree of homology between the two sequences is sufficient to allow homologous recombination therebetween, utilizing normal cellular mechanisms.
  • Two homologous non-identical sequences can be any length and their degree of non-homology can be as small as a single nucleotide (e.g., for correction of a genomic point mutation by targeted homologous recombination) or as large as 10 or more kilobases (e.g., for insertion of a gene at a predetermined ectopic site in a chromosome).
  • Two polynucleotides comprising the homologous non-identical sequences need not be the same length.
  • an exogenous polynucleotide i.e., donor polynucleotide
  • an exogenous polynucleotide i.e., donor polynucleotide of between 20 and 10,000 nucleotides or nucleotide pairs can be used.
  • 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, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986).
  • the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects sequence identity.
  • 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.
  • the degree of sequence similarity between polynucleotides can be determined by hybridization of polynucleotides under conditions that allow formation of stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
  • Two nucleic acid, or two polypeptide sequences are substantially homologous to each other when the sequences exhibit at least about 70%-75%, preferably 80%-82%, more preferably 85%-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity over a defined length of the molecules, as determined using the methods above.
  • substantially homologous also refers to sequences showing complete identity to a specified DNA or polypeptide sequence.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).
  • Selective hybridization of two nucleic acid fragments can be determined as follows. The degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit the hybridization of a completely identical sequence to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern (DNA) blot, Northern (RNA) blot, solution hybridization, or the like, see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.).
  • hybridization assays that are well known in the art (e.g., Southern (DNA) blot, Northern (RNA) blot, solution hybridization, or the like, see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.).
  • Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency. If conditions of low stringency are employed, the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.
  • a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.
  • a nucleic acid probe When utilizing a hybridization-based detection system, a nucleic acid probe is chosen that is complementary to a reference nucleic acid sequence, and then by selection of appropriate conditions the probe and the reference sequence selectively hybridize, or bind, to each other to form a duplex molecule.
  • a nucleic acid molecule that is capable of hybridizing selectively to a reference sequence under moderately stringent hybridization conditions typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe.
  • Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence of the selected nucleic acid probe.
  • Hybridization conditions useful for probe/reference sequence hybridization where the probe and reference sequence have a specific degree of sequence identity, can be determined as is known in the art (see, for example, Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).
  • Hybridization stringency refers to the degree to which hybridization conditions disfavor the formation of hybrids containing mismatched nucleotides, with higher stringency correlated with a lower tolerance for mismatched hybrids.
  • Factors that affect the stringency of hybridization include, but are not limited to, temperature, pH, ionic strength, and concentration of organic solvents such as, for example, formamide and dimethylsulfoxide. As is known to those of skill in the art, hybridization stringency is increased by higher temperatures, lower ionic strength and lower solvent concentrations.
  • stringency conditions for hybridization it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of the sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions.
  • the selection of a particular set of hybridization conditions is selected following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.).
  • “Recombination” refers to a process of exchange of genetic information between two polynucleotides.
  • “homologous recombination (HR)” refers to the specialized form of such exchange that takes place, for example, during repair of double-strand breaks in cells. This process requires nucleotide sequence homology, uses a “donor” molecule to template repair of a “target” molecule (i.e., the one that experienced the double-strand break), and is variously known as “non-crossover gene conversion” or “short tract gene conversion,” because it leads to the transfer of genetic information from the donor to the target.
  • such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and/or “synthesis-dependent strand annealing,” in which the donor is used to resynthesize genetic information that will become part of the target, and/or related processes.
  • Such specialized HR often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.
  • “Cleavage” refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, fusion polypeptides are used for targeted double-stranded DNA cleavage.
  • a “cleavage domain” comprises one or more polypeptide sequences which possesses catalytic activity for DNA cleavage.
  • a cleavage domain can be contained in a single polypeptide chain or cleavage activity can result from the association of two (or more) polypeptides.
  • a “cleavage half-domain” is a polypeptide sequence which, in conjunction with a second polypeptide (either identical or different) forms a complex having cleavage activity (preferably double-strand cleavage activity).
  • Chromatin is the nucleoprotein structure comprising the cellular genome.
  • Cellular chromatin comprises nucleic acid, primarily DNA, and protein, including histones and non-histone chromosomal proteins.
  • the majority of eukaryotic cellular chromatin exists in the form of nucleosomes, wherein a nucleosome core comprises approximately 150 base pairs of DNA associated with an octamer comprising two each of histones H2A, H2B, H3 and H4; and linker DNA (of variable length depending on the organism) extends between nucleosome cores.
  • a molecule of histone H1 is generally associated with the linker DNA.
  • chromatin is meant to encompass all types of cellular nucleoprotein, both prokaryotic and eukaryotic.
  • Cellular chromatin includes both chromosomal and episomal chromatin.
  • a “chromosome,” is a chromatin complex comprising all or a portion of the genome of a cell.
  • the genome of a cell is often characterized by its karyotype, which is the collection of all the chromosomes that comprise the genome of the cell.
  • the genome of a cell can comprise one or more chromosomes.
  • an “episome” is a replicating nucleic acid, nucleoprotein complex or other structure comprising a nucleic acid that is not part of the chromosomal karyotype of a cell.
  • Examples of episomes include plasmids and certain viral genomes.
  • an “accessible region” is a site in cellular chromatin in which a target site present in the nucleic acid can be bound by an exogenous molecule which recognizes the target site. Without wishing to be bound by any particular theory, it is believed that an accessible region is one that is not packaged into a nucleosomal structure. The distinct structure of an accessible region can often be detected by its sensitivity to chemical and enzymatic probes, for example, nucleases.
  • a “target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist.
  • the sequence 5′-GAATTC-3′ is a target site for the Eco RI restriction endonuclease.
  • exogenous molecule is a molecule that is not normally present in a cell, but can be introduced into a cell by one or more genetic, biochemical or other methods. “Normal presence in the cell” is determined with respect to the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic development of muscle is an exogenous molecule with respect to an adult muscle cell. Similarly, a molecule induced by heat shock is an exogenous molecule with respect to a non-heat-shocked cell.
  • An exogenous molecule can comprise, for example, a functioning version of a malfunctioning endogenous molecule or a malfunctioning version of a normally-functioning endogenous molecule.
  • An exogenous molecule can be, among other things, a small molecule, such as is generated by a combinatorial chemistry process, or a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules.
  • Nucleic acids include DNA and RNA, can be single- or double-stranded; can be linear, branched or circular; and can be of any length. Nucleic acids include those capable of forming duplexes, as well as triplex-forming nucleic acids. See, for example, U.S. Pat. Nos. 5,176,996 and 5,422,251.
  • Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases and helicases.
  • exogenous molecule can be the same type of molecule as an endogenous molecule, e.g., an exogenous protein or nucleic acid.
  • an exogenous nucleic acid can comprise an infecting viral genome, a plasmid or episome introduced into a cell, or a chromosome that is not normally present in the cell.
  • Methods for the introduction of exogenous molecules into cells include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer.
  • an “endogenous” molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions.
  • an endogenous nucleic acid can comprise a chromosome, the genome of a mitochondrion, chloroplast or other organelle, or a naturally-occurring episomal nucleic acid.
  • Additional endogenous molecules can include proteins, for example, transcription factors and enzymes.
  • a “fusion” molecule is a molecule in which two or more subunit molecules are linked, preferably covalently.
  • the subunit molecules can be the same chemical type of molecule, or can be different chemical types of molecules.
  • Examples of the first type of fusion molecule include, but are not limited to, fusion proteins (for example, a fusion between a ZFP DNA-binding domain and a cleavage domain) and fusion nucleic acids (for example, a nucleic acid encoding the fusion protein described supra).
  • Examples of the second type of fusion molecule include, but are not limited to, a fusion between a triplex-forming nucleic acid and a polypeptide, and a fusion between a minor groove binder and a nucleic acid.
  • Fusion protein in a cell can result from delivery of the fusion protein to the cell or by delivery of a polynucleotide encoding the fusion protein to a cell, wherein the polynucleotide is transcribed, and the transcript is translated, to generate the fusion protein.
  • Trans-splicing, polypeptide cleavage and polypeptide ligation can also be involved in expression of a protein in a cell. Methods for polynucleotide and polypeptide delivery to cells are presented elsewhere in this disclosure.
  • Gene expression refers to the conversion of the information, contained in a gene, into a gene product.
  • a gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA.
  • Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.
  • Modulation of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression.
  • Eucaryotic cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells.
  • a “region of interest” is any region of cellular chromatin, such as, for example, a gene or a non-coding sequence within or adjacent to a gene, in which it is desirable to bind an exogenous molecule. Binding can be for the purposes of targeted DNA cleavage and/or targeted recombination.
  • a region of interest can be present in a chromosome, an episome, an organellar genome (e.g., mitochondrial, chloroplast), or an infecting viral genome, for example.
  • a region of interest can be within the coding region of a gene, within transcribed non-coding regions such as, for example, leader sequences, trailer sequences or introns, or within non-transcribed regions, either upstream or downstream of the coding region.
  • a region of interest can be as small as a single nucleotide pair or up to 2,000 nucleotide pairs in length, or any integral value of nucleotide pairs.
  • operative linkage and “operatively linked” (or “operably linked”) are used interchangeably with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components.
  • a transcriptional regulatory sequence such as a promoter
  • a transcriptional regulatory sequence is generally operatively linked in cis with a coding sequence, but need not be directly adjacent to it.
  • an enhancer is a transcriptional regulatory sequence that is operatively linked to a coding sequence, even though they are not contiguous.
  • the term “operatively linked” can refer to the fact that each of the components performs the same function in linkage to the other component as it would if it were not so linked.
  • the ZFP DNA-binding domain and the cleavage domain are in operative linkage if, in the fusion polypeptide, the ZFP DNA-binding domain portion is able to bind its target site and/or its binding site, while the cleavage domain is able to cleave DNA in the vicinity of the target site.
  • a “functional fragment” of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, yet retains the same function as the full-length protein, polypeptide or nucleic acid.
  • a functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one ore more amino acid or nucleotide substitutions.
  • DNA-binding function of a polypeptide can be determined, for example, by filter-binding, electrophoretic mobility-shift, or immunoprecipitation assays. DNA cleavage can be assayed by gel electrophoresis. See Ausubel et al., supra.
  • the ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays or complementation, both genetic and biochemical. See, for example, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No. 5,585,245 and PCT WO 98/44350.
  • the disclosed methods and compositions include fusion proteins comprising a cleavage domain (or a cleavage half-domain) and a zinc finger domain, in which the zinc finger domain, by binding to a sequence in cellular chromatin (e.g., a target site or a binding site), directs the activity of the cleavage domain (or cleavage half-domain) to the vicinity of the sequence and, hence, induces cleavage in the vicinity of the target sequence.
  • a zinc finger domain can be engineered to bind to virtually any desired sequence.
  • one or more zinc finger binding domains can be engineered to bind to one or more sequences in the region of interest.
  • Expression of a fusion protein comprising a zinc finger binding domain and a cleavage domain (or of two fusion proteins, each comprising a zinc finger binding domain and a cleavage half-domain), in a cell effects cleavage in the region of interest.
  • Selection of a sequence in cellular chromatin for binding by a zinc finger domain can be accomplished, for example, according to the methods disclosed in co-owned U.S. Pat. No. 6,453,242 (Sep. 17, 2002), which also discloses methods for designing ZFPs to bind to a selected sequence. It will be clear to those skilled in the art that simple visual inspection of a nucleotide sequence can also be used for selection of a target site. Accordingly, any means for target site selection can be used in the claimed methods.
  • Target sites are generally composed of a plurality of adjacent target subsites.
  • a target subsite refers to the sequence (usually either a nucleotide triplet, or a nucleotide quadruplet that can overlap by one nucleotide with an adjacent quadruplet) bound by an individual zinc finger. See, for example, WO 02/077227. If the strand with which a zinc finger protein makes most contacts is designated the target strand “primary recognition strand,” or “primary contact strand,” some zinc finger proteins bind to a three base triplet in the target strand and a fourth base on the non-target strand.
  • a target site generally has a length of at least 9 nucleotides and, accordingly, is bound by a zinc finger binding domain comprising at least three zinc fingers.
  • a zinc finger binding domain comprising at least three zinc fingers.
  • binding of, for example, a 4-finger binding domain to a 12-nucleotide target site, a 5-finger binding domain to a 15-nucleotide target site or a 6-finger binding domain to an 18-nucleotide target site is also possible.
  • binding of larger binding domains e.g., 7-, 8-, 9-finger and more
  • a target site it is not necessary for a target site to be a multiple of three nucleotides.
  • a target site it is not necessary for a target site to be a multiple of three nucleotides.
  • one or more of the individual zinc fingers of a multi-finger binding domain can bind to overlapping quadruplet subsites.
  • a three-finger protein can bind a 1 0-nucleotide sequence, wherein the tenth nucleotide is part of a quadruplet bound by a terminal finger
  • a four-finger protein can bind a 13-nucleotide sequence, wherein the thirteenth nucleotide is part of a quadruplet bound by a terminal finger, etc.
  • the length and nature of amino acid linker sequences between individual zinc fingers in a multi-finger binding domain also affects binding to a target sequence.
  • a so-called “non-canonical linker,” “long linker” or “structured linker” between adjacent zinc fingers in a multi-finger binding domain can allow those fingers to bind subsites which are not immediately adjacent.
  • Non-limiting examples of such linkers are described, for example, in U.S. Pat. No. 6,479,626 and WO 01/53480. Accordingly, one or more subsites, in a target site for a zinc finger binding domain, can be separated from each other by 1, 2, 3, 4, 5 or more nucleotides.
  • a four-finger binding domain can bind to a 13-nucleotide target site comprising, in sequence, two contiguous 3-nucleotide subsites, an intervening nucleotide, and two contiguous triplet subsites.
  • Distance between sequences refers to the number of nucleotides or nucleotide pairs intervening between two sequences, as measured from the edges of the sequences nearest each other.
  • the two target sites can be on opposite DNA strands. In other embodiments, both target sites are on the same DNA strand.
  • a zinc finger binding domain comprises one or more zinc fingers. Miller et al. (1985) EMBO J 4:1609-1614; Rhodes (1993) Scientific American February: 56-65; U.S. Pat. No. 6,453,242. Typically, a single zinc finger domain is about 30 amino acids in length. Structural studies have demonstrated that each zinc finger domain (motif) contains two beta sheets (held in a beta turn which contains the two invariant cysteine residues) and an alpha helix (containing the two invariant histidine residues), which are held in a particular conformation through coordination of a zinc atom by the two cysteines and the two histidines.
  • Zinc fingers include both canonical C 2 H 2 zinc fingers (i.e., those in which the zinc ion is coordinated by two cysteine and two histidine residues) and non-canonical zinc fingers such as, for example, C 3 H zinc fingers (those in which the zinc ion is coordinated by three cysteine residues and one histidine residue) and C 4 zinc fingers (those in which the zinc ion is coordinated by four cysteine residues). See also WO 02/057293.
  • Zinc finger binding domains can be engineered to bind to a sequence of choice. See, for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature 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.
  • An engineered zinc finger binding domain can have a novel binding specificity, compared to a naturally-occurring zinc finger protein.
  • Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each 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. See, for example, co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261.
  • Exemplary selection methods including phage display and two-hybrid systems, 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.
  • an individual zinc finger binds to a three-nucleotide (i.e., triplet) sequence (or a four-nucleotide sequence which can overlap, by one nucleotide, with the four-nucleotide binding site of an adjacent zinc finger)
  • the length of a sequence to which a zinc finger binding domain is engineered to bind e.g., a target sequence
  • binding sites for individual zinc fingers (i.e., subsites) in a target site need not be contiguous, but can be separated by one or several nucleotides, depending on the length and nature of the amino acids sequences between the zinc fingers (i.e., the inter-finger linkers) in a multi-finger binding domain.
  • adjacent zinc fingers can be separated by amino acid linker sequences of approximately 5 amino acids (so-called “canonical” inter-finger linkers) or, alternatively, by one or more non-canonical linkers.
  • canonical inter-finger linkers amino acid linker sequences of approximately 5 amino acids
  • non-canonical linkers amino acid linker sequences of approximately 5 amino acids
  • non-canonical linkers amino acid linker sequences of approximately 5 amino acids
  • non-canonical linkers e.g., co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261.
  • insertion of longer (“non-canonical”) inter-finger linkers between certain of the zinc fingers may be preferred as it may increase the affinity and/or specificity of binding by the binding domain. See, for example, U.S. Pat. No. 6,479,626 and WO 01/53480.
  • multi-finger zinc finger binding domains can also be characterized with respect to the presence and location of non-canonical inter-finger linkers.
  • a six-finger zinc finger binding domain comprising three fingers (joined by two canonical inter-finger linkers), a long linker and three additional fingers (joined by two canonical inter-finger linkers) is denoted a 2 ⁇ 3 configuration.
  • a binding domain comprising two fingers (with a canonical linker therebetween), a long linker and two additional fingers (joined by a canonical linker) is denoted a 2 ⁇ 2 protein.
  • a protein comprising three two-finger units (in each of which the two fingers are joined by a canonical linker), and in which each two-finger unit is joined to the adjacent two finger unit by a long linker, is referred to as a 3 ⁇ 2 protein.
  • a long or non-canonical inter-finger linker between two adjacent zinc fingers in a multi-finger binding domain often allows the two fingers to bind to subsites which are not immediately contiguous in the target sequence. Accordingly, there can be gaps of one or more nucleotides between subsites in a target site; i.e., a target site can contain one or more nucleotides that are not contacted by a zinc finger.
  • a 2 ⁇ 2 zinc finger binding domain can bind to two six-nucleotide sequences separated by one nucleotide, i.e., it binds to a 13-nucleotide target site. See also Moore et al. (2001a) Proc. Natl. Acad. Sci. USA 98:1432-1436; Moore et al. (2001b) Proc. Natl. Acad. Sci. USA 98:1437-1441 and WO 01/53480.
  • a target subsite is a three- or four-nucleotide sequence that is bound by a single zinc finger.
  • a two-finger unit is denoted a binding module.
  • a binding module can be obtained by, for example, selecting for two adjacent fingers in the context of a multi-finger protein (generally three fingers) which bind a particular six-nucleotide target sequence.
  • modules can be constructed by assembly of individual zinc fingers. See also WO 98/53057 and WO 01/53480.
  • the cleavage domain portion of the fusion proteins disclosed herein can be obtained from any endonuclease or exonuclease.
  • Exemplary endonucleases from which a cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388.
  • Additional enzymes which 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).
  • 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
  • a cleavage half-domain e.g., fusion proteins comprising a zinc finger binding domain and a cleavage half-domain
  • a cleavage half-domain can be derived from any nuclease or portion thereof, as set forth above, that requires dimerization for cleavage activity.
  • two fusion proteins are required for cleavage if the fusion proteins comprise cleavage half-domains.
  • a single protein comprising two cleavage half-domains can be used.
  • the two cleavage half-domains can be derived from the same endonuclease (or functional fragments thereof), or each cleavage half-domain can be derived from a different endonuclease (or functional fragments thereof).
  • the target sites for the two fusion proteins are preferably disposed, with respect to each other, such that binding of the two fusion proteins to their respective target sites places the cleavage half-domains in a spatial orientation to each other that allows the cleavage half-domains to form a functional cleavage domain, e.g., by dimerizing.
  • the near edges of the target sites are separated by 5-8 nucleotides or by 15-18 nucleotides.
  • any integral number of nucleotides or nucleotide pairs can intervene between two target sites (e.g., from 2 to 50 nucleotides or more). In general, the point of cleavage lies between the target 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
  • Fok I 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. 5,356,802; 5,436,150 and 5,487,994; as well as Li et al.
  • fusion proteins comprise the cleavage domain (or cleavage half-domain) from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered.
  • Fok I An exemplary Type IIS restriction enzyme, whose cleavage domain is separable from the binding domain, is Fok I.
  • This particular enzyme is active as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575. Accordingly, for the purposes of the present disclosure, the portion of the Fok I enzyme used in the disclosed fusion proteins is considered a cleavage half-domain.
  • two fusion proteins, each comprising a FokI cleavage half-domain can be used to reconstitute a catalytically active cleavage domain.
  • a single polypeptide molecule containing a zinc finger binding domain and two Fok I cleavage half-domains can also be used. Parameters for targeted cleavage and targeted sequence alteration using zinc finger-Fok I fusions are provided elsewhere in this disclosure.
  • a cleavage domain or cleavage half-domain can be any portion of a protein that retains cleavage activity, or that retains the ability to multimerize (e.g., dimerize) to form a functional cleavage domain.
  • Type IIS restriction enzymes are listed in Table 1. Additional restriction enzymes also contain separable binding and cleavage domains, and these are contemplated by the present disclosure. See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.
  • fusion proteins and polynucleotides encoding same are known to those of skill in the art. For example, methods for the design and construction of fusion protein comprising zinc finger proteins (and polynucleotides encoding same) are described in co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261. In certain embodiments, polynucleotides encoding such fusion proteins are constructed. These polynucleotides can be inserted into a vector and the vector can be introduced into a cell (see below for additional disclosure regarding vectors and methods for introducing polynucleotides into cells).
  • a fusion protein comprises a zinc finger binding domain and a cleavage half-domain from the Fok I restriction enzyme, and two such fusion proteins are expressed in a cell.
  • Expression of two fusion proteins in a cell can result from delivery of the two proteins to the cell; delivery of one protein and one nucleic acid encoding one of the proteins to the cell; delivery of two nucleic acids, each encoding one of the proteins, to the cell; or by delivery of a single nucleic acid, encoding both proteins, to the cell.
  • a fusion protein comprises a single polypeptide chain comprising two cleavage half domains and a zinc finger binding domain. In this case, a single fusion protein is expressed in a cell and, without wishing to be bound by theory, is believed to cleave DNA as a result of formation of an intramolecular dimer of the cleavage half-domains.
  • the components of the fusion proteins are arranged such that the zinc finger domain is nearest the amino terminus of the fusion protein, and the cleavage half-domain is nearest the carboxy-terminus. This mirrors the relative orientation of the cleavage domain in naturally-occurring dimerizing cleavage domains such as those derived from the Fok I enzyme, in which the DNA-binding domain is nearest the amino terminus and the cleavage half-domain is nearest the carboxy terminus.
  • dimerization of the cleavage half-domains to form a functional nuclease is brought about by binding of the fusion proteins to sites on opposite DNA strands, with the 5′ ends of the binding sites being proximal to each other. See FIG. 43A .
  • the components of the fusion proteins are arranged such that the cleavage half-domain is nearest the amino terminus of the fusion protein, and the zinc finger domain is nearest the carboxy-terminus.
  • dimerization of the cleavage half-domains to form a functional nuclease is brought about by binding of the fusion proteins to sites on opposite DNA strands, with the 3′ ends of the binding sites being proximal to each other. See FIG. 43B .
  • a first fusion protein contains the cleavage half-domain nearest the amino terminus of the fusion protein, and the zinc finger domain nearest the carboxy-terminus
  • a second fusion protein is arranged such that the zinc finger domain is nearest the amino terminus of the fusion protein, and the cleavage half-domain is nearest the carboxy-terminus.
  • both fusion proteins bind to the same DNA strand, with the binding site of the first fusion protein containing the zinc finger domain nearest the carboxy terminus located to the 5′ side of the binding site of the second fusion protein containing the zinc finger domain nearest the amino terminus. See FIG. 43C .
  • the amino acid sequence between the zinc finger domain and the cleavage domain is denoted the “ZC linker.”
  • the ZC linker is to be distinguished from the inter-finger linkers discussed above.
  • the zinc finger structure described by Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340 is used: X-X-C-X 2-4 -C-X 12 -H-X 3-5 -H (SEQ ID NO:201)
  • the first residue of a zinc finger is the amino acid located two residues amino-terminal to the first conserved cysteine residue. In the majority of naturally-occurring zinc finger proteins, this position is occupied by a hydrophobic amino acid (usually either phenylalanine or tyrosine). In the disclosed fusion proteins, the first residue of a zinc finger will thus often be a hydrophobic residue, but it can be any amino acid.
  • the final amino acid residue of a zinc finger, as shown above, is the second conserved histidine residue.
  • the ZC linker is the amino acid sequence between the second conserved histidine residue of the C-terminal-most zinc finger and the N-terminal-most amino acid of the cleavage domain (or cleavage half-domain).
  • the N-terminal-most amino acid of a cleavage half-domain is a glutamine (Q) residue corresponding to amino acid number 384 in the FokI sequence of Looney et al. (1989) Gene 80:193-208.
  • the ZC linker is the amino acid sequence between the C-terminal-most amino acid residue of the cleavage domain (or half-domain) and the first residue of the N-terminal-most zinc finger of the zinc finger binding domain (i.e., the residue located two residues upstream of the first conserved cysteine residue).
  • the C-terminal-most amino acid of a cleavage half-domain is a phenylalanine (F) residue corresponding to amino acid number 579 in the FokI sequence of Looney et al. (1989) Gene 80:193-208.
  • the ZC linker can be any amino acid sequence.
  • the length of the ZC linker and the distance between the target sites (binding sites) are interrelated. See, for example, Smith et al. (2000) Nucleic Acids Res. 28:3361-3369; Bibikova et al. (2001) Mol. Cell. Biol. 21:289-297, noting that their notation for linker length differs from that given here.
  • the disclosed methods and compositions can be used to cleave DNA at a region of interest in cellular chromatin (e.g., at a desired or predetermined site in a genome, for example, in a gene, either mutant or wild-type).
  • a zinc finger binding domain is engineered to bind a target site at or near the predetermined cleavage site, and a fusion protein comprising the engineered zinc finger binding domain and a cleavage domain is expressed in a cell.
  • the DNA is cleaved near the target site by the cleavage domain.
  • the exact site of cleavage can depend on the length of the ZC linker.
  • two fusion proteins each comprising a zinc finger binding domain and a cleavage half-domain, are expressed in a cell, and bind to target sites which are juxtaposed in such a way that a functional cleavage domain is reconstituted and DNA is cleaved in the vicinity of the target sites.
  • cleavage occurs between the target sites of the two zinc finger binding domains.
  • One or both of the zinc finger binding domains can be engineered.
  • the binding site can encompass the cleavage site, or the near edge of the binding site can be 1, 2, 3, 4, 5, 6, 10, 25, 50 or more nucleotides (or any integral value between 1 and 50 nucleotides) from the cleavage site.
  • the exact location of the binding site, with respect to the cleavage site, will depend upon the particular cleavage domain, and the length of the ZC linker.
  • the binding sites generally straddle the cleavage site.
  • the near edge of the first binding site can be 1, 2, 3, 4, 5, 6, 10, 25 or more nucleotides (or any integral value between 1 and 50 nucleotides) on one side of the cleavage site
  • the near edge of the second binding site can be 1, 2, 3, 4, 5, 6, 10, 25 or more nucleotides (or any integral value between 1 and 50 nucleotides) on the other side of the cleavage site.
  • the methods described herein can employ an engineered zinc finger binding domain fused to a cleavage domain.
  • the binding domain is engineered to bind to a target sequence, at or near which cleavage is desired.
  • the fusion protein, or a polynucleotide encoding same is introduced into a cell. Once introduced into, or expressed in, the cell, the fusion protein binds to the target sequence and cleaves at or near the target sequence. The exact site of cleavage depends on the nature of the cleavage domain and/or the presence and/or nature of linker sequences between the binding and cleavage domains.
  • the distance between the near edges of the binding sites can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25 or more nucleotides (or any integral value between 1 and 50 nucleotides).
  • Optimal levels of cleavage can also depend on both the distance between the binding sites of the two fusion proteins (See, for example, Smith et al. (2000) Nucleic Acids Res. 28:3361-3369; Bibikova et al. (2001) Mol. Cell. Biol. 21:289-297) and the length of the ZC linker in each fusion protein.
  • the length of the linker between the ZFP and the FokI cleavage half-domain can influence cleavage efficiency.
  • the ZC linker In one experimental system utilizing a ZFP-FokI fusion with a ZC linker of 4 amino acid residues, optimal cleavage was obtained when the near edges of the binding sites for two ZFP-FokI nucleases were separated by 6 base pairs.
  • This particular fusion nuclease comprised the following amino acid sequence between the zinc finger portion and the nuclease half-domain: H QRT H QNKK QLV (SEQ ID NO:26) in which the two conserved histidines in the C-terminal portion of the zinc finger and the first three residues in the FokI cleavage half-domain are underlined. Accordingly, the ZC linker sequence in this construct is QNKK. Bibikova et al. (2001) Mol. Cell. Biol. 21:289-297.
  • the present inventors have constructed a number of ZFP-FokI fusion nucleases having a variety of ZC linker lengths and sequences, and analyzed the cleavage efficiencies of these nucleases on a series of substrates having different distances between the ZFP binding sites. See Example 4.
  • the cleavage domain comprises two cleavage half-domains, both of which are part of a single polypeptide comprising a binding domain, a first cleavage half-domain and a second cleavage half-domain.
  • the cleavage half-domains can have the same amino acid sequence or different amino acid sequences, so long as they function to cleave the DNA.
  • Cleavage half-domains may also be provided in separate molecules.
  • two fusion polypeptides may be introduced into a cell, wherein each polypeptide comprises a binding domain and a cleavage half-domain.
  • the cleavage half-domains can have the same amino acid sequence or different amino acid sequences, so long as they function to cleave the DNA.
  • the binding domains bind to target sequences which are typically disposed in such a way that, upon binding of the fusion polypeptides, the two cleavage half-domains are presented in a spatial orientation to each other that allows reconstitution of a cleavage domain (e.g., by dimerization of the half-domains), thereby positioning the half-domains relative to each other to form a functional cleavage domain, resulting in cleavage of cellular chromatin in a region of interest.
  • cleavage by the reconstituted cleavage domain occurs at a site located between the two target sequences.
  • One or both of the proteins can be engineered to bind to its target site.
  • the two fusion proteins can bind in the region of interest in the same or opposite polarity, and their binding sites (i.e., target sites) can be separated by any number of nucleotides, e.g., from 0 to 200 nucleotides or any integral value therebetween.
  • the binding sites for two fusion proteins, each comprising a zinc finger binding domain and a cleavage half-domain can be located between 5 and 18 nucleotides apart, for example, 5-8 nucleotides apart, or 15-18 nucleotides apart, or 6 nucleotides apart, or 16 nucleotides apart, as measured from the edge of each binding site nearest the other binding site, and cleavage occurs between the binding sites.
  • the site at which the DNA is cleaved generally lies between the binding sites for the two fusion proteins. Double-strand breakage of DNA often results from two single-strand breaks, or “nicks,” offset by 1, 2, 3, 4, 5, 6 or more nucleotides, (for example, cleavage of double-stranded DNA by native Fok I results from single-strand breaks offset by 4 nucleotides). Thus, cleavage does not necessarily occur at exactly opposite sites on each DNA strand.
  • the structure of the fusion proteins and the distance between the target sites can influence whether cleavage occurs adjacent a single nucleotide pair, or whether cleavage occurs at several sites. However, for many applications, including targeted recombination and targeted mutagenesis (see infra) cleavage within a range of nucleotides is generally sufficient, and cleavage between particular base pairs is not required.
  • the fusion protein(s) can be introduced as polypeptides and/or polynucleotides.
  • two polynucleotides, each comprising sequences encoding one of the aforementioned polypeptides, can be introduced into a cell, and when the polypeptides are expressed and each binds to its target sequence, cleavage occurs at or near the target sequence.
  • a single polynucleotide comprising sequences encoding both fusion polypeptides is introduced into a cell.
  • Polynucleotides can be DNA, RNA or any modified forms or analogues or DNA and/or RNA.
  • single cleavage half-domains can exhibit limited double-stranded cleavage activity.
  • either protein specifies an approximately 9-nucleotide target site.
  • the aggregate target sequence of 18 nucleotides is likely to be unique in a mammalian genome, any given 9-nucleotide target site occurs, on average, approximately 23,000 times in the human genome.
  • non-specific cleavage due to the site-specific binding of a single half-domain, may occur.
  • the methods described herein contemplate the use of a dominant-negative mutant of a cleavage half-domain such as Fok I (or a nucleic acid encoding same) that is expressed in a cell along with the two fusion proteins.
  • the dominant-negative mutant is capable of dimerizing but is unable to cleave, and also blocks the cleavage activity of a half-domain to which it is dimerized.
  • By providing the dominant-negative mutant in molar excess to the fusion proteins only regions in which both fusion proteins are bound will have a high enough local concentration of functional cleavage half-domains for dimerization and cleavage to occur. At sites where only one of the two fusion proteins is bound, its cleavage half-domain forms a dimer with the dominant negative mutant half-domain, and undesirable, non-specific cleavage does not occur.
  • Methods for targeted cleavage which involve the use of fusions between a ZFP and a cleavage half-domain (such as, e.g., a ZFP/FokI fusion) require the use of two such fusion molecules, each generally directed to a distinct target sequence.
  • Target sequences for the two fusion proteins can be chosen so that targeted cleavage is directed to a unique site in a genome, as discussed above.
  • a potential source of reduced cleavage specificity could result from homodimerization of one of the two ZFP/cleavage half-domain fusions.
  • One approach for reducing the probability of this type of aberrant cleavage at sequences other than the intended target site involves generating variants of the cleavage half-domain that minimize or prevent homodimerization. Preferably, one or more amino acids in the region of the half-domain involved in its dimerization are altered.
  • the structure of the cleavage half-domains is reported to be similar to the arrangement of the cleavage half-domains during cleavage of DNA by FokI. Wah et al. (1998) Proc. Natl. Acad. Sci. USA 95:10564-10569.
  • This structure indicates that amino acid residues at positions 483 and 487 play a key role in the dimerization of the FokI cleavage half-domains.
  • the structure also indicates that amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 are all close enough to the dimerization interface to influence dimerization. Accordingly, amino acid sequence alterations at one or more of the aforementioned positions will likely alter the dimerization properties of the cleavage half-domain.
  • Such changes can be introduced, for example, by constructing a library containing (or encoding) different amino acid residues at these positions and selecting variants with the desired properties, or by rationally designing individual mutants. In addition to preventing homodimerization, it is also possible that some of these mutations may increase the cleavage efficiency above that obtained with two wild-type cleavage half-domains.
  • alteration of a FokI cleavage half-domain at any amino acid residue which affects dimerization can be used to prevent one of a pair of ZFP/FokI fusions from undergoing homodimerization which can lead to cleavage at undesired sequences.
  • one or both of the fusion proteins can comprise one or more amino acid alterations that inhibit self-dimerization, but allow heterodimerization of the two fusion proteins to occur such that cleavage occurs at the desired target site.
  • alterations are present in both fusion proteins, and the alterations have additive effects; i.e., homodimerization of either fusion, leading to aberrant cleavage, is minimized or abolished, while heterodimerization of the two fusion proteins is facilitated compared to that obtained with wild-type cleavage half-domains. See Example 5.
  • a genomic sequence e.g., a region of interest in cellular chromatin
  • a homologous non-identical sequence i.e., targeted recombination.
  • Previous attempts to replace particular sequences have involved contacting a cell with a polynucleotide comprising sequences bearing homology to a chromosomal region (i.e., a donor DNA), followed by selection of cells in which the donor DNA molecule had undergone homologous recombination into the genome.
  • the success rate of these methods is low, due to poor efficiency of homologous recombination and a high frequency of non-specific insertion of the donor DNA into regions of the genome other than the target site.
  • the present disclosure provides methods of targeted sequence alteration characterized by a greater efficiency of targeted recombination and a lower frequency of non-specific insertion events.
  • the methods involve making and using engineered zinc finger binding domains fused to cleavage domains (or cleavage half-domains) to make one or more targeted double-stranded breaks in cellular DNA. Because double-stranded breaks in cellular DNA stimulate cellular repair mechanisms several thousand-fold in the vicinity of the cleavage site, such targeted cleavage allows for the alteration or replacement (via homology-directed repair) of sequences at virtually any site in the genome.
  • targeted replacement of a selected genomic sequence also requires the introduction of the replacement (or donor) sequence.
  • the donor sequence can be introduced into the cell prior to, concurrently with, or subsequent to, expression of the fusion protein(s).
  • the donor polynucleotide contains sufficient homology to a genomic sequence to support homologous recombination (or homology-directed repair) between it and the genomic sequence to which it bears homology. Approximately 25, 50 100, 200, 500, 750, 1,000, 1,500, 2,000 nucleotides or more of sequence homology between a donor and a genomic sequence (or any integral value between 10 and 2,000 nucleotides, or more) will support homologous recombination therebetween.
  • Donor sequences can range in length from 10 to 5,000 nucleotides (or any integral value of nucleotides therebetween) or longer. It will be readily apparent that the donor sequence is typically not identical to the genomic sequence that it replaces.
  • the sequence of the donor polynucleotide can contain one or more single base changes, insertions, deletions, inversions or rearrangements with respect to the genomic sequence, so long as sufficient homology with chromosomal sequences is present.
  • a donor sequence can contain a non-homologous sequence flanked by two regions of homology.
  • donor sequences can comprise a vector molecule containing sequences that are not homologous to the region of interest in cellular chromatin.
  • the homologous region(s) of a donor sequence will have at least 50% sequence identity to a genomic sequence with which recombination is desired. In certain embodiments, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity is present. Any value between 1% and 100% sequence identity can be present, depending upon the length of the donor polynucleotide.
  • a donor molecule can contain several, discontinuous regions of homology to cellular chromatin. For example, for targeted insertion of sequences not normally present in a region of interest, said sequences can be present in a donor nucleic acid molecule and flanked by regions of homology to sequence in the region of interest.
  • certain sequence differences may be present in the donor sequence as compared to the genomic sequence.
  • such nucleotide sequence differences will not change the amino acid sequence, or will make silent amino acid changes (i.e., changes which do not affect the structure or function of the protein).
  • the donor polynucleotide can optionally contain changes in sequences corresponding to the zinc finger domain binding sites in the region of interest, to prevent cleavage of donor sequences that have been introduced into cellular chromatin by homologous recombination.
  • the donor polynucleotide can be DNA or RNA, single-stranded or double-stranded and can be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3′ terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls et al.
  • Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.
  • a polynucleotide can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance.
  • donor polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus).
  • viruses e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus.
  • Applicants' methods advantageously combine the powerful targeting capabilities of engineered ZFPs with a cleavage domain (or cleavage half-domain) to specifically target a double-stranded break to the region of the genome at insertion of exogenous sequences is desired.
  • the efficiency of insertion of donor sequences by homologous recombination is inversely related to the distance, in the cellular DNA, between the double-stranded break and the site at which recombination is desired. In other words, higher homologous recombination efficiencies are observed when the double-stranded break is closer to the site at which recombination is desired.
  • a precise site of recombination is not predetermined (e.g., the desired recombination event can occur over an interval of genomic sequence)
  • the length and sequence of the donor nucleic acid, together with the site(s) of cleavage are selected to obtain the desired recombination event.
  • cellular chromatin is cleaved within 10,000 nucleotides on either side of that nucleotide pair. In certain embodiments, cleavage occurs within 1,000, 500, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, or 2 nucleotides, or any integral value between 2 and 1,000 nucleotides, on either side of the nucleotide pair whose sequence is to be changed.
  • the binding sites for two fusion proteins each comprising a zinc finger binding domain and a cleavage half-domain, can be located 5-8 or 15-18 nucleotides apart, as measured from the edge of each binding site nearest the other binding site, and cleavage occurs between the binding sites. Whether cleavage occurs at a single site or at multiple sites between the binding sites is immaterial, since the cleaved genomic sequences are replaced by the donor sequences.
  • the midpoint of the region between the binding sites is within 10,000 nucleotides of that nucleotide pair, preferably within 1,000 nucleotides, or 500 nucleotides, or 200 nucleotides, or 100 nucleotides, or 50 nucleotides, or 20 nucleotides, or 10 nucleotides, or 5 nucleotide, or 2 nucleotides, or one nucleotide, or at the nucleotide pair of interest.
  • a homologous chromosome can serve as the donor polynucleotide.
  • correction of a mutation in a heterozygote can be achieved by engineering fusion proteins which bind to and cleave the mutant sequence on one chromosome, but do not cleave the wild-type sequence on the homologous chromosome.
  • the double-stranded break on the mutation-bearing chromosome stimulates a homology-based “gene conversion” process in which the wild-type sequence from the homologous chromosome is copied into the cleaved chromosome, thus restoring two copies of the wild-type sequence.
  • Methods and compositions are also provided that may enhance levels of targeted recombination including, but not limited to, the use of additional ZFP-functional domain fusions to activate expression of genes involved in homologous recombination, such as, for example, members of the RAD52 epistasis group (e.g., Rad50, Rad51, Rad51B, Rad51C, Rad51D, Rad52, Rad54, Rad54B, Mre11, XRCC2, XRCC3), genes whose products interact with the aforementioned gene products (e.g., BRCA1, BRCA2) and/or genes in the NBS1 complex.
  • members of the RAD52 epistasis group e.g., Rad50, Rad51, Rad51B, Rad51C, Rad51D, Rad52, Rad54, Rad54B, Mre11, XRCC2, XRCC3
  • genes whose products interact with the aforementioned gene products e.g., BRCA1, BRCA2
  • genes in the NBS1 complex e.
  • ZFP-functional domain fusions can be used, in combination with the methods and compositions disclosed herein, to repress expression of genes involved in non-homologous end joining (e.g., Ku70/80, XRCC4, poly(ADP ribose) polymerase, DNA ligase 4).
  • non-homologous end joining e.g., Ku70/80, XRCC4, poly(ADP ribose) polymerase, DNA ligase 4
  • Non-homologous end joining e.g., Ku70/80, XRCC4, poly(ADP ribose) polymerase, DNA ligase 4
  • Non-homologous end joining e.g., Ku70/80, XRCC4, poly(ADP ribose) polymerase, DNA ligase 4
  • Yanez et al. 1998 Gene Therapy 5:149-159; Hoeijmakers (2001) Nature 411:366-374; Johnson et
  • Additional repression methods include the use of antisense oligonucleotides and/or small interfering RNA (siRNA or RNAi) targeted to the sequence of the gene to be repressed.
  • siRNA or RNAi small interfering RNA
  • fusions of these protein (or functional fragments thereof) with a zinc finger binding domain targeted to the region of interest can be used to recruit these proteins (recombination proteins) to the region of interest, thereby increasing their local concentration and further stimulating homologous recombination processes.
  • a polypeptide involved in homologous recombination as described above (or a functional fragment thereof) can be part of a triple fusion protein comprising a zinc finger binding domain, a cleavage domain (or cleavage half-domain) and the recombination protein (or functional fragment thereof).
  • Additional proteins involved in gene conversion and recombination-related chromatin remodeling include histone acetyltransferases (e.g., Esa1p, Tip60), histone methyltransferases (e.g., Dot1p), histone kinases and histone phosphatases.
  • histone acetyltransferases e.g., Esa1p, Tip60
  • histone methyltransferases e.g., Dot1p
  • histone kinases e.g., histone kinases and histone phosphatases.
  • the p53 protein has been reported to play a central role in repressing homologous recombination (HR). See, for example, Valerie et al., (2003) Oncogene 22:5792-5812; Janz, et al. (2002) Oncogene 21:5929-5933.
  • HR homologous recombination
  • the rate of HR in p53-deficient human tumor lines is 10,000-fold greater than in primary human fibroblasts, and there is a 100-fold increase in HR in tumor cells with a non-functional p53 compared to those with functional p53.
  • Mekeel et al. (1997) Oncogene 14:1847-1857.
  • Any method for downregulation of p53 activity can be used, including but not limited to cotransfection and overexpression of a p53 dominant negative mutant or targeted repression of p53 gene expression according to methods disclosed, e.g., in co-owned U.S. Pat. No. 6,534,261.
  • Exemplary molecules of this type include, but are not limited to, compounds which affect microtubule polymerization (e.g., vinblastine, nocodazole, Taxol), compounds that interact with DNA (e.g., cis-platinum(II) diamine dichloride, Cisplatin, doxorubicin) and/or compounds that affect DNA synthesis (e.g., thymidine, hydroxyurea, L-mimosine, etoposide, 5-fluorouracil).
  • compounds which affect microtubule polymerization e.g., vinblastine, nocodazole, Taxol
  • compounds that interact with DNA e.g., cis-platinum(II) diamine dichloride, Cisplatin, doxorubicin
  • compounds that affect DNA synthesis e.g., thymidine, hydroxyurea, L-mimosine, etoposide, 5-fluorouracil.
  • HDAC histone deacetylase
  • Additional methods for cell-cycle arrest include overexpression of proteins which inhibit the activity of the CDK cell-cycle kinases, for example, by introducing a cDNA encoding the protein into the cell or by introducing into the cell an engineered ZFP which activates expression of the gene encoding the protein.
  • Cell-cycle arrest is also achieved by inhibiting the activity of cyclins and CDKs, for example, using RNAi methods (e.g., U.S. Pat. No. 6,506,559) or by introducing into the cell an engineered ZFP which represses expression of one or more genes involved in cell-cycle progression such as, for example, cyclin and/or CDK genes. See, e.g., co- owned U.S. Pat. No. 6,534,261 for methods for the synthesis of engineered zinc finger proteins for regulation of gene expression.
  • targeted cleavage is conducted in the absence of a donor polynucleotide (preferably in S or G 2 phase), and recombination occurs between homologous chromosomes.
  • a donor polynucleotide preferably in S or G 2 phase
  • homologous recombination is a multi-step process requiring the modification of DNA ends and the recruitment of several cellular factors into a protein complex
  • addition of one or more exogenous factors, along with donor DNA and vectors encoding zinc finger-cleavage domain fusions, can be used to facilitate targeted homologous recombination.
  • An exemplary method for identifying such a factor or factors employs analyses of gene expression using microarrays (e.g., Affymetrix Gene Chip® arrays) to compare the mRNA expression patterns of different cells.
  • cells that exhibit a higher capacity to stimulate double strand break-driven homologous recombination in the presence of donor DNA and zinc finger-cleavage domain fusions can be analyzed for their gene expression patterns compared to cells that lack such capacity.
  • Genes that are upregulated or downregulated in a manner that directly correlates with increased levels of homologous recombination are thereby identified and can be cloned into any one of a number of expression vectors.
  • These expression constructs can be co-transfected along with zinc finger-cleavage domain fusions and donor constructs to yield improved methods for achieving high-efficiency homologous recombination.
  • expression of such genes can be appropriately regulated using engineered zinc finger proteins which modulate expression (either activation or repression) of one or more these genes. See, e.g., co-owned U.S. Pat. No. 6,534,261 for methods for the synthesis of engineered zinc finger proteins for regulation of gene expression.
  • Candidate genes that are differentially expressed in cells that carry out homologous recombination at a higher rate, either unaided or in response to compounds that arrest the cells in G2, are identified, cloned, and re-introduced into cells to determine whether their expression is sufficient to re-capitulate the improved rates.
  • expression of said candidate genes is activated using engineered zinc finger transcription factors as described, for example, in co-owned U.S. Pat. No. 6,534,261.
  • a nucleic acid encoding one or more ZFPs or ZFP fusion proteins can be cloned into a vector for transformation into prokaryotic or eukaryotic cells for replication and/or expression.
  • Vectors can be prokaryotic vectors, e.g., plasmids, or shuttle vectors, insect vectors, or eukaryotic vectors.
  • a nucleic acid encoding a ZFP can also be cloned into an expression vector, for administration to a plant cell, animal cell, preferably a mammalian cell or a human cell, fungal cell, bacterial cell, or protozoal cell.
  • sequences encoding a ZFP or ZFP fusion protein are typically subcloned into an expression vector that contains a promoter to direct transcription.
  • Suitable bacterial and eukaryotic promoters are well known in the art and described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989; 3 rd ed., 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., supra.
  • Bacterial expression systems for expressing the ZFP are available in, e.g., E.
  • Kits for such expression systems are commercially available.
  • Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known by those of skill in the art and are also commercially available.
  • the promoter used to direct expression of a ZFP-encoding nucleic acid depends on the particular application. For example, a strong constitutive promoter is typically used for expression and purification of ZFP. In contrast, when a ZFP is administered in vivo for gene regulation, either a constitutive or an inducible promoter is used, depending on the particular use of the ZFP.
  • a preferred promoter for administration of a ZFP can be a weak promoter, such as HSV TK or a promoter having similar activity.
  • the promoter typically can also include elements that are responsive to transactivation, e.g., hypoxia response elements, Ga14 response elements, lac repressor response element, and small molecule control systems such as tet-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard, PNAS 89:5547 (1992); Oligino et al., Gene Ther. 5:491-496 (1998); Wang et al., Gene Ther. 4:432-441 (1997); Neering et al., Blood 88:1147-1155 (1996); and Rendahl et al., Nat. Biotechnol. 16:757-761 (1998)).
  • the MNDU3 promoter can also be used, and is preferentially active in CD34 + hematopoietic stem cells.
  • the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in host cells, either prokaryotic or eukaryotic.
  • a typical expression cassette thus contains a promoter operably linked, e.g., to a nucleic acid sequence encoding the ZFP, and signals required, e.g., for efficient polyadenylation of the transcript, transcriptional termination, ribosome binding sites, or translation termination. Additional elements of the cassette may include, e.g., enhancers, and heterologous splicing signals.
  • the particular expression vector used to transport the genetic information into the cell is selected with regard to the intended use of the ZFP, e.g., expression in plants, animals, bacteria, fungus, protozoa, etc. (see expression vectors described below).
  • Standard bacterial expression vectors include plasmids such as pBR322-based plasmids, pSKF, pET23D, and commercially available fusion expression systems such as GST and LacZ.
  • An exemplary fusion protein is the maltose binding protein, “MBP.” Such fusion proteins are used for purification of the ZFP.
  • Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, for monitoring expression, and for monitoring cellular and subcellular localization, e.g., c-myc or FLAG.
  • Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus.
  • eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • Some expression systems have markers for selection of stably transfected cell lines such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase.
  • High yield expression systems are also suitable, such as using a baculovirus vector in insect cells, with a ZFP encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.
  • the elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of recombinant sequences.
  • Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, which are then purified using standard techniques (see, e.g., Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, J. Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds., 1983).
  • Any of the well known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, ultrasonic methods (e.g., sonoporation), liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the protein of choice.
  • Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • Methods of non-viral delivery of nucleic acids encoding engineered ZFPs include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids.
  • nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.) and BTX Molecular Delivery Systems (Holliston, Mass.).
  • Lipofection is described in e.g., US 5,049,386, US 4,946,787; and US 4,897,355) and lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM ).
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration).
  • lipid:nucleic acid complexes including targeted liposomes such as immunolipid complexes
  • the preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
  • RNA or DNA viral based systems for the delivery of nucleic acids encoding engineered ZFPs take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus.
  • Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo).
  • Conventional viral based systems for the delivery of ZFPs include, but are not limited to, retroviral, lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
  • Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system depends on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression.
  • Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700).
  • MiLV murine leukemia virus
  • GaLV gibbon ape leukemia virus
  • SIV Simian Immunodeficiency virus
  • HAV human immunodeficiency virus
  • Adenoviral based systems can be used.
  • Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and high levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system.
  • Adeno-associated virus (“AAV”) vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No.
  • At least six viral vector approaches are currently available for gene transfer in clinical trials, which utilize approaches that involve complementation of defective vectors by genes inserted into helper cell lines to generate the transducing agent.
  • pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn et al., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138 (1997)).
  • PA317/pLASN was the first therapeutic vector used in a gene therapy trial. (Blaese et al., Science 270:475-480 (1995)). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors. (Ellem et al., Immunol Immunother. 44(l):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).
  • Recombinant adeno-associated virus vectors are a promising alternative gene delivery systems based on the defective and nonpathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from a plasmid that retains only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system. (Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther. 9:748-55 (1996)).
  • Ad Replication-deficient recombinant adenoviral vectors
  • Ad can be produced at high titer and readily infect a number of different cell types.
  • Most adenovirus vectors are engineered such that a transgene replaces the Ad E1a, E1b, and/or E3 genes; subsequently the replication defective vector is propagated in human 293 cells that supply deleted gene function in trans.
  • Ad vectors can transduce multiple types of tissues in vivo, including nondividing, differentiated cells such as those found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity.
  • Ad vector An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et al., Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).
  • Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and ⁇ 2 cells or PA317 cells, which package retrovirus.
  • Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host (if applicable), other viral sequences being replaced by an expression cassette encoding the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line.
  • AAV vectors used in gene therapy typically only possess inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and integration into the host genome.
  • ITR inverted terminal repeat
  • Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
  • the cell line is also infected with adenovirus as a helper.
  • the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
  • the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.
  • a viral vector can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus.
  • the ligand is chosen to have affinity for a receptor known to be present on the cell type of interest.
  • Han et al., Proc. Natl. Acad. Sci. USA 92:9747-9751 (1995) reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor.
  • filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor.
  • Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below.
  • vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
  • Ex vivo cell transfection for diagnostics, research, or for gene therapy is well known to those of skill in the art.
  • cells are isolated from the subject organism, transfected with a ZFP nucleic acid (gene or cDNA), and re-infused back into the subject organism (e.g., patient).
  • a ZFP nucleic acid gene or cDNA
  • Various cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et al., Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) and the references cited therein for a discussion of how to isolate and culture cells from patients).
  • stem cells are used in ex vivo procedures for cell transfection and gene therapy.
  • the advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow.
  • Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such a GM-CSF, IFN- ⁇ and TNF- ⁇ are known (see Inaba et al., J. Exp. Med. 176:1693-1702 (1992)).
  • Stem cells are isolated for transduction and differentiation using known methods. For example, stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD8+ (T cells), CD45+ (panb cells), GR-1 (granulocytes), and Iad (differentiated antigen presenting cells) (see Inaba et al., J. Exp. Med. 176:1693-1702 (1992)).
  • T cells CD4+ and CD8+
  • CD45+ panb cells
  • GR-1 granulocytes
  • Iad differentiated antigen presenting cells
  • Vectors e.g., retroviruses, adenoviruses, liposomes, etc.
  • therapeutic ZFP nucleic acids can also be administered directly to an organism for transduction of cells in vivo.
  • naked DNA can be administered.
  • Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • Vectors useful for introduction of transgenes into hematopoietic stem cells include adenovirus Type 35.
  • Vectors suitable for introduction of transgenes into immune cells include non-integrating lentivirus vectors. See, for example, Ory et al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al. (1998) J. Virol. 72:8463-8471; Zuffery et al. (1998) J. Virol. 72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions available, as described below (see, e.g., Remington 's Pharmaceutical Sciences, 17th ed., 1989).
  • DNA constructs may be introduced into the genome of a desired plant host by a variety of conventional techniques. For reviews of such techniques see, for example, Weissbach & Weissbach Methods for Plant Molecular Biology (1988, Academic Press, N.Y.) Section VIII, pp. 421-463; and Grierson & Corey, Plant Molecular Biology (1988, 2d Ed.), Blackie, London, Ch. 7-9.
  • the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using biolistic methods, such as DNA particle bombardment (see, e.g., Klein et al (1987) Nature 327:70-73).
  • the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector.
  • Agrobacterium tumefaciens -mediated transformation techniques including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et al (1984) Science 233:496-498, and Fraley et al (1983) Proc. Nat'l. Acad. Sci. USA 80:4803.
  • the virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria using binary T DNA vector (Bevan (1984) Nuc. Acid Res.
  • Agrobacterium transformation system is used to engineer dicotyledonous plants (Bevan et al (1982) Ann. Rev. Genet 16:357-384; Rogers et al (1986) Methods Enzymol. 118:627-641).
  • the Agrobacterium transformation system may also be used to transform, as well as transfer, DNA to monocotyledonous plants and plant cells.
  • Alternative gene transfer and transformation methods include, but are not limited to, protoplast transformation through calcium-, polyethylene glycol (PEG)- or electroporation-mediated uptake of naked DNA (see Paszkowski et al. (1984) EMBO J 3:2717-2722, Potrykus et al. (1985) Molec. Gen. Genet. 199:169-177; Fromm et al. (1985) Proc. Nat. Acad. Sci. USA 82:5824-5828; and Shimamoto (1989) Nature 338:274-276) and electroporation of plant tissues (D' Halluin et al. (1992) Plant Cell 4:1495-1505).
  • PEG polyethylene glycol
  • Additional methods for plant cell transformation include microinjection, silicon carbide mediated DNA uptake (Kaeppler et al. (1990) Plant Cell Reporter 9:415-418), and microprojectile bombardment (see Klein et al. (1988) Proc. Nat. Acad. Sci. USA 85:4305-4309; and Gordon-Kamm et al. (1990) Plant Cell 2:603-618).
  • the disclosed methods and compositions can be used to insert exogenous sequences into a predetermined location in a plant cell genome. This is useful inasmuch as expression of an introduced transgene into a plant genome depends critically on its integration site. Accordingly, genes encoding, e.g., nutrients, antibiotics or therapeutic molecules can be inserted, by targeted recombination, into regions of a plant genome favorable to their expression.
  • Transformed plant cells which are produced by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype.
  • Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences.
  • Plant regeneration from cultured protoplasts is described in Evans, et al., “Protoplasts Isolation and Culture” in Handbook of Plant Cell Culture, pp. 124-176, Macmillian Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, pollens, embryos or parts thereof. Such regeneration techniques are described generally in Klee et al (1987) Ann. Rev. of Plant Phys. 38:467-486.
  • Nucleic acids introduced into a plant cell can be used to confer desired traits on essentially any plant.
  • a wide variety of plants and plant cell systems may be engineered for the desired physiological and agronomic characteristics described herein using the nucleic acid constructs of the present disclosure and the various transformation methods mentioned above.
  • target plants and plant cells for engineering include, but are not limited to, those monocotyledonous and dicotyledonous plants, such as crops including grain crops (e.g., wheat, maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear, strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops (e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g., lettuce, spinach); flowering plants (e.g., petunia, rose, chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plants used in phytoremediation (e.g., heavy metal accumulating plants); oil crops (e.g., sunflower, rape seed) and plants used for experimental purposes (e.g., Arabidopsis ).
  • crops including grain crops (e.g., wheat, maize, rice, millet, barley), fruit
  • the disclosed methods and compositions have use over a broad range of plants, including, but not limited to, species from the genera Asparagus, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucurbita, Daucus, Glycine, Hordeum, Lactuca, Lycopersicon, Malus, Manihot, Nicotiana, Oryza, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Solanum, Sorghum, Triticum, Vitis, Vigna, and Zea.
  • the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
  • a transformed plant cell, callus, tissue or plant may be identified and isolated by selecting or screening the engineered plant material for traits encoded by the marker genes present on the transforming DNA. For instance, selection may be performed by growing the engineered plant material on media containing an inhibitory amount of the antibiotic or herbicide to which the transforming gene construct confers resistance. Further, transformed plants and plant cells may also be identified by screening for the activities of any visible marker genes (e.g., the ⁇ -glucuronidase, luciferase, B or C1 genes) that may be present on the recombinant nucleic acid constructs. Such selection and screening methodologies are well known to those skilled in the art.
  • any visible marker genes e.g., the ⁇ -glucuronidase, luciferase, B or C1 genes
  • Physical and biochemical methods also may be used to identify plant or plant cell transformants containing inserted gene constructs. These methods include but are not limited to: 1) Southern analysis or PCR amplification for detecting and determining the structure of the recombinant DNA insert; 2) Northern blot, S 1 RNase protection, primer-extension or reverse transcriptase-PCR amplification for detecting and examining RNA transcripts of the gene constructs; 3) enzymatic assays for detecting enzyme or ribozyme activity, where such gene products are encoded by the gene construct; 4) protein gel electrophoresis, Western blot techniques, immunoprecipitation, or enzyme-linked immunoassays, where the gene construct products are proteins.
  • RNA e.g., mRNA
  • Effects of gene manipulation using the methods disclosed herein can be observed by, for example, northern blots of the RNA (e.g., mRNA) isolated from the tissues of interest. Typically, if the amount of mRNA has increased, it can be assumed that the corresponding endogenous gene is being expressed at a greater rate than before. Other methods of measuring gene and/or CYP74B activity can be used. Different types of enzymatic assays can be used, depending on the substrate used and the method of detecting the increase or decrease of a reaction product or by-product.
  • the levels of and/or CYP74B protein expressed can be measured immunochemically, i.e., ELISA, RIA, EIA and other antibody based assays well known to those of skill in the art, such as by electrophoretic detection assays (either with staining or western blotting).
  • the transgene may be selectively expressed in some tissues of the plant or at some developmental stages, or the transgene may be expressed in substantially all plant tissues, substantially along its entire life cycle. However, any combinatorial expression mode is also applicable.
  • the present disclosure also encompasses seeds of the transgenic plants described above wherein the seed has the transgene or gene construct.
  • the present disclosure further encompasses the progeny, clones, cell lines or cells of the transgenic plants described above wherein said progeny, clone, cell line or cell has the transgene or gene construct.
  • polypeptide compounds such as ZFP fusion proteins
  • ZFP fusion proteins An important factor in the administration of polypeptide compounds, such as ZFP fusion proteins, is ensuring that the polypeptide has the ability to traverse the plasma membrane of a cell, or the membrane of an intra-cellular compartment such as the nucleus.
  • Cellular membranes are composed of lipid-protein bilayers that are freely permeable to small, nonionic lipophilic compounds and are inherently impermeable to polar compounds, macromolecules, and therapeutic or diagnostic agents.
  • proteins and other compounds such as liposomes have been described, which have the ability to translocate polypeptides such as ZFPs across a cell membrane.
  • membrane translocation polypeptides have amphiphilic or hydrophobic amino acid subsequences that have the ability to act as membrane-translocating carriers.
  • homeodomain proteins have the ability to translocate across cell membranes.
  • the shortest internalizable peptide of a homeodomain protein, Antennapedia was found to be the third helix of the protein, from amino acid position 43 to 58 (see, e.g., Prochiantz, Current Opinion in Neurobiology 6:629-634 (1996)).
  • Another subsequence, the h (hydrophobic) domain of signal peptides was found to have similar cell membrane translocation characteristics (see, e.g., Lin et al., J. Biol. Chem. 270:1 4255-14258 (1995)).
  • Examples of peptide sequences which can be linked to a protein, for facilitating uptake of the protein into cells include, but are not limited to: an 11 amino acid peptide of the tat protein of HIV; a 20 residue peptide sequence which corresponds to amino acids 84-103 of the p16 protein (see Fahraeus et al., Current Biology 6:84 (1996)); the third helix of the 60-amino acid long homeodomain of Antennapedia (Derossi et al., J. Biol. Chem.
  • Membrane translocation domains can also be selected from libraries of randomized peptide sequences. See, for example, Yeh et al. (2003) Molecular Therapy 7(5):S461, Abstract #1191.
  • Toxin molecules also have the ability to transport polypeptides across cell membranes. Often, such molecules (called “binary toxins”) are composed of at least two parts: a translocation/binding domain or polypeptide and a separate toxin domain or polypeptide. Typically, the translocation domain or polypeptide binds to a cellular receptor, and then the toxin is transported into the cell.
  • binary toxins are composed of at least two parts: a translocation/binding domain or polypeptide and a separate toxin domain or polypeptide. Typically, the translocation domain or polypeptide binds to a cellular receptor, and then the toxin is transported into the cell.
  • Clostridium perfringens iota toxin diphtheria toxin (DT), Pseudomonas exotoxin A (PE), pertussis toxin (PT), Bacillus anthracis toxin, and pertussis adenylate cyclase (CYA)
  • DT diphtheria toxin
  • PE Pseudomonas exotoxin A
  • PT pertussis toxin
  • Bacillus anthracis toxin Bacillus anthracis toxin
  • pertussis adenylate cyclase CYA
  • Such peptide sequences can be used to translocate ZFPs across a cell membrane.
  • ZFPs can be conveniently fused to or derivatized with such sequences.
  • the translocation sequence is provided as part of a fusion protein.
  • a linker can be used to link the ZFP and the translocation sequence. Any suitable linker can be used, e.g., a peptide linker.
  • the ZFP can also be introduced into an animal cell, preferably a mammalian cell, via a liposomes and liposome derivatives such as immunoliposomes.
  • liposome refers to vesicles comprised of one or more concentrically ordered lipid bilayers, which encapsulate an aqueous phase.
  • the aqueous phase typically contains the compound to be delivered to the cell, i.e., a ZFP.
  • the liposome fuses with the plasma membrane, thereby releasing the drug into the cytosol.
  • the liposome is phagocytosed or taken up by the cell in a transport vesicle. Once in the endosome or phagosome, the liposome either degrades or fuses with the membrane of the transport vesicle and releases its contents.
  • the liposome In current methods of drug delivery via liposomes, the liposome ultimately becomes permeable and releases the encapsulated compound (in this case, a ZFP) at the target tissue or cell.
  • the encapsulated compound in this case, a ZFP
  • this can be accomplished, for example, in a passive manner wherein the liposome bilayer degrades over time through the action of various agents in the body.
  • active drug release involves using an agent to induce a permeability change in the liposome vesicle.
  • Liposome membranes can be constructed so that they become destabilized when the environment becomes acidic near the liposome membrane (see, e.g., PNAS 84:7851 (1987); Biochemistry 28:908 (1989)).
  • DOPE Dioleoylphosphatidylethanolamine
  • Such liposomes typically comprise a ZFP and a lipid component, e.g., a neutral and/or cationic lipid, optionally including a receptor-recognition molecule such as an antibody that binds to a predetermined cell surface receptor or ligand (e.g., an antigen).
  • a lipid component e.g., a neutral and/or cationic lipid, optionally including a receptor-recognition molecule such as an antibody that binds to a predetermined cell surface receptor or ligand (e.g., an antigen).
  • Suitable methods include, for example, sonication, extrusion, high pressure/homogenization, microfluidization, detergent dialysis, calcium-induced fusion of small liposome vesicles and ether-fusion methods, all of which are known to those of skill in the art.
  • targeting moieties that are specific to a particular cell type, tissue, and the like.
  • targeting moieties e.g., ligands, receptors, and monoclonal antibodies
  • targeting moieties include monoclonal antibodies specific to antigens associated with neoplasms, such as prostate cancer specific antigen and MAGE. Tumors can also be diagnosed by detecting gene products resulting from the activation or over-expression of oncogenes, such as ras or c-erbB2. In addition, many tumors express antigens normally expressed by fetal tissue, such as the alphafetoprotein (AFP) and carcinoembryonic antigen (CEA).
  • AFP alphafetoprotein
  • CEA carcinoembryonic antigen
  • Sites of viral infection can be diagnosed using various viral antigens such as hepatitis B core and surface antigens (HBVc, HBVs) hepatitis C antigens, Epstein-Barr virus antigens, human immunodeficiency type-1 virus (HIV1) and papilloma virus antigens.
  • Inflammation can be detected using molecules specifically recognized by surface molecules which are expressed at sites of inflammation such as integrins (e.g., VCAM-1), selectin receptors (e.g., ELAM-1) and the like.
  • Standard methods for coupling targeting agents to liposomes can be used. These methods generally involve incorporation into liposomes of lipid components, e.g., phosphatidylethanolamine, which can be activated for attachment of targeting agents, or derivatized lipophilic compounds, such as lipid derivatized bleomycin.
  • lipid components e.g., phosphatidylethanolamine
  • Antibody targeted liposomes can be constructed using, for instance, liposomes which incorporate protein A (see Renneisen et al., J. Biol. Chem., 265:16337-16342 (1990) and Leonetti et al., PNAS 87:2448-2451 (1990).
  • the dose administered to a patient, or to a cell which will be introduced into a patient, in the context of the present disclosure, should be sufficient to effect a beneficial therapeutic response in the patient over time.
  • particular dosage regimens can be useful for determining phenotypic changes in an experimental setting, e.g., in functional genomics studies, and in cell or animal models.
  • the dose will be determined by the efficacy and K d of the particular ZFP employed, the nuclear volume of the target cell, and the condition of the patient, as well as the body weight or surface area of the patient to be treated.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound or vector in a particular patient.
  • the maximum therapeutically effective dosage of ZFP for approximately 99% binding to target sites is calculated to be in the range of less than about 1.5 ⁇ 10 5 to 1.5 ⁇ 10 6 copies of the specific ZFP molecule per cell.
  • the number of ZFPs per cell for this level of binding is calculated as follows, using the volume of a HeLa cell nucleus (approximately 1000 ⁇ m 3 or 10 ⁇ 12 L; Cell Biology, (Altman & Katz, eds. (1976)). As the HeLa nucleus is relatively large, this dosage number is recalculated as needed using the volume of the target cell nucleus. This calculation also does not take into account competition for ZFP binding by other sites. This calculation also assumes that essentially all of the ZFP is localized to the nucleus.
  • a value of 100 ⁇ K d is used to calculate approximately 99% binding of to the target site, and a value of 10 ⁇ K d is used to calculate approximately 90% binding of to the target site.
  • K d 25 nM ZFP + target ⁇ ⁇ site ⁇ complex i . e .
  • the appropriate dose of an expression vector encoding a ZFP can also be calculated by taking into account the average rate of ZFP expression from the promoter and the average rate of ZFP degradation in the cell.
  • a weak promoter such as a wild-type or mutant HSV TK promoter is used, as described above.
  • the dose of ZFP in micrograms is calculated by taking into account the molecular weight of the particular ZFP being employed.
  • the physician evaluates circulating plasma levels of the ZFP or nucleic acid encoding the ZFP, potential ZFP toxicities, progression of the disease, and the production of anti-ZFP antibodies. Administration can be accomplished via single or divided doses.
  • ZFPs and expression vectors encoding ZFPs can be administered directly to the patient for targeted cleavage and/or recombination, and for therapeutic or prophylactic applications, for example, cancer, ischemia, diabetic retinopathy, macular degeneration, rheumatoid arthritis, psoriasis, HIV infection, sickle cell anemia, Alzheimer's disease, muscular dystrophy, neurodegenerative diseases, vascular disease, cystic fibrosis, stroke, and the like.
  • Administration of therapeutically effective amounts is by any of the routes normally used for introducing ZFP into ultimate contact with the tissue to be treated.
  • the ZFPs are administered in any suitable manner, preferably with pharmaceutically acceptable carriers. Suitable methods of administering such modulators are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions that are available (see, e.g., Remington 's Pharmaceutical Sciences, 17 th ed. 1985)).
  • the ZFPs can be made into aerosol formulations (i.e., they can be “nebulized”) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the disclosed compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally.
  • the formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the disclosed methods and compositions for targeted cleavage can be used to induce mutations in a genomic sequence, e.g., by cleaving at two sites and deleting sequences in between, by cleavage at a single site followed by non-homologous end joining, cleaving at one or two sites with insertion of an exogenous sequence between the breaks and/or by cleaving at a site so as to remove one or two or a few nucleotides.
  • Targeted cleavage can also be used to create gene knock-outs (e.g., for functional genomics or target validation) and to facilitate targeted insertion of a sequence into a genome (i.e., gene knock-in); e.g., for purposes of cell engineering or protein overexpression.
  • Insertion can be by means of replacements of chromosomal sequences through homologous recombination or by targeted integration, in which a new sequence (i.e., a sequence not present in the region of interest), flanked by sequences homologous to the region of interest in the chromosome, is inserted at a predetermined target site.
  • a new sequence i.e., a sequence not present in the region of interest
  • the same methods can also be used to replace a wild-type sequence with a mutant sequence, or to convert one allele to a different allele.
  • Targeted cleavage of infecting or integrated viral genomes can be used to treat viral infections in a host. Additionally, targeted cleavage of genes encoding receptors for viruses can be used to block expression of such receptors, thereby preventing viral infection and/or viral spread in a host organism. Targeted mutagenesis of genes encoding viral receptors (e.g., the CCR5 and CXCR4 receptors for HIV) can be used to render the receptors unable to bind to virus, thereby preventing new infection and blocking the spread of existing infections.
  • Targeted mutagenesis of genes encoding viral receptors e.g., the CCR5 and CXCR4 receptors for HIV
  • viruses or viral receptors that may be targeted include herpes simplex virus (HSV), such as HSV-1 and HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV), HHV6 and HHV7.
  • HSV herpes simplex virus
  • VZV varicella zoster virus
  • EBV Epstein-Barr virus
  • CMV cytomegalovirus
  • HHV6 and HHV7 herpes simplex virus
  • the hepatitis family of viruses includes hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitis virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV).
  • viruses or their receptors may be targeted, including, but not limited to, Picomaviridae (e.g., polioviruses, etc.); Caliciviridae; Togaviridae (e.g., rubella virus, dengue virus, etc.); Flaviviridae; Coronaviridae; Reoviridae; Bimaviridae; Rhabodoviridae (e.g., rabies virus, etc.); Filoviridae; Paramyxoviridae (e.g., mumps virus, measles virus, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g., influenza virus types A, B and C, etc.); Bunyaviridae; Arenaviridae; Retroviradae; lentiviruses (e.g., HTLV-I; HTLV-II; HIV-1 (also known as HTLV-III, LAV, ARV, hTLR, etc.) HIV-II
  • Receptors for HIV include CCR-5 and CXCR-4.
  • the genome of an infecting bacterium can be mutagenized by targeted DNA cleavage followed by non-homologous end joining, to block or ameliorate bacterial infections.
  • the disclosed methods for targeted recombination can be used to replace any genomic sequence with a homologous, non-identical sequence.
  • a mutant genomic sequence can be replaced by its wild-type counterpart, thereby providing methods for treatment of e.g., genetic disease, inherited disorders, cancer, and autoimmune disease.
  • one allele of a gene can be replaced by a different allele using the methods of targeted recombination disclosed herein.
  • Exemplary genetic diseases include, but are not limited to, achondroplasia, achromatopsia, acid maltase deficiency, adenosine deaminase deficiency (OMIM No.102700), adrenoleukodystrophy, aicardi syndrome, alpha-I antitrypsin deficiency, alpha-thalassemia, androgen insensitivity syndrome, apert syndrome, arrhythmogenic right ventricular, dysplasia, ataxia telangictasia, barth syndrome, beta-thalassemia, blue rubber bleb nevus syndrome, canavan disease, chronic granulomatous diseases (CGD), cri du chat syndrome, cystic fibrosis, dercum's disease, ectodermal dysplasia, fanconi anemia, fibrodysplasia ossificans progressive, fragile X syndrome, galactosemis, Gaucher's disease, generalized gangliosidoses (e.g
  • leukodystrophy long QT syndrome, Marfan syndrome, Moebius syndrome, mucopolysaccharidosis (MPS), nail patella syndrome, nephrogenic diabetes insipdius, neurofibromatosis, Neimann-Pick disease, osteogenesis imperfecta, porphyria, Prader-Willi syndrome, progeria, Proteus syndrome, retinoblastoma, Rett syndrome, Rubinstein-Taybi syndrome, Sanfilippo syndrome, severe combined immunodeficiency (SCID), Shwachman syndrome, sickle cell disease (sickle cell anemia), Smith-Magenis syndrome, Stickler syndrome, Tay-Sachs disease, Thrombocytopenia Absent Radius (TAR) syndrome, Treacher Collins syndrome, trisomy, tuberous sclerosis, Turner's syndrome, urea cycle disorder, von Hippel-Landau disease, Waardenburg syndrome, Williams syndrome, Wilson's disease, Wiskott-Aldrich syndrome
  • Additional exemplary diseases that can be treated by targeted DNA cleavage and/or homologous recombination include acquired immunodeficiencies, lysosomal storage diseases (e.g., Gaucher's disease, GM1, Fabry disease and Tay-Sachs disease), mucopolysaccahidosis (e.g. Hunter's disease, Hurler's disease), hemoglobinopathies (e.g., sickle cell diseases, HbC, ⁇ -thalassemia, ⁇ -thalassemia) and hemophilias.
  • acquired immunodeficiencies e.g., Gaucher's disease, GM1, Fabry disease and Tay-Sachs disease
  • mucopolysaccahidosis e.g. Hunter's disease, Hurler's disease
  • hemoglobinopathies e.g., sickle cell diseases, HbC, ⁇ -thalassemia, ⁇ -thalassemia
  • hemophilias e.g., sickle cell
  • a region of interest comprises a mutation
  • the donor polynucleotide comprises the corresponding wild-type sequence.
  • a wild-type genomic sequence can be replaced by a mutant sequence, if such is desirable.
  • overexpression of an oncogene can be reversed either by mutating the gene or by replacing its control sequences with sequences that support a lower, non-pathologic level of expression.
  • the wild-type allele of the ApoAI gene can be replaced by the ApoAI Milano allele, to treat atherosclerosis. Indeed, any pathology dependent upon a particular genomic sequence, in any fashion, can be corrected or alleviated using the methods and compositions disclosed herein.
  • Targeted cleavage and targeted recombination can also be used to alter non-coding sequences (e.g., regulatory sequences such as promoters, enhancers, initiators, terminators, splice sites) to alter the levels of expression of a gene product.
  • non-coding sequences e.g., regulatory sequences such as promoters, enhancers, initiators, terminators, splice sites
  • Such methods can be used, for example, for therapeutic purposes, functional genomics and/or target validation studies.
  • compositions and methods described herein also allow for novel approaches and systems to address immune reactions of a host to allogeneic grafts.
  • a major problem faced when allogeneic stem cells (or any type of allogeneic cell) are grafted into a host recipient is the high risk of rejection by the host's immune system, primarily mediated through recognition of the Major Histocompatibility Complex (MHC) on the surface of the engrafted cells.
  • MHC Major Histocompatibility Complex
  • the MHC comprises the HLA class I protein(s) that function as heterodimers that are comprised of a common ⁇ subunit and variable ⁇ subunits. It has been demonstrated that tissue grafts derived from stem cells that are devoid of HLA escape the host's immune response.
  • genes encoding HLA proteins involved in graft rejection can be cleaved, mutagenized or altered by recombination, in either their coding or regulatory sequences, so that their expression is blocked or they express a non-functional product.
  • HLA class I can be removed from the cells to rapidly and reliably generate HLA class I null stem cells from any donor, thereby reducing the need for closely matched donor/recipient MHC haplotypes during stem cell grafting.
  • Inactivation of any gene can be achieved, for example, by a single cleavage event, by cleavage followed by non-homologous end joining, by cleavage at two sites followed by joining so as to delete the sequence between the two cleavage sites, by targeted recombination of a missense or nonsense codon into the coding region, or by targeted recombination of an irrelevant sequence (i.e., a “stuffer” sequence) into the gene or its regulatory region, so as to disrupt the gene or regulatory region.
  • a single cleavage event by cleavage followed by non-homologous end joining, by cleavage at two sites followed by joining so as to delete the sequence between the two cleavage sites, by targeted recombination of a missense or nonsense codon into the coding region, or by targeted recombination of an irrelevant sequence (i.e., a “stuffer” sequence) into the gene or its regulatory region, so as to disrupt the gene or regulatory region
  • Targeted modification of chromatin structure can be used to facilitate the binding of fusion proteins to cellular chromatin.
  • one or more fusions between a zinc finger binding domain and a recombinase can be used, in addition to or instead of the zinc finger-cleavage domain fusions disclosed herein, to facilitate targeted recombination. See, for example, co-owned U.S. Pat. No. 6,534,261 and Akopian et al. (2003) Proc. Natl. Acad. Sci. USA 100:8688-8691.
  • fusion polypeptide comprises a zinc finger binding domain and a functional domain monomer (e.g., a monomer from a dimeric transcriptional activation or repression domain). Binding of two such fusion polypeptides to properly situated target sites allows dimerization so as to reconstitute a functional transcription activation or repression domain.
  • the methods and compositions set forth herein can be used for targeted integration of exogenous sequences into a region of interest in the genome of a cell.
  • Targeted integration of an exogenous sequence at a double-strand break in a genome can occur by both homology-dependent and homology-independent mechanisms.
  • targeted integration by both homology-dependent and homology-independent mechanisms involves insertion of an exogenous sequence between the ends generated by cleavage.
  • the exogenous sequence inserted can be any length, for example, a relatively short “patch” sequence of between 1 and 50 nucleotides in length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 nucleotide sequence).
  • a donor nucleic acid or donor sequence comprises an exogenous sequence together with one or more sequences that are either identical, or homologous but non-identical, with a predetermined genomic sequence (i.e., a target site).
  • a predetermined genomic sequence i.e., a target site.
  • two of the identical sequences or two of the homologous but non-identical sequences (or one of each) are present, flanking the exogenous sequence.
  • An exogenous sequence is one that contains a nucleotide sequence that is not normally present in the region of interest.
  • exogenous sequences include, but are not limited to, cDNAs, promoter sequences, enhancer sequences, epitope tags, marker genes, cleavage enzyme recognition sites and various types of expression constructs.
  • Marker genes include, but are not limited to, sequences encoding proteins that mediate antibiotic resistance (e.g., ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), sequences encoding colored or fluorescent or luminescent proteins (e.g., green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase), and proteins which mediate enhanced cell growth and/or gene amplification (e.g., dihydrofolate reductase).
  • Epitope tags include, for example, one or more copies of FLAG, His, myc, Tap, HA or any detectable amino acid sequence.
  • Protein expression constructs include, but are not limited to, cDNAs and transcriptional control sequences in operative linkage with cDNA sequences.
  • Transcriptional control sequences include promoters, enhancers and insulators.
  • Additional transcriptional and translational regulatory sequences which can be included in expression constructs include, e.g., internal ribosome entry sites, sequences encoding 2A peptides and polyadenylation signals.
  • An exemplary protein expression construct is an antibody expression construct comprising a sequence encoding an antibody heavy chain and a sequence encoding an antibody light chain, each sequence operatively linked to a promoter (the promoters being the same or different) and either or both sequences optionally operatively linked to an enhancer (and, in the case of both coding sequences being linked to enhancers, the enhancers being the same or different).
  • Cleavage enzyme recognition sites include, for example, sequences recognized by restriction endonucleases, homing endonucleases and/or meganucleases.
  • Targeted integration of a cleavage enzyme recognition site is useful for generating cells whose genome contains only a single site that can be cleaved by a particular enzyme. Contacting such cells with an enzyme that recognizes and cleaves at the single site facilitates subsequent targeted integration of exogenous sequences (by either homology-dependent or homology-independent mechanisms) and/or targeted mutagenesis at the site that is cleaved.
  • a cleavage enzyme recognition site is that recognized by the homing endonuclease I-SceI, which has the following sequence: TAGGGATAACAGGGTAAT (SEQ ID NO:213) See, for example, U.S. Pat. No. 6,833,252.
  • Additional exemplary homing endonucleases include I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII. Their recognition sequences are known. See also U.S. Pat. No.
  • cleavage specificity of most homing endonucleases is not absolute with respect to their recognition sites, the sites are of sufficient length that a single cleavage event per mammalian-sized genome can be obtained by expressing a homing endonuclease in a cell containing a single copy of its recognition site. It has also been reported that cleavage enzymes can be engineered to bind non-natural target sites. See, for example, Chevalier et al. (2002) Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res. 31:2952-2962; Ashworth et al. (2006) Nature 441:656-659.
  • targeted integration is used to insert a RNA expression construct, e.g., sequences responsible for regulated expression of micro RNA or siRNA. Promoters, enhancers and additional transcription regulatory sequences, as described above, can also be incorporated in a RNA expression construct.
  • the donor sequence contains sufficient homology, in the regions flanking the exogenous sequence, to support homology-directed repair of a double-strand break in a genomic sequence, thereby inserting the exogenous sequence at the genomic target site. Therefore, the donor nucleic acid can be of any size sufficient to support integration of the exogenous sequence by homology-dependent repair mechanisms (e.g., homologous recombination).
  • homology-dependent repair mechanisms e.g., homologous recombination.
  • the regions of homology flanking the exogenous sequence are thought to provide the broken chromosome ends with a template for re-synthesis of the genetic information at the site of the double-stranded break.
  • Targeted integration of exogenous sequences can be used to generate cells and cell lines for protein expression. See, for example, co-owned U.S. Patent Application Publication No. 2006/0063231 (the disclosure of which is hereby incorporated by reference herein, in its entirety, for all purposes).
  • the chromosomal integration site should be compatible with high-level transcription of the integrated sequences, preferably in a wide range of cell types and developmental states.
  • transcription of integrated sequences varies depending on the integration site due to, among other things, the chromatin structure of the genome at the integration site. Accordingly, genomic target sites that support high-level transcription of integrated sequences are desirable.
  • exogenous sequences not result in ectopic activation of one or more cellular genes (e.g., oncogenes).
  • ectopic expression may be desired.
  • an integration site is not present in an essential gene (e.g., a gene essential for cell viability), so that inactivation of said essential gene does not result from integration of the exogenous sequences.
  • an essential gene e.g., a gene essential for cell viability
  • the exogenous sequence can be any sequence capable of blocking transcription of the endogenous gene or of generating a non-functional translation product, for example a short patch of amino acid sequence, which is optionally detectable (see above).
  • the exogenous sequences can comprise a marker gene (described above), allowing selection of cells that have undergone targeted integration.
  • Non-limiting examples of chromosomal regions that do not encode an essential gene and support high-level transcription of sequences integrated therein (“safe harbor” integration sites) include the Rosa26 and CCR5 loci.
  • the Rosa26 locus has been identified in the murine genome. Zambrowicz et al. (1997) Proc. Natl. Acad. Sci. USA 94:3789-3794. The sequence of mouse Rosa26 mRNA was compared to data from a human cDNA screen (Strausberg et al. (2002) Proc. Natl. Acad. Sci. USA 99:16899-16903), and a homologous human transcript was detected by the present inventors. Accordingly, the human homologue of Rosa26 can be used as a target site for integration of exogenous sequences into the genome of human cells and cell lines, using the methods and compositions disclosed herein.
  • CCR5 genomic sequences including allelic variants such as CCR5- ⁇ 32 are well known in the art. See, e.g., Liu et al. (1996) Cell 367-377.
  • Additional genomic target sites supporting high-level transcription of integrated sequences can be identified as regions of open chromatin or ‘accessible regions” as described, for example in co-owned U.S. Patent Application Publications 2002/0064802 (May 30, 2002) and 2002/0081603 (Jun. 27, 2002).
  • compositions and methods disclosed herein can be used for targeted cleavage of a genomic sequence, followed by non-homology-dependent integration of an exogenous sequence at or near the targeted cleavage site.
  • a cell can be contacted with one or more ZFP-cleavage domain (or cleavage half-domain) fusion proteins engineered to cleave in a region of interest in a genome as described herein (or one or more polynucleotides encoding such fusion proteins), and a polynucleotide comprising an exogenous sequence lacking homology to the region of interest, to obtain a cell in which all or a portion of the exogenous sequence is integrated in the region of interest.
  • ZFP-cleavage domain or cleavage half-domain
  • the methods of targeted integration i.e., insertion of an exogenous sequence into a genome
  • both homology-dependent and -independent, disclosed herein can be used for a number of purposes. These include, but are not limited to, insertion of a gene or cDNA sequence into the genome of a cell to enable expression of the transcription and/or translation products of the gene or cDNA by the cell.
  • targeted integration either homology-dependent or homology-independent
  • a cDNA copy of the wild-type gene is particularly effective.
  • such a wild-type cDNA is inserted into an untranslated leader sequence or into the first exon of a gene upstream of all known mutations.
  • the result is that the wild-type cDNA is expressed and its expression is regulated by the appropriate endogenous transcriptional regulatory sequences.
  • such integrated cDNA sequences can include transcriptional (and/or translational) termination signals disposed downstream of the wild-type cDNA and upstream of the mutant endogenous gene. In this way, a wild-type copy of the disease-causing gene is expressed, and the mutant endogenous gene is not expressed.
  • a portion of a wild-type cDNA is inserted into the appropriate region of a gene (for example, a gene in which disease-causing mutations are clustered).
  • the hSMC1L1 gene is the human orthologue of the budding yeast gene structural maintenance of chromosomes 1.
  • a region of this gene encoding an amino-terminal portion of the protein which includes the Walker ATPase domain was mutagenized by targeted cleavage and recombination. Cleavage was targeted to the region of the methionine initiation codon (nucleotides 24-26, FIG. 1 ), by designing chimeric nucleases, comprising a zinc finger DNA-binding domain and a FokI cleavage half-domain, which bind in the vicinity of the codon.
  • two zinc finger binding domains were designed, one of which recognizes nucleotides 23-34 (primary contacts along the top strand as shown in FIG.
  • Zinc finger proteins were designed as described in co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261. See Table 2 for the amino acid sequences of the recognition regions of the zinc finger proteins.
  • Sequences encoding each of these two ZFP binding domains were fused to sequences encoding a FokI cleavage half-domain (amino acids 384-579 of the native FokI sequence; Kita et al. (1989) J. Biol. Chem. 264:5751-5756), such that the encoded protein contained FokI sequences at the carboxy terminus and ZFP sequences at the amino terminus.
  • Each of these fusion sequences was then cloned in a modified mammalian expression vector pcDNA3 ( FIG. 2 ).
  • a donor DNA molecule was obtained as follows. First, a 700 base pair fragment of human genomic DNA representing nucleotides 52415936-52416635 of the “ ⁇ ” strand of the X chromosome (UCSC human genome release July, 2003), which includes the first exon of the human hSMC1L1 gene, was amplified, using genomic DNA from HEK293 cells as template. Sequences of primers used for amplification are shown in Table 3 (“Initial amp 1” and “Initial amp 2”). The PCR product was then altered, using standard overlap extension PCR methodology (see, e.g., Ho, et al. (1989) Gene 77:51-59), resulting in replacement of the sequence ATGGGG (nucleotides 24-29 in FIG.
  • FIG. 3 A schematic diagram of the hSMC1 gene, including sequences of the chromosomal DNA in the region of the initiation codon, and sequences in the donor DNA that differ from the chromosomal sequence, is given in FIG. 3 .
  • the resulting 700 base pair donor fragment was cloned into pCR4BluntTopo, which does not contain any sequences homologous to the human genome. See FIG. 4 .
  • the two plasmids encoding ZFP-FokI fusions and the donor plasmid were introduced into 1 ⁇ 10 6 HEK293 cells by transfection using Lipofectamine 2000® (Invitrogen). Controls included cells transfected only with the two plasmids encoding the ZFP-FokI fusions, cells transfected only with the donor plasmid and cells transfected with a control plasmid (pEGFP-N1, Clontech). Cells were cultured in 5% CO 2 at 37° C.
  • genomic DNA was isolated from the cells, and 200 ng was used as template for PCR amplification, using one primer complementary to a region of the gene outside of its region of homology with the donor sequences (nucleotides 52416677-52416701 on the “ ⁇ ” STRAND of the X chromosome; UCSC July 2003), and a second primer complementary to a region of the donor molecule into which distinguishing mutations were introduced.
  • an amplification product of 400 base pairs will be obtained from genomic DNA if a targeted recombination event has occurred.
  • the sequences of these primers are given in Table 3 (labeled “chromosome-specific” and “donor-specific,” respectively).
  • Conditions for amplification were: 94° C., 2 min, followed by 40 cycles of 94° C., 30 sec, 60° C., 1 min, 72° C, 1 min; and a final step of 72° C., 7 min.
  • FIG. 5 The results of this analysis ( FIG. 5 ) indicate that a 400 base pair amplification product (labeled “Chimeric DNA” in the Figure) was obtained only with DNA extracted from cells which had been transfected with the donor plasmid and both ZFP-FokI plasmids.
  • the amplification product was cloned into pCR4Blunt-Topo (Invitrogen) and its nucleotide sequence was determined.
  • FIG. 6 SEQ ID NO: 6
  • the amplified sequence obtained from chromosomal DNA of cells transfected with the two ZFP-FokI-encoding plasmids and the donor plasmid contains the AAGAAGC sequence that is unique to the donor (nucleotides 395-401 of the sequence presented in FIG. 6 ) covalently linked to chromosomal sequences not present in the donor molecule (nucleotides 32-97 of FIG. 6 ), indicating that donor sequences have been recombined into the chromosome.
  • the G ⁇ A mutation converting the initiation codon to an isoleucine codon is observed at position 395 in the sequence.
  • chromosomal DNA from cells transfected only with donor plasmid, cells transfected with both ZFP-FokI fusion plasmids, cells transfected with the donor plasmid and both ZFP-FokI fusion plasmids or cells transfected with the EGFP control plasmid was used as template for amplification, using primers complementary to sequences outside of the 700-nucleotide region of homology between donor and chromosomal sequences (identified as “Outside 1” and “Outside 2” in Table 3).
  • the resulting amplification product was purified and used as template for a second amplification reaction using the donor-specific and chromosome-specific primers described above (Table 3). This amplification yielded a 400 nucleotide product only from cells transfected with the donor construct and both ZFP-FokI fusion constructs, a result consistent with the replacement of genomic sequences by targeted recombination in these cells.
  • the IL-2R ⁇ gene encodes a protein, known as the “common cytokine receptor gamma chain,” that functions as a subunit of several interleukin receptors (including IL-2R, IL-4R, IL-7R, IL-9R, IL-15R and IL-21R). Mutations in this gene, including those surrounding the 5′ end of the third exon (e.g. the tyrosine 91 codon), can cause X-linked severe combined immunodeficiency (SCID). See, for example, Puck et al. (1997) Blood 89:1968-1977. A mutation in the tyrosine 91 codon (nucleotides 23-25 of SEQ ID NO: 7; FIG.
  • the first pair (first two rows of Table 4) comprises a zinc finger protein designed to bind to nucleotides 29-40 (primary contacts along the top strand as shown in FIG. 7 ) and a zinc finger protein designed to bind to nucleotides 8-20 (primary contacts along the bottom strand).
  • the second pair (third and fourth rows of Table 4) comprises two zinc finger proteins, the first of which recognizes nucleotides 23-34 (primary contacts along the top strand as shown in FIG. 7 ) and the second of which recognizes nucleotides 8-16 (primary contacts along the bottom strand).
  • Zinc finger proteins were designed as described in co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261. See Table 4 for the amino acid sequences of the recognition regions of the zinc finger proteins.
  • Sequences encoding the ZFP binding domains were fused to sequences encoding a FokI cleavage half-domain (amino acids 384-579 of the native FokI sequence, Kita et al., supra), such that the encoded protein contained FokI sequences at the carboxy terminus and ZFP sequences at the amino terminus.
  • Each of these fusion sequences was then cloned in a modified mammalian expression vector pcDNA3. See FIG. 8 for a schematic diagram of the constructs.
  • a donor DNA molecule was obtained as follows. First, a 700 base pair fragment of human DNA corresponding to positions 69196910-69197609 on the “ ⁇ ” strand of the X chromosome (UCSC, July 2003), which includes exon 3 of the of the IL2R ⁇ gene, was amplified, using genomic DNA from K562 cells as template. See FIG. 9 . Sequences of primers used for amplification are shown in Table 5 (labeled initial amp 1 and initial amp 2). The PCR product was then altered via standard overlap extension PCR methodology (Ho, et al., supra) to replace the sequence TACAAGAACTCGGATAAT (SEQ ID NO:62) with the sequence TAAAAGAATTCCGACAAC (SEQ ID NO:63).
  • the donor plasmid For targeted mutation of the chromosomal IL2R ⁇ gene, the donor plasmid, along with two plasmids each encoding one of a pair of ZFP-FokI fusions, were introduced into 2 ⁇ 10 6 K652 cells using mixed lipofection/electroporation (Amaxa). Each of the ZFP/FokI pairs (see Table 4) was tested in separate experiments. Controls included cells transfected only with two plasmids encoding ZFP-FokI fusions, and cells transfected only with the donor plasmid. Cells were cultured in 5% CO 2 at 37° C.
  • genomic DNA was isolated from the cells, and 200 ng was used as template for PCR amplification, using one primer complementary to a region of the gene outside of its region of homology with the donor sequences (nucleotides 69196839-69196863 on the “+” strand of the X chromosome; UCSC, July 2003), and a second primer complementary to a region of the donor molecule into which distinguishing mutations were introduced (see above) and whose sequence therefore diverges from that of chromosomal DNA. See Table 5 for primer sequences, labeled “chromosome-specific” and “donor-specific,” respectively.
  • an amplification product of 500 bp is obtained from genomic DNA in which a targeted recombination event has occurred.
  • Conditions for amplification were: 94° C., 2 min, followed by 35 cycles of 94° C., 30 sec, 62° C., 1 min, 72° C., 45 sec; and a final step of 72° C., 7 min.
  • FIG. 11 The results of this analysis ( FIG. 11 ) indicate that an amplification product of the expected size (500 base pairs) is obtained with DNA extracted from cells which had been transfected with the donor plasmid and either of the pairs of ZFP-FokI-encoding plasmids. DNA from cells transfected with plasmids encoding a pair of ZFPs only (no donor plasmid) did not result in generation of the 500 bp product, nor did DNA from cells transfected only with the donor plasmid.
  • the amplification product obtained from the experiment using the second pair of ZFP/FokI fusions was cloned into pCR4Blunt-Topo (Invitrogen) and its nucleotide sequence was determined.
  • the sequence consists of a fusion between chromosomal sequences and sequences from the donor plasmid.
  • the G to A mutation converting tyrosine 91 to a stop codon is observed at position 43 in the sequence.
  • Positions 43-58 contain nucleotides unique to the donor; nucleotides 32-42 and 59-459 are sequences common to the donor and the chromosome, and nucleotides 460-552 are unique to the chromosome.
  • the presence of donor-unique sequences covalently linked to sequences present in the chromosome but not in the donor indicates that DNA from the donor plasmid was introduced into the chromosome by homologous recombination.
  • the human beta globin gene is one of two gene products responsible for the structure and function of hemoglobin in adult human erythrocytes. Mutations in the beta-globin gene can result in sickle cell anemia. Two zinc finger proteins were designed to bind within this sequence, near the location of a nucleotide which, when mutated, causes sickle cell anemia.
  • FIG. 13 shows the nucleotide sequence of a portion of the human beta-globin gene, and the target sites for the two zinc finger proteins are underlined in the sequence presented in FIG. 13 . Amino acid sequences of the recognition regions of the two zinc finger proteins are shown in Table 6.
  • Sequences encoding each of these two ZFP binding domains were fused to sequences encoding a FokI cleavage half-domain, as described above, to create engineered ZFP-nucleases that targeted the endogenous beta globin gene. Each of these fusion sequences was then cloned in the mammalian expression vector pcDNA3.1 ( FIG. 14 ).
  • a donor DNA molecule was obtained as follows. First, a 700 base pair fragment of human genomic DNA corresponding to nucleotides 5212134- 5212833 on the “ ⁇ ” strand of Chromosome 11 (BLAT, UCSC Human Genome site) was amplified by PCR, using genomic DNA from K562 cells as template. Sequences of primers used for amplification are shown in Table 7 (labeled initial amp 1 and initial amp 2). The resulting amplified fragment contains sequences corresponding to the promoter, the first two exons and the first intron of the human beta globin gene. See FIG. 15 for a schematic illustrating the locations of exons 1 and 2, the first intron, and the primer binding sites in the beta globin sequence.
  • the cloned product was then further modified by PCR to introduce a set of sequence changes between nucleotides 305-336 (as shown in FIG. 13 ), which replaced the sequence CCGTTACTGCCCTGTGGGGCAAGGTGAACGTG (SEQ ID NO: 78) with gCGTTAgTGCCCGAATTCCGAtcGTcAACcac (SEQ ID NO: 79) (changes in bold). Certain of these changes (shown in lowercase) were specifically engineered to prevent the ZFP/FokI fusion proteins from binding to and cleaving the donor sequence, once integrated into the chromosome. In addition, all of the sequence changes enable discrimination between donor and endogenous chromosomal sequences following recombination. The resulting 700 base pair fragment was cloned into pCR4-TOPO, which does not contain any sequences homologous to the human genome ( FIG. 16 ).
  • the two plasmids encoding ZFP-FokI fusions and the donor plasmid were introduced into 1 ⁇ 10 6 K562 cells by transfection using NucleofectorTM Solution (Amaxa Biosystems). Controls included cells transfected only with 100 ng (low) or 200 ng (high) of the two plasmids encoding the ZFP-FokI fusions, cells transfected only with 200 ng (low) or 600 ng (high) of the donor plasmid, cells transfected with a GFP-encoding plasmid, and mock transfected cells.
  • FIG. 17 The results of this analysis ( FIG. 17 ) indicate that a 415 base pair amplification product was obtained only with DNA extracted from cells which had been transfected with the “high” concentration of donor plasmid and both ZFP-FokI plasmids, consistent with targeted recombination of donor sequences into the chromosomal beta-globin locus.
  • the amplification product was cloned into pCR4-TOPO (Invitrogen) and its nucleotide sequence was determined.
  • the sequence consists of a fusion between chromosomal sequences not present on the donor plasmid and sequences unique to the donor plasmid. For example, two C ⁇ G mutations which disrupt ZFP-binding are observed at positions 377 and 383 in the sequence.
  • Nucleotides 377-408 represent sequence obtained from the donor plasmid containing the sequence changes described above; nucleotides 73-376 are sequences common to the donor and the chromosome, and nucleotides 1-72 are unique to the chromosome.
  • the covalent linkage of donor-specific and chromosome-specific sequences in the genome confirms the successful recombination of the donor sequence at the correct locus within the genome of K562 cells.
  • the target site for the ZFP is 5′-AACTCGGATAAT-3′ (SEQ ID NO:84) and the amino acid sequences of the recognition regions (positions -1 through +6 with respect to the start of the alpha-helix) of each of the zinc fingers were as follows (wherein F1 is the N-most, and F4 is the C-most zinc finger): F1: DRSTLIE (SEQ ID NO:85) F2: SSSNLSR (SEQ ID NO:86) F3: RSDDLSK (SEQ ID NO:87) F4: DNSNRIIK (SEQ ID NO:88)
  • ZFP-FokI fusions in which the aforementioned ZFP binding domain and a FokI cleavage half-domain were separated by 2, 3, 4, 5, 6, or 10 amino acid residues, were constructed. Each of these proteins was tested for cleavage of substrates having an inverted repeat of the ZFP target site, with repeats separated by 4, 5, 6, 7, 8, 9, 12, 15, 16, 17, 22, or 26 basepairs.
  • amino acid sequences of the fusion constructs are as follows: 10-residue linker HTKIH LRQKDAARGS QLV (SEQ ID NO:89) 6-residue linker HTKIH LRQKGS QLV (SEQ ID NO:90) 5-residue linker HTKIH LRQGS QLV (SEQ ID NO:91) 4-residue linker HTKIH LRGS QLV (SEQ ID NO:92) 3-residue linker HTKIH LGS QLV (SEQ ID NO:93) 2-residue linker HTKIH GS QLV (SEQ ID NO:94)
  • sequences of the various cleavage substrates, with the ZFP target sites underlined are as follows: 4bp separation CTAGCATTATCCGAGTTACAC AACTCGGATAAT G CTAGGATCG TAATAGGCTCAA TGTGTTGAGCCTA TTACGATC (SEQ ID NO:95) 5bp separation CTAGCATTATCCGAGTTCACAC AACTCGGATAAT G CTAGGATCG TAATAGGCTCAA GTGTGTTGAGCCTA TTACGATC (SEQ ID NO:96) 6bp separation CTAGGCATTATCCGAGTTCACCAC AACTCGGATAA T GACTAGGATCCG TAATAGGCTCAA GTGGTGTTG AGCCTATTACTGATC (SEQ ID NO:97) 7bp separation CTAGCATTATCCGAGTTCACACAC AACTCGGATA AT GCTAGGATCG TAATAGGCTCAA GTGTGTGTTG AGCCTATTACGATC (SEQ ID NO:98) 8bp separation CTAGCATTATCCGA
  • Plasmids encoding the different ZFP-FokI fusion proteins were constructed by standard molecular biological techniques, and an in vitro coupled transcription/translation system was used to express the encoded proteins. For each construct, 200 ng linearized plasmid DNA was incubated in 20 ⁇ L TnT mix and incubated at 30° C. for 1 hour and 45 minutes. TnT mix contains 100 ⁇ l TnT lysate (Promega, Madison, Wis.) with 4 ⁇ l T7 RNA polymerase (Promega)+2 ⁇ l Methionine (1 mM)+2.5 ⁇ l ZnCl 2 (20 mM).
  • FokI Cleavage buffer contains 20 mM Tris-HCl pH 8.5, 75 mM NaCl, 10 ⁇ M ZnCl 2 , 1 mM DTT, 5% glycerol, 500 ⁇ g/ml BSA. The mixture was incubated for 1 hour at 37° C.
  • the gel was subjected to autoradiography, and the cleavage efficiency for each ZFP-FokI fusion/substrate pair was calculated by quantifying the radioactivity in bands corresponding to uncleaved and cleaved substrate, summing to obtain total radioactivity, and determining the percentage of the total radioactivity present in the bands representing cleavage products.
  • the amino acid sequence of the linker was also varied.
  • the original LRGS linker sequence (SEQ ID NO:107) was changed to LGGS (SEQ ID NO:108), TGGS (SEQ ID NO:109), GGGS (SEQ ID NO:110), LPGS (SEQ ID NO: 111), LRKS (SEQ ID NO:112), and LRWS (SEQ ID NO:113); and the resulting fusions were tested on substrates having a six-basepair separation between binding sites. Fusions containing the LGGS (SEQ ID NO:108) linker sequence were observed to cleave more efficiently than those containing the original LRGS sequence(SEQ ID NO:107).
  • a pair of ZFP/FokI fusion proteins (denoted 5-8 and 5-10) were designed to bind to target sites in the fifth exon of the IL-2R ⁇ gene, to promote cleavage in the region between the target sites.
  • the amino acid sequence of the 5-8 protein is shown in FIG. 20
  • the amino acid sequence of the 5-10 protein is shown in FIG. 21 .
  • Both proteins contain a 10 amino acid ZC linker.
  • the DNA target sequences, as well as amino acid sequences of the recognition regions in the zinc fingers are given in Table 9.
  • 5-10 was modified in its FokI cleavage half-domain by converting amino acid residue 490 from glutamic acid (E) to lysine (K). (Numbering of amino acid residues in the FokI protein is according to Wah et al., supra.) This modification was designed to prevent homodimerization by altering an amino acid residue in the dimerization interface.
  • the 5-10 (E490K) mutant unlike the parental 5-10 protein, was unable to cleave at aberrant sites in the absence of the 5-8 fusion protein (Table 10, Row 3). However, the 5-10 (E490K) mutant, together with the 5-8 protein, catalyzed specific cleavage of the substrate (Table 10, Row 4).
  • the 5-8 protein was modified in its dimerization interface by replacing the glutamine (Q) residue at position 486 with glutamic acid (E).
  • This 5-8 (Q486E) mutant was tested for its ability to catalyze targeted cleavage in the presence of either the wild-type 5-10 protein or the 5-10 (E490K) mutant. DNA cleavage was not observed when the labeled substrate was incubated in the presence of both 5-8 (Q486E) and wild-type 5-10 (Table 10, Row 5). However, cleavage was obtained when the 5-8 (Q486E) and 5-10 (E490K) mutants were used in combination (Table 10, Row 6).
  • One of the fusion proteins contained the 5-8 DNA binding domain, and the other fusion protein contained the 5-10 DNA binding domain (See Table 9 and FIG. 19 ).
  • the cleavage half-domain portion of the fusion proteins was as indicated in the Table. #
  • the entries in the ZFP 5-8 column indicate the type of FokI cleavage domain fused to ZFP 5-8; and the entries in the ZFP 5-10 column indicates the type of FokI cleavage domain fused to ZFP 5-10.
  • the number refers to the amino acid residue in the FokI protein; the letter preceding the number refers to the amino acid present in the wild-type protein # and the letter following the number denotes the amino acid to which the wild-type residue was changed in generating the modified protein. ‘Not present’ indicates that the entire ZFP/FokI fusion protein was omitted from that particular experiment.
  • the DNA substrate used in this experiment was an approximately 400 bp PCR product containing the target sites for both ZFP 5-8 and ZFP 5-10. See FIG. 19 for the sequences and relative orientation of the two target sites.
  • eGFP Enhanced Green Fluorescent Protein
  • the enhanced Green Fluorescent Protein is a modified form of the Green Fluorescent Protein (GFP; see, e.g., Tsien (1998) Ann. Rev. Biochem. 67:509-544) containing changes at amino acid 64 (phe to leu) and 65 (ser to thr).
  • eGFP-based reporter system was constructed by generating a defective form of the eGFP gene, which contained a stop codon and a 2-bp frameshift mutation. The sequence of the eGFP gene is shown in FIG. 22 .
  • the mutations were inserted by overlapping PCR mutagenesis, using the Platinum® Taq DNA Polymerase High Fidelity kit (Invitrogen) and the oligonucleotides GFP-Bam, GFP-Xba, stop sense2, and stop anti2 as primers (oligonucleotide sequences are listed below in Table 11).
  • GFP-Bam and GFP-Xba served as the external primers, while the primers stop sense2 and stop anti2 served as the internal primers encoding the nucleotide changes.
  • the peGFP-NI vector (BD Biosciences), encoding a full-length eGFP gene, was used as the DNA template in two separate amplification reactions, the first utilizing the GFP-Bam and stop anti2 oligonucleotides as primers and the second using the GFP-Xba and stop sense2 oligonucleotides as primers. This generated two amplification products whose sequences overlapped.
  • ZFP 287A binds nucleotides 271-279 on the non-coding strand
  • ZFP 296 binds nucleotides 286-294 on the coding strand.
  • the DNA target and amino acid sequence for the recognition regions of the ZFPs are listed below, and in Table 12: TABLE 12 Zinc finger designs for the GFP gene Protein Target sequence F1 F2 F3 287A GGGGTAGCGg RSDDLTR QSGALAR RSDHLSR (SEQ ID NO:136) (SEQ ID NO:137) (SEQ ID NO:138) (SEQ ID NO:139) 296S GAAGCAGCA QSGSLTR QSGDLTR QSGNLAR (SEQ ID NO:140) (SEQ ID NO:141) (SEQ ID NO:142) (SEQ ID NO:143)
  • the zinc finger amino acid sequences shown above represent residues ⁇ 1 through +6, with respect to the start of the alpha-helical portion of each zinc finger.
  • Finger F1 is closest to the amino terminus of the protein, and Finger F3 is closest to the carboxy terminus.
  • F1 GCGg
  • RSDDLTR SEQ ID NO:130
  • F2 GTA
  • QSGALAR SEQ ID NO:131
  • F3 GGG
  • RSDHLSR SEQ ID NO:132
  • Sequences encoding these proteins were generated by PCR assembly (e.g., U.S. Pat. No. 6,534,261), cloned between the KpnI and BamHI sites of the pcDNA3.1 vector (Invitrogen), and fused in frame with the catalytic domain of the FokI endonuclease (amino acids 384-579 of the sequence of Looney et al. (1989) Gene 80:193-208).
  • the resulting constructs were named pcDNA3.1-GFP287-FokI and pcDNA3.1-GFP296-FokI ( FIG. 24 ).
  • the pCR(R)4-TOPO-GFPmut construct (Example 6) was used to provide a template for testing the ability of the 287 and 296 zinc finger proteins to specifically recognize their target sites and cleave this modified form of eGFP in vitro.
  • a DNA fragment containing the defective eGFP-encoding insert was obtained by PCR amplification, using the T7 and T3 universal primers and pCR(R)4-TOPO-GFPmut as template. This fragment was end-labeled using ⁇ - 32 P-ATP and T4 polynucleotide kinase. Unincorporated nucleotide was removed using a microspin G-50 column (Amersham).
  • TnT mix contains 100 ⁇ l TnT lysate (which includes T7 RNA polymerase, Promega, Madison, Wis.) supplemented with 2 ⁇ l Methionine (1 mM) and 2.5 ⁇ l ZnCl 2 (20 mM).
  • Cleavage buffer contains 20 mM Tris-HCl pH 8.5, 75 mM NaCl, 10 mM MgCl 2 , 10 ⁇ M ZnCl 2 , 1 mM DTT, 5% glycerol, 500 ⁇ g/ml BSA.
  • each dilution was combined with approximately 1 ng DNA substrate (end-labeled with 32 P using T4 polynucleotide kinase as described above), and each mixture was further diluted to generate a 20 ⁇ l cleavage reaction having the following composition: 20 mM Tris-HCl pH 8.5, 75 mM NaCl, 10 mM MgCl 2 , 10 ⁇ M ZnCl 2 , 1 mM DTT, 5% glycerol, 500 ⁇ g/ml BSA. Cleavage reactions were incubated for 1 hour at 37° C. Protein was extracted by adding 10 ⁇ l phenol-chloroform solution to each reaction, mixing, and centrifuging to separate the phases. Ten microliters of the aqueous phase from each reaction was analyzed by electrophoresis on a 10% polyacrylamide gel.
  • the gel was subjected to autoradiography, and the results of this experiment are shown in FIG. 25 .
  • the four left-most lanes show the results of reactions in which the final dilution of each coupled transcription/translation reaction mixture (in the cleavage reaction) was 1/156.25, 1/31.25, 1/12.5 and 1/5, respectively, resulting in effective volumes of 0.032, 0.16, 04. and 1 ul, respectively of each coupled transcription/translation reaction.
  • the appearance of two DNA fragments having lower molecular weights than the starting fragment (lane labeled “uncut control” in FIG. 25 ) is correlated with increasing amounts of the 287 and 296 zinc finger endonucleases in the reaction mixture, showing that DNA cleavage at the expected target site was obtained.
  • a DNA fragment encoding the mutated eGFP, eGFPmut was cleaved out of the pCR(R)4-TOPO-GFPmut vector (Example 6) and cloned into the HindIII and NotI sites of pcDNA4/TO, thereby placing this gene under control of a tetracycline-inducible CMV promoter.
  • the resulting plasmid was named pcDNA4/TO/GFPmut ( FIG. 26 ).
  • T-Rex 293 cells (Invitrogen) were grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% Tet-free fetal bovine serum (FBS) (HyClone).
  • Cells were plated into a 6-well dish at 50% confluence, and two wells were each transfected with pcDNA4/TO/GFPmut. The cells were allowed to recover for 48 hours, then cells from both wells were combined and split into 10 ⁇ 15-cm 2 dishes in selective medium, i.e., medium supplemented with 400 ug/ml Zeocin (Invitrogen). The medium was changed every 3 days, and after 10 days single colonies were isolated and expanded further. Each clonal line was tested individually for doxycycline(dox)-inducible expression of the eGFPmut gene by quantitative RT-PCR (TaqMan®).
  • PCR Polymerase chain reaction
  • eGFP primer 1 CTGCTGCCCGACAACCA (SEQ ID NO:144)
  • eGFP primer 2 (3T) CCATGTGATCGCGCTTCTC (SEQ ID NO:145) eGFP probe CCCAGTCCGCCCTGAGCAAAGA (SEQ ID NO:146)
  • GAPDH primer 1 CCATGTTCGTCATGGGTGTGA (SEQ ID NO:147)
  • GAPDH primer 2 CATGGACTGTGGTCATGAGT (SEQ ID NO:148) GAPDH probe TCCTGCACCACCAACTGCTTAGCA (SEQ ID NO:149)
  • a donor construct containing the genetic information for correcting the defective eGFPmut gene was constructed by PCR.
  • the PCR reaction was carried out as described above, using the peGFP-NI vector as the template.
  • the first 12 bp and start codon were removed from the donor by PCR using the primers GFPnostart and GFP-Xba (sequences provided in Table 14).
  • the resulting PCR fragment (734 bp) was cloned into the pCR(R)4-TOPO vector, which does not contain a mammalian cell promoter, by TOPO-TA cloning to create pCR(R)4-TOPO-GFPdonor5 ( FIG. 28 ).
  • FIG. 29 The sequence of the eGFP insert of this construct (corresponding to nucleotides 64-797 of the sequence shown in FIG. 22 ) is shown in FIG. 29 (SEQ ID NO:20).
  • Oligonucleotides for construction of donor molecule Oligonucleotide Sequence 5′-3′ GFPnostart GGCGAGGAGCTGTTCAC (SEQ ID NO:150) GFP-Xba GATTATGATCTAGAGTCG (SEQ ID NO:151)
  • the T18 stable cell line (Example 9) was transfected with one or both of the ZFP-FokI expression plasmid (pcDNA3.1-GFP287-FokI and pcDNA3.1-GFP296-FokI, Example 7) and 300 ng of the donor plasmid pCR(R)4-TOPO-GFPdonor5 (Example 10) using LipofectAMINE 2000 Reagent (Invitrogen) in Opti-MEM I reduced serum medium, according to the manufacturer's protocol. Expression of the defective chromosomal eGFP gene was induced 5-6 hours after transfection by the addition of 2 ng/ml doxycycline to the culture medium.
  • the cells were arrested in the G2 phase of the cell cycle by the addition, at 24 hours post-transfection, of 100 ng/ml Nocodazole ( FIG. 30 ) or 0.2 ⁇ M Vinblastine ( FIG. 31 ). G2 arrest was allowed to continue for 24-48 hours, and was then released by the removal of the medium. The cells were washed with PBS and the medium was replaced with DMEM containing tetracycline-free FBS and 2 ng/ml doxycycline. The cells were allowed to recover for 24-48 hours, and gene correction efficiency was measured by monitoring the number of cells exhibiting eGFP fluorescence, by fluorescence-activated cell sorting (FACS) analysis.
  • FACS fluorescence-activated cell sorting
  • FACS analysis was carried out using a Beckman-Coulter EPICS XL-MCL instrument and System II Data Acquisition and Display software, version 2.0.
  • eGFP fluorescence was detected by excitation at 488 nm with an argon laser and monitoring emissions at 525 nm (x-axis). Background or autofluorescence was measured by monitoring emissions at 570 nm (y-axis). Cells exhibiting high fluorescent emission at 525 nm and low emission at 570 nm (region E) were scored positive for gene correction.
  • FIGS. 30 and 31 show results in which T18 cells were transfected with the pcDNA3.1-GFP287-FokI and pcDNA3.
  • Cells were optionally arrested in G2 phase of the cell cycle after eGFP induction. FACS analysis was conducted 5 days after transfection. 2 The number is the percent of total fluorescence exhibiting high emission at 525 nm and low emission at 570 nm (region E of the FACS trace).
  • Zinc finger nucleases whose sequences had been altered in the dimerization interface were tested for their ability to catalyze correction of a defective chromosomal eGFP gene.
  • the protocol described in Example 11 was used, except that the nuclease portion of the ZFP nucleases (i.e., the FokI cleavage half-domains) were altered as described in Example 5.
  • an E490K cleavage half-domain was fused to the GFP96 ZFP domain (Table 12), and a Q486E cleavage half-domain was fused to the GFP287 ZFP (Table 12).
  • T18 cells were transfected with the two ZFP nucleases, and eGFP expression was induced with doxycycline, as in Example 11.
  • Cells were also transfected with either the pCR(R)4-TOPO-GFPdonor5 plasmid ( FIG. 28 ) containing a 734 bp eGFP insert ( FIG. 29 ) as in Example 11, or a similar plasmid containing a 1527 bp sequence insert ( FIG. 32 ) homologous to the mutated chromosomal eGFP gene. Additionally, the effect of G2 arrest with nocodazole on recombination frequency was assessed.
  • T18 cells were transfected with 50 ng of the 287-FokI and 296-FokI expression plasmids (Example 7, Table 12) and 500 ng of a 0.7 kbp, 1.08 kbp, or 1.5 kbp donors, as described in Example 11.
  • Four days after transfection cells were assayed for correction of the defective eGFP gene by FACS, monitoring GFP fluorescence.
  • Each ZFP-nuclease contained a zinc finger protein-based DNA binding domain (see Table 17) fused to the nuclease domain of the type IIS restriction enzyme FokI (amino acids 384-579 of the sequence of Looney et al. (1989) Gene 80:193-208) via a four amino acid ZC linker (see Example 4).
  • FokI amino acids 384-579 of the sequence of Looney et al. (1989) Gene 80:193-208
  • the nucleases were designed to bind to positions in exon 5 of the chromosomal IL-2R ⁇ gene surrounding codons 228 and 229 (a mutational hotspot in the gene) and to introduce a double-strand break in the DNA between their binding sites.
  • each of the chimeric endonucleases was as follows: (SEQ ID NO:152) Nuclease targeted to ACTCTGTGGAAG (SEQ ID NO:162) MAERPFQCRICMRINFSRSDNLSVHIRTHTGEKPFACDICGRKFARNAHR INHTKIHTGSQKPFQCRICMRNFSRSDTLSEHIRTHTGEKPFACDICGRK FAARSTRTNHTKIHLRGS (SEQ ID NO:157) Nuclease targeted to AAAGCGGCTCCG (SEQ ID NO:163) MAERPFQCRICMRNFSRSDTLSEHIRTHTGEKPFACDICGRKFAARSTRT THTKIHTGSQKPFQCRICMRNFSRSDSLSKHIRTHTGEKPFACDICGRKF AQRSNLKVHTKIHLRGS
  • Human embryonic kidney 293 cells were transfected (Lipofectamine 2000; Invitrogen) with two expression constructs, each encoding one of the ZFP-nucleases described in the preceding paragraph.
  • the cells were also transfected with a donor construct carrying as an insert a 1,543 bp fragment of the IL2R ⁇ locus corresponding to positions 69195166-69196708 of the “minus” strand of the X chromosome (UCSC human genome release July 2003), in the pCR4Blunt Topo (Invitrogen) vector.
  • the IL-2R ⁇ insert sequence contained the following two point mutations in the sequence of exon 5 (underlined): F R V R S R F N P L C G S (SEQ ID NO:164) TTTCGTGTTCGGAGCCG G TTTAACCC G CTCTGTGGAAGT (SEQ ID NO:165)
  • the first mutation does not change the amino acid sequence (upper line) and serves to adversely affect the ability of the ZFP-nuclease to bind to the donor DNA, and to chromosomal DNA following recombination.
  • the second mutation does not change the amino acid sequence and creates a recognition site for the restriction enzyme BsrBI.
  • This treatment was performed to enhance the frequency of targeting because the homology-directed double-stranded break repair pathway is more active than non-homologous end-joining in the G 2 phase of the cell cycle.
  • growth medium was replaced, and the cells were allowed to recover from vinblastine treatment for an additional 24 hours.
  • Genomic DNA was then isolated from all cell samples using the DNEasy Tissue Kit (Qiagen). Five hundred nanograms of genomic DNA from each sample was then assayed for frequency of gene targeting, by testing for the presence of a new BsrBI site in the chromosomal IL-2R ⁇ locus, using the assay described schematically in FIG. 33 .
  • K562 is a cell line derived from a human chronic myelogenous leukemia.
  • the proteins used for targeted cleavage were FokI fusions to the 5-8G and 5-9D zinc finger DNA-binding domains (Example 14, Table 17).
  • the donor sequence was the 1.5 kbp fragment of the human IL-2R ⁇ gene containing a BsrBI site introduced by mutation, described in Example 14.
  • K562 cells were cultured in RPMI Medium 1640 (Invitrogen), supplemented with 10% fetal bovine serum (FBS) (Hyclone) and 2 mM L-glutamine. All cells were maintained at 37° C. in an atmosphere of 5% CO 2 . These cells were transfected by NucleofectionTM (Solution V, Program T16) (Amaxa Biosystems), according to the manufacturers' protocol, transfecting 2 million cells per sample.
  • FBS fetal bovine serum
  • DNAs for transfection used in various combinations as described below, were a plasmid encoding the 5-8G ZFP-FokI fusion endonuclease, a plasmid encoding the 5-9D ZFP-FokI fusion endonuclease, a plasmid containing the donor sequence (described above and in Example 14) and the peGFP-N1 vector (BD Biosciences) used as a control.
  • Vinblastine-treated cells were exposed to 0.2 ⁇ M vinblastine at 24 hours after transfection for 30 hours. The cells were collected, washed twice with PBS, and re-growth plated in growth medium. Cells were harvested 4 days after transfection for analysis of genomic DNA.
  • Genomic DNA was extracted from the cells using the DNEasy kit (Qiagen). One hundred nanograms of genomic DNA from each sample were used in a PCR reaction with the following primers: (SEQ ID NO:168) Exon 5 forward: GCTAAGGCCAAGAAAGTAGGGCTAAAG (SEQ ID NO:169) Exon 5 reverse: TTCCTTCCATCACCAAACCCTCTTG
  • primers amplify a 1,669 bp fragment of the X chromosome corresponding to positions 69195100-69196768 on the “ ⁇ ” strand (UCSC human genome release July 2003) that contain exon 5 of the IL2R ⁇ gene.
  • Amplification of genomic DNA which has undergone homologous recombination with the donor DNA yields a product containing a BsrBI site; whereas the amplification product of genomic DNA which has not undergone homologous recombination with donor DNA will not contain this restriction site.
  • DNA from transfected cells in this second experiment was analyzed by Southern blotting.
  • twelve micrograms of genomic DNA from each sample were digested with 100 units EcoRI, 50 units BsrBI, and 40 units of DpnI (all from New England Biolabs) for 12 hours at 37° C. This digestion generates a 7.7 kbp Eco RI fragment from the native IL-2R ⁇ gene (lacking a BsrBI site) and fragments of 6.7 and 1.0 kbp from a chromosomal IL-2R ⁇ gene whose sequence has been altered, by homologous recombination, to include the BsrBI site.
  • DpnI a methylation-dependent restriction enzyme, was included to destroy the dam-methylated donor DNA. Unmethylated K562 cell genomic DNA is resistant to DpnI digestion.
  • genomic DNA was purified by phenol-chloroform extraction and ethanol precipitation, resuspended in TE buffer, and resolved on a 0.8% agarose gel along with a sample of genomic DNA digested with EcoRI and SphI to generate a size marker.
  • the gel was processed for alkaline transfer following standard procedure and DNA was transferred to a nylon membrane (Schleicher and Schuell). Hybridization to the blot was then performed by using a radiolabelled fragment of the IL-2R ⁇ locus corresponding to positions 69198428-69198769 of the “ ⁇ ” strand of the X chromosome (UCSC human genome July 2003 release). This region of the gene is outside of the region homologous to donor DNA.
  • the membrane was exposed to a Phosphorimager plate and the data quantitated using Molecular Dynamics software. Alteration of the chromosomal IL-2R ⁇ sequence was measured by analyzing the intensity of the band corresponding to the EcoRI-BsrBI fragment (arrow next to autoradiograph; BsrBI site indicated by filled triangle in the map above the autoradiograph).
  • results, shown in FIG. 37 indicate up to 15% of chromosomal IL-2R ⁇ sequences were altered by homologous recombination, thereby confirming the results obtained by PCR analysis that the targeted recombination event was stable through multiple rounds of cell division.
  • the Southern blot results also indicate that the results shown in FIG. 36 do not result from an amplification artifact.
  • Bone marrow-derived human CD34 cells were purchased from AllCells, LLC and shipped as frozen stocks. These cells were thawed and allowed to stand for 2 hours at 37° C. in an atmosphere of 5% CO 2 in RPMI Medium 1640 (Invitrogen), supplemented with 10% fetal bovine serum (FBS) (Hyclone) and 2 mM L-glutamine. Cell samples (1 ⁇ 10 6 or 2 ⁇ 10 6 cells) were transfected by NucleofectionTM (amaxa biosystems) using the Human CD34 Cell NucleofectorTM Kit, according to the manufacturers' protocol.
  • FBS fetal bovine serum
  • Genomic DNA was extracted from the cells using the MasterPure DNA Purification Kit (Epicentre). Due to the presence of glycogen in the precipitate, accurate quantitation of this DNA used as input in the PCR reaction is impossible; estimates using analysis of ethidium bromide-stained agarose gels indicate that ca. 50 ng genomic DNA was used in each sample.
  • ⁇ - 32 PdCTP and ⁇ - 32 PdATP were included in each PCR reaction to allow detection of PCR products.
  • this SNP creates a RFLP by destroying a MaeII site that is present in normal human DNA.
  • a reference standard was therefore created by adding 1 or 10 nanograms of normal human genomic DNA (obtained from Clontech, Palo Alto, Calif.) to 100 or 90 ng of Jurkat genomic DNA, respectively, and performing the PCR as described above.
  • PCR reactions were desalted on a G-50 column (Amersham), and digested for 1 hour with restriction enzyme: experimental samples were digested with 10 units of BsrBI (New England Biolabs); the “reference standard” reactions were digested with MaeII. The digestion products were resolved on a 10% non-denaturing PAGE (BioRad), the gel dried and analyzed by exposure to a PhosphorImager plate (Molecular Dynamics).
  • the targeting frequency was estimated, by comparison to the reference standard (left panel), to be between 1-5%.
  • T18 cell line contains a chromosomally integrated defective eGFP gene, and the donor DNA contains sequence changes, with respect to the chromosomal gene, that correct the defect.
  • the donor sequence described in Example 10 was modified, by PCR mutagenesis, to generate a series of ⁇ 700 bp donor constructs with different degrees of non-homology to the target.
  • All of the modified donors contained sequence changes that corrected the defect in the chromosomal eGFP gene and contained additional silent mutations (DNA mutations that do not change the sequence of the encoded protein) inserted into the coding region surrounding the cleavage site.
  • These silent mutations were intended to prevent the binding to, and cleavage of, the donor sequence by the zinc finger-cleavage domain fusions, thereby reducing competition between the intended chromosomal target and the donor plasmid for binding by the chimeric nucleases.
  • Donor 1 contains 8 mismatches with respect to the chromosomal defective eGFP target sequence
  • Donor 2 has 10 mismatches
  • Donor 3 has 6 mismatches
  • Donor 5 has 4 mismatches.
  • the sequence of donor 5 is identical to wild-type eGFP sequence, but contains 4 mismatches with respect to the defective chromosomal eGFP sequence in the T18 cell line.
  • Table 22 provides the sequence of each donor between nucleotides 201-242. Nucleotides that are divergent from the sequence of the defective eGFP gene integrated into the genome of the T18 cell line are shown in bold and underlined.
  • the T18 cell line was transfected, as described in Example 11, with 50 ng of the 287-FokI and 296-FokI expression constructs (Example 7 and Table 12) and 500 ng of each donor construct. FACS analysis was conducted as described in Example 11.
  • the number is the percent of total fluorescence exhibiting high emission at 525 nm and low emission at 570 nm (region E of the FACS trace).
  • a cleaved donor could help facilitate homologous recombination by increasing the rate of strand invasion or could aid in the recognition of the cleaved donor end as a homologous stretch of DNA during homology search by the homologous recombination machinery. Moreover, these possibilities are not mutually exclusive.
  • siRNA molecules targeted to the Ku70 gene were generated by transcription of Ku70 cDNA followed by cleavage of double-stranded transcript with Dicer enzyme.
  • a cDNA pool generated from 293 and U2OS cells was used in five separate amplification reactions, each using a different set of amplification primers specific to the Ku70 gene, to generate five pools of cDNA fragments (pools A-E), ranging in size from 500-750 bp. Fragments in each of these five pools were then re-amplified using primers containing the bacteriophage T7 RNA polymerase promoter element, again using a different set of primers for each cDNA pool.
  • cDNA generation and PCR reactions were performed using the Superscript Choice cDNA system and Platinum Taq High Fidelity Polymerase (both from Invitrogen, Carlsbad, Calif.), according to manufacturers protocols and recommendations.
  • Each of the amplified DNA pools was then transcribed in vitro with bacteriophage T7 RNA polymerase to generate five pools (A-E) of double stranded RNA (dsRNA), using the RNAMAXX in vitro transcription kit (Stratagene, San Diego, Calif.) according to the manufacturer's instructions. After precipitation with ethanol, the RNA in each of the pools was resuspended and cleaved in vitro using recombinant Dicer enzyme (Stratagene, San Diego, Calif.) according to the manufacturer's instructions.
  • dsRNA double stranded RNA
  • siRNA products in each of the five pools were purified by a two-step method, first using a Microspin G-25 column (Amershan), followed by a Microcon YM-100 column (Amicon). Each pool of siRNA products was transiently transfected into the T7 cell line using Lipofectamone 2000®.
  • the upper portion of the blot was exposed to an anti-Ku70 antibody (Santa Cruz sc-5309) and the lower portion exposed to an anti-TF IIB antibody (Santa Cruz sc-225, used as an input control).
  • the blot was then exposed to horseradish peroxidase-conjugated goat anti-mouse secondary antibody and processed for electrochemiluminescent (ECL) detection using a kit from Pierce Chemical Co. according to the manufacturer's instructions.
  • FIG. 39 shows representative results following transfection of two of the siRNA pools (pools D and E) into T7 cells. Transfection with 70 ng of siRNA E results in a significant decrease in Ku70 protein levels ( FIG. 39 , lane 3).
  • T7 cell line (see Example 9 and FIG. 27 ) was used. These cells contain a chromosomally-integrated defective eGFP gene, but have been observed to exhibit lower levels of targeted homologous recombination than the T18 cell line used in Examples 11-13.
  • T7 cells were transfected, as described in Example 11, with either 70 or 140 ng of one of two pools of dicer product targeting Ku70 (see Example 18). Protein blot analysis was performed on extracts derived from the transfected cells to determine whether the treatment of cells with siRNA resulted in a decrease in the levels of the Ku70 protein (see previous Example).
  • FIG. 39 shows that levels of the Ku70 protein were reduced in cells that had been treated with 70 ng of siRNA from pool E.
  • the percent correction of the defective eGFP gene in the transfected T7 cells is shown in the right-most column of Table 24.
  • the highest frequency of targeted recombination is observed in Experiment 6, in which cells were transfected with donor DNA, plasmids encoding the two eGFP-targeted fusion nucleases and 70 ng of siRNA Pool E.
  • Reference to Example 18 and FIG. 39 indicates that 70 ng of Pool E siRNA significantly depressed Ku70 protein levels.
  • Each zinc finger domain contained four zinc fingers and recognized a 12 bp target site in the region of the human ⁇ -globin gene encoding the mutation responsible for Sickle Cell Anemia.
  • the binding affinity of each of these proteins to its target sequence was assessed, and four proteins exhibiting strong binding (sca-r29b, sca-36a, sca-36b, and sca-36c) were used for construction of FokI fusion endonucleases.
  • the translational start codon (ATG) is in bold and underlined, as is the A-T substitution causing Sickle Cell Anemia.
  • sca-36a GAAGTCTGCCGT (SEQ ID NO:178) sca-36b GAAGTCtGCCGTT (SEQ ID NO:179) sca-36c GAAGTCtGCCGTT (SEQ ID NO:180) CAAACAGACACC ATG GTGCATCTG (SEQ ID NO:181) ACTCCTG T GGAGAAG T CTGC C GT T ACTGGTTTGTCTGTGGTACCACGT AGACTGAGGAC A CCTCTTCAGACG GCAATGAC sca-r29b ACGTAGaCTGAGG (SEQ ID NO:182)
  • Amino acid sequences of the recognition regions of the zinc fingers in these four proteins are shown in Table 25.
  • the complete amino acid sequences of these zinc finger domains are shown in FIG. 40 .
  • the sca-36a domain recognizes a target site having 12 contiguous nucleotides (shown in upper case above), while the other three domain recognize a thirteen nucleotide sequence consisting of two six-nucleotide target sites (shown in upper case) separated by a single nucleotide (shown in lower case).
  • the sca-r29b, sca-36b and sca-36c domains contain a non-canonical inter-finger linker having the amino acid sequence TGGGGSQKP (SEQ ID NO: 183) between the second and the third of their four fingers.
  • Fusion proteins containing a FokI cleavage half-domain and one the four ZFP DNA binding domains described in the previous example were tested for their ability to cleave DNA in vitro with the predicted sequence specificity. These ZFP domains were cloned into the pcDNA3.1 expression vector via KpnI and BamHI sites and fused in-frame to the FokI cleavage domain via a 4 amino acid ZC linker, as described above.
  • a DNA fragment containing 700 bp of the human ⁇ -globin gene was cloned from genomic DNA obtained from K562 cells. The isolation and sequence of this fragment was described in Example 3, supra.
  • ZFNs fusion endonucleases
  • the probe was end-labeled with 32 P using polynucleotide kinase. This reaction was incubated for 1 hour at room temperature to allow binding of the ZFNs. Cleavage was stimulated by the addition of 8 ul of 8 mM MgCl 2 , diluted in cleavage buffer, to a final concentration of approximately 2.5 mM. The cleavage reaction was incubated for 1 hour at 37° C. and stopped by the addition of 11 ul of phenol/chloroform. The DNA was isolated by phenol/chloroform extraction and analyzed by gel electrophoresis, as described in Example 4. As a control, 3 ul of probe was analyzed on the gel to mark the migration of uncut DNA (labeled “U” in FIG. 41 ).
  • a DNA fragment containing the human P-globin gene sequence targeted by the ZFNs described in Example 20 was synthesized and cloned into a SpeI site in an eGFP reporter gene thereby, disrupting eGFP expression.
  • the fragment contained the following sequence, in which the nucleotide responsible for the sickle cell mutation is in bold and underlined): (SEQ ID NO:200) CTAGACACCATGGTGCATCTGACTCCTG T GGAGAAGTCTGCCGTTACTGC CCTAG
  • This disrupted eGFP gene containing inserted ⁇ -globin sequences was cloned into pcDNA4/TO (Invitrogen, Carlsbad, Calif.) using the HindIII and NotI sites, and the resulting vector was transfected into HEK293 TRex cells (Invitrogen). Individual stable clones were isolated and grown up, and the clones were tested for targeted homologous recombination by transfecting each of the sca-36 proteins (sca-36a, sca-36b, sca-36c) paired with sca-29b (See Example 20 and Table 25 for sequences and binding sites of these chimeric nucleases).
  • Cells were transfected with 50 ng of plasmid encoding each of the ZFNs and with 500 ng of the 1.5-kb GFP Donor (Example 13). Five days after transfection, cells were tested for homologous recombination at the inserted defective eGFP locus. Initially, cells were examined by fluorescence microscopy for eGFP function. Cells exhibiting fluorescence were then analyzed quantitatively using a FACS assay for eGFP fluorescence, as described in Example 11.
  • T18 cells were transfected with plasmids encoding the eGFP-targeted 287 and 296 zinc finger/FokI fusion proteins (Example 7) and a 1.5 kbp donor DNA molecule containing sequences that correct the defect in the chromosomal eGFP gene (Example 9).
  • transfected cells were treated with different concentrations of doxycycline, then eGFP mRNA levels were measured 48 hours after addition of doxycycline.
  • eGFP fluorescence at 520 nm (indicative of targeted recombination of the donor sequence into the chromosome to replace the inserted ⁇ -globin sequences) was measured by FACS at 4 days after transfection.
  • results are shown in FIG. 42 .
  • Increasing steady-state levels of eGFP mRNA normalized to GAPDH mRNA are indicated by the bars.
  • the number above each bar indicate the percent of cells exhibiting eGFP fluorescence.
  • the results show that increasing transcription rate of the target gene is accompanied by higher frequencies of targeted recombination. This suggests that targeted activation of transcription (as disclosed, e.g. in co-owned U.S. Pat. Nos. 6,534,261 and 6,607,882) can be used, in conjunction with targeted DNA cleavage, to stimulate targeted homologous recombination in cells.
  • K562 cells were transfected with plasmids encoding the 5-8GL0 and the 5-9DL0 zinc finger nucleases (ZFNs) (see Example 14; Table 17) and with a 1.5 kbp DraI donor construct.
  • the DraI donor is comprised of a sequence with homology to the region encoding the 5 th exon of the IL2R ⁇ gene, but inserts an extra base between the ZFN-binding sites to create a frameshift and generate a DraI site.
  • the cells are grown for about 3 weeks, and cells homozygous for the DraI mutant phenotype are isolated.
  • the cells are tested for genome modification (by testing for the presence of a DraI site in exon 5 of the IL-2R ⁇ gene) and for levels of IL-2R ⁇ mRNA (by real-time PCR) and protein (by Western blotting) to determine the effect of the mutation on gene expression.
  • Cells are tested for function by FACS analysis.
  • Cells containing the DraI frameshift mutation in the IL-2R ⁇ gene are transfected with plasmids encoding the 5-8GL0 and 5-9DL0 fusion proteins and a 1.5 kb BsrBI donor construct (Example 14) to replace the DraI frameshift mutation with a sequence encoding a functional protein.
  • Levels of homologous recombination greater than 1% are obtained in these cells, as measured by assaying for the presence of a BsrBI site as described in Example 14. Recovery of gene function is demonstrated by measuring mRNA and protein levels and by FACS analysis.
  • a vector encoding a ZFP/FokI fusion in which the ZFP domain was N-terminal to the FokI domain, was constructed.
  • the ZFP domain denoted IL2-1, contained four zinc fingers, and was targeted to the sequence AACTCGGATAAT (SEQ ID NO:202), located in the third exon of the IL-2R ⁇ gene.
  • the amino acid sequences of the recognition regions of the zinc fingers are given in Table 27.
  • Sequences encoding this zinc finger domain were joined to sequences encoding the cleavage half-domain of the FokI restriction endonuclease (amino acids 384-579 according to Looney et al. (1989) Gene 80:193-208) such that a four amino acid linker was present between the ZFP domain and the cleavage half-domain (i.e., a four amino acid ZC linker).
  • the FokI cleavage half-domain was obtained by PCR amplification of genomic DNA isolated from the bacterial strain Planomicrobium okeanokoites (ATCC 33414) using the following primers: (SEQ ID NO:208) 5′-GGATCCCAACTAGTCAAAAGTGAAC (SEQ ID NO:209) 5′-CTCGAGTTAAAAGTTTATCTCGCCG.
  • PCR product was digested with BamHI and XhoI (sites underlined in sequences shown above) and then ligated with a vector fragment prepared from the plasmid pcDNA-nls-ZFP1656-VP16-flag after BamHI and XhoI digestion.
  • the resulting construct, pcDNA-nls-ZFP1656-FokI encodes a fusion protein containing, from N-terminus to C-terminus, a SV40 large T antigen-derived nuclear localization signal (NLS, Kalderon et al.
  • pIL2-1C encodes a fusion protein comprising, from N- to C-terminus, a nuclear localization signal, the four-finger IL2-1 zinc finger binding domain and a FokI cleavage half-domain, with a four amino acid ZC linker.
  • the IL2-1 four-finger zinc finger domain was inserted, as a KpnI/BamHI fragment, into a vector encoding a fusion protein containing a NLS, the KOX-1 repression domain, EGFP and a FLAG epitope tag, that had been digested with KpnI and BamHI to release the EGFP-encoding sequences.
  • This construct was then digested with EcoRI and KpnI to release the NLS- and KOX-encoding sequences, and an EcoRI/KpnI fragment (generated by PCR using, as template, a vector encoding FokI) encoding amino acids 384-579 of the FokI restriction enzyme and a NLS was inserted.
  • pIL2-1R encodes a fusion protein containing, from N-terminus to C-terminus, a FokI cleavage half-domain, a NLS, and the four-finger IL2-1 ZFP binding domain.
  • the ZC linker in this construct is 21 amino acids long and includes the seven amino acid nuclear localization sequence (PKKKRKV; SEQ ID NO: 210).
  • the 5-9D zinc finger domain binds the 12-nucleotide target sequence AAAGCGGCTCCG (SEQ ID NO: 157) located in the fifth exon of the IL-2R ⁇ gene. See Example 14 (Table 17). Sequences encoding the 5-9D zinc finger domain were inserted into a vector to generate a FokI/ZFP fusion, in which the FokI sequences were N-terminal to the ZFP sequences.
  • the pIL2-1R plasmid described in the previous paragraph was digested with KpnI and BamHI to release a fragment containing sequences encoding the IL2-1 zinc finger binding domain, and a KpnI/BamHI fragment encoding the 5-9D zinc finger binding domain was inserted in its place.
  • the resulting construct, p5-9DR encodes a fusion protein containing, from N-terminus to C-terminus, a FokI cleavage half-domain, a NLS, and the four-finger 5-9D zinc finger binding domain.
  • the ZC linker in this construct is 22 amino acids long and includes the seven amino acid nuclear localization sequence (PKKKRKV; SEQ ID NO: 210).
  • the target sequences bound by the IL2-1 and 5-9D fusion proteins described above were introduced into double-stranded DNA fragments in a variety of orientations, to test the cleavage ability of zinc finger/FokI fusion proteins having an altered polarity in which the FokI domain is N-terminal to the ZFP domain.
  • the 5-9D target site is present in one strand and the IL2-1 target site is present on the complementary strand, with the 3′ ends of the binding sites being proximal to each other and separated by six intervening nucleotide pairs.
  • the 5-9D and IL2-1 target sites are present on the same DNA strand, with the 3′ end of the 5-9D binding site separated by six nucleotide pairs from the 5′ end of the IL2-1 binding site.
  • DNA fragments of approximately 442 base pairs, containing the sequences described above, were obtained as amplification products of plasmids into which the templates had been cloned.
  • the IL2-1 and 5-9D target sites were located within these fragments such that double-stranded DNA cleavage between the two target sites would generate DNA fragments of approximately 278 and 164 base pairs.
  • Amplification products were radioactively labeled by transfer of orthophosphate from ⁇ -32P-ATP using T4 polynucleotide kinase.
  • the IL2-1C, IL2-1R and 5-9DR fusion proteins were obtained by incubating plasmids encoding these proteins in a TNT coupled reticulocyte lysate (Promega, Madison, Wis.). Cleavage reactions were conducted in 23 ⁇ l of a mixture containing 1 ⁇ l of TNT reaction for each fusion protein, 1 ⁇ l labeled digestion substrate and 20 ⁇ l cleavage buffer.
  • Cleavage buffer was prepared by adding 1 ⁇ l of 1M dithiothreitol and 50 ⁇ l of bovine serum albumin (10 mg/ml) to 1 ml of 20 mM Tris-Cl, pH 8.5, 75 mM NaCl, 10 ⁇ M ZnCl 2 , 5% (v/v) glycerol. Cleavage reactions were incubated at 37° C. for 2 hours, then shaken with 13 ⁇ l phenol/chloroform/isoamyl alcohol (25:24:1). After centrifugation, 10 ⁇ l of the aqueous phase was analyzed on a 10% polyacrylamide gel. Radioactivity in the gel was detected using a Phosphorimager (Molecular Dynamics) and quantitated using ImageQuant software (Molecular Dynamics).
  • FIG. 44 shows the results obtained using two chimeric nucleases having a NH 2 -FokI domain-zinc finger domain-COOH polarity to cleave a substrate in which the binding sites for the two chimeric nucleases are located on opposite strands and the 3′ ends of the binding sites are proximal to each other and separated by six nucleotide pairs.
  • Incubation of the substrate with either of the IL2-1R or 5-9DR nucleases alone does not result in cleavage of the substrate (compare lanes 2 and 3 with lane 1), while incubation of both nucleases results in almost complete cleavage of the DNA substrate at the intended target site (lane 4).
  • FIG. 45 shows the ability of a first chimeric nuclease having a NH 2 -zinc finger domain-FokI domain-COOH polarity, and a second chimeric nuclease having a NH 2 -FokI domain-zinc finger domain-COOH polarity, to cleave a substrate in which the binding sites for the two chimeric nucleases are located on the same strand, and the 3′ end of the first binding site is proximal to the 5′ end of the second binding site and separated from it by six nucleotide pairs. Only the combination of the 5-9DR and the IL2-1C nucleases (i.e. each nuclease having a different polarity) was successful in cleaving the substrate having both target sites on the same strand (compare lane 6 with lanes 1-5).
  • the FokI domain is amino acids 384-579 according to Looney et al. (1989) Gene 80:193-208.
  • the ZFP domain was selected from the IL1-2 (Table 27), 5-8G (Table 17) and 5-9D (Table 17) domains.
  • the first set had the structure NH 2 -NLS-FokI-ZFP-Flag-COOH. In this set, proteins having ZC linker lengths of 13, 14, 18, 19,28 and 29 amino acids were designed.
  • the second set had the structure NH 2 -FokI-NLS-ZFP-Flag-COOH and proteins with ZC linkers of 21, 22, 23, 24, 28, 29, 38 and 39 amino acids were designed. Note that, in the second set, the NLS is part of the ZC linker. Plasmids encoding these fusion proteins are also constructed.
  • Model DNA sequences were designed to test the cleavage activity of these fusion proteins and to determine optimal ZC linker lengths as a function of distance between the target sites for the two fusion proteins. The following sequences were designed:
  • sequences are constructed in which the separation between the two target sites is 4, 5, 6 or 7 base pairs.
  • Example 26 These sequences are introduced into labeled substrates as described in Example 26 and are used to test the various fusion proteins described in this example for their ability to cleave DNA, according to the methods described in Example 27.
  • eGFP enhanced green fluorescent protein
  • the resulting plasmid contained sequences encoding mutant eGFP containing a fragment of DNA sequence from exon 5 of the IL2R ⁇ gene.
  • This plasmid was digested with HindIII and NotI, releasing a fragment containing the mutated eGFP sequence (including the inserted IL-2R ⁇ exon 5 sequences).
  • This fragment was inserted into the HindIII and NotI sites of the pcDNA4/TO vector (Invitrogen), resulting in a construct in which expression of eGFP sequence is controlled by a 2 ⁇ tet-operator-regulated CMV promoter.
  • FIG. 46 A schematic diagram of this plasmid is shown in FIG. 46 .
  • This construct was used to transform HEK293 TRex cells (Invitrogen), and a stable cell line containing an integrated copy of this construct was isolated.
  • the eGFP coding sequences are transcribed upon addition of doxycycline, but because of the frameshift mutation and the IL-2R ⁇ insertion, no functional protein is expressed.
  • a promoterless donor was constructed that contained sequences encoding puromycin resistance (denoted “puro sequences”), flanked by sequences homologous to the eGFP cDNA construct, as follows. Sequences were PCR amplified from the pTRE2pur-HA vector (BD Biosciences) to generate a puro sequence with flanking SpeI sites and a consensus Kozak sequence upstream of the ATG initiation codon. Amplification primers were: (SEQ ID NO:215) puro-5′: ACTAGTGCCGCCACCATGACCGAGTACAAGCCCA (SEQ ID NO:216) puro-3′: ACTAGTCAGGCACCGGGCTT
  • This PCR fragment was cloned into the pEGFP-N1 vector containing a modified eGFP gene that encoded a novel SpeI restriction site and a frameshift mutation that prevented functional expression of the gene (see Example 29).
  • This eGFP/Puromycin gene was cloned into the pcDNA4/TO vector, via HindIII and NotI sites, to create the vector pcDNA4/TO/GFPpuro, which also served as the positive control in experiments to obtain Puromycin resistant cells by targeted integration.
  • the pcDNA4/TO/GFPpuro vector was PCR amplified with the following primers: (SEQ ID NO:217) GFP-Bam CGAATTCTGCAGTCGAC (SEQ ID NO:218) pcDNA42571 TGCATACTTCTGCCTGC
  • the resulting amplification product was Topo cloned into the pCR4-TOPO vector and its sequence was confirmed. This created a donor with 413 bp of sequence homologous to the chromosomal eGFP construct upstream of the puro sequences and 1285 bp of sequence homologous to the chromosomal eGFP construct downstream of the puro sequences.
  • Example 29 To test for targeted integration of puro sequences, the cell line described in Example 29 was subjected to targeted DNA cleavage by zinc finger/FokI fusion proteins in the presence of the donor construct described in this example, transfected cells were selected for puromycin resistance and their chromosomal DNA was analyzed.
  • the two zinc finger/FokI fusion proteins (ZFNs), designed to cleave within the exon 5 sequences inserted into the eGFP gene (5-8G and 5-9D) have been described in Example 14, Table 17, supra.
  • Puromycin resistance can arise from either homology-dependent or homology-independent integration of donor sequences at the cleavage site located within the IL-2R ⁇ sequences inserted into the eGFP coding sequences. Homology-dependent integration of the donor construct will result in replacement of IL-2R ⁇ sequences by the puro sequences.
  • HEK 293 cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen), supplemented with 10% fetal bovine serum (FBS) (Hyclone) and 2 mM L-glutamine and maintained at 37° C. in an atmosphere of 5% CO 2 .
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • 2 mM L-glutamine 2 mM L-glutamine
  • pcDNA4/TO/GFPpuro vector 500 ng was transfected into HEK293 cells.
  • Cells were transfected using LipofectAMINE 2000 Reagent (Invitrogen) in Opti-MEM I reduced serum medium. Puromycin resistance was assayed by the addition of doxycycline to 2 ng/ml (to activate transcription of the integrated sequences) and puromycin to 2 ug/ml (final concentration) in the growth medium.
  • One of the primers is complementary to the exogenous puro sequences and the other is complementary to sequences in the CMV promoter present in the integrated reporter construct. Twenty-one out of 24 colonies yielded amplification products whose sizes were consistent with targeted integration of puro sequences. These fragments were cloned and their nucleotide sequences were determined. Sequence analysis indicated that eight out of the 24 clones had undergone homology-directed integration of puro sequences into the chromosomal eGFP construct, while 13 had undergone homology-independent integration of donor DNA into chromosomal sequences, accompanied by partial duplication of the puro sequences.
  • Fusion proteins containing the 5-8G and 5-9D zinc finger binding domains (Table 17) joined to a FokI cleavage half-domain by a 4 amino acid ZC linker (L0) have been described supra. See, e.g., Example 14 and Example 24. Polynucleotides encoding these two fusion proteins were designed so that the codons were optimized for expression in mammalian cells.
  • the codon-optimized nucleotide sequences encoding these two fusion proteins are as follows: 5-8G LO FokI (SEQ ID NO:221) aattcgctagcgccaccatggcccccaagaagaagaggaaagtgggaatc cacggggtacccgccgctatggccgagaggcccttccagtgtcggatctg catgcggaacttcagccggagcgacaacctgagcgtgcacatccgcaccc acacaggcgagaagccttttgcctgtgacatttgtgggaggaaatttgcc cgcaacgcccaccgcatcaaccacaccaagatccacaccggatctcagaa gcctttcagtgcagaatctgcatgagaaacttctcccggtcgacacccgg
  • Human K-562 erythroleukemia cells were cultured at 37° C. in DMEM supplemented with 10% fetal bovine serum, penicillin and streptomycin, and transfected (Nucleofector; Amaxa) with 2.5 ⁇ g of an expression vector encoding two zinc finger nucleases (ZFN) designed to introduce a double-strand break at a position surrounding the codon for arginine 226 in the endogenous IL2R ⁇ gene.
  • ZFN zinc finger nucleases
  • the two nucleases (5-8G and 5-9D) have been described in Example 14, Table 17, supra. Sequences encoding the nucleases were separated by sequences encoding a 2A peptide. See, e.g., Szymczak et al.
  • genomic DNA was isolated (DNEasy; Qiagen) and cell genotype at the IL2R ⁇ locus was determined by PCR of the exon 5-containing stretch of the X chromosome, using primers that anneal outside of the 1.5 kb region of donor homology and generate a 1.6 kilobase pair amplification product from the wild-type IL2R ⁇ sequence (Umov et al., supra). PCR products were analyzed by gel electrophoresis and, where indicated, by restriction digestion. Control samples included: (1) cells transfected with a GFP-encoding expression vector, (2) cells transfected solely with the expression vector encoding the ZFNs, and (3) cells transfected solely with the donor DNA molecule.
  • Example 32 Cell growth and transfection were conducted as described in Example 32.
  • the donor DNA molecule was engineered to contain a 12 nucleotide pair sequence tag containing a novel diagnostic recognition site for the restriction enzyme StuI.
  • Cellular DNA was isolated and used as a template for amplification as described in Example 32, then digested with StuI.
  • FIG. 47 all control samples carried chromosomes yielding amplification products that were resistant to cleavage by the restriction enzyme.
  • 15% of all amplification products in the cell sample transfected both with the donor DNA molecule and the ZFN expression construct were sensitive to the restriction enzyme, indicating integration of the donor DNA. Direct nucleotide sequence determination of the chromosome-derived PCR product confirmed that integration was homology-dependent.
  • Donor DNA molecule #1 was engineered to contain the entire 720 bp ORF of enhanced green fluorescent protein (eGFP) flanked by sequences homologous to the chromosomal IL2R ⁇ locus (see Example 32).
  • Donor DNA molecule #2 contained a 924 bp sequence consisting of the entire eGFP ORF followed by a polyadenylation signal; this sequence was flanked by IL2R ⁇ -homologous sequences (see Example 32).
  • eGFP enhanced green fluorescent protein
  • Donor DNA molecule #2 contained a 924 bp sequence consisting of the entire eGFP ORF followed by a polyadenylation signal; this sequence was flanked by IL2R ⁇ -homologous sequences (see Example 32).
  • cellular DNA was isolated and used as a template for amplification as described in Example 32. As shown in FIG.
  • the donor DNA molecule consisted of a 720 nucleotide-pair partial IL2R ⁇ cDNA containing the downstream portion of exon 5, and complete copies of exons 6, 7 and 8, (including a translation termination codon and a polyadenylation signal within exon 8). These cDNA sequences were flanked, on one side, by sequences homologous to the upstream portion of exon 5 and the adjoining portion of intron 4 and, on the other side, by sequences homologous to the downstream portion of exon 5 and the adjoining portion of intron 5 (see FIG. 49 ).
  • a control sample in which cells were transfected with a GFP-encoding plasmid contained chromosomes yielding PCR products of wild-type size only ( ⁇ 1.6 kb).
  • 6% of all chromosomes in the cell samples transfected both with the therapeutic half-gene-carrying donor and the ZFN expression construct were larger than the wild-type-chromosome-derived PCR product, and the difference in size was consistent with the notion that ZFN-driven targeted integration of the “therapeutic half-gene” had occurred.
  • Direct nucleotide sequence determination of the larger PCR product confirmed that homology-dependent integration of the donor construct had occurred.
  • Example 32 Cell growth and transfection were conducted as described in Example 32.
  • a donor DNA molecule was constructed that contained a 7.7 kbp antibody expression construct flanked by sequences homologous to IL2R ⁇ exon 5 and adjacent sequences (See Example 32).
  • two topological forms of the donor were used: a plasmid donor, in which the vector backbone abuts an insert with two homology arms interrupted by the expression construct (“circular”); and a linear donor, which contains two homology arms interrupted by the expression construct (“linear”).
  • DNA was isolated from transfected cells as described in Example 32.
  • the DNA was analyzed by PCR, using two primer pairs designed to detect the junction between integrated exogenous sequences and endogenous IL-2R ⁇ exon 5 sequences.
  • one of the primers was complementary to endogenous exon 5 sequence
  • the other primer was complementary to the expression construct (see upper portion of FIG. 51 ).
  • no PCR product was observed in control samples transfected only with donor DNA.
  • PCR products of the expected size were observed in cell samples transfected with donor DNA (either linear or circular) and the ZFN-encoding plasmid.
  • a pair of zinc finger/FokI fusion proteins were constructed to bind to two target sites, separated by six nucleotide pairs, in the Chinese hamster dihydrofolate reductase (DHFR) gene and cleave the gene between the two target sites.
  • DHFR Chinese hamster dihydrofolate reductase
  • CHO cells Chinese hamster ovary (CHO) cells were cultured in adherent medium (DMEM+10% FBS supplemented with 2 mM L-glutamine plus non-essential amino acids) at 37° C. 3 ⁇ 10 5 cells were grown to 70% confluence in 12-well plates and transiently transfected (using Lipofectamine 2000®) with 100 ng each of the two fusion protein-encoding plasmids. 24 hours after transfection, 20 ⁇ M vinblastine was added to the growth medium. Medium was replaced 24 hours after addition of vinblastine. 24 hours after replacement of medium, cellular DNA was purified (Qiagen) and DHFR gene sequences surrounding the target sites were amplified by PCR.
  • adherent medium DMEM+10% FBS supplemented with 2 mM L-glutamine plus non-essential amino acids
  • Primers were designed such that DNA from cells containing a wild-type DHFR gene were expected to yield a 383 nucleotide pair amplification product. Unexpectedly, two amplification products were obtained, one of the expected size and another approximately 150 nucleotide pairs larger.
  • the amplification product was analyzed using a Cel-1 assay, in which the amplification product is denatured and renatured, followed by treatment with the mismatch-specific Cel-1 nuclease. See, for example, Oleykowski et al. (1998) Nucleic Acids res. 26:4597-4602; Qui et al. (2004) BioTechniques 36:702-707; Yeung et al. (2005) BioTechniques 38:749-758. The results of the Cel-1 assay ( FIG.
  • the nucleotide sequences of the two amplification products described above were determined, to characterize the nature of the insertion, and are shown in FIG. 53 .
  • the sequence shown on the top line (SEQ ID NO:233) is the wild-type DHFR sequence
  • the sequence shown on the bottom line (SEQ ID NO:234) consists of the DHFR sequence containing, at the cleavage site, an insertion of 157 base pairs and a deletion of a single nucleotide pair.
  • the inserted 157 base pairs correspond to a portion of the vector plasmid encoding the zinc finger/FokI fusion proteins.
  • cells containing this mutation showed 53% mean methotrexate uptake, compared to wild-type CHO cells, consistent with loss of function of one copy of the DHFR gene.
  • Plasmids were constructed in which sequences encoding these mutant cleavage half-domains were fused to sequences encoding the 5-8G and 5-9D zinc finger domains (see Example 14). Various combinations of these mutants were then assayed for their ability to stimulate homology-directed repair of a double-stranded break in exon 5 of the IL-2R ⁇ gene, in the presence of a donor DNA sequence containing a BsrBI site. The assay system and procedures described in Example 15 were used, except that cells were not treated with vinblastine and 20 Units of BsrBI was used for digestion.
  • the Phosphorimager screen was read and the intensity of the RFLP-derived and wild-type bands were quantified using ImageQuant software (MolecularDynamics).
  • the intensity of the RFLP-derived band is given in Table 29. The results indicates that the X3 FokI mutant functions significantly better when paired with the Q486E mutant than when paired with a second copy of itself.
  • the second and third columns identify the nature of the FokI cleavage half-domain in the 5-8 and 5-9 zinc finger fusion proteins, as follows: WT (wild-type FokI cleavage half-domain); Q486E mutant cleavage half-domain (containing a single amino acid change compared to wild-type, described in Example 5); X3 mutant cleavage half-domain (containing three amino acid changes compared to wild-type, shown in FIG. 54 ).
  • “% GC” refers to fraction of total amplification product that is cleaved by BsrBI, as measured by radioactivity in BsrBI digestion products.
  • Example 29 The cell line described in Example 29, containing a chromosomally integrated mutant eGFP coding sequence operatively linked to a tetracycline-regulated CMU promoter, was used in experiments to test different combinations of zinc finger/FokI fusion proteins (ZFNs) containing amino acid sequence alterations in the dimerization interface of the FokI cleavage half-domain. See Example 38 and FIG. 54 .
  • ZFNs zinc finger/FokI fusion proteins
  • the exogenous donor DNA construct previously described in Example 13 and FIG. 32 , contained a 1527 nucleotide pair insert homologous to wild-type eGFP coding sequences.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • Opti-MEM I reduced serum medium 10% fetal bovine serum
  • Cells were transfected with plasmids encoding the ZFNs only (5 ng each), donor plasmid only (500 ng), or plasmids encoding the ZFNs (5 ng each) +donor plasmid (500 ng).
  • Expression of the chromosomal eGFP coding sequences was activated by the addition of 2 ng/ml doxycycline (final concentration) to the growth medium 5 hours post-transfection. The cells were harvested 3 days post-transfection and assayed by flow cytometry for eGFP expression.
  • the nature of the cleavage half-domain fused to the 5-8 zinc finger binding domain is given across the top row; the nature of the cleavage half- domain fused to the 5-9 zinc finger binding # domain is given down the leftmost column. Numbers indicate percentage of cells exhibiting eGFP fluorescence for each pair of ZFNs tested.
  • K562 cells were conducted as described in Example 32.
  • Cells were transfected with 2.5 ⁇ g of a construct encoding two zinc finger/FokI fusion proteins (separated by a 2A peptide sequence) in which the zinc finger domains (7568 and 7296) were designed to bind target sites in the human CCR-5 gene, and 50 ⁇ g of donor construct (see below).
  • the target sites for the zinc finger domains (boxed) are separated by 5 nucleotide pairs, as shown below.
  • the donor DNA molecule comprised a ⁇ 2 kilobase-pair portion of the human CCR-5 gene engineered to contain a 41 nucleotide pair sequence tag containing a novel diagnostic recognition site for the restriction enzyme BglI.
  • the donor molecule was constructed by mutagenizing the CCR-5 gene fragment to create a XbaI site, and introducing the 41-nucleotide tag into that XbaI site.
  • the 41-nucleotide tag was flanked by approximately 0.5 kilobase pairs of CCR-5 sequence on one side and approximately 1.5 kilobase pairs of CCR-5 sequence on the other.
  • DNA from cells transfected with both the donor construct and the construct encoding two zinc finger/FokI fusion proteins approximately 1% of the amplification products were cleaved by BglI, indicative of targeted insertion of the sequence tag into the CCR-5 gene.
  • DNA from untransfected cells, cells that were transfected only with the donor construct, and cells that were transfected only with the construct encoding two zinc finger/FokI fusion proteins did not yield amplification products that were cleaved by BglI. It is significant that the targeted insertion of this 41-nucleotide sequence tag generates a frameshift mutation in the CCR-5 gene, thereby inactivating gene function, including its function as a receptor for HIV.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Mycology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
US11/493,423 2003-08-08 2006-07-26 Targeted integration and expression of exogenous nucleic acid sequences Abandoned US20070134796A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
US11/493,423 US20070134796A1 (en) 2005-07-26 2006-07-26 Targeted integration and expression of exogenous nucleic acid sequences
US12/381,217 US8313925B2 (en) 2005-07-26 2009-03-09 Zinc finger proteins and method for inactivating a dhfr gene in a chinese hamster ovary cell
US13/134,766 US9045763B2 (en) 2005-07-26 2011-06-16 Linear donor constructs for targeted integration
US13/787,473 US9260726B2 (en) 2003-08-08 2013-03-06 Targeted integration and expression on exogenous nucleic acid sequences
US14/699,908 US9376685B2 (en) 2005-07-26 2015-04-29 Linear donor constructs for targeted integration
US15/146,276 US9765360B2 (en) 2005-07-26 2016-05-04 Linear donor constructs for targeted integration

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US70239405P 2005-07-26 2005-07-26
US72105405P 2005-09-26 2005-09-26
US11/493,423 US20070134796A1 (en) 2005-07-26 2006-07-26 Targeted integration and expression of exogenous nucleic acid sequences

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US12/804,234 Continuation-In-Part US8524500B2 (en) 2003-08-08 2010-07-16 Methods and compositions for targeted cleavage and recombination

Related Child Applications (4)

Application Number Title Priority Date Filing Date
US12/381,217 Continuation US8313925B2 (en) 2005-07-26 2009-03-09 Zinc finger proteins and method for inactivating a dhfr gene in a chinese hamster ovary cell
US12/386,059 Continuation-In-Part US20090263900A1 (en) 2005-07-26 2009-04-13 Linear donor constructs for targeted integration
US13/134,766 Continuation-In-Part US9045763B2 (en) 2005-07-26 2011-06-16 Linear donor constructs for targeted integration
US13/787,473 Continuation US9260726B2 (en) 2003-08-08 2013-03-06 Targeted integration and expression on exogenous nucleic acid sequences

Publications (1)

Publication Number Publication Date
US20070134796A1 true US20070134796A1 (en) 2007-06-14

Family

ID=37683957

Family Applications (3)

Application Number Title Priority Date Filing Date
US11/493,423 Abandoned US20070134796A1 (en) 2003-08-08 2006-07-26 Targeted integration and expression of exogenous nucleic acid sequences
US12/381,217 Expired - Fee Related US8313925B2 (en) 2005-07-26 2009-03-09 Zinc finger proteins and method for inactivating a dhfr gene in a chinese hamster ovary cell
US13/787,473 Expired - Lifetime US9260726B2 (en) 2003-08-08 2013-03-06 Targeted integration and expression on exogenous nucleic acid sequences

Family Applications After (2)

Application Number Title Priority Date Filing Date
US12/381,217 Expired - Fee Related US8313925B2 (en) 2005-07-26 2009-03-09 Zinc finger proteins and method for inactivating a dhfr gene in a chinese hamster ovary cell
US13/787,473 Expired - Lifetime US9260726B2 (en) 2003-08-08 2013-03-06 Targeted integration and expression on exogenous nucleic acid sequences

Country Status (10)

Country Link
US (3) US20070134796A1 (fr)
EP (1) EP1913149A4 (fr)
JP (3) JP2009502170A (fr)
KR (2) KR20080033455A (fr)
CN (1) CN101273141B (fr)
AU (1) AU2006272634B2 (fr)
CA (1) CA2615532C (fr)
IL (1) IL188966A (fr)
SG (1) SG10201508995QA (fr)
WO (1) WO2007014275A2 (fr)

Cited By (121)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060063231A1 (en) * 2004-09-16 2006-03-23 Sangamo Biosciences, Inc. Compositions and methods for protein production
US20080015164A1 (en) * 2006-05-19 2008-01-17 Sangamo Biosciences, Inc. Methods and compositions for inactivation of dihydrofolate reductase
WO2008021207A2 (fr) 2006-08-11 2008-02-21 Dow Agrosciences Llc Recombinaison homologue médiée par une nucléase à doigts de zinc
US20080159996A1 (en) * 2006-05-25 2008-07-03 Dale Ando Methods and compositions for gene inactivation
US20080182332A1 (en) * 2006-12-14 2008-07-31 Cai Qihua C Optimized non-canonical zinc finger proteins
US20080188000A1 (en) * 2006-11-13 2008-08-07 Andreas Reik Methods and compositions for modification of the human glucocorticoid receptor locus
US20090042250A1 (en) * 2007-07-12 2009-02-12 Sangamo Biosciences, Inc. Methods and compositions for inactivating alpha 1,6 fucosyltransferase (FUT8) gene expression
US20090111119A1 (en) * 2007-09-27 2009-04-30 Yannick Doyon Rapid in vivo identification of biologically active nucleases
US20090117617A1 (en) * 2007-10-25 2009-05-07 Sangamo Biosciences, Inc. Methods and compositions for targeted integration
US20090205083A1 (en) * 2007-09-27 2009-08-13 Manju Gupta Engineered zinc finger proteins targeting 5-enolpyruvyl shikimate-3-phosphate synthase genes
US20090305419A1 (en) * 2008-05-28 2009-12-10 Sangamo Biosciences, Inc. Compositions for linking DNA-binding domains and cleavage domains
US20100011455A1 (en) * 2007-01-19 2010-01-14 Mount Sinai School Of Medicine Of New York Univers Stem cell gene targeting
WO2010021692A1 (fr) 2008-08-22 2010-02-25 Sangamo Biosciences, Inc. Procédés et compositions pour un clivage simple brin ciblé et une intégration ciblée
WO2010019386A3 (fr) * 2008-08-12 2010-04-08 Dow Agrosciences Llc Production de protéine dans des cellules végétales et procédés et compositions apparentés
WO2010053518A2 (fr) 2008-10-29 2010-05-14 Sangamo Biosciences, Inc. Procédés et compositions pour inactiver l'expression du gène de la glutamine synthétase
WO2010065123A1 (fr) 2008-12-04 2010-06-10 Sangamo Biosciences, Inc. Édition de génome chez des rats au moyen de nucléases en doigt de zinc
WO2010077319A1 (fr) 2008-12-17 2010-07-08 Dow Agrosciences Llc Intégration ciblée dans le locus zp15
WO2010107493A2 (fr) 2009-03-20 2010-09-23 Sangamo Biosciences, Inc. Modification de cxcr4 en utilisant des protéines à doigt de zinc modifiées
US20110016541A1 (en) * 2008-12-04 2011-01-20 Sigma-Aldrich Co. Genome editing of sensory-related genes in animals
US20110014616A1 (en) * 2009-06-30 2011-01-20 Sangamo Biosciences, Inc. Rapid screening of biologically active nucleases and isolation of nuclease-modified cells
US20110016539A1 (en) * 2008-12-04 2011-01-20 Sigma-Aldrich Co. Genome editing of neurotransmission-related genes in animals
US20110016546A1 (en) * 2008-12-04 2011-01-20 Sigma-Aldrich Co. Porcine genome editing with zinc finger nucleases
US20110016542A1 (en) * 2008-12-04 2011-01-20 Sigma-Aldrich Co. Canine genome editing with zinc finger nucleases
US20110016543A1 (en) * 2008-12-04 2011-01-20 Sigma-Aldrich Co. Genomic editing of genes involved in inflammation
US20110016540A1 (en) * 2008-12-04 2011-01-20 Sigma-Aldrich Co. Genome editing of genes associated with trinucleotide repeat expansion disorders in animals
US20110023158A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Bovine genome editing with zinc finger nucleases
US20110023153A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved in alzheimer's disease
US20110023140A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Rabbit genome editing with zinc finger nucleases
US20110023156A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Feline genome editing with zinc finger nucleases
US20110023152A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genome editing of cognition related genes in animals
US20110023159A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Ovine genome editing with zinc finger nucleases
US20110023144A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved in amyotrophyic lateral sclerosis disease
US20110023146A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved in secretase-associated disorders
US20110023157A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Equine genome editing with zinc finger nucleases
US20110023151A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genome editing of abc transporters
US20110023148A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genome editing of addiction-related genes in animals
US20110023141A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved with parkinson's disease
US20110023143A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of neurodevelopmental genes in animals
US20110023147A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of prion disorder-related genes in animals
US20110023150A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genome editing of genes associated with schizophrenia in animals
US20110023154A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Silkworm genome editing with zinc finger nucleases
US20110023149A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved in tumor suppression in animals
US20110023145A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved in autism spectrum disorders
US20110023139A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved in cardiovascular disease
US20110030072A1 (en) * 2008-12-04 2011-02-03 Sigma-Aldrich Co. Genome editing of immunodeficiency genes in animals
US20110027235A1 (en) * 2009-04-09 2011-02-03 Sangamo Biosciences, Inc. Targeted integration into stem cells
US20110041195A1 (en) * 2009-08-11 2011-02-17 Sangamo Biosciences, Inc. Organisms homozygous for targeted modification
WO2011048600A1 (fr) 2009-10-21 2011-04-28 Danziger Innovations Ltd. Génération de variations génotypiques dans des génomes de plantes par infection d'un gamète
WO2011049627A1 (fr) 2009-10-22 2011-04-28 Dow Agrosciences Llc Protéines à doigt de zinc modifiées ciblant des gènes de plante impliqués dans la biosynthèse des acides gras
US20110126317A1 (en) * 2008-04-21 2011-05-26 Danziger Innovations Ltd. Plant viral expression vectors and use of same for generating genotypic variations in plant genomes
US20110158957A1 (en) * 2009-11-10 2011-06-30 Sangamo Biosciences, Inc. Targeted disruption of T cell receptor genes using engineered zinc finger protein nucleases
WO2011090804A1 (fr) 2010-01-22 2011-07-28 Dow Agrosciences Llc Altération génomique ciblée
US20110191899A1 (en) * 2010-01-22 2011-08-04 Dow Agrosciences Llc Engineered landing pads for gene targeting in plants
US20110191877A1 (en) * 2010-01-22 2011-08-04 Dow Agrosciences Llc Excision of transgenes in genetically modified organisms
WO2011100058A1 (fr) 2010-02-09 2011-08-18 Sangamo Biosciences, Inc. Modification génomique ciblée avec des molécules donneuses partiellement monocaténaires
WO2011139335A1 (fr) * 2010-04-26 2011-11-10 Sangamo Biosciences, Inc. Édition du génome d'un locus de rosa en utilisant des nucléases à doigt de zinc
WO2011139349A1 (fr) 2010-05-03 2011-11-10 Sangamo Biosciences, Inc. Compositions pour relier des modules en doigt de zinc
WO2012012738A1 (fr) 2010-07-23 2012-01-26 Sigma-Aldrich Co., Llc Modifications du génome à l'aide d'endonucléases de ciblage et d'acides nucléiques simples brins
WO2012018726A1 (fr) * 2010-08-02 2012-02-09 Cellectis Sa Procédé d'augmentation du ciblage de gènes à cassure double-brin induite
WO2012047598A1 (fr) 2010-09-27 2012-04-12 Sangamo Biosciences, Inc. Procédés et compositions d'inhibition d'entrée de virus dans cellules
US20120171771A1 (en) * 2009-07-08 2012-07-05 Cellular Dynamics International, Inc. Modified ips cells having a mutant form of a human immunodeficiency virus (hiv) cellular entry gene
US20120192301A1 (en) * 2011-01-05 2012-07-26 Sangamo BioSciences, Inc. and Whitehead Institute for Biomedical Research Methods and compositions for gene correction
WO2012139045A1 (fr) 2011-04-08 2012-10-11 Gilead Biologics, Inc. Procédés et compositions pour normaliser le système vasculaire tumoral par inhibition de la loxl2
US20130122591A1 (en) * 2011-10-27 2013-05-16 The Regents Of The University Of California Methods and compositions for modification of the hprt locus
WO2013074999A1 (fr) 2011-11-16 2013-05-23 Sangamo Biosciences, Inc. Protéines de liaison d'adn modifiées et utilisations de celles-ci
US20130143204A1 (en) * 2010-01-14 2013-06-06 Ospedale San Raffaele Srl Determination of in vivo dna double-strand break localization and application thereof
US8563314B2 (en) 2007-09-27 2013-10-22 Sangamo Biosciences, Inc. Methods and compositions for modulating PD1
WO2013166315A1 (fr) 2012-05-02 2013-11-07 Dow Agrosciences Llc Modification ciblée de malate déshydrogénase
US8586526B2 (en) 2010-05-17 2013-11-19 Sangamo Biosciences, Inc. DNA-binding proteins and uses thereof
US20140017214A1 (en) * 2012-07-11 2014-01-16 Sangamo Biosciences, Inc. Methods and compositions for delivery of biologics
WO2014039702A2 (fr) 2012-09-07 2014-03-13 Dow Agrosciences Llc Loci de performance fad2 et protéines correspondantes de liaison spécifique d'un site cible capables d'induire des cassures ciblées
US20140080216A1 (en) * 2012-08-29 2014-03-20 Sangamo Biosciences, Inc. Methods and compositions for treatment of a genetic condition
WO2014071219A1 (fr) * 2012-11-01 2014-05-08 Factor Bioscience Inc. Procédés et produits pour l'expression de protéines dans des cellules
WO2014127287A1 (fr) * 2013-02-14 2014-08-21 Massachusetts Institute Of Technology Procédé pour la mutagenèse ciblée in vivo
WO2014165612A2 (fr) 2013-04-05 2014-10-09 Dow Agrosciences Llc Procédés et compositions permettant d'intégrer une séquence exogène au sein du génome de plantes
US20150128300A1 (en) * 2012-06-12 2015-05-07 Genentech, Inc. Methods and compositions for generating conditional knock-out alleles
US9175280B2 (en) 2010-10-12 2015-11-03 Sangamo Biosciences, Inc. Methods and compositions for treating hemophilia B
WO2015167959A1 (fr) * 2014-04-28 2015-11-05 Sigma-Aldrich Co. Llc Modification épigénétique de génomes de mammifères à l'aide d'endonucléases ciblées
WO2016011029A2 (fr) 2014-07-14 2016-01-21 Washington State University Knock-out nanos qui permet de pratiquer l'ablation des cellules de la lignée germinale
US20160022737A1 (en) * 2014-07-25 2016-01-28 Sangamo Biosciences, Inc. Gene editing for hiv gene therapy
US9249428B2 (en) 2003-08-08 2016-02-02 Sangamo Biosciences, Inc. Methods and compositions for targeted genomic deletion
US9260752B1 (en) 2013-03-14 2016-02-16 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
US20160160238A1 (en) * 2014-11-12 2016-06-09 Recombinetics, Inc. Heterozygous modifications of tumor suppressor genes
US20160168593A1 (en) * 2014-12-15 2016-06-16 Sangamo Biosciences, Inc. Methods and compositions for enhancing targeted transgene integration
US9422577B2 (en) 2011-12-05 2016-08-23 Factor Bioscience Inc. Methods and products for transfecting cells
WO2016170484A1 (fr) 2015-04-21 2016-10-27 Novartis Ag Système d'édition de gènes guidée par arn et utilisations associées
WO2017023570A1 (fr) 2015-08-06 2017-02-09 The Curators Of The University Of Missouri Animaux résistant aux pathogènes ayant des gènes cd163 modifiés
US9567609B2 (en) 2013-08-28 2017-02-14 Sangamo Biosciences, Inc. Compositions for linking DNA-binding domains and cleavage domains
US9574211B2 (en) 2014-05-13 2017-02-21 Sangamo Biosciences, Inc. Methods and compositions for prevention or treatment of a disease
US9624498B2 (en) 2014-03-18 2017-04-18 Sangamo Biosciences, Inc. Methods and compositions for regulation of zinc finger protein expression
US9770489B2 (en) 2014-01-31 2017-09-26 Factor Bioscience Inc. Methods and products for nucleic acid production and delivery
US9885026B2 (en) 2011-12-30 2018-02-06 Caribou Biosciences, Inc. Modified cascade ribonucleoproteins and uses thereof
US9909131B2 (en) 2013-11-04 2018-03-06 Dow Agrosciences Llc Optimal soybean loci
WO2018052919A1 (fr) * 2016-09-14 2018-03-22 Monsanto Technology Llc Méthodes et compositions d'édition génomique par induction d'haploïde
US9937207B2 (en) 2013-03-21 2018-04-10 Sangamo Therapeutics, Inc. Targeted disruption of T cell receptor genes using talens
US10000772B2 (en) 2012-05-25 2018-06-19 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10093940B2 (en) 2013-11-04 2018-10-09 Dow Agrosciences Llc Optimal maize loci
US10137206B2 (en) 2016-08-17 2018-11-27 Factor Bioscience Inc. Nucleic acid products and methods of administration thereof
EP3406715A1 (fr) 2012-09-07 2018-11-28 Dow AgroSciences LLC Loci de performance fad3 et site cible correspondant, protéines de liaison spécifiques capables d'induire des ruptures ciblées
US10179918B2 (en) 2015-05-07 2019-01-15 Sangamo Therapeutics, Inc. Methods and compositions for increasing transgene activity
US10233465B2 (en) 2013-11-04 2019-03-19 Dow Agrosciences Llc Optimal soybean loci
US10266851B2 (en) 2016-06-02 2019-04-23 Sigma-Aldrich Co. Llc Using programmable DNA binding proteins to enhance targeted genome modification
US10273493B2 (en) 2013-11-04 2019-04-30 Dow Agrosciences Llc Optimal maize loci
US10415046B2 (en) 2012-12-13 2019-09-17 Dow Agrosciences Llc Precision gene targeting to a particular locus in maize
US10501404B1 (en) 2019-07-30 2019-12-10 Factor Bioscience Inc. Cationic lipids and transfection methods
US10604771B2 (en) 2013-05-10 2020-03-31 Sangamo Therapeutics, Inc. Delivery methods and compositions for nuclease-mediated genome engineering
US10731181B2 (en) * 2012-12-06 2020-08-04 Sigma, Aldrich Co. LLC CRISPR-based genome modification and regulation
US10883111B2 (en) 2014-11-27 2021-01-05 Danziger Innovations Ltd. Nucleic acid constructs for genome editing
US11235026B2 (en) 2007-09-27 2022-02-01 Sangamo Therapeutics, Inc. Methods and compositions for modulating PD1
US11241505B2 (en) 2015-02-13 2022-02-08 Factor Bioscience Inc. Nucleic acid products and methods of administration thereof
WO2022101641A1 (fr) 2020-11-16 2022-05-19 Pig Improvement Company Uk Limited Animaux résistants à la grippe a ayant des gènes anp32 modifiés
WO2023081756A1 (fr) 2021-11-03 2023-05-11 The J. David Gladstone Institutes, A Testamentary Trust Established Under The Will Of J. David Gladstone Édition précise du génome à l'aide de rétrons
WO2023105244A1 (fr) 2021-12-10 2023-06-15 Pig Improvement Company Uk Limited Édition de tmprss2/4 pour la résistance aux maladies chez le bétail
WO2023141602A2 (fr) 2022-01-21 2023-07-27 Renagade Therapeutics Management Inc. Rétrons modifiés et méthodes d'utilisation
US11781248B2 (en) 2019-06-13 2023-10-10 Allogene Therapeutics, Inc. Anti-TALEN antibodies and uses thereof
WO2024013514A2 (fr) 2022-07-15 2024-01-18 Pig Improvement Company Uk Limited Animaux d'élevage ayant subi une édition génique et présentant une résistance aux coronavirus
WO2024044723A1 (fr) 2022-08-25 2024-02-29 Renagade Therapeutics Management Inc. Rétrons modifiés et méthodes d'utilisation
US12227768B2 (en) 2011-12-05 2025-02-18 Factor Bioscience Inc. Methods and products for transfection
WO2025049959A2 (fr) 2023-09-01 2025-03-06 Renagade Therapeutics Management Inc. Systèmes et compositions d'édition génique, et méthodes de traitement du syndrome vexas
US12344572B2 (en) 2019-07-03 2025-07-01 Factor Bioscience Inc. Cationic lipids and transfection methods
WO2025174765A1 (fr) 2024-02-12 2025-08-21 Renagade Therapeutics Management Inc. Nanoparticules lipidiques comprenant des molécules d'arn codant destinées à être utilisées dans l'édition génique et comme vaccins et agents thérapeutiques

Families Citing this family (207)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2027262B1 (fr) 2006-05-25 2010-03-31 Sangamo Biosciences Inc. Demi-domaines résultant de clivage par génie génétique
WO2008102199A1 (fr) * 2007-02-20 2008-08-28 Cellectis Variants de méganucléase clivant une séquence cible d'adn provenant du gène de la bêta-2-microglobuline et utilisations de ceux-ci
DE602008003684D1 (de) * 2007-04-26 2011-01-05 Sangamo Biosciences Inc Gezielte integration in die ppp1r12c-position
WO2008153742A2 (fr) 2007-05-23 2008-12-18 Sangamo Biosciences, Inc. Méthodes et compositions assurant une expression transgénique accrue
WO2008149176A1 (fr) * 2007-06-06 2008-12-11 Cellectis Variants de méganucléase clivant une séquence cible d'adn issue du locus rosa26 de souris et leurs utilisations
MX341747B (es) 2008-02-29 2016-08-31 Monsanto Technology Llc Evento de maiz mon87460 y composiciones y metodos para detectarlo.
EP2251423B1 (fr) * 2008-03-07 2016-01-13 National University Corporation University Of Toyama Procédé de recombinaison homologue, procédé de clonage et kit
CA2720903C (fr) 2008-04-14 2019-01-15 Sangamo Biosciences, Inc. Constructions donneuses lineaires pour une integration ciblee
WO2009151591A2 (fr) 2008-06-10 2009-12-17 Sangamo Biosciences, Inc. Procédés et compositions pour la génération de lignées cellulaires déficientes en bax et bak
CN102123778B (zh) * 2008-08-14 2014-10-29 李伟德 利用离心力的动态过滤装置
KR20100080068A (ko) * 2008-12-31 2010-07-08 주식회사 툴젠 신규한 징크 핑거 뉴클레아제 및 이의 용도
US8772008B2 (en) 2009-05-18 2014-07-08 Sangamo Biosciences, Inc. Methods and compositions for increasing nuclease activity
CA2769262C (fr) 2009-07-28 2019-04-30 Sangamo Biosciences, Inc. Procedes et compositions de traitement de troubles de repetition tri-nucleotidique
AU2010325563B2 (en) 2009-11-27 2017-02-02 Basf Plant Science Company Gmbh Chimeric endonucleases and uses thereof
CN102762726A (zh) 2009-11-27 2012-10-31 巴斯夫植物科学有限公司 嵌合内切核酸酶及其用途
ES2751916T3 (es) * 2010-02-08 2020-04-02 Sangamo Therapeutics Inc Semidominios de escisión genomanipulados
WO2011104382A1 (fr) * 2010-02-26 2011-09-01 Cellectis Utilisation d'endonucléases pour insérer des transgènes dans des locus safe harbor
AU2011265733B2 (en) * 2010-06-14 2014-04-17 Iowa State University Research Foundation, Inc. Nuclease activity of TAL effector and Foki fusion protein
AU2011281062B2 (en) * 2010-07-21 2015-01-22 Board Of Regents, The University Of Texas System Methods and compositions for modification of a HLA locus
CN102559704B (zh) * 2010-12-23 2014-08-27 中国科学院上海生命科学研究院 一种在丙酮丁醇梭菌中敲除基因的方法
US20130316391A1 (en) 2010-12-29 2013-11-28 Sigmaaldrich Co., LLC Cells having disrupted expression of proteins involved in adme and toxicology processes
CN102358902B (zh) * 2011-04-02 2013-01-02 西南大学 家蚕丝素重链基因突变序列及突变的方法和应用
US9062106B2 (en) 2011-04-27 2015-06-23 Abbvie Inc. Methods for controlling the galactosylation profile of recombinantly-expressed proteins
US9708672B2 (en) 2011-05-02 2017-07-18 Nutech Ventures Plants with useful traits and related methods
US8980583B2 (en) 2011-06-30 2015-03-17 Sigma-Aldrich Co. Llc Cells deficient in CMP-N-acetylneuraminic acid hydroxylase and/or glycoprotein alpha-1,3-galactosyltransferase
US9161995B2 (en) 2011-07-25 2015-10-20 Sangamo Biosciences, Inc. Methods and compositions for alteration of a cystic fibrosis transmembrane conductance regulator (CFTR) gene
CA2848417C (fr) 2011-09-21 2023-05-02 Sangamo Biosciences, Inc. Procedes et compositions de regulation de l'expression d'un transgene
JP2014530603A (ja) * 2011-10-06 2014-11-20 サンガモ バイオサイエンシーズ， インコーポレイテッド Ｈｉｖ感染を制御するための方法および組成物
US10391126B2 (en) 2011-11-18 2019-08-27 Board Of Regents, The University Of Texas System CAR+ T cells genetically modified to eliminate expression of T-cell receptor and/or HLA
WO2013101877A2 (fr) 2011-12-29 2013-07-04 Iowa State University Research Foundation, Inc. Plantes génétiquement modifiées résistantes à xanthomonas et à d'autres agents pathogènes bactériens des plantes
US9376484B2 (en) 2012-01-11 2016-06-28 Sigma-Aldrich Co. Llc Production of recombinant proteins with simple glycoforms
WO2013130824A1 (fr) 2012-02-29 2013-09-06 Sangamo Biosciences, Inc. Procédés et compositions permettant de traiter la maladie de huntington
EP2839013B1 (fr) * 2012-04-18 2020-08-26 The Board of Trustees of the Leland Stanford Junior University Ciblage génique non disruptif
WO2013158279A1 (fr) 2012-04-20 2013-10-24 Abbvie Inc. Procédés de purification de protéines pour réduire des espèces acides
US9067990B2 (en) 2013-03-14 2015-06-30 Abbvie, Inc. Protein purification using displacement chromatography
WO2013158273A1 (fr) 2012-04-20 2013-10-24 Abbvie Inc. Procédés de modulation de la distribution de variant de lysine c-terminal
AU2013259647B2 (en) 2012-05-07 2018-11-08 Corteva Agriscience Llc Methods and compositions for nuclease-mediated targeted integration of transgenes
US11120889B2 (en) 2012-05-09 2021-09-14 Georgia Tech Research Corporation Method for synthesizing a nuclease with reduced off-site cleavage
EP2711428A1 (fr) 2012-09-21 2014-03-26 Lonza Biologics plc. Intégration spécifique d'un site
US10648001B2 (en) 2012-07-11 2020-05-12 Sangamo Therapeutics, Inc. Method of treating mucopolysaccharidosis type I or II
EP3196301B1 (fr) 2012-07-11 2018-10-17 Sangamo Therapeutics, Inc. Compositions et procédés pour le traitement de maladies génétiques monofactorielles
US9512214B2 (en) 2012-09-02 2016-12-06 Abbvie, Inc. Methods to control protein heterogeneity
WO2014039513A2 (fr) 2012-09-04 2014-03-13 The Trustees Of The University Of Pennsylvania Inhibition de la diacylglycérol kinase pour augmenter le transfert de lymphocyte t adoptif
UA119135C2 (uk) * 2012-09-07 2019-05-10 ДАУ АГРОСАЙЄНСІЗ ЕлЕлСі Спосіб отримання трансгенної рослини
AU2013329186B2 (en) 2012-10-10 2019-02-14 Sangamo Therapeutics, Inc. T cell modifying compounds and uses thereof
US9255250B2 (en) 2012-12-05 2016-02-09 Sangamo Bioscience, Inc. Isolated mouse or human cell having an exogenous transgene in an endogenous albumin gene
EP3031921B1 (fr) * 2012-12-12 2025-03-12 The Broad Institute, Inc. Administration, ingénierie et optimisation de systèmes, procédés et compositions pour manipulation de séquence et applications thérapeutiques
EP2840140B2 (fr) * 2012-12-12 2023-02-22 The Broad Institute, Inc. Procédé utilisant de composants CRISPR-cas pour la mutation de cellules procaryotiques
IL239344B2 (en) * 2012-12-12 2024-06-01 Broad Inst Inc Systems engineering, methods and optimal guiding components for sequence manipulation
US8697359B1 (en) * 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
EP2931899A1 (fr) 2012-12-12 2015-10-21 The Broad Institute, Inc. Génomique fonctionnelle employant des systèmes crispr-cas, des compositions, des procédés, des banques d'inactivation et leurs applications
WO2014093709A1 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Procédés, modèles, systèmes et appareil pour identifier des séquences cibles pour les enzymes cas ou des systèmes crispr-cas pour des séquences cibles et transmettre les résultats associés
US10227610B2 (en) 2013-02-25 2019-03-12 Sangamo Therapeutics, Inc. Methods and compositions for enhancing nuclease-mediated gene disruption
HK1207960A1 (en) 2013-03-12 2016-02-19 Abbvie Inc. Human antibodies that bind human tnf-alpha and methods of preparing the same
US9017687B1 (en) 2013-10-18 2015-04-28 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same using displacement chromatography
US9499614B2 (en) 2013-03-14 2016-11-22 Abbvie Inc. Methods for modulating protein glycosylation profiles of recombinant protein therapeutics using monosaccharides and oligosaccharides
WO2014152832A1 (fr) 2013-03-14 2014-09-25 Immusoft Corporation Procédé de différentiation et de transduction in vitro de lymphocytes b à mémoire avec des vecteurs viraux pseudotypés vsv-g
CN112301024A (zh) 2013-03-15 2021-02-02 通用医疗公司 使用RNA引导的FokI核酸酶(RFN)提高RNA引导的基因组编辑的特异性
CN105209060B (zh) * 2013-03-15 2018-12-07 高等教育联邦系统-匹兹堡大学 用于治疗碳氧血红蛋白血症的组合物和方法
US10760064B2 (en) 2013-03-15 2020-09-01 The General Hospital Corporation RNA-guided targeting of genetic and epigenomic regulatory proteins to specific genomic loci
WO2014204578A1 (fr) * 2013-06-21 2014-12-24 The General Hospital Corporation Utilisation de nucléases foki à guidage arn (rfn) afin d'augmenter la spécificité pour l'édition de génome par guidage arn
EP3783098A1 (fr) 2013-05-14 2021-02-24 Board Of Regents, The University Of Texas System Application à des humains de lymphocytes t comprenant un récepteur antigénique chimérique (car)
CN105683376A (zh) 2013-05-15 2016-06-15 桑格摩生物科学股份有限公司 用于治疗遗传病状的方法和组合物
CA2913052A1 (fr) 2013-05-24 2014-11-27 Board Of Regents, The University Of Texas System Anticorps monoclonaux ciblant un recepteur d'antigene chimerique
RU2716420C2 (ru) 2013-06-17 2020-03-11 Те Брод Инститьют Инк. Доставка и применение систем crispr-cas, векторов и композиций для целенаправленного воздействия и терапии в печени
CN105492611A (zh) 2013-06-17 2016-04-13 布罗德研究所有限公司 用于序列操纵的优化的crispr-cas双切口酶系统、方法以及组合物
MX374532B (es) 2013-06-17 2025-03-06 Broad Inst Inc Suministro, uso y aplicaciones terapéuticas de los sistemas y composiciones crispr-cas, para actuar sobre trastornos y enfermedades utilizando componentes víricos.
EP3725885A1 (fr) 2013-06-17 2020-10-21 The Broad Institute, Inc. Génomique fonctionnelle utilisant des systèmes crispr-cas, compositions, procédés, cribles et applications de ces systèmes
WO2014204724A1 (fr) 2013-06-17 2014-12-24 The Broad Institute Inc. Administration, modification et optimisation de systèmes guides tandems, méthodes et compositions pour la manipulation de séquence
US9944925B2 (en) 2013-08-02 2018-04-17 Enevolv, Inc. Processes and host cells for genome, pathway, and biomolecular engineering
US9765404B2 (en) 2013-09-04 2017-09-19 Dow Agrosciences Llc Rapid assay for identifying transformants having targeted donor insertion
US10767188B2 (en) 2013-09-25 2020-09-08 Nutech Ventures Methods and compositions for obtaining useful plant traits
US9598667B2 (en) 2013-10-04 2017-03-21 Abbvie Inc. Use of metal ions for modulation of protein glycosylation profiles of recombinant proteins
EP3441468B1 (fr) 2013-10-17 2021-05-19 Sangamo Therapeutics, Inc. Procédés d'administration et compositions pour génie génomique médié par nucléase
US10117899B2 (en) 2013-10-17 2018-11-06 Sangamo Therapeutics, Inc. Delivery methods and compositions for nuclease-mediated genome engineering in hematopoietic stem cells
US9181337B2 (en) 2013-10-18 2015-11-10 Abbvie, Inc. Modulated lysine variant species compositions and methods for producing and using the same
US9085618B2 (en) 2013-10-18 2015-07-21 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same
JP5900942B2 (ja) * 2013-11-06 2016-04-06 国立大学法人広島大学 核酸挿入用ベクター
CN106459995B (zh) 2013-11-07 2020-02-21 爱迪塔斯医药有限公司 使用统治型gRNA的CRISPR相关方法和组合物
WO2015070212A1 (fr) 2013-11-11 2015-05-14 Sangamo Biosciences, Inc. Méthodes et compositions pour traiter la maladie de huntington
DK3492593T3 (da) 2013-11-13 2021-11-08 Childrens Medical Center Nukleasemedieret regulering af genekspression
US20150139988A1 (en) 2013-11-15 2015-05-21 Abbvie, Inc. Glycoengineered binding protein compositions
CN105940013B (zh) 2013-12-09 2020-03-27 桑格摩生物科学股份有限公司 用于治疗血友病的方法和组合物
EP3080259B1 (fr) 2013-12-12 2023-02-01 The Broad Institute, Inc. Ingénierie de systèmes, procédés et compositions guides optimisées avec de nouvelles architectures pour la manipulation de séquences
JP6712948B2 (ja) 2013-12-12 2020-06-24 ザ・ブロード・インスティテュート・インコーポレイテッド 組成物、及びヌクレオチドリピート障害におけるｃｒｉｓｐｒ−ｃａｓ系の使用方法
WO2015089364A1 (fr) 2013-12-12 2015-06-18 The Broad Institute Inc. Structure cristalline d'un système crispr-cas, et ses utilisations
JP6793547B2 (ja) 2013-12-12 2020-12-02 ザ・ブロード・インスティテュート・インコーポレイテッド 最適化機能ＣＲＩＳＰＲ−Ｃａｓ系による配列操作のための系、方法および組成物
SG10201804975PA (en) 2013-12-12 2018-07-30 Broad Inst Inc Delivery, Use and Therapeutic Applications of the Crispr-Cas Systems and Compositions for HBV and Viral Diseases and Disorders
JP7103750B2 (ja) 2013-12-12 2022-07-20 ザ・ブロード・インスティテュート・インコーポレイテッド ゲノム編集のためのＣＲＩＳＰＲ－Ｃａｓ系及び組成物の送達、使用及び治療適用
US10774338B2 (en) 2014-01-16 2020-09-15 The Regents Of The University Of California Generation of heritable chimeric plant traits
US10072066B2 (en) 2014-02-03 2018-09-11 Sangamo Therapeutics, Inc. Methods and compositions for treatment of a beta thalessemia
CA2931140C (fr) 2014-02-05 2022-08-30 Fraunhofer-Gesellschaft Zur Forderung Der Angewandten Forschung E.V. Sequencage sans erreur d'adn
WO2015121454A1 (fr) 2014-02-14 2015-08-20 Cellectis Cellules destinées à l'immunothérapie modifiées pour cibler un antigène présent à la fois sur les cellules immunitaires et les cellules pathologiques
KR102375998B1 (ko) 2014-02-14 2022-03-21 더 보드 오브 리젠츠 오브 더 유니버시티 오브 텍사스 시스템 키메라 항원 수용체 및 제조방법
WO2015127439A1 (fr) 2014-02-24 2015-08-27 Sangamo Biosciences, Inc. Méthodes et compositions pour l'intégration ciblée médiée par les nucléases
KR20220013460A (ko) 2014-03-04 2022-02-04 시그마-알드리치 컴퍼니., 엘엘씨 바이러스 내성 세포 및 이의 용도
WO2015164748A1 (fr) 2014-04-24 2015-10-29 Sangamo Biosciences, Inc. Protéines effectrices de type activateur de transcription (tale) obtenues par génie génétique
US20170159065A1 (en) 2014-07-08 2017-06-08 Vib Vzw Means and methods to increase plant yield
WO2016014794A1 (fr) 2014-07-25 2016-01-28 Sangamo Biosciences, Inc. Procédés et compositions pour moduler l'ingénierie génomique médiée par les nucléases dans des cellules souches hématopoïétiques
WO2016019144A2 (fr) 2014-07-30 2016-02-04 Sangamo Biosciences, Inc. Correction génique de gènes, liés à l'alymphocytose, de cellules souches hématopoïétiques et progénitrices
WO2016025759A1 (fr) * 2014-08-14 2016-02-18 Shen Yuelei Système "knock-in" d'adn
DK3194570T3 (da) 2014-09-16 2021-09-13 Sangamo Therapeutics Inc Fremgangsmåder og sammensætninger til nukleasemedieret genommodificering og -korrektion i hæmatopoietiske stamceller
EP3889260A1 (fr) 2014-12-12 2021-10-06 The Broad Institute, Inc. Arn guides protégés (pgrnas)
CA2970370A1 (fr) 2014-12-24 2016-06-30 Massachusetts Institute Of Technology Crispr presentant ou associe avec un domaine de destabilisation
HK1246690A1 (zh) 2015-01-21 2018-09-14 Sangamo Therapeutics, Inc. 用於鉴定高度特异性核酸酶的方法和组合物
CA2981077A1 (fr) 2015-04-03 2016-10-06 Dana-Farber Cancer Institute, Inc. Composition et procedes de correction genomique de cellules b
AU2016261927B2 (en) * 2015-05-12 2022-04-07 Sangamo Therapeutics, Inc. Nuclease-mediated regulation of gene expression
CN107614680A (zh) * 2015-05-14 2018-01-19 南加利福尼亚大学 利用重组核酸内切酶系统的最佳化基因编辑
WO2016205759A1 (fr) 2015-06-18 2016-12-22 The Broad Institute Inc. Modification et optimisation de systèmes, de méthodes, d'enzymes et d'échafaudages guides d'orthologues de cas9 et variant pour la manipulation de séquences
TWI906646B (zh) 2015-06-18 2025-12-01 美商博得學院股份有限公司 降低脫靶效應的ｃｒｉｓｐｒ酶突變
AU2016291778B2 (en) 2015-07-13 2021-05-06 Sangamo Therapeutics, Inc. Delivery methods and compositions for nuclease-mediated genome engineering
US9512446B1 (en) 2015-08-28 2016-12-06 The General Hospital Corporation Engineered CRISPR-Cas9 nucleases
US9926546B2 (en) 2015-08-28 2018-03-27 The General Hospital Corporation Engineered CRISPR-Cas9 nucleases
WO2017075538A1 (fr) 2015-10-29 2017-05-04 Amyris, Inc. Compositions et procédés permettant la production de myrcène
BR112018012235A2 (pt) 2015-12-18 2018-12-04 Sangamo Therapeutics Inc rompimento alvejado do receptor de células de mhc
EP3389677B1 (fr) 2015-12-18 2024-06-26 Sangamo Therapeutics, Inc. Disruption ciblée du récepteur des lymphocytes t
WO2017123757A1 (fr) 2016-01-15 2017-07-20 Sangamo Therapeutics, Inc. Méthodes et compositions pour le traitement d'une maladie neurologique
KR20180101442A (ko) 2016-02-02 2018-09-12 상가모 테라퓨틱스, 인코포레이티드 Dna-결합 도메인 및 절단 도메인을 연결시키기 위한 조성물
US11674952B2 (en) 2016-02-24 2023-06-13 The Rockefeller University Embryonic cell-based therapeutic candidate screening systems, models for Huntington's Disease and uses thereof
CN106148286B (zh) * 2016-06-29 2019-10-29 牛刚 一种用于检测热原的细胞模型的构建方法和细胞模型及热原检测试剂盒
MA45670A (fr) 2016-07-13 2019-05-22 Vertex Pharma Procédés, compositions et kits pour augmenter l'efficacité d'édition du génome
KR20190031306A (ko) 2016-07-21 2019-03-25 맥스시티 인코포레이티드 게놈 dna를 변경하기 위한 방법 및 조성물
WO2018029034A1 (fr) 2016-08-09 2018-02-15 Vib Vzw Inhibiteurs de cellulose synthase et plantes mutantes
SG11201901107RA (en) 2016-08-11 2019-03-28 Jackson Lab Methods and compositions relating to improved human red blood cell survival in genetically modified immunodeficient non-human animals
CA3033372A1 (fr) 2016-08-15 2018-02-22 Enevolv, Inc. Systemes de capteurs sans cellule
KR102455249B1 (ko) 2016-08-24 2022-10-17 상가모 테라퓨틱스, 인코포레이티드 가공된 표적 특이적 뉴클레아제
DK3523326T3 (da) 2016-10-04 2020-08-03 Prec Biosciences Inc Samstimulerende domæner til anvendelse i genetisk modificerede celler
KR102712926B1 (ko) 2016-10-20 2024-10-07 상가모 테라퓨틱스, 인코포레이티드 파브리병의 치료를 위한 방법 및 조성물
WO2018081775A1 (fr) 2016-10-31 2018-05-03 Sangamo Therapeutics, Inc. Correction génique de gènes liés à la scid dans des cellules souches hématopoïétiques et progénitrices
GB201619876D0 (en) 2016-11-24 2017-01-11 Cambridge Entpr Ltd Controllable transcription
EP3551754B1 (fr) 2016-12-08 2023-08-30 Case Western Reserve University Procédés et compositions pour améliorer la production de myéline fonctionnelle
WO2018140478A1 (fr) 2017-01-24 2018-08-02 Sigma-Aldrich Co. Llc Cellules résistantes aux virus et systèmes de culture
CN110891417B (zh) 2017-03-21 2023-05-23 杰克逊实验室 表达人APOE4和小鼠Trem2 p.R47H的遗传修饰小鼠及其使用方法
US11655275B2 (en) 2017-05-03 2023-05-23 Sangamo Therapeutics, Inc. Methods and compositions for modification of a cystic fibrosis transmembrane conductance regulator (CFTR) gene
EP4029943A1 (fr) 2017-05-08 2022-07-20 Precision Biosciences, Inc. Molécules d'acide nucléique codant pour un récepteur d'antigène modifié et molécule d'acide nucléique inhibitrice et leurs méthodes d'utilisation
RU2019140867A (ru) 2017-05-12 2021-06-15 Зе Джексон Лаборатори Nsg мыши, не имеющие mhc класса i и класса ii
EP3645038B1 (fr) 2017-06-30 2026-02-18 Precision Biosciences, Inc. Lymphocytes t génétiquement modifiés comprenant un intron modifié dans le gène alpha du récepteur des lymphocytes t
AU2018301393B2 (en) 2017-07-11 2025-02-27 Compass Therapeutics Llc Agonist antibodies that bind human CD137 and uses thereof
EP4269560A3 (fr) 2017-10-03 2024-01-17 Precision Biosciences, Inc. Peptides de récepteur de facteur de croissance épidermique modifiés destinés à être utilisés dans des cellules génétiquement modifiées
WO2019089753A2 (fr) 2017-10-31 2019-05-09 Compass Therapeutics Llc Anticorps cd137 et antagonistes pd-1 et leurs utilisations
WO2019089913A1 (fr) 2017-11-01 2019-05-09 Precision Biosciences, Inc. Nucléases génétiquement modifiées ciblant des gènes du facteur viii humain et canin en tant que traitement de l'hémophilie a
EP3713961A2 (fr) 2017-11-20 2020-09-30 Compass Therapeutics LLC Anticorps cd137 et anticorps ciblant un antigène tumoral et leurs utilisations
US11459577B2 (en) 2017-12-18 2022-10-04 Syngenta Participations Ag Targeted insertion sites in the maize genome
MA51619A (fr) 2018-01-17 2021-04-14 Vertex Pharma Inhibiteurs de la protéine kinase dépendante de l'adn
JP7466448B2 (ja) 2018-01-17 2024-04-12 バーテックス ファーマシューティカルズ インコーポレイテッド Ｄｎａ－ｐｋ阻害剤
BR112020013626A2 (pt) 2018-01-17 2020-12-01 Vertex Pharmaceuticals Incorporated compostos de quinoxalinona, composições, métodos e kits para aumentar a eficiência de edição de genoma
CA3089587A1 (fr) 2018-02-08 2019-08-15 Sangamo Therapeutics, Inc. Nucleases specifiques de cible specifiquement modifiees
EP3765601A1 (fr) 2018-03-16 2021-01-20 Immusoft Corporation Lymphocytes b génétiquement modifiées pour sécréter de la follistatine et leurs méthodes d'utilisation pour traiter des maladies, des états, des troubles liés à la follistatine et pour améliorer la croissance et la résistance musculaires
US11690921B2 (en) 2018-05-18 2023-07-04 Sangamo Therapeutics, Inc. Delivery of target specific nucleases
GB201809273D0 (en) 2018-06-06 2018-07-25 Vib Vzw Novel mutant plant cinnamoyl-coa reductase proteins
US20210195879A1 (en) 2018-06-21 2021-07-01 The Jackson Laboratory Genetically modified mouse models of alzheimer's disease
US11708569B2 (en) 2018-08-29 2023-07-25 University Of Copenhagen Modified recombinant lysosomal alpha-galactosidase A and aspartylglucoaminidase having low mannose-6-phosphate and high sialic acid
KR20210060533A (ko) 2018-09-18 2021-05-26 상가모 테라퓨틱스, 인코포레이티드 프로그램화된 세포 사멸 1 (pd1) 특이적 뉴클레아제
IL281615B2 (en) 2018-09-21 2026-01-01 Acuitas Therapeutics Inc Systems and methods for producing lipid nanoparticles and liposomes
CA3116576A1 (fr) 2018-10-18 2020-04-23 Acuitas Therapeutics, Inc. Lipides pour l'administration de nanoparticules lipidiques d'agents actifs
WO2020084034A1 (fr) 2018-10-26 2020-04-30 F. Hoffmann-La Roche Ag Procédé de criblage d'anticorps multispécifiques utilisant un échange de cassette à médiation par recombinase
US11046769B2 (en) 2018-11-13 2021-06-29 Compass Therapeutics Llc Multispecific binding constructs against checkpoint molecules and uses thereof
GB201820109D0 (en) 2018-12-11 2019-01-23 Vib Vzw Plants with a lignin trait and udp-glycosyltransferase mutation
WO2020132659A1 (fr) 2018-12-21 2020-06-25 Precision Biosciences, Inc. Modification génétique du gène de l'hydroxyacide oxydase 1 pour le traitement de l'hyperoxalurie primaire
PT3908568T (pt) 2019-01-11 2024-09-30 Acuitas Therapeutics Inc Lípidos para a administração de agentes ativos por nanopartículas lipídicas
AU2019428629A1 (en) 2019-02-06 2021-01-28 Sangamo Therapeutics, Inc. Method for the treatment of mucopolysaccharidosis type I
CA3132167A1 (fr) 2019-04-02 2020-10-08 Weston P. MILLER IV Procedes pour le traitement de beta-thalassemie
KR102691932B1 (ko) 2019-04-03 2024-08-06 프리시젼 바이오사이언시스 인코포레이티드 마이크로RNA-적응 shRNA(shRNAmiR)를 포함하는 유전자-변형 면역 세포
EP3947646A1 (fr) 2019-04-05 2022-02-09 Precision BioSciences, Inc. Procédés de préparation de populations de cellules immunitaires génétiquement modifiées
WO2020252455A1 (fr) 2019-06-13 2020-12-17 The General Hospital Corporation Pseudo-particules virales endogènes humaines génétiquement modifiées et leurs méthodes d'utilisation en vue d'une administration à des cellules
US20220273715A1 (en) 2019-07-25 2022-09-01 Precision Biosciences, Inc. Compositions and methods for sequential stacking of nucleic acid sequences into a genomic locus
CA3148179A1 (fr) 2019-08-20 2021-02-25 Bruce J. Mccreedy Jr. Schemas posologiques de lymphodepletion pour immunotherapies cellulaires
WO2021035170A1 (fr) 2019-08-21 2021-02-25 Precision Biosciences, Inc. Compositions et procédés de reprogrammation tcr à l'aide de protéines de fusion
US20220411479A1 (en) 2019-10-30 2022-12-29 Precision Biosciences, Inc. Cd20 chimeric antigen receptors and methods of use for immunotherapy
WO2021113543A1 (fr) 2019-12-06 2021-06-10 Precision Biosciences, Inc. Méthodes d'immunothérapie anticancéreuse utilisant des régimes de lymphodéplétion et des lymphocytes car-t allogéniques cd19, cd20 ou bcma
WO2021158915A1 (fr) 2020-02-06 2021-08-12 Precision Biosciences, Inc. Compositions de virus adéno-associé recombinant ainsi que leurs méthodes de production et d'utilisation
CA3175071A1 (fr) 2020-03-11 2021-09-16 Bit Bio Limited Procede de generation de cellules hepatiques
US20230263121A1 (en) 2020-03-31 2023-08-24 Elo Life Systems Modulation of endogenous mogroside pathway genes in watermelon and other cucurbits
CN115803435A (zh) 2020-05-06 2023-03-14 塞勒克提斯公司 用于在细胞基因组中靶向插入外源序列的方法
EP4146284A1 (fr) 2020-05-06 2023-03-15 Cellectis S.A. Procédés de modification génétique de cellules pour l'administration de protéines thérapeutiques
US20230183664A1 (en) 2020-05-11 2023-06-15 Precision Biosciences, Inc. Self-limiting viral vectors encoding nucleases
CN111518220A (zh) * 2020-05-14 2020-08-11 重庆英茂盛业生物科技有限公司 一种融合蛋白及其设计方法
ES3054438T3 (en) 2020-07-16 2026-02-03 Acuitas Therapeutics Inc Cationic lipids for use in lipid nanoparticles
CA3189601A1 (fr) 2020-07-24 2022-01-27 The General Hospital Corporation Pseudo-particules virales ameliorees et leurs methodes d'utilisation pour l'administration a des cellules
EP4192875A1 (fr) 2020-08-10 2023-06-14 Precision BioSciences, Inc. Anticorps et fragments spécifiques de l'antigène de maturation des lymphocytes b et leurs utilisations
US12152251B2 (en) 2020-08-25 2024-11-26 Kite Pharma, Inc. T cells with improved functionality
WO2022076547A1 (fr) 2020-10-07 2022-04-14 Precision Biosciences, Inc. Compositions de nanoparticules lipidiques
US20240060079A1 (en) 2020-10-23 2024-02-22 Elo Life Systems Methods for producing vanilla plants with improved flavor and agronomic production
US20250127811A1 (en) 2021-01-28 2025-04-24 Precision Biosciences, Inc. Modulation of tgf beta signaling in genetically-modified eukaryotic cells
US20240141311A1 (en) 2021-04-22 2024-05-02 North Carolina State University Compositions and methods for generating male sterile plants
US20220411826A1 (en) * 2021-06-21 2022-12-29 The Penn State Research Foundation Co-opting regulatory bypass repair of genetic diseases
WO2023064872A1 (fr) 2021-10-14 2023-04-20 Precision Biosciences, Inc. Associations de lymphocytes t car anti-bcma et d'inhibiteurs de gamma secrétase
IL312244A (en) 2021-10-19 2024-06-01 Prec Biosciences Inc Gene editing methods for treating alpha-1 antitrypsin (aat) deficiency
WO2023070043A1 (fr) 2021-10-20 2023-04-27 Yale University Compositions et procédés pour l'édition et l'évolution ciblées d'éléments génétiques répétitifs
AU2022386314A1 (en) 2021-11-09 2024-06-06 Amgen Inc. Method for producing an antibody peptide conjugate
WO2023091910A1 (fr) 2021-11-16 2023-05-25 Precision Biosciences, Inc. Méthodes d'immunothérapie anticancéreuse
CA3242402A1 (fr) 2021-12-16 2023-06-22 Acuitas Therapeutics, Inc. Lipides destines a etre utilises dans des formulations de nanoparticules lipidiques
EP4460571A1 (fr) 2022-01-05 2024-11-13 Vib Vzw Moyens et procédés pour augmenter la tolérance au stress abiotique dans des plantes
WO2023131637A1 (fr) 2022-01-06 2023-07-13 Vib Vzw Graminées d'ensilage améliorées
WO2023144199A1 (fr) 2022-01-26 2023-08-03 Vib Vzw Plantes ayant des niveaux réduits de métabolites de goût amer
CA3244807A1 (fr) 2022-03-04 2023-09-07 Sigma-Aldrich Co. Llc Sélection métabolique au moyen de la voie de biosynthèse de l’asparagine
AR128959A1 (es) * 2022-04-04 2024-06-26 Spark Therapeutics Inc Mejora inmunológica del tratamiento del cáncer
EP4594506A1 (fr) 2022-09-30 2025-08-06 Sigma-Aldrich Co., LLC Sélection métabolique par l'intermédiaire de la voie de biosynthèse de la sérine
CN120283058A (zh) 2022-09-30 2025-07-08 西格马-奥尔德里奇有限责任公司 经由甘氨酸-甲酸盐生物合成途径的代谢选择
CN115786375A (zh) * 2022-12-12 2023-03-14 烟台药物研究所 一种双荧光素酶报告基因载体、其构建方法及其应用
WO2024216118A1 (fr) 2023-04-14 2024-10-17 Precision Biosciences, Inc. Cassettes d'expression spécifiques des muscles
WO2024216116A1 (fr) 2023-04-14 2024-10-17 Precision Biosciences, Inc. Cassettes d'expression spécifiques des muscles
KR20260046192A (ko) 2023-07-31 2026-04-06 시그마-알드리치 컴퍼니., 엘엘씨 알라닌 생합성 경로를 통한 대사 선택
WO2025147573A2 (fr) 2024-01-05 2025-07-10 Immusoft Corporation Lymphocytes b modifiés exprimant le glp-1 pour le traitement d'une maladie métabolique
WO2026030277A1 (fr) 2024-08-01 2026-02-05 Amgen Inc. Procédé pour réduire les activités proteasiques
WO2026083328A1 (fr) 2024-10-18 2026-04-23 Precision Biosciences, Inc. Édition de bases au niveau des séquences nucléotidiques par l'utilisation de la réparation guidée par homologie
WO2026083329A1 (fr) 2024-10-18 2026-04-23 Precision Biosciences, Inc. Procédés de réparation et de remplacement dirigés par homologie initiée par nucléase et compositions et utilisations associées

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6063630A (en) * 1991-11-05 2000-05-16 Transkaryotic Therapies, Inc. Targeted introduction of DNA into primary or secondary cells and their use for gene therapy
US6395523B1 (en) * 2001-06-01 2002-05-28 New England Biolabs, Inc. Engineering nicking endonucleases from type IIs restriction endonucleases
US20020064802A1 (en) * 2000-04-28 2002-05-30 Eva Raschke Methods for binding an exogenous molecule to cellular chromatin
US20020081603A1 (en) * 2000-04-28 2002-06-27 Alan Wolffe Databases of regulatory sequences; methods of making and using same
US6534261B1 (en) * 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6645489B2 (en) * 1997-09-25 2003-11-11 Cytomatrix, Llc Methods and devices for the long-term culture of hematopoietic progenitor cells
US6667064B2 (en) * 2000-08-30 2003-12-23 Pilot Therapeutics, Inc. Composition and method for treatment of hypertriglyceridemia
US20040002092A1 (en) * 2002-03-15 2004-01-01 Sylvain Arnould Hybrid and single chain meganucleases and use thereof
US20050064474A1 (en) * 2003-08-08 2005-03-24 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US20050208489A1 (en) * 2002-01-23 2005-09-22 Dana Carroll Targeted chromosomal mutagenasis using zinc finger nucleases
US20060188987A1 (en) * 2003-08-08 2006-08-24 Dmitry Guschin Targeted deletion of cellular DNA sequences

Family Cites Families (65)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4217344A (en) * 1976-06-23 1980-08-12 L'oreal Compositions containing aqueous dispersions of lipid spheres
US4235871A (en) * 1978-02-24 1980-11-25 Papahadjopoulos Demetrios P Method of encapsulating biologically active materials in lipid vesicles
US4186183A (en) * 1978-03-29 1980-01-29 The United States Of America As Represented By The Secretary Of The Army Liposome carriers in chemotherapy of leishmaniasis
US4261975A (en) * 1979-09-19 1981-04-14 Merck & Co., Inc. Viral liposome particle
US4485054A (en) * 1982-10-04 1984-11-27 Lipoderm Pharmaceuticals Limited Method of encapsulating biologically active materials in multilamellar lipid vesicles (MLV)
US4603044A (en) * 1983-01-06 1986-07-29 Technology Unlimited, Inc. Hepatocyte Directed Vesicle delivery system
US4501728A (en) * 1983-01-06 1985-02-26 Technology Unlimited, Inc. Masking of liposomes from RES recognition
US4897355A (en) * 1985-01-07 1990-01-30 Syntex (U.S.A.) Inc. N[ω,(ω-1)-dialkyloxy]- and N-[ω,(ω-1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US4946787A (en) * 1985-01-07 1990-08-07 Syntex (U.S.A.) Inc. N-(ω,(ω-1)-dialkyloxy)- and N-(ω,(ω-1)-dialkenyloxy)-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US5049386A (en) * 1985-01-07 1991-09-17 Syntex (U.S.A.) Inc. N-ω,(ω-1)-dialkyloxy)- and N-(ω,(ω-1)-dialkenyloxy)Alk-1-YL-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US4797368A (en) * 1985-03-15 1989-01-10 The United States Of America As Represented By The Department Of Health And Human Services Adeno-associated virus as eukaryotic expression vector
US4774085A (en) * 1985-07-09 1988-09-27 501 Board of Regents, Univ. of Texas Pharmaceutical administration systems containing a mixture of immunomodulators
US5422251A (en) * 1986-11-26 1995-06-06 Princeton University Triple-stranded nucleic acids
US4837028A (en) * 1986-12-24 1989-06-06 Liposome Technology, Inc. Liposomes with enhanced circulation time
US5176996A (en) * 1988-12-20 1993-01-05 Baylor College Of Medicine Method for making synthetic oligonucleotides which bind specifically to target sites on duplex DNA molecules, by forming a colinear triplex, the synthetic oligonucleotides and methods of use
US4957773A (en) * 1989-02-13 1990-09-18 Syracuse University Deposition of boron-containing films from decaborane
FR2646438B1 (fr) * 1989-03-20 2007-11-02 Pasteur Institut Procede de remplacement specifique d'une copie d'un gene present dans le genome receveur par l'integration d'un gene different de celui ou se fait l'integration
US5061620A (en) * 1990-03-30 1991-10-29 Systemix, Inc. Human hematopoietic stem cell
US5264618A (en) 1990-04-19 1993-11-23 Vical, Inc. Cationic lipids for intracellular delivery of biologically active molecules
WO1991017424A1 (fr) 1990-05-03 1991-11-14 Vical, Inc. Acheminement intracellulaire de substances biologiquement actives effectue a l'aide de complexes de lipides s'auto-assemblant
US5173414A (en) * 1990-10-30 1992-12-22 Applied Immune Sciences, Inc. Production of recombinant adeno-associated virus vectors
US5420032A (en) * 1991-12-23 1995-05-30 Universitge Laval Homing endonuclease which originates from chlamydomonas eugametos and recognizes and cleaves a 15, 17 or 19 degenerate double stranded nucleotide sequence
US5436150A (en) * 1992-04-03 1995-07-25 The Johns Hopkins University Functional domains in flavobacterium okeanokoities (foki) restriction endonuclease
US5487994A (en) * 1992-04-03 1996-01-30 The Johns Hopkins University Insertion and deletion mutants of FokI restriction endonuclease
US5356802A (en) * 1992-04-03 1994-10-18 The Johns Hopkins University Functional domains in flavobacterium okeanokoites (FokI) restriction endonuclease
US5587308A (en) 1992-06-02 1996-12-24 The United States Of America As Represented By The Department Of Health & Human Services Modified adeno-associated virus vector capable of expression from a novel promoter
CA2170357A1 (fr) * 1993-08-25 1995-03-02 David Digiusto Methode pour l'obtention d'une population tres enrichie en cellules souches hematopoietiques
DE69534629D1 (de) 1994-01-18 2005-12-29 Scripps Research Inst Derivate von zinkfingerproteinen und methoden
US6242568B1 (en) * 1994-01-18 2001-06-05 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
US6140466A (en) * 1994-01-18 2000-10-31 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
US5585245A (en) * 1994-04-22 1996-12-17 California Institute Of Technology Ubiquitin-based split protein sensor
GB9824544D0 (en) 1998-11-09 1999-01-06 Medical Res Council Screening system
USRE39229E1 (en) * 1994-08-20 2006-08-08 Gendaq Limited Binding proteins for recognition of DNA
US5789538A (en) * 1995-02-03 1998-08-04 Massachusetts Institute Of Technology Zinc finger proteins with high affinity new DNA binding specificities
US5928638A (en) 1996-06-17 1999-07-27 Systemix, Inc. Methods for gene transfer
US5925523A (en) * 1996-08-23 1999-07-20 President & Fellows Of Harvard College Intraction trap assay, reagents and uses thereof
US6335195B1 (en) * 1997-01-28 2002-01-01 Maret Corporation Method for promoting hematopoietic and mesenchymal cell proliferation and differentiation
GB2338237B (en) 1997-02-18 2001-02-28 Actinova Ltd In vitro peptide or protein expression library
GB9703369D0 (en) 1997-02-18 1997-04-09 Lindqvist Bjorn H Process
US6342345B1 (en) 1997-04-02 2002-01-29 The Board Of Trustees Of The Leland Stanford Junior University Detection of molecular interactions by reporter subunit complementation
GB9710809D0 (en) 1997-05-23 1997-07-23 Medical Res Council Nucleic acid binding proteins
GB9710807D0 (en) 1997-05-23 1997-07-23 Medical Res Council Nucleic acid binding proteins
US5928628A (en) * 1997-10-23 1999-07-27 Pellico; Michael A. Two-component dental bleaching system and method
AU757930B2 (en) * 1997-12-01 2003-03-13 Roche Diagnostics Gmbh Optimization of cells for endogenous gene activation
US6506559B1 (en) * 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
US6410248B1 (en) * 1998-01-30 2002-06-25 Massachusetts Institute Of Technology General strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites
ES2341926T3 (es) * 1998-03-02 2010-06-29 Massachusetts Institute Of Technology Poliproteinas con dedos de cinc que tienen enlazadores mejorados.
US6140081A (en) 1998-10-16 2000-10-31 The Scripps Research Institute Zinc finger binding domains for GNN
US6453242B1 (en) * 1999-01-12 2002-09-17 Sangamo Biosciences, Inc. Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites
US6794136B1 (en) 2000-11-20 2004-09-21 Sangamo Biosciences, Inc. Iterative optimization in the design of binding proteins
UA74557C2 (en) * 1999-09-03 2006-01-16 Applied Research Systems A method for producing a heterologous secreted protein from chinese hamster ovaries cells grown on microcarriers
ATE355368T1 (de) 2000-01-24 2006-03-15 Gendaq Ltd Nucleinsäure bindende polypeptide gekennzeichnet durch flexible linker verbundene nucleinsäuredomäne
US20020061512A1 (en) 2000-02-18 2002-05-23 Kim Jin-Soo Zinc finger domains and methods of identifying same
AU2001263155A1 (en) 2000-05-16 2001-11-26 Massachusetts Institute Of Technology Methods and compositions for interaction trap assays
JP2002060786A (ja) 2000-08-23 2002-02-26 Kao Corp 硬質表面用殺菌防汚剤
WO2002057293A2 (fr) 2001-01-22 2002-07-25 Sangamo Biosciences, Inc. Protéines de liaison en doigt de gant modifiées
GB0108491D0 (en) 2001-04-04 2001-05-23 Gendaq Ltd Engineering zinc fingers
JP2005500061A (ja) 2001-08-20 2005-01-06 ザ スクリップス リサーチ インスティテュート Ｃｎｎについての亜鉛フィンガー結合ドメイン
KR100454016B1 (ko) * 2002-01-05 2004-10-26 한국과학기술원 항예정사 유전자로 형질전환되고 ｄｈｆｒ 유전자가결핍된 신규한 ｃｈｏ 세포주, 그의 제조 방법 및 상기형질전환된 ｃｈｏ 숙주 세포를 이용한 목적단백질의 생산방법
US6845734B2 (en) 2002-04-11 2005-01-25 Micron Technology, Inc. Deposition apparatuses configured for utilizing phased microwave radiation
EP2806025B1 (fr) * 2002-09-05 2019-04-03 California Institute of Technology Utilisation de nucléases à doigt de zinc pour stimuler le ciblage de gènes
EP1439234A1 (fr) * 2003-01-08 2004-07-21 ARTEMIS Pharmaceuticals GmbH Transgénèse ciblée utilisant le locus 26
AU2004263865B2 (en) * 2003-08-08 2007-05-17 Sangamo Therapeutics, Inc. Methods and compositions for targeted cleavage and recombination
AU2005287278B2 (en) * 2004-09-16 2011-08-04 Sangamo Biosciences, Inc. Compositions and methods for protein production
JP2010516250A (ja) * 2007-01-19 2010-05-20 マウント シナイ スクール オブ メディスン オブ ニューヨーク ユニバーシティー 幹細胞遺伝子標的化法

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6063630A (en) * 1991-11-05 2000-05-16 Transkaryotic Therapies, Inc. Targeted introduction of DNA into primary or secondary cells and their use for gene therapy
US6645489B2 (en) * 1997-09-25 2003-11-11 Cytomatrix, Llc Methods and devices for the long-term culture of hematopoietic progenitor cells
US6824978B1 (en) * 1999-01-12 2004-11-30 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6933113B2 (en) * 1999-01-12 2005-08-23 Sangamo Biosciences, Inc. Modulation of endogenous gene expression in cells
US6534261B1 (en) * 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US20020064802A1 (en) * 2000-04-28 2002-05-30 Eva Raschke Methods for binding an exogenous molecule to cellular chromatin
US20020081603A1 (en) * 2000-04-28 2002-06-27 Alan Wolffe Databases of regulatory sequences; methods of making and using same
US6667064B2 (en) * 2000-08-30 2003-12-23 Pilot Therapeutics, Inc. Composition and method for treatment of hypertriglyceridemia
US6395523B1 (en) * 2001-06-01 2002-05-28 New England Biolabs, Inc. Engineering nicking endonucleases from type IIs restriction endonucleases
US20050208489A1 (en) * 2002-01-23 2005-09-22 Dana Carroll Targeted chromosomal mutagenasis using zinc finger nucleases
US20040002092A1 (en) * 2002-03-15 2004-01-01 Sylvain Arnould Hybrid and single chain meganucleases and use thereof
US20050064474A1 (en) * 2003-08-08 2005-03-24 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US20060188987A1 (en) * 2003-08-08 2006-08-24 Dmitry Guschin Targeted deletion of cellular DNA sequences

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
Edelstein et al. Gene therapy clinical trials worldwide 1989-2004 -- an overview. J. Gene Med. Vol. 6, pages 597-602, 2004. *
Guo et al. Group II introns designed to insert into therapeutically relevant DNA target sites in human cells. Science, Vol. 289, pages 452-457, July 2000. *
High, KA. The moving finger. Nature, Vol. 435, pages 577 and 579, 2005. *
Luo et al. synthetic DNA delivery systems. Nature Biotechnology, Vol. 18, pages 33-37, 2000. *
Palù et al. In pursuit of new developments for gene therapy of human diseases. J. Biotechnol. Vol. 68, pages 1-13, 1999. *
Porteus et al. Gene targeting using zinc finger nucleases. Nature Biotechnology, Vol. 23, No. 8, pages 967-973, August 2005. *
Verma and Weitzman, Gene Therapy: Twenty-first century medicine. Annual Review of Biochemistry, Vol. 74, pages 711-738, 2005. *
Verma et al. Gene therapy--promises, problems and prospects. Nature, Vol. 389, pages 239-242, 1997. *

Cited By (392)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9249428B2 (en) 2003-08-08 2016-02-02 Sangamo Biosciences, Inc. Methods and compositions for targeted genomic deletion
US9752140B2 (en) 2003-08-08 2017-09-05 Sangamo Therapeutics, Inc. Methods and compostions for targeted genomic deletion
US20060063231A1 (en) * 2004-09-16 2006-03-23 Sangamo Biosciences, Inc. Compositions and methods for protein production
US20080015164A1 (en) * 2006-05-19 2008-01-17 Sangamo Biosciences, Inc. Methods and compositions for inactivation of dihydrofolate reductase
US8569253B2 (en) 2006-05-25 2013-10-29 Sangamo Biosciences, Inc. Methods and compositions for gene inactivation
US20080159996A1 (en) * 2006-05-25 2008-07-03 Dale Ando Methods and compositions for gene inactivation
US9434776B2 (en) 2006-05-25 2016-09-06 Sangamo Biosciences, Inc. Methods and compositions for gene inactivation
US11648267B2 (en) 2006-05-25 2023-05-16 Sangamo Therapeutics, Inc. Methods and compositions for gene inactivation
US7951925B2 (en) * 2006-05-25 2011-05-31 Sangamo Biosciences, Inc. Methods and compositions for gene inactivation
US8524221B2 (en) * 2006-05-25 2013-09-03 Sangamo Biosciences, Inc. Methods and compositions for gene inactivation
EP2395081A1 (fr) 2006-08-11 2011-12-14 Dow AgroSciences LLC Recombinaison homologue médiée par une nucléase à doigts de zinc
WO2008021207A2 (fr) 2006-08-11 2008-02-21 Dow Agrosciences Llc Recombinaison homologue médiée par une nucléase à doigts de zinc
US9217026B2 (en) 2006-11-13 2015-12-22 Sangamo Biosciences, Inc. Method of inactivating a glucocorticoid receptor gene in an isolated cell
US11884930B2 (en) 2006-11-13 2024-01-30 Sangamo Biosciences, Inc. Method of inactivating a glucocorticoid receptor gene in an isolated cell
US10907175B2 (en) 2006-11-13 2021-02-02 Sangamo Therapeutics, Inc. Isolated human cell with an inactivated glucocorticoid receptor gene
US20080188000A1 (en) * 2006-11-13 2008-08-07 Andreas Reik Methods and compositions for modification of the human glucocorticoid receptor locus
US10662434B2 (en) 2006-12-14 2020-05-26 Dow Agrosciences Llc Optimized non-canonical zinc finger proteins
US8921112B2 (en) 2006-12-14 2014-12-30 Dow Agrosciences Llc Optimized non-canonical zinc finger proteins
US20080182332A1 (en) * 2006-12-14 2008-07-31 Cai Qihua C Optimized non-canonical zinc finger proteins
US9187758B2 (en) 2006-12-14 2015-11-17 Sangamo Biosciences, Inc. Optimized non-canonical zinc finger proteins
US11078494B2 (en) 2007-01-19 2021-08-03 Icahn School Of Medicine At Mount Sinai Vector for targeting the human Rosa26 gene
US9062122B2 (en) 2007-01-19 2015-06-23 Mount Sinai School Of Medicine Of New York University Targeting the Rosa26 gene in human embryonic stem cells
US20100011455A1 (en) * 2007-01-19 2010-01-14 Mount Sinai School Of Medicine Of New York Univers Stem cell gene targeting
US9481896B2 (en) 2007-01-19 2016-11-01 Icahn School Of Medicine At Mount Sinai Stem cell gene targeting
US7919313B2 (en) 2007-07-12 2011-04-05 Sangamo Biosciences, Inc. Methods and compositions for inactivating alpha 1,6 fucosyltransferase (FUT8) gene expression
US9322036B2 (en) 2007-07-12 2016-04-26 Sangamo Biosciences, Inc. Methods and compositions for inactivating alpha 1,6 fucosyltransferase (FUT8) gene expression
US20090042250A1 (en) * 2007-07-12 2009-02-12 Sangamo Biosciences, Inc. Methods and compositions for inactivating alpha 1,6 fucosyltransferase (FUT8) gene expression
US20110159541A1 (en) * 2007-07-12 2011-06-30 Sangamo Biosciences, Inc. Methods and compositions for inactivating alpha 1,6 fucosyltransferase (FUT8) gene expression
US9890395B2 (en) 2007-07-12 2018-02-13 Sangamo Therapeutics, Inc. Methods and compositions for inactivating alpha 1,6 fucosyltransferase (FUT8) gene expression
US8772025B2 (en) 2007-07-12 2014-07-08 Sangamo Biosciences, Inc. Methods and compositions for inactivating alpha 1,6 fucosyltransferase (FUT8) gene expression
KR101657504B1 (ko) * 2007-09-27 2016-09-19 상가모 바이오사이언스 인코포레이티드 생물학적으로 활성인 뉴클레아제의 신속한 생체내 확인
US9402879B2 (en) 2007-09-27 2016-08-02 Sangamo Biosciences, Inc. Methods and compositions for modulating PD1
US8399218B2 (en) 2007-09-27 2013-03-19 Dow Agrosciences, Llc Engineered zinc finger proteins targeting 5-enolpyruvyl shikimate-3-phosphate synthase genes
EP3072973A1 (fr) 2007-09-27 2016-09-28 Dow AgroSciences LLC Protéines à doigts de zinc synthétisées ciblant des gènes de la synthase shikimate-3-phosphate5-énolpyruvyl
US9506120B2 (en) 2007-09-27 2016-11-29 Sangamo Biosciences, Inc. Rapid in vivo identification of biologically active nucleases
KR20100087286A (ko) * 2007-09-27 2010-08-04 상가모 바이오사이언스 인코포레이티드 생물학적으로 활성인 뉴클레아제의 신속한 생체내 확인
US20090111119A1 (en) * 2007-09-27 2009-04-30 Yannick Doyon Rapid in vivo identification of biologically active nucleases
US10344289B2 (en) 2007-09-27 2019-07-09 Dow Agrosciences Llc Engineered zinc finger proteins targeting 5-enolpyruvyl shikimate-3-phosphate synthase genes
WO2009042163A3 (fr) * 2007-09-27 2009-05-22 Sangamo Biosciences Inc Identification in vivo rapide de nucléases biologiquement actives
US8563314B2 (en) 2007-09-27 2013-10-22 Sangamo Biosciences, Inc. Methods and compositions for modulating PD1
US9121072B2 (en) 2007-09-27 2015-09-01 Sangamo Biosciences, Inc. Rapid screening of biologically active nucleases and isolation of nuclease-modified cells
US9115409B2 (en) 2007-09-27 2015-08-25 Sangamo Biosciences, Inc. Rapid screening of biologically active nucleases and isolation of nuclease-modified cells
US20090205083A1 (en) * 2007-09-27 2009-08-13 Manju Gupta Engineered zinc finger proteins targeting 5-enolpyruvyl shikimate-3-phosphate synthase genes
US10046028B2 (en) 2007-09-27 2018-08-14 Sangamo Therapeutics, Inc. Methods and compositions for modulating PD1
US11235026B2 (en) 2007-09-27 2022-02-01 Sangamo Therapeutics, Inc. Methods and compositions for modulating PD1
US8889390B2 (en) 2007-09-27 2014-11-18 Dow Agrosciences Llc Engineered zinc finger proteins targeting 5-enolpyruvyl shikimate-3-phosphate synthase genes
US20090117617A1 (en) * 2007-10-25 2009-05-07 Sangamo Biosciences, Inc. Methods and compositions for targeted integration
US8936936B2 (en) 2007-10-25 2015-01-20 Sangamo Biosciences, Inc. Methods and compositions for targeted integration
US8791324B2 (en) 2008-04-21 2014-07-29 Danziger Innovations Ltd. Plant viral expression vectors and use of same for generating genotypic variations in plant genomes
US20110126317A1 (en) * 2008-04-21 2011-05-26 Danziger Innovations Ltd. Plant viral expression vectors and use of same for generating genotypic variations in plant genomes
EP2936976A1 (fr) 2008-04-21 2015-10-28 Danziger Innovations Ltd. Vecteurs d'expression de virus de plante et leur utilisation pour générer des variations génotypiques dans des génomes de plantes
US11608492B2 (en) 2008-05-28 2023-03-21 Sangamo Therapeutics, Inc. Compositions for linking DNA-binding domains and cleavage domains
US9394531B2 (en) 2008-05-28 2016-07-19 Sangamo Biosciences, Inc. Compositions for linking DNA-binding domains and cleavage domains
US20090305419A1 (en) * 2008-05-28 2009-12-10 Sangamo Biosciences, Inc. Compositions for linking DNA-binding domains and cleavage domains
US9982245B2 (en) 2008-05-28 2018-05-29 Sangamo Therapeutics, Inc. Compositions for linking DNA-binding domains and cleavage domains
US10822599B2 (en) 2008-05-28 2020-11-03 Sangamo Therapeutics, Inc. Compositions for linking DNA-binding domains and cleavage domains
US20110008833A1 (en) * 2008-08-12 2011-01-13 Dow Agrosciences Llc Protein production in plant cells and associated methods and compositions
US9249422B2 (en) 2008-08-12 2016-02-02 Dow Agrosciences Llc Protein production in plant cells and associated methods and compositions
WO2010019386A3 (fr) * 2008-08-12 2010-04-08 Dow Agrosciences Llc Production de protéine dans des cellules végétales et procédés et compositions apparentés
US8703489B2 (en) 2008-08-22 2014-04-22 Sangamo Biosciences, Inc. Methods and compositions for targeted single-stranded cleavage and targeted integration
US11149321B2 (en) 2008-08-22 2021-10-19 Sangamo Therapeutics, Inc. Methods and compositions for targeted single-stranded cleavage and targeted integration
WO2010021692A1 (fr) 2008-08-22 2010-02-25 Sangamo Biosciences, Inc. Procédés et compositions pour un clivage simple brin ciblé et une intégration ciblée
AU2009283194B2 (en) * 2008-08-22 2014-10-16 Sangamo Therapeutics, Inc. Methods and compositions for targeted single-stranded cleavage and targeted integration
US9631186B2 (en) 2008-08-22 2017-04-25 Sangamo Biosciences, Inc. Methods and compositions for targeted single-stranded cleavage and targeted integration
US20100047805A1 (en) * 2008-08-22 2010-02-25 Sangamo Biosciences, Inc. Methods and compositions for targeted single-stranded cleavage and targeted integration
US10113207B2 (en) 2008-08-22 2018-10-30 Sangamo Therapeutics, Inc. Methods and compositions for targeted single-stranded cleavage and targeted integration
EP2789691A1 (fr) 2008-08-22 2014-10-15 Sangamo BioSciences, Inc. Procédés et compositions pour un clivage simple brin ciblé et une intégration ciblée
US9200266B2 (en) 2008-08-22 2015-12-01 Sangamo Biosciences, Inc. Methods and compositions for targeted single-stranded cleavage and targeted integration
US10689717B2 (en) 2008-08-22 2020-06-23 Sangamo Therapeutics, Inc. Methods and compositions for targeted single-stranded cleavage and targeted integration
US20100129869A1 (en) * 2008-10-29 2010-05-27 Sangamo Biosciences, Inc. Methods and compositions for inactivating glutamine synthetase gene expression
US20100311124A1 (en) * 2008-10-29 2010-12-09 Sangamo Biosciences, Inc. Methods and compositions for inactivating glutamine synthetase gene expression
WO2010053518A2 (fr) 2008-10-29 2010-05-14 Sangamo Biosciences, Inc. Procédés et compositions pour inactiver l'expression du gène de la glutamine synthétase
US9388426B2 (en) 2008-10-29 2016-07-12 Sangamo Biosciences, Inc. Methods and compositions for inactivating glutamine synthetase gene expression
US8153399B2 (en) 2008-10-29 2012-04-10 Sangamo Biosciences, Inc. Methods and compositions for inactivating glutamine synthetase gene expression
US20110023159A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Ovine genome editing with zinc finger nucleases
US9206404B2 (en) 2008-12-04 2015-12-08 Sangamo Biosciences, Inc. Method of deleting an IgM gene in an isolated rat cell
WO2010065123A1 (fr) 2008-12-04 2010-06-10 Sangamo Biosciences, Inc. Édition de génome chez des rats au moyen de nucléases en doigt de zinc
US20110016541A1 (en) * 2008-12-04 2011-01-20 Sigma-Aldrich Co. Genome editing of sensory-related genes in animals
US20110016539A1 (en) * 2008-12-04 2011-01-20 Sigma-Aldrich Co. Genome editing of neurotransmission-related genes in animals
US20110016546A1 (en) * 2008-12-04 2011-01-20 Sigma-Aldrich Co. Porcine genome editing with zinc finger nucleases
US20110016542A1 (en) * 2008-12-04 2011-01-20 Sigma-Aldrich Co. Canine genome editing with zinc finger nucleases
US20110016543A1 (en) * 2008-12-04 2011-01-20 Sigma-Aldrich Co. Genomic editing of genes involved in inflammation
US20110016540A1 (en) * 2008-12-04 2011-01-20 Sigma-Aldrich Co. Genome editing of genes associated with trinucleotide repeat expansion disorders in animals
US20110023158A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Bovine genome editing with zinc finger nucleases
US20110023153A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved in alzheimer's disease
US20110023140A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Rabbit genome editing with zinc finger nucleases
US20110023156A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Feline genome editing with zinc finger nucleases
US20110023152A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genome editing of cognition related genes in animals
EP3156494A1 (fr) 2008-12-04 2017-04-19 Sangamo BioSciences, Inc. Édition de génome chez des rats au moyen de nucléases en doigt de zinc
US20110023144A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved in amyotrophyic lateral sclerosis disease
US20110023146A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved in secretase-associated disorders
US20110023157A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Equine genome editing with zinc finger nucleases
US20110023151A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genome editing of abc transporters
US20110023148A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genome editing of addiction-related genes in animals
US20110023141A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved with parkinson's disease
US20110023143A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of neurodevelopmental genes in animals
US20110023147A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of prion disorder-related genes in animals
US20110023150A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genome editing of genes associated with schizophrenia in animals
US20110023154A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Silkworm genome editing with zinc finger nucleases
US20110023149A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved in tumor suppression in animals
US20110023145A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved in autism spectrum disorders
US20110023139A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved in cardiovascular disease
US20110030072A1 (en) * 2008-12-04 2011-02-03 Sigma-Aldrich Co. Genome editing of immunodeficiency genes in animals
WO2010077319A1 (fr) 2008-12-17 2010-07-08 Dow Agrosciences Llc Intégration ciblée dans le locus zp15
US8329986B2 (en) 2008-12-17 2012-12-11 Dow Agrosciences, Llc Targeted integration into the Zp15 locus
US20100199389A1 (en) * 2008-12-17 2010-08-05 Dow Agrosciences Llc Targeted integration into the Zp15 locus
US9717759B2 (en) 2009-03-20 2017-08-01 Sangamo Therapeutics, Inc. Modification of CXCR4 using engineered zinc finger proteins
US20100291048A1 (en) * 2009-03-20 2010-11-18 Sangamo Biosciences, Inc. Modification of CXCR4 using engineered zinc finger proteins
US8871905B2 (en) 2009-03-20 2014-10-28 Sangamo Biosciences, Inc. Modification of CXCR4 using engineered zinc finger proteins
WO2010107493A2 (fr) 2009-03-20 2010-09-23 Sangamo Biosciences, Inc. Modification de cxcr4 en utilisant des protéines à doigt de zinc modifiées
US9834787B2 (en) 2009-04-09 2017-12-05 Sangamo Therapeutics, Inc. Targeted integration into stem cells
US20110027235A1 (en) * 2009-04-09 2011-02-03 Sangamo Biosciences, Inc. Targeted integration into stem cells
US20110014616A1 (en) * 2009-06-30 2011-01-20 Sangamo Biosciences, Inc. Rapid screening of biologically active nucleases and isolation of nuclease-modified cells
US20120171771A1 (en) * 2009-07-08 2012-07-05 Cellular Dynamics International, Inc. Modified ips cells having a mutant form of a human immunodeficiency virus (hiv) cellular entry gene
EP3156504A1 (fr) 2009-08-11 2017-04-19 Sangamo BioSciences, Inc. Organismes homozygotes destinés à une modification ciblée
US20110041195A1 (en) * 2009-08-11 2011-02-17 Sangamo Biosciences, Inc. Organisms homozygous for targeted modification
US10827731B2 (en) 2009-08-11 2020-11-10 Sangamo Therapeutics, Inc. Method of inactivating the IPK1 gene in corn
EP2464750A4 (fr) * 2009-08-11 2013-10-02 Sangamo Biosciences Inc Organismes homozygotes destinés à une modification ciblée
EP3428289A1 (fr) 2009-08-11 2019-01-16 Sangamo Therapeutics, Inc. Organismes homozygotes destinés à une modification ciblée
US9476060B2 (en) 2009-10-21 2016-10-25 Danziger Innovations Ltd. Generating genotypic variations in plant genomes by gamete infection
WO2011048600A1 (fr) 2009-10-21 2011-04-28 Danziger Innovations Ltd. Génération de variations génotypiques dans des génomes de plantes par infection d'un gamète
US9631201B2 (en) 2009-10-22 2017-04-25 Sangamo Biosciences, Inc. Engineered zinc finger proteins targeting plant genes involved in fatty acid biosynthesis
US20110167521A1 (en) * 2009-10-22 2011-07-07 Dow Agrosciences Llc Engineered zinc finger proteins targeting plant genes involved in fatty acid biosynthesis
US10017775B2 (en) 2009-10-22 2018-07-10 Dow Agrosciences Llc Engineered zinc finger proteins targeting plant genes involved in fatty acid biosynthesis
US8592645B2 (en) 2009-10-22 2013-11-26 Dow Agrosciences Llc Engineered zinc finger proteins targeting plant genes involved in fatty acid biosynthesis
WO2011049627A1 (fr) 2009-10-22 2011-04-28 Dow Agrosciences Llc Protéines à doigt de zinc modifiées ciblant des gènes de plante impliqués dans la biosynthèse des acides gras
EP2722392A2 (fr) 2009-10-22 2014-04-23 Dow AgroSciences LLC Protéines à doigt de zinc modifiées ciblant des gènes de plante impliqués dans la biosynthèse des acides gras
US11439666B2 (en) 2009-11-10 2022-09-13 Sangamo Therapeutics, Inc. Targeted disruption of T cell receptor genes using engineered zinc finger protein nucleases
US8956828B2 (en) 2009-11-10 2015-02-17 Sangamo Biosciences, Inc. Targeted disruption of T cell receptor genes using engineered zinc finger protein nucleases
US20110158957A1 (en) * 2009-11-10 2011-06-30 Sangamo Biosciences, Inc. Targeted disruption of T cell receptor genes using engineered zinc finger protein nucleases
US10155011B2 (en) 2009-11-10 2018-12-18 Sangamo Therapeutics, Inc. Targeted disruption of T cell receptor genes using engineered zinc finger protein nucleases
US9493827B2 (en) * 2010-01-14 2016-11-15 Deutsches Krebsforschungszentrum Determination of in vivo DNA double-strand break localization and application thereof
US20130143204A1 (en) * 2010-01-14 2013-06-06 Ospedale San Raffaele Srl Determination of in vivo dna double-strand break localization and application thereof
EA031429B1 (ru) * 2010-01-22 2018-12-28 ДАУ АГРОСАЙЕНСИЗ ЭлЭлСи Сконструированные посадочные площадки для направленного воздействия на ген в растениях
CN102821598B (zh) * 2010-01-22 2016-01-20 陶氏益农公司 供植物中基因靶向用的工程化降落场
JP2013517775A (ja) * 2010-01-22 2013-05-20 ダウ アグロサイエンシィズ エルエルシー 植物における遺伝子ターゲティング用の操作されたランディングパッド
US10160975B2 (en) 2010-01-22 2018-12-25 Dow Agrosciences Llc Engineered landing pads for gene targeting in plants
WO2011091317A3 (fr) * 2010-01-22 2011-11-17 Dow Agrosciences Llc Sites d'entrée génétiquement modifiés pour ciblage de gènes dans des plantes
CN102821598A (zh) * 2010-01-22 2012-12-12 陶氏益农公司 供植物中基因靶向用的工程化降落场
JP2013517774A (ja) * 2010-01-22 2013-05-20 ダウ アグロサイエンシィズ エルエルシー 遺伝子改変生物における導入遺伝子の切除
JP2016163572A (ja) * 2010-01-22 2016-09-08 ダウ アグロサイエンシィズ エルエルシー 植物における遺伝子ターゲティング用の操作されたランディングパッド
US10260062B2 (en) 2010-01-22 2019-04-16 Sangamo Therapeutics, Inc. Targeted genomic alteration
EA031356B1 (ru) * 2010-01-22 2018-12-28 ДАУ АГРОСАЙЕНСИЗ ЭлЭлСи Вырезание трансгенов в генетически измененных организмах
US20110191877A1 (en) * 2010-01-22 2011-08-04 Dow Agrosciences Llc Excision of transgenes in genetically modified organisms
CN105671073A (zh) * 2010-01-22 2016-06-15 陶氏益农公司 供植物中基因靶向用的工程化降落场
WO2011091311A3 (fr) * 2010-01-22 2011-11-17 Dow Agrosciences Llc Excision de transgènes dans organismes génétiquement modifiés
US20110189775A1 (en) * 2010-01-22 2011-08-04 Dow Agrosciences Llc Targeted genomic alteration
US20110191899A1 (en) * 2010-01-22 2011-08-04 Dow Agrosciences Llc Engineered landing pads for gene targeting in plants
WO2011090804A1 (fr) 2010-01-22 2011-07-28 Dow Agrosciences Llc Altération génomique ciblée
US9695432B2 (en) 2010-01-22 2017-07-04 Dow Agrosciences Llc Excision of transgenes in genetically modified organisms
US8802921B2 (en) 2010-01-22 2014-08-12 Dow Agrosciences, Llc. Engineered landing pads for gene targeting in plants
US11008578B2 (en) 2010-01-22 2021-05-18 Corteva Agriscience Llc Engineered landing pads for gene targeting in plants
EA027914B1 (ru) * 2010-01-22 2017-09-29 ДАУ АГРОСАЙЕНСИЗ ЭлЭлСи Сконструированные посадочные площадки для направленного воздействия на ген в растениях
WO2011100058A1 (fr) 2010-02-09 2011-08-18 Sangamo Biosciences, Inc. Modification génomique ciblée avec des molécules donneuses partiellement monocaténaires
WO2011139335A1 (fr) * 2010-04-26 2011-11-10 Sangamo Biosciences, Inc. Édition du génome d'un locus de rosa en utilisant des nucléases à doigt de zinc
US9567573B2 (en) 2010-04-26 2017-02-14 Sangamo Biosciences, Inc. Genome editing of a Rosa locus using nucleases
US8771985B2 (en) 2010-04-26 2014-07-08 Sangamo Biosciences, Inc. Genome editing of a Rosa locus using zinc-finger nucleases
WO2011139349A1 (fr) 2010-05-03 2011-11-10 Sangamo Biosciences, Inc. Compositions pour relier des modules en doigt de zinc
EP3636766A1 (fr) 2010-05-03 2020-04-15 Sangamo Therapeutics, Inc. Compositions pour relier des modules en doigt de zinc
US8772453B2 (en) 2010-05-03 2014-07-08 Sangamo Biosciences, Inc. Compositions for linking zinc finger modules
US9163245B2 (en) 2010-05-03 2015-10-20 Sangamo Biosciences, Inc. Compositions for linking zinc finger modules
US8586526B2 (en) 2010-05-17 2013-11-19 Sangamo Biosciences, Inc. DNA-binding proteins and uses thereof
US9322005B2 (en) 2010-05-17 2016-04-26 Sangamo Biosciences, Inc. DNA-binding proteins and uses thereof
US8912138B2 (en) 2010-05-17 2014-12-16 Sangamo Biosciences, Inc. DNA-binding proteins and uses thereof
US9493750B2 (en) 2010-05-17 2016-11-15 Sangamo Biosciences, Inc. DNA-binding proteins and uses thereof
US10253333B2 (en) 2010-05-17 2019-04-09 Sangamo Therapeutics, Inc. DNA-binding proteins and uses thereof
US9783827B2 (en) 2010-05-17 2017-10-10 Sangamo Therapeutics, Inc. DNA-binding proteins and uses thereof
US11661612B2 (en) 2010-05-17 2023-05-30 Sangamo Therapeutics, Inc. DNA-binding proteins and uses thereof
KR101971741B1 (ko) * 2010-07-23 2019-08-14 시그마-알드리치 컴퍼니., 엘엘씨 표적화 엔도뉴클레아제 및 단일-가닥 핵산을 사용하는 게놈 편집
US9512444B2 (en) 2010-07-23 2016-12-06 Sigma-Aldrich Co. Llc Genome editing using targeting endonucleases and single-stranded nucleic acids
CN103168101A (zh) * 2010-07-23 2013-06-19 西格马-奥尔德里奇有限责任公司 使用靶向核酸内切酶和单链核酸的基因组编辑
KR20130099916A (ko) * 2010-07-23 2013-09-06 시그마-알드리치 컴퍼니., 엘엘씨 표적화 엔도뉴클레아제 및 단일-가닥 핵산을 사용하는 게놈 편집
WO2012012738A1 (fr) 2010-07-23 2012-01-26 Sigma-Aldrich Co., Llc Modifications du génome à l'aide d'endonucléases de ciblage et d'acides nucléiques simples brins
EP3489359A1 (fr) * 2010-07-23 2019-05-29 Sigma Aldrich Co. LLC Modifications du genome a l'aide d'endonuclease de ciblage et d'acides nucleiques simples brins
WO2012018726A1 (fr) * 2010-08-02 2012-02-09 Cellectis Sa Procédé d'augmentation du ciblage de gènes à cassure double-brin induite
WO2012047598A1 (fr) 2010-09-27 2012-04-12 Sangamo Biosciences, Inc. Procédés et compositions d'inhibition d'entrée de virus dans cellules
US9566352B2 (en) * 2010-09-27 2017-02-14 Sangamo Biosciences, Inc. Methods and compositions for inhibiting viral entry into cells
EP3511420A1 (fr) 2010-09-27 2019-07-17 Sangamo Therapeutics, Inc. Procédés et compositions pour l'inhibition de l'entrée de virus dans des cellules
US20120093787A1 (en) * 2010-09-27 2012-04-19 Sangamo Biosciences, Inc. Methods and compositions for inhibiting viral entry into cells
US9175280B2 (en) 2010-10-12 2015-11-03 Sangamo Biosciences, Inc. Methods and compositions for treating hemophilia B
US9629930B2 (en) 2010-10-12 2017-04-25 Sangamo Biosciences, Inc. Methods and compositions for treating hemophilia B
US9631187B2 (en) 2011-01-05 2017-04-25 Sangamo Biosciences, Inc. Methods and compositions for gene correction
US20120192301A1 (en) * 2011-01-05 2012-07-26 Sangamo BioSciences, Inc. and Whitehead Institute for Biomedical Research Methods and compositions for gene correction
US9267123B2 (en) * 2011-01-05 2016-02-23 Sangamo Biosciences, Inc. Methods and compositions for gene correction
WO2012139045A1 (fr) 2011-04-08 2012-10-11 Gilead Biologics, Inc. Procédés et compositions pour normaliser le système vasculaire tumoral par inhibition de la loxl2
US20130122591A1 (en) * 2011-10-27 2013-05-16 The Regents Of The University Of California Methods and compositions for modification of the hprt locus
US8895264B2 (en) * 2011-10-27 2014-11-25 Sangamo Biosciences, Inc. Methods and compositions for modification of the HPRT locus
WO2013074999A1 (fr) 2011-11-16 2013-05-23 Sangamo Biosciences, Inc. Protéines de liaison d'adn modifiées et utilisations de celles-ci
US9458205B2 (en) 2011-11-16 2016-10-04 Sangamo Biosciences, Inc. Modified DNA-binding proteins and uses thereof
US12391961B2 (en) 2011-12-05 2025-08-19 Factor Bioscience Inc. Methods and products for transfecting cells
US12227757B2 (en) 2011-12-05 2025-02-18 Factor Bioscience Inc. Methods and products for transfecting cells
US10662410B1 (en) 2011-12-05 2020-05-26 Factor Bioscience Inc. Methods and products for transfecting cells
US11708586B2 (en) 2011-12-05 2023-07-25 Factor Bioscience Inc. Methods and products for transfecting cells
US9422577B2 (en) 2011-12-05 2016-08-23 Factor Bioscience Inc. Methods and products for transfecting cells
US11692203B2 (en) 2011-12-05 2023-07-04 Factor Bioscience Inc. Methods and products for transfecting cells
US9605277B2 (en) 2011-12-05 2017-03-28 Factor Bioscience, Inc. Methods and products for transfecting cells
US10982229B2 (en) 2011-12-05 2021-04-20 Factor Bioscience Inc. Methods and products for transfecting cells
US12227768B2 (en) 2011-12-05 2025-02-18 Factor Bioscience Inc. Methods and products for transfection
US10472611B2 (en) 2011-12-05 2019-11-12 Factor Bioscience Inc. Methods and products for transfecting cells
US9605278B2 (en) 2011-12-05 2017-03-28 Factor Bioscience Inc. Methods and products for transfecting cells
US12559772B2 (en) 2011-12-05 2026-02-24 Factor Bioscience Inc. Methods and products for transfecting cells
US10829738B2 (en) 2011-12-05 2020-11-10 Factor Bioscience Inc. Methods and products for transfecting cells
US11466293B2 (en) 2011-12-05 2022-10-11 Factor Bioscience Inc. Methods and products for transfecting cells
US10435678B2 (en) 2011-12-30 2019-10-08 Caribou Biosciences, Inc. Modified cascade ribonucleoproteins and uses thereof
US10954498B2 (en) 2011-12-30 2021-03-23 Caribou Biosciences, Inc. Modified cascade ribonucleoproteins and uses thereof
US11939604B2 (en) 2011-12-30 2024-03-26 Caribou Biosciences, Inc. Modified cascade ribonucleoproteins and uses thereof
US9885026B2 (en) 2011-12-30 2018-02-06 Caribou Biosciences, Inc. Modified cascade ribonucleoproteins and uses thereof
US10711257B2 (en) 2011-12-30 2020-07-14 Caribou Biosciences, Inc. Modified cascade ribonucleoproteins and uses thereof
WO2013166315A1 (fr) 2012-05-02 2013-11-07 Dow Agrosciences Llc Modification ciblée de malate déshydrogénase
US10982230B2 (en) 2012-05-25 2021-04-20 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10358659B2 (en) 2012-05-25 2019-07-23 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10988780B2 (en) 2012-05-25 2021-04-27 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10988782B2 (en) 2012-05-25 2021-04-27 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10982231B2 (en) 2012-05-25 2021-04-20 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11001863B2 (en) 2012-05-25 2021-05-11 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10000772B2 (en) 2012-05-25 2018-06-19 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11008589B2 (en) 2012-05-25 2021-05-18 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11008590B2 (en) 2012-05-25 2021-05-18 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11028412B2 (en) 2012-05-25 2021-06-08 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US12215343B2 (en) 2012-05-25 2025-02-04 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11186849B2 (en) 2012-05-25 2021-11-30 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10900054B2 (en) 2012-05-25 2021-01-26 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US12180504B2 (en) 2012-05-25 2024-12-31 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11242543B2 (en) 2012-05-25 2022-02-08 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10227611B2 (en) 2012-05-25 2019-03-12 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11274318B2 (en) 2012-05-25 2022-03-15 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US12180503B2 (en) 2012-05-25 2024-12-31 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11293034B2 (en) 2012-05-25 2022-04-05 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10266850B2 (en) 2012-05-25 2019-04-23 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11332761B2 (en) 2012-05-25 2022-05-17 The Regenis of Wie University of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10793878B1 (en) 2012-05-25 2020-10-06 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10774344B1 (en) 2012-05-25 2020-09-15 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10301651B2 (en) 2012-05-25 2019-05-28 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11401532B2 (en) 2012-05-25 2022-08-02 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10308961B2 (en) 2012-05-25 2019-06-04 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10337029B2 (en) 2012-05-25 2019-07-02 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10597680B2 (en) 2012-05-25 2020-03-24 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10752920B2 (en) 2012-05-25 2020-08-25 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10351878B2 (en) 2012-05-25 2019-07-16 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US12123015B2 (en) 2012-05-25 2024-10-22 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10113167B2 (en) 2012-05-25 2018-10-30 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10358658B2 (en) 2012-05-25 2019-07-23 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11970711B2 (en) 2012-05-25 2024-04-30 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11473108B2 (en) 2012-05-25 2022-10-18 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11479794B2 (en) 2012-05-25 2022-10-25 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10385360B2 (en) 2012-05-25 2019-08-20 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10400253B2 (en) 2012-05-25 2019-09-03 The Regents Of The University Of California Methods and compositions or RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10407697B2 (en) 2012-05-25 2019-09-10 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10415061B2 (en) 2012-05-25 2019-09-17 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10676759B2 (en) 2012-05-25 2020-06-09 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10669560B2 (en) 2012-05-25 2020-06-02 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10421980B2 (en) 2012-05-25 2019-09-24 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10428352B2 (en) 2012-05-25 2019-10-01 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11549127B2 (en) 2012-05-25 2023-01-10 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10443076B2 (en) 2012-05-25 2019-10-15 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11814645B2 (en) 2012-05-25 2023-11-14 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10487341B2 (en) 2012-05-25 2019-11-26 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10577631B2 (en) 2012-05-25 2020-03-03 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11634730B2 (en) 2012-05-25 2023-04-25 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10513712B2 (en) 2012-05-25 2019-12-24 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10519467B2 (en) 2012-05-25 2019-12-31 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10526619B2 (en) 2012-05-25 2020-01-07 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10640791B2 (en) 2012-05-25 2020-05-05 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10533190B2 (en) 2012-05-25 2020-01-14 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10626419B2 (en) 2012-05-25 2020-04-21 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10550407B2 (en) 2012-05-25 2020-02-04 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11674159B2 (en) 2012-05-25 2023-06-13 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10563227B2 (en) 2012-05-25 2020-02-18 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10570419B2 (en) 2012-05-25 2020-02-25 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10612045B2 (en) 2012-05-25 2020-04-07 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US20150128300A1 (en) * 2012-06-12 2015-05-07 Genentech, Inc. Methods and compositions for generating conditional knock-out alleles
US20140017214A1 (en) * 2012-07-11 2014-01-16 Sangamo Biosciences, Inc. Methods and compositions for delivery of biologics
WO2014011901A2 (fr) 2012-07-11 2014-01-16 Sangamo Biosciences, Inc. Procédés et compositions pour la délivrance d'agents biologiques
US10883119B2 (en) * 2012-07-11 2021-01-05 Sangamo Therapeutics, Inc. Methods and compositions for delivery of biologics
US11492643B2 (en) * 2012-08-29 2022-11-08 Sangamo Therapeutics, Inc. Methods and compositions for treatment of a genetic condition
US20140080216A1 (en) * 2012-08-29 2014-03-20 Sangamo Biosciences, Inc. Methods and compositions for treatment of a genetic condition
US20180223311A1 (en) * 2012-08-29 2018-08-09 Sangamo Therapeutics, Inc. Methods and compositions for treatment of a genetic condition
US9650648B2 (en) * 2012-08-29 2017-05-16 Sangamo Biosciences, Inc. Methods and compositions for treatment of a genetic condition
EP3406715A1 (fr) 2012-09-07 2018-11-28 Dow AgroSciences LLC Loci de performance fad3 et site cible correspondant, protéines de liaison spécifiques capables d'induire des ruptures ciblées
US9963711B2 (en) 2012-09-07 2018-05-08 Sangamo Therapeutics, Inc. FAD2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
US9493779B2 (en) 2012-09-07 2016-11-15 Dow Agrosciences Llc FAD2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
US10577616B2 (en) 2012-09-07 2020-03-03 Dow Agrosciences Llc FAD2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
US10844389B2 (en) 2012-09-07 2020-11-24 Dow Agrosciences Llc FAD2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
EP3431600A1 (fr) 2012-09-07 2019-01-23 Dow AgroSciences LLC Loci de performance fad2 et site cible correspondant, protéines de liaison spécifiques capables d'induire des ruptures ciblées
EP3404099A1 (fr) 2012-09-07 2018-11-21 Dow AgroSciences LLC Loci de performance fad2 et protéines de liaison spécifiques à un site cible correspondant pouvant induire des ruptures ciblées
WO2014039702A2 (fr) 2012-09-07 2014-03-13 Dow Agrosciences Llc Loci de performance fad2 et protéines correspondantes de liaison spécifique d'un site cible capables d'induire des cassures ciblées
US10287595B2 (en) 2012-09-07 2019-05-14 Dow Agrosciences Llc Fad2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
WO2014039692A2 (fr) 2012-09-07 2014-03-13 Dow Agrosciences Llc Loci de performance fad2 et protéines se liant à un site spécifique cible correspondantes pouvant induire des cassures ciblées
US10590437B2 (en) 2012-11-01 2020-03-17 Factor Bioscience Inc. Methods and products for expressing proteins in cells
KR102315098B1 (ko) 2012-11-01 2021-10-21 팩터 바이오사이언스 인크. 세포에서 단백질을 발현시키는 방법들과 생성물들
US9657282B2 (en) 2012-11-01 2017-05-23 Factor Bioscience, Inc. Methods and products for expressing proteins in cells
US10724053B2 (en) 2012-11-01 2020-07-28 Factor Bioscience Inc. Methods and products for expressing proteins in cells
US11339410B2 (en) 2012-11-01 2022-05-24 Factor Bioscience Inc. Methods and products for expressing proteins in cells
US10752918B2 (en) 2012-11-01 2020-08-25 Factor Bioscience Inc. Methods and products for expressing proteins in cells
US10767195B2 (en) 2012-11-01 2020-09-08 Factor Bioscience Inc. Methods and products for expressing proteins in cells
US12006508B2 (en) 2012-11-01 2024-06-11 Factor Bioscience Inc. Methods and products for expressing proteins in cells
US10752919B2 (en) 2012-11-01 2020-08-25 Factor Bioscience Inc. Methods and products for expressing proteins in cells
US10752917B2 (en) 2012-11-01 2020-08-25 Factor Bioscience Inc. Methods and products for expressing proteins in cells
WO2014071219A1 (fr) * 2012-11-01 2014-05-08 Factor Bioscience Inc. Procédés et produits pour l'expression de protéines dans des cellules
US11332758B2 (en) 2012-11-01 2022-05-17 Factor Bioscience Inc. Methods and products for expressing proteins in cells
KR20200067921A (ko) * 2012-11-01 2020-06-12 팩터 바이오사이언스 인크. 세포에서 단백질을 발현시키는 방법들과 생성물들
US10415060B2 (en) 2012-11-01 2019-09-17 Factor Bioscience Inc. Methods and products for expressing proteins in cells
US9758797B2 (en) 2012-11-01 2017-09-12 Factor Bioscience, Inc. Methods and products for expressing proteins in cells
US9376669B2 (en) 2012-11-01 2016-06-28 Factor Bioscience Inc. Methods and products for expressing proteins in cells
US11332759B2 (en) 2012-11-01 2022-05-17 Factor Bioscience Inc. Methods and products for expressing proteins in cells
US11339409B2 (en) 2012-11-01 2022-05-24 Factor Bioscience Inc. Methods and products for expressing proteins in cells
US9447395B2 (en) 2012-11-01 2016-09-20 Factor Bioscience Inc. Methods and products for expressing proteins in cells
US9464285B2 (en) 2012-11-01 2016-10-11 Factor Bioscience Inc. Methods and products for expressing proteins in cells
US9487768B2 (en) 2012-11-01 2016-11-08 Factor Bioscience Inc. Methods and products for expressing proteins in cells
US10731181B2 (en) * 2012-12-06 2020-08-04 Sigma, Aldrich Co. LLC CRISPR-based genome modification and regulation
US10745716B2 (en) 2012-12-06 2020-08-18 Sigma-Aldrich Co. Llc CRISPR-based genome modification and regulation
US10415046B2 (en) 2012-12-13 2019-09-17 Dow Agrosciences Llc Precision gene targeting to a particular locus in maize
WO2014127287A1 (fr) * 2013-02-14 2014-08-21 Massachusetts Institute Of Technology Procédé pour la mutagenèse ciblée in vivo
US9260752B1 (en) 2013-03-14 2016-02-16 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
US10125361B2 (en) 2013-03-14 2018-11-13 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
US9725714B2 (en) 2013-03-14 2017-08-08 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
US9809814B1 (en) 2013-03-14 2017-11-07 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
US11312953B2 (en) 2013-03-14 2022-04-26 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
US9803194B2 (en) 2013-03-14 2017-10-31 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
US9909122B2 (en) 2013-03-14 2018-03-06 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
US9410198B2 (en) 2013-03-14 2016-08-09 Caribou Biosciences, Inc. Compostions and methods of nucleic acid-targeting nucleic acids
US10918668B2 (en) 2013-03-21 2021-02-16 Sangamo Therapeutics, Inc. Targeted disruption of T cell receptor genes using engineered zinc finger protein nucleases
US9937207B2 (en) 2013-03-21 2018-04-10 Sangamo Therapeutics, Inc. Targeted disruption of T cell receptor genes using talens
WO2014165612A2 (fr) 2013-04-05 2014-10-09 Dow Agrosciences Llc Procédés et compositions permettant d'intégrer une séquence exogène au sein du génome de plantes
US11198883B2 (en) 2013-04-05 2021-12-14 Dow Agrosciences Llc Methods and compositions for integration of an exogenous sequence within the genome of plants
US10501748B2 (en) 2013-04-05 2019-12-10 Dow Agrosciences Llc Methods and compositions for integration of an exogenous sequence within the genome of plants
EP3679785A2 (fr) 2013-04-05 2020-07-15 Dow AgroSciences LLC Procédés et compositions pour l'intégration d'une séquence exogène dans le génome de plantes
US10604771B2 (en) 2013-05-10 2020-03-31 Sangamo Therapeutics, Inc. Delivery methods and compositions for nuclease-mediated genome engineering
US11041174B2 (en) 2013-08-28 2021-06-22 Sangamo Therapeutics, Inc. Compositions for linking DNA-binding domains and cleavage domains
US9567609B2 (en) 2013-08-28 2017-02-14 Sangamo Biosciences, Inc. Compositions for linking DNA-binding domains and cleavage domains
EP3988654A1 (fr) 2013-08-28 2022-04-27 Sangamo Therapeutics, Inc. Compositions de liaison de domaines de liaison à l'adn et de domaines de clivage
US10538787B2 (en) 2013-08-28 2020-01-21 Sangamo Therapeutics, Inc. Compositions for linking DNA-binding domains and cleavage domains
EP3591045A1 (fr) 2013-08-28 2020-01-08 Sangamo Therapeutics, Inc. Compositions de liaison de domaines de liaison à l'adn et de domaines de clivage
US11149287B2 (en) 2013-11-04 2021-10-19 Corteva Agriscience Llc Optimal soybean loci
US10093940B2 (en) 2013-11-04 2018-10-09 Dow Agrosciences Llc Optimal maize loci
US10273493B2 (en) 2013-11-04 2019-04-30 Dow Agrosciences Llc Optimal maize loci
US9909131B2 (en) 2013-11-04 2018-03-06 Dow Agrosciences Llc Optimal soybean loci
US11098317B2 (en) 2013-11-04 2021-08-24 Corteva Agriscience Llc Optimal maize loci
US10106804B2 (en) 2013-11-04 2018-10-23 Dow Agrosciences Llc Optimal soybean loci
US10233465B2 (en) 2013-11-04 2019-03-19 Dow Agrosciences Llc Optimal soybean loci
US11198882B2 (en) 2013-11-04 2021-12-14 Corteva Agriscience Llc Optimal maize loci
US11098316B2 (en) 2013-11-04 2021-08-24 Corteva Agriscience Llc Optimal soybean loci
US9770489B2 (en) 2014-01-31 2017-09-26 Factor Bioscience Inc. Methods and products for nucleic acid production and delivery
US12201675B2 (en) 2014-01-31 2025-01-21 Factor Bioscience Inc. Methods and products for nucleic acid production and delivery
US10124042B2 (en) 2014-01-31 2018-11-13 Factor Bioscience Inc. Methods and products for nucleic acid production and delivery
EP3929279A1 (fr) 2014-03-18 2021-12-29 Sangamo Therapeutics, Inc. Procédés et compositions de régulation de l'expression de protéines en doigt de zinc
US9624498B2 (en) 2014-03-18 2017-04-18 Sangamo Biosciences, Inc. Methods and compositions for regulation of zinc finger protein expression
WO2015167959A1 (fr) * 2014-04-28 2015-11-05 Sigma-Aldrich Co. Llc Modification épigénétique de génomes de mammifères à l'aide d'endonucléases ciblées
US9574211B2 (en) 2014-05-13 2017-02-21 Sangamo Biosciences, Inc. Methods and compositions for prevention or treatment of a disease
EP4335926A2 (fr) 2014-07-14 2024-03-13 Washington State University Nanos knock-out qui ablit des cellules de lignée germinale
WO2016011029A2 (fr) 2014-07-14 2016-01-21 Washington State University Knock-out nanos qui permet de pratiquer l'ablation des cellules de la lignée germinale
US20160022737A1 (en) * 2014-07-25 2016-01-28 Sangamo Biosciences, Inc. Gene editing for hiv gene therapy
US9757420B2 (en) * 2014-07-25 2017-09-12 Sangamo Therapeutics, Inc. Gene editing for HIV gene therapy
US20160160238A1 (en) * 2014-11-12 2016-06-09 Recombinetics, Inc. Heterozygous modifications of tumor suppressor genes
US10883111B2 (en) 2014-11-27 2021-01-05 Danziger Innovations Ltd. Nucleic acid constructs for genome editing
US10889834B2 (en) * 2014-12-15 2021-01-12 Sangamo Therapeutics, Inc. Methods and compositions for enhancing targeted transgene integration
US20160168593A1 (en) * 2014-12-15 2016-06-16 Sangamo Biosciences, Inc. Methods and compositions for enhancing targeted transgene integration
US11241505B2 (en) 2015-02-13 2022-02-08 Factor Bioscience Inc. Nucleic acid products and methods of administration thereof
WO2016170484A1 (fr) 2015-04-21 2016-10-27 Novartis Ag Système d'édition de gènes guidée par arn et utilisations associées
US10179918B2 (en) 2015-05-07 2019-01-15 Sangamo Therapeutics, Inc. Methods and compositions for increasing transgene activity
WO2017023570A1 (fr) 2015-08-06 2017-02-09 The Curators Of The University Of Missouri Animaux résistant aux pathogènes ayant des gènes cd163 modifiés
EP4361279A2 (fr) 2015-08-06 2024-05-01 The Curators of the University of Missouri Animaux résistants aux pathogènes ayant des gènes cd163 modifiés
US12084675B2 (en) 2016-06-02 2024-09-10 Sigma-Aldrich Co. Llc Using programmable DNA binding proteins to enhance targeted genome modification
US10266851B2 (en) 2016-06-02 2019-04-23 Sigma-Aldrich Co. Llc Using programmable DNA binding proteins to enhance targeted genome modification
US12275952B2 (en) 2016-06-02 2025-04-15 Sigma-Aldrich Co. Llc Using programmable DNA binding proteins to enhance targeted genome modification
US10888627B2 (en) 2016-08-17 2021-01-12 Factor Bioscience Inc. Nucleic acid products and methods of administration thereof
US10369233B2 (en) 2016-08-17 2019-08-06 Factor Bioscience Inc. Nucleic acid products and methods of administration thereof
US10363321B2 (en) 2016-08-17 2019-07-30 Factor Bioscience Inc. Nucleic acid products and methods of administration thereof
US10894092B2 (en) 2016-08-17 2021-01-19 Factor Bioscience Inc. Nucleic acid products and methods of administration thereof
US11904023B2 (en) 2016-08-17 2024-02-20 Factor Bioscience Inc. Nucleic acid products and methods of administration thereof
US10137206B2 (en) 2016-08-17 2018-11-27 Factor Bioscience Inc. Nucleic acid products and methods of administration thereof
US10576167B2 (en) 2016-08-17 2020-03-03 Factor Bioscience Inc. Nucleic acid products and methods of administration thereof
US10350304B2 (en) 2016-08-17 2019-07-16 Factor Bioscience Inc. Nucleic acid products and methods of administration thereof
WO2018052919A1 (fr) * 2016-09-14 2018-03-22 Monsanto Technology Llc Méthodes et compositions d'édition génomique par induction d'haploïde
US11401524B2 (en) 2016-09-14 2022-08-02 Monsanto Technology, Llc Methods and compositions for genome editing via haploid induction
US11781248B2 (en) 2019-06-13 2023-10-10 Allogene Therapeutics, Inc. Anti-TALEN antibodies and uses thereof
US12344572B2 (en) 2019-07-03 2025-07-01 Factor Bioscience Inc. Cationic lipids and transfection methods
US11814333B2 (en) 2019-07-30 2023-11-14 Factor Bioscience Inc. Cationic lipids and transfection methods
US11242311B2 (en) 2019-07-30 2022-02-08 Factor Bioscience Inc. Cationic lipids and transfection methods
US10752576B1 (en) 2019-07-30 2020-08-25 Factor Bioscience Inc. Cationic lipids and transfection methods
US10611722B1 (en) 2019-07-30 2020-04-07 Factor Bioscience Inc. Cationic lipids and transfection methods
US12384740B2 (en) 2019-07-30 2025-08-12 Factor Bioscience Inc. Cationic lipids and transfection methods
US10556855B1 (en) 2019-07-30 2020-02-11 Factor Bioscience Inc. Cationic lipids and transfection methods
US10501404B1 (en) 2019-07-30 2019-12-10 Factor Bioscience Inc. Cationic lipids and transfection methods
WO2022101641A1 (fr) 2020-11-16 2022-05-19 Pig Improvement Company Uk Limited Animaux résistants à la grippe a ayant des gènes anp32 modifiés
WO2023081756A1 (fr) 2021-11-03 2023-05-11 The J. David Gladstone Institutes, A Testamentary Trust Established Under The Will Of J. David Gladstone Édition précise du génome à l'aide de rétrons
WO2023105244A1 (fr) 2021-12-10 2023-06-15 Pig Improvement Company Uk Limited Édition de tmprss2/4 pour la résistance aux maladies chez le bétail
WO2023141602A2 (fr) 2022-01-21 2023-07-27 Renagade Therapeutics Management Inc. Rétrons modifiés et méthodes d'utilisation
WO2024013514A2 (fr) 2022-07-15 2024-01-18 Pig Improvement Company Uk Limited Animaux d'élevage ayant subi une édition génique et présentant une résistance aux coronavirus
WO2024044723A1 (fr) 2022-08-25 2024-02-29 Renagade Therapeutics Management Inc. Rétrons modifiés et méthodes d'utilisation
WO2025049959A2 (fr) 2023-09-01 2025-03-06 Renagade Therapeutics Management Inc. Systèmes et compositions d'édition génique, et méthodes de traitement du syndrome vexas
WO2025174765A1 (fr) 2024-02-12 2025-08-21 Renagade Therapeutics Management Inc. Nanoparticules lipidiques comprenant des molécules d'arn codant destinées à être utilisées dans l'édition génique et comme vaccins et agents thérapeutiques

Also Published As

Publication number Publication date
US20130244332A1 (en) 2013-09-19
US20090258363A1 (en) 2009-10-15
US8313925B2 (en) 2012-11-20
CN101273141A (zh) 2008-09-24
CA2615532C (fr) 2016-06-28
JP2009502170A (ja) 2009-01-29
EP1913149A2 (fr) 2008-04-23
WO2007014275A2 (fr) 2007-02-01
CN101273141B (zh) 2013-03-27
AU2006272634A1 (en) 2007-02-01
IL188966A (en) 2015-10-29
KR101419729B1 (ko) 2014-07-17
SG10201508995QA (en) 2015-11-27
KR20130136590A (ko) 2013-12-12
US9260726B2 (en) 2016-02-16
JP2013031447A (ja) 2013-02-14
JP2015165804A (ja) 2015-09-24
JP5788847B2 (ja) 2015-10-07
JP6122062B2 (ja) 2017-04-26
IL188966A0 (en) 2008-08-07
AU2006272634B2 (en) 2013-01-24
EP1913149A4 (fr) 2009-08-05
WO2007014275A3 (fr) 2007-05-24
KR20080033455A (ko) 2008-04-16
CA2615532A1 (fr) 2007-02-01

Similar Documents

Publication Publication Date Title
US10675302B2 (en) Methods and compositions for targeted cleavage and recombination
US9260726B2 (en) Targeted integration and expression on exogenous nucleic acid sequences
US8349810B2 (en) Methods for targeted cleavage and recombination of CCR5
EP1651660B1 (fr) Methodes et compositions permettant un clivage et une recombinaison cibles
US11311574B2 (en) Methods and compositions for targeted cleavage and recombination
AU2012245168B2 (en) Targeted Integration and Expression of Exogenous Nucleic Acid Sequences
AU2007201649B2 (en) Methods and Compositions for Targeted Cleavage and Recombination
HK1094009B (en) Methods and compostions for targeted cleavage and recombination
HK1085484A (en) Methods and compositions for targeted cleavage and recombination
HK1085484B (en) Methods and compositions for targeted cleavage and recombination

Legal Events

Date Code Title Description
AS Assignment

Owner name: SANGAMO BIOSCIENCES, INC., CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HOLMES, MICHAEL C.;URNOV, FYODOR;GREGORY, PHILIP D.;AND OTHERS;REEL/FRAME:018494/0190

Effective date: 20061101

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION

AS Assignment

Owner name: SANGAMO THERAPEUTICS, INC., CALIFORNIA

Free format text: CHANGE OF NAME;ASSIGNOR:SANGAMO BIOSCIENCES, INC.;REEL/FRAME:051071/0336

Effective date: 20170105