USRE45721E1 - Relating to binding proteins for recognition of DNA - Google Patents

Relating to binding proteins for recognition of DNA Download PDF

Info

Publication number
USRE45721E1
USRE45721E1 US10/400,017 US40001703A USRE45721E US RE45721 E1 USRE45721 E1 US RE45721E1 US 40001703 A US40001703 A US 40001703A US RE45721 E USRE45721 E US RE45721E
Authority
US
United States
Prior art keywords
zinc finger
binding
zinc
library
sequence
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.)
Expired - Lifetime
Application number
US10/400,017
Other languages
English (en)
Inventor
Yen Choo
Aaron Klug
Isidro Sanchez-Garcia
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.)
Gendaq Ltd
United Kingdom Research and Innovation
Original Assignee
Gendaq Ltd
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
Priority claimed from GB9416880A external-priority patent/GB9416880D0/en
Priority claimed from GB9422534A external-priority patent/GB9422534D0/en
Priority claimed from GBGB9514698.1A external-priority patent/GB9514698D0/en
Application filed by Gendaq Ltd filed Critical Gendaq Ltd
Priority to US10/400,017 priority Critical patent/USRE45721E1/en
Anticipated expiration legal-status Critical
Application granted granted Critical
Publication of USRE45721E1 publication Critical patent/USRE45721E1/en
Assigned to UNITED KINGDOM RESEARCH AND INNOVATION reassignment UNITED KINGDOM RESEARCH AND INNOVATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MEDICAL RESEARCH COUNCIL
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • 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

  • This invention relates inter alia to methods of selecting and designing polypeptides comprising zinc finger binding motifs, polypeptides made by the method(s) of the invention and to various applications thereof.
  • Zf zinc finger
  • DNA-binding protein domains are able to discriminate between different DNA sequences.
  • the zinc finger motif has been studied extensively, with a view to providing some insight into this problem, owing to its remarkable prevalence in the eukaryotic genome, and its important role in proteins which control gene expression in Drosophila (e.g. Harrison & Travers 1990 EMBO J. 9, 207-216), the mouse (Christy et al., 1988 Proc. Natl. Acad. Sci. USA 85, 7857-7861) and humans (Kinzler et al., 1988 Nature (London) 332, 371).
  • sequence-specific DNA-binding proteins bind to the DNA double helix by inserting an ⁇ -helix into the major groove (Pabo & Sauer 1992 Annu. Rev. Biochem. 61, 1053-1095; Harrison 1991 Nature (London) 353, 715-719; and Klug 1993 Gene 135, 83-92). Sequence specificity results from the geometrical and chemical complementarity between the amino acid side chains of the ⁇ -helix and the accessible groups exposed on the edges of base-pairs. In addition to this direct reading of the DNA sequence, interactions with the DNA backbone stabilise the complex and are sensitive to the conformation of the nucleic acid, which in turn depends on the base sequence (Dickerson & Drew 1981 J. Mol. Biol. 149, 761-786).
  • the zinc finger of the TFIIIA class is a good candidate for deriving a set of more generally applicable specificity rules owing to its great simplicity of structure and interaction with DNA.
  • the zinc finger is an independently folding domain which uses a zinc ion to stabilise the packing of an antiparallel ⁇ -sheet against an ⁇ -helix (Miller et al., 1985 EMBO J. 4, 1609-1614; Berg 1988 Proc. Natl. Acad. Sci. USA 85, 99-102; and Lee et al., 1989 Science 245, 635-637).
  • each finger is folded so that three amino acids are presented for binding to the DNA target sequence, although binding may be directly through only two of these positions.
  • the protein is made up of three fingers which contact a 9 base pair contiguous sequence of target DNA. A linker sequence is found between fingers which appears to make no direct contact with the nucleic acid.
  • Randomised mutagenesis (at the same postions as those selected by Rebar & Pabo) of finger 1 of Zif 268 with phage display has also been used by Jamieson et al., (1994 Biochemistry 33, 5689-5695) to create novel binding specificity and affinity.
  • Zf protein motifs are widespread in DNA binding proteins and that binding is via three key amino acids, each one contacting a single base pair in the target DNA sequence.
  • Motifs are modular and may be linked together to form a set of fingers which recognise a contiguous DNA sequence (e.g. a three fingered protein will recognise a 9 mer etc).
  • the key residues involved in DNA binding have been identified through sequence data and from structural information. Directed and random mutagenesis has confirmed the role of these amino acids in determining specificity and affinity.
  • Phage display has been used to screen for new binding specificities of random mutants of fingers.
  • a recognition code, to aid design of new finger specificities, has been worked towards although it has been suggested that specificity may be difficult to predict.
  • the invention provides a library of DNA sequences, each sequence encoding at least one zinc finger binding motif for display on a viral particle, the sequences coding for zinc finger binding motifs having random allocation of amino acids at positions ⁇ 1, +2, +3, +6 and at least at one of positions +1, +5 and +8.
  • a zinc finger binding motif is the ⁇ -helical structural motif found in zinc finger binding proteins, well known to those skilled in the art. The above numbering is based on the first amino acid in the ⁇ -helix of the zinc finger binding motif being position +1. It will be apparent to those skilled in the art that the amino acid residue at position ⁇ 1 does not, strictly speaking, form part of the ⁇ -helix of the zinc binding finger motif. Nevertheless, the residue at ⁇ 1 is shown to be very important functionally and is therefore considered as part of the binding motif ⁇ -helix for the purposes of the present invention.
  • the sequences may code for zinc finger binding motifs having random allocation at all of positions +1, +5 and +8.
  • the sequences may also be randomised at other positions (e.g. at position +9, although it is generally preferred to retain an arginine or a lysine residue at this position).
  • allocation of amino acids at the designated “random” positions may be genuinely random, it is preferred to avoid a hydrophobic residue (Phe, Trp or Tyr) or a cysteine residue at such positions.
  • the zinc finger binding motif is present within the context of other amino acids (which may be present in zinc finger proteins), so as to form a zinc finger (which includes an antiparallel ⁇ -sheet).
  • the zinc finger is preferably displayed as part of a zinc finger polypeptide, which polypeptide comprises a plurality of zinc fingers joined by an intervening linker peptide.
  • the library of sequences is such that the zinc finger polypeptide will comprise two or more zinc fingers of defined amino acid sequence (generally the wild type sequence) and one zinc finger having a zinc finger binding motif randomised in the manner defined above. It is preferred that the randomised finger of the polypeptide is positioned between the two or more fingers having defined sequence. The defined fingers will establish the “phase” of binding of the polypeptide to DNA, which helps to increase the binding specificity of the randomised finger.
  • sequences encode the randomised binding motif of the middle finger of the Zif268 polypeptide.
  • sequences also encode those amino acids N-terminal and C-terminal of the middle finger in wild type Zif268, which encode the first and third zinc fingers respectively.
  • the sequence encodes the whole of the Zif268 polypeptide.
  • the invention provides a library of DNA sequences, each sequence encoding the zinc finger binding motif of at least a middle finger of a zinc finger binding polypeptide for display on a viral particle, the sequences coding for the binding motif having random allocation of amino acids at positions ⁇ 1, +2, +3 and +6.
  • the zinc finger polypeptide will be Zif268.
  • the sequences of either library are such that the zinc finger binding domain can be cloned as a fusion with the minor coat protein (pIII) of bacteriophage fd.
  • the encoded polypeptide includes the tripeptide sequence Met-Ala-Glu as the N terminal of the zinc finger domain, which is known to allow expression and display using the bacteriophage fd system.
  • the library comprises 10 6 or more different sequences (ideally, as many as is practicable).
  • the invention provides a method of designing a zinc finger polypeptide for binding to a particular target DNA sequence, comprising screening each of a plurality of zinc finger binding motifs against at least an effective portion of the target DNA sequence and selecting those motifs which bind to the target DNA sequence.
  • An effective portion of the target DNA sequence is a sufficient length of DNA to allow binding of the zinc binding motif to the DNA. This is the minimum sequence information (concerning the target DNA sequence) that is required. Desirably at least two, preferably three or more, rounds of screening are performed.
  • the invention also provides a method of designing a zinc finger polypeptide for binding to a particular target DNA sequence, comprising comparing the binding of each of a plurality of zinc finger binding motifs to one or more DNA triplets, and selecting those motifs exhibiting preferable binding characteristics.
  • the method defined immediately above is preceded by a screening step according to the method defined in the previous paragraph.
  • the first step comprising screening each of a plurality of zinc finger binding motifs (typically in the form of a display library), mainly or wholly on the basis of affinity for the target sequence; the second step comprising comparing binding characteristics of those motifs selected by the initial screening step, and selecting those having preferable binding characteristics for a particular DNA triplet.
  • the plurality of zinc finger binding motifs is screened against a single DNA triplet, it is preferred that the triplet is represented in the target DNA sequence at the appropriate postion. However, it is also desirable to compare the binding of the plurality of zinc binding motifs to one or more DNA triplets not represented in the target DNA sequence (e.g. differing by just one of the three base pairs) in order to compare the specificity of binding of the various binding motifs.
  • the plurality of zinc finger binding motifs may be screened against all 64 possible permutations of 3 DNA bases.
  • zinc finger binding motifs Once suitable zinc finger binding motifs have been identified and obtained, they will advantageously be combined in a single zinc finger polypeptide. Typically this will be accomplished by use of recombinant DNA technology; conveniently a phage display system may be used.
  • the invention provides a DNA library consisting of 64 sequences, each sequence comprising a different one of the 64 possible permutations of three DNA bases in a form suitable for use in the selection method defined above.
  • sequences are associated, or capable of being associated, with separation means.
  • the separation means is selected from one of the following: microtitre plate; magnetic beads; or affinity chromatography column.
  • the sequences are biotinylated.
  • sequences are contained within 12 mini-libraries, as explained elsewhere.
  • the invention provides a zinc finger polypeptide designed by one or both of the methods defined above.
  • the zinc finger polypeptide designed by the method comprises a combination of a plurality of zinc fingers (adjacent zinc fingers being joined by an intervening linker peptide), each finger comprising a zinc finger binding motif.
  • each zinc finger binding motif in the zinc finger polypeptide has been selected for preferable binding characteristics by the method defined above.
  • the intervening linker peptide may be the same between each adjacent zinc finger or, alternatively, the same zinc finger polypeptide may contain a number of different linker peptides.
  • the intervening linker peptide may be one that is present in naturally-occurring zinc finger polypeptides or may be an artificial sequence. In particular, the sequence of the intervening linker peptide may be varied, for example, to optimise binding of the zinc finger polypeptide to the target sequence.
  • each motif binds to those DNA triplets which represent contiguous or substantially contiguous DNA in the sequence of interest.
  • candidate binding motifs or candidate combinations of motifs may be screened against the actual target sequence to determine the optimum composition of the polypeptide. Competitor DNA may be included in the screening assay for comparison, as described below.
  • the non-specific component of all protein-DNA interactions which includes contacts to the sugar-phosphate backbone as well as ambiguous contacts to base-pairs, is a considerable driving force towards complex formation and can result in the selection of DNA-binding proteins with reasonable affinity but without specificity for a given DNA sequence. Therefore, in order to minimise these non-specific interactions when designing a polypeptide, selections should preferably be performed with low concentrations of specific binding site in a background of competitor DNA, and binding should desirably take place in solution to avoid local concentration effects and the avidity of multivalent phage for ligands immobilised on solid surfaces.
  • the known sequence is only 9 bases long then three zinc finger binding motifs can be included in the polypeptide. If the known sequence is 27 bases long then, in theory, up to nine binding motifs could be included in the polypeptide. The longer the target DNA sequence, the lower the probability of its occurrence in any given portion of DNA.
  • those motifs selected for inclusion in the polypeptide could be artificially modified (e.g. by directed mutagenesis) in order to optimise further their binding characteristics.
  • the length and amino acid sequence of the linker peptide joining adjacent zinc binding fingers could be varied, as outlined above. This may have the effect of altering the position of the zinc finger binding motif relative to the DNA sequence of interest, and thereby exert a further influence on binding characteristics.
  • the invention provides a kit for making a zinc finger polypeptide for binding to a nucleic acid sequence of interest, comprising: a library of DNA sequences encoding zinc finger binding motifs of known binding characteristics in a form suitable for cloning into a vector; a vector molecule suitable for accepting one or more sequences from the library; and instructions for use.
  • the vector is capable of directing the expression of the cloned sequences as a single zinc finger polypeptide.
  • the vector is capable of directing the expression of the cloned sequences as a single zinc finger polypeptide displayed on the surface of a viral particle, typically of the sort of viral display particle which are known to those skilled in the art.
  • the DNA sequences are preferably in such a form that the expressed polypeptides are capable of self-assembling into a number of zinc finger polypeptides.
  • the kit defined above will be of particular use in designing a zinc finger polypeptide comprising a plurality of zinc finger binding motifs, the binding characteristics of which are already known.
  • the invention provides a kit for use when zinc finger binding motifs with suitable binding characteristics have not yet been identified, such that the invention provides a kit for making a zinc finger polypeptide for binding to a nucleic acid sequence of interest, comprising: a library of DNA sequences, each encoding a zinc finger binding motif in a form suitable for screening and/or selecting according to the methods defined above; and instructions for use.
  • the library of DNA sequences in the kit will be a library in accordance with the first aspect of the invention.
  • the kit may also comprise a library of 64 DNA sequences, each sequence comprising a different one of the 64 possible permutations of three DNA bases, in a form suitable for use in the selection method defined previously.
  • the 64 sequences are present in 12 separate mini-libraries, each mini-library having one postion in the relevant triplet fixed and two postions randomised.
  • the kit will also comprise appropriate buffer solutions, and/or reagents for use in the detection of bound zinc fingers.
  • the kit may also usefully include a vector suitable for accepting one or more sequences selected from the library of DNA sequences encoding zinc finger binding motifs.
  • the present teaching will be used for isolating the genes for the middle zinc fingers which, having been previously selected by one of the 64 triplets, are thought to have specific DNA binding activity.
  • the mixture of genes specifying fingers which bind to a given triplet will be amplified by PCR using three sets of primers. The sets will have unique restriction sites, which will define the assembly of zinc fingers into three finger polypeptides.
  • the appropriate reagents are preferably provided in kit form.
  • the first set of primers might have SfiI and AgeI sites, the second set AgeI and EagI sites and third set EagI and NotI sites.
  • the “first” site will preferably be SfiI, and the “last” site NotI, so as to facilitate cloning into the SfiI and NotI sites of the phage vector.
  • the fingers selected by the triplet GGG are amplified using the first two sets of primers and ligated to the fingers selected by the triplet AAA amplified using the third set of primers.
  • the combinatorial library is cloned on the surface of phage and a nine base-pair site can be used to select the best combination of fingers en bloc.
  • genes for fingers which bind to each of the 64 triplets can be amplified by each set of primers and cut using the appropriate restriction enzymes.
  • These building blocks for three-finger proteins can be sold as components of a kit for use as described above. The same could be done for the library amplified with different primers so that 4- or 5-finger proteins could be built.
  • a large (pre-assembled) library of all combinations of the fingers selected by all triplets can also be developed for single-step selection of DNA-binding proteins using 9 bp, or much longer. DNA fragments.
  • methods of selection other than phage display for example stalled polysomes (developed by Affimax) where protein and mRNA become linked.
  • the invention provides a method of altering the expression of a gene of interest in a target cell, comprising: determining (if necessary) at least part of the DNA sequence of the structural region and/or a regulatory region of the gene of interest; designing a zinc finger polypeptide to bind to the DNA of known sequence, and causing said zinc finger polypeptide to be present in the target cell, (preferably in the nucleus thereof). (It will be apparent that the DNA sequence need not be determined if it is already known.)
  • the regulatory region could be quite remote from the structural region of the gene of interest (e.g. a distant enhancer sequence or similar).
  • the zinc finger polypeptide is designed by one or both of the methods of the invention defined above.
  • Binding of the zinc finger polypeptide to the target sequence may result in increased or reduced expression of the gene of interest depending, for example, on the nature of the target sequence (e.g. structural or regulatory) to which the polypeptide binds.
  • the zinc finger polypeptide may advantageously comprise functional domains from other proteins (e.g. catalytic domains from restriction enzymes, rccombinases, replicases, integrases and the like) or even “synthetic” effector domains.
  • the polypeptide may also comprise activation or processing signals, such as nuclear localisation signals. These are of particular usefulness in targtetting the polypeptide to the nucleus of the cell in order to enhance the binding of the polypeptide to an intranuclear target (such as genomic DNA).
  • a particular example of such a localisation signal is that from the large T antigen of SV40.
  • Such other functional domains/signals and the like are conveniently present as a fusion with the zinc finger polypeptide.
  • Other desirable fusion partners comprise immunoglobulins or fragments thereof (eg. Fab, scFv) having binding activity.
  • the zinc finger polypeptide may be synthesised in situ in the cell as a result of delivery to the cell of DNA directing expression of the polypeptide.
  • Methods of facilitating delivery of DNA include, for example, recombinant viral vectors (e.g. retroviruses, adenoviruses), liposomes and the like.
  • the zinc finger polypeptide could be made outside the cell and then delivered thereto. Delivery could be facilitated by incorporating the polypeptide into liposomes etc. or by attaching the polypeptide to a targetting moiety (such as the binding portion of an antibody or hormone molecule).
  • Media e.g. microtitre wells, resins etc.
  • NIP nanoparticles
  • the invention provides a method of inhibiting cell division by causing the presence in a cell of a zinc finger polypeptide which inhibits the expression of a gene enabling the cell to divide.
  • the invention provides a method of treating a cancer, comprising delivering to a patient, or causing to be present therein, a zinc finger polypeptide which inhibits the expression of a gene enabling the cancer cells to divide.
  • the target could be, for example, an oncogene or a normal gene which is overexpressed in the cancer cells.
  • zinc finger polypeptides could be designed for therapeutic and/or prophylactic use in regulating the expression of disease-associated genes.
  • zinc finger polypeptides could be used to inhibit the expression of foreign genes (e.g, the genes of bacterial or viral pathogens) in man or animals, or to modify the expression of mutated host genes (such
  • the invention therefore provides a zinc finger polypeptide capable of inhibiting the expression of a disease-associated gene.
  • the zinc finger polypeptide will not be a naturally-occurring polypeptide but will be specifically designed to inhibit the expression of the disease-associated gene.
  • the polypeptide will be designed by one or both of the methods of the invention defined above.
  • the disease-associated gene will be an oncogene, typically the BCR-ABL fusion oncogene or a ras oncogene.
  • the invention provides a zinc finger polypeptide designed to bind to the DNA sequence GCAGAAGCC and capable of inihibting the expression of the BCR-ABL fusion oncogene.
  • the invention provides a method of modifying a nucleic acid sequence of interest present in a sample mixture by binding thereto a zinc finger polypeptide, comprising contacting the sample mixture with a zinc finger polypeptide having affinity for at least a portion of the sequence of interest, so as to allow the zinc finger polypeptide to bind specifically to the sequence of interest.
  • modifying as used herein is intended to mean that the sequence is considered modified simply by the binding of the zinc finger polypeptide. It is not intended to suggest that the sequence of nucleotides is changed, although such changes (and others) could ensue following binding of the zinc finger polypeptide to the nucleic acid of interest. Conveniently the nucleic acid sequence is DNA.
  • Modification of the nucleic acid of interest could be detected in any of a number of methods (e.g. gel mobility shift assays, use of labelled zinc finger polypeptides—labels could include radioactive, fluorescent, enzyme or biotin/streptavidin labels).
  • Modification of the nucleic acid sequence of interest may be all that is required (e.g. in diagnosis of disease). Desirably however, further processing of the sample is performed. Conveniently the zinc finger polypeptide (and nucleic acid sequences specifically bound thereto) are separated from the rest of the sample.
  • the zinc finger polypeptide is bound to a solid phase support, to facilitate such separation.
  • the zinc finger polypeptide may be present in an acrylamide or agarose gel matrix or, more preferably, is immobilised on the surface of a membrane or in the wells of a microtitre place.
  • a) Therapy e.g. targetting to double stranded DNA
  • Diagnosis e.g. detecting mutations in gene sequences: the present work has shown that “tailor made” zinc finger polypeptides can distinguish DNA sequences differing by one base pair).
  • DNA purification the zinc finger polypeptide could be used to purify restriction fragments from solution, or to visualise DNA fragments on a gel [for example, where the polypeptide is linked to an appropriate fusion partner, or is detected by probing with an antibody ]).
  • zinc finger polypeptides could even be targeted to other nucleic acids such as ss or ds RNA (e.g. self-complementary RNA such as is present in many RNA molecules) or to RNA-DNA hybrids, which would present another possible mechanism of affecting cellular events at the molecular level.
  • nucleic acids such as ss or ds RNA (e.g. self-complementary RNA such as is present in many RNA molecules) or to RNA-DNA hybrids, which would present another possible mechanism of affecting cellular events at the molecular level.
  • Example 1 the inventors describe and successfully demonstrate the use of the phage display technique to construct and screen a random zinc finger binding motif library, using a defined oligonucleotide target sequence.
  • Example 2 is disclosed the analysis of zinc finger binding motif sequences selected by the screening procedure of Example 1, the DNA-specificity of the motifs being studied by binding to a mini-library of randomised DNA target sequences to reveal a pattern of acceptable bases at each position in the target triplet—a “binding site signature”.
  • Example 3 the findings of the first two sections are used to select and modify rationally a zinc finger binding polypeptide in order to bind to a particular DNA target with high affinity: it is convincingly shown that the peptide binds to the target sequence and can modify gene expression in cells cultured in vitro.
  • Example 4 describes the development of an alternative zinc finger binding motif library.
  • Example 5 describes the design of a zinc finger binding polypeptide which binds to a DNA sequence of special clinical significance.
  • FIG. 1 is a schematic representation of affinity purification of phage particles displaying zinc finger binding motifs fused to phage coat proteins
  • FIG. 2 shows three zinc fingers (Seq ID No. 2) used in the phage display library
  • FIG. 3 shows the DNA sequences of three oligonucleotides (Seq ID Nos. 3-8) used in the affinity purification of phage display particles;
  • FIG. 4 is a “checker board” of binding site signatures determined for various zinc finger binding motifs (Seq ID Nos. 19-51);
  • FIG. 5A-5F show graphs fractional saturation against concentration of DNA (nM) for various binding motifs and target DNA triplets
  • FIG. 6 shows the nucleotide sequence of the fusion between BCR and ABL sequences in p190 cDNA (Seq ID No. 9) and the corresponding exon boundaries in the BCR and ABL genes (Seq ID Nos. 10-11),
  • FIG. 7 shows the amino acid sequences of various zinc finger binding motifs (Seq ID Nos. 12-17) designed to test for binding to the BCR/ABL fusion;
  • FIG. 8 is a graph of peptide binding (as measured by A 450-460 nm) against DNA concentration ( ⁇ M) of target or control DNA sequences;
  • FIG. 9 is a graph showing percentage viability against time for various transfected cells.
  • FIGS. 10A-10C and 11 illustrate schematically different methods of designing zinc finger binding polypeptides.
  • FIG. 12 shows the amino acid sequence of zinc fingers in a polypeptide (Seq ID No. 18) designed to bind to a particular DNA sequence (a ras oncogene).
  • the inventors have used a screening technique to study sequence-specific DNA recognition by zinc finger binding motifs.
  • the example describes how a library of zinc finger binding motifs displayed on the surface of bacteriophage enables selection of fingers capable of binding to given DNA triplets.
  • the amino acid sequences of selected fingers which bind the same triplet were compared to examine how sequence-specific DNA recognition occurs.
  • the results can be rationalised in terms of coded interactions between zinc fingers and DNA, involving base contacts from a few ⁇ -helical positions.
  • the inventors applied this technology to the study of zinc finger-DNA interactions after demonstrating that functional zinc finger proteins can be displayed on the surface of fd phage, and that the engineered phage can be captured on a solid support coated with specific DNA.
  • a phage display library was created comprising variants of the middle finger from the DNA binding domain of Zif268 (a mouse transcription factor containing 3 zinc fingers—Christy et al., 1988). DNA of fixed sequence was used to purify phage from this library over several rounds of selection, returning a number of different but related zinc fingers which bind the given DNA. By comparing similarities in the amino acid sequences of functionally equivalent fingers we deduce the likely mode of interaction of these fingers with DNA.
  • TFIIIA Transcription Factor IIIA
  • the gene for the first three fingers (residues 3-101) of Transcription Factor IIIA (TFIIIA) was amplified by PCR from the cDNA clone of TFIIIA using forward and backward primers which contain restriction sites for L NotI and SfiI respectively.
  • the gene for the Zif268 fingers (residues 333-420) was assembled from 8 overlapping synthetic oligonucleotides, giving SfiI and NotI overhangs.
  • the genes for fingers of the phage library were synthesised from 4 oligonucleotides by directional end to end ligation using 3 short complementary linkers, and amplified by PCR from the single strand using forward and backward primers which contained sites for NotI and SfiI respectively.
  • Backward PCR primers in addition introduced Met-Ala-Glu as the first three amino acids of the zinc finger peptides, and these were followed by the residues of the wild type or library fingers as discussed in the text. Cloning overhangs were produced by digestion with SfiI and NotI where necessary. Fragments were ligated to 1 ⁇ g similarly prepared Fd-Tet-SN vector.
  • Electrocompetent DH5 ⁇ cells were transformed with recombinant vector in 200 ng aliquots, grown for 1 hour in 2xTY medium with 1% glucose, and plated on TYE containing 15 ⁇ g/ml tetracycline and 1% glucose.
  • FIG. 2 shows the amino acid sequence (Seq ID No. 2) of the three zinc fingers from Zif268 used in the phage display library.
  • the top and bottom rows represent the sequence of the first and third fingers respectively.
  • the middle row represents the sequence of the middle finger.
  • the randomised positions in the ⁇ -helix of the middle finger have residues marked ‘X’.
  • the amino acid positions are numbered relative to the first helical residue (position 1).
  • codons are equal mixtures of (G,A,C)NN: T in the first base position is omitted in order to avoid stop codons, but this has the unfortunate effect that the codons for Trp, Phe, Tyr and Cys are not represented.
  • Position +9 is specified by the codon A(G,A)G, allowing either Arg or Lys. Residues of the hydrophobic core are circled, whereas the zinc ligands are written as white letters on black circles. The positions forming the ⁇ -sheets and the ⁇ -helix of the zinc fingers are marked below the sequence.
  • Colonies were transferred from plates to 200 ml 2xTY/Zn/Tet (2xTY containing 50 ⁇ M Zn(CH3.C00) 2 and 15 ⁇ g/ml tetracycline) and grown overnight. Phage were purified from the culture supernatant by two rounds of precipitation using 0.2 volumes of 20% PEG/2.5M NaCl containing 50, ⁇ M Zn(CH3.C00) 2 , and resuspended in zinc finger phage buffer (20 mM HEPES pH7.5, 50 mM NaCl, 1 mM MgCl 2 and 50 ⁇ M Zn(CH3.C00) 2 ).
  • Streptavidin-coated paramagnetic beads (Dynal) were washed in zinc finger phage buffer and blocked for 1 hour at room temperature with the same buffer made up to 6% in fat-free dried milk (Marvel). Selection of phage was over three rounds: in the first round, beads (1 mg) were saturated with biotinylated oligonucleotide ( ⁇ 80 nM) and then washed prior to phage binding, but in the second and third rounds 1.7 nM oligonucleotide and 5 ⁇ g poly dGC (Sigma) were added to the beads with the phage. Binding reactions (1.5 ml) for 1 hour at 15° C.
  • the phage selection procedure based on affinity purification, is illustrated schematically in FIG. 1 : zinc fingers (A) are expressed on the surface of fd phage(B) as fusions to the the minor coat protein (C). The third finger is mainly obscured by the DNA helix. Zinc finger phage are bound to 5′-biotinylated DNA oligonucleotide [D ] attached to streptavidin-coated paramagnetic beads [E ], and captured using a magnet [F ]. ( Figure adapted from Dynal AS and also Marks et al. (1992 J. Biol. Chem. 267, 16007-16105).
  • FIG. 3 shows sequences (Seq ID No.s 3-8) of DNA oligonucleotides used to purify (i) phage displaying the first three fingers of TFIIIA, (ii) phage displaying the three fingers of Zif268, and (iii) zinc finger phage from the phage display library.
  • the Zif268 consensus operator sequence used in the X-ray crystal structure (Pavletich & Pabo 1991 Science 252, 809-817) is highlighted in (ii), and in (iii) where “X” denotes a base change from the ideal operator in oligonucleotides used to purify phage with new specificities. Biotinylation of one strand is shown by a circled “B”.
  • Phage display of 3-finger DNA-Binding Domains from TFIIIA or Zif268 Prior to the construction of a phage display library, the inventors demonstrated that peptides containing three fully functional zinc fingers could be displayed on the surface of viable fd phage when cloned in the vector Fd-Tet-SN. In preliminary experiments, the inventors cloned as fusions to pill firstly the three N-terminal fingers from TFIIIA (Ginsberg et al., 1984 Cell 39, 479-489), and secondly the three fingers from Zif268 (Christy et al., 1988), for both of which the DNA binding sites are known.
  • Peptide fused to the minor coat protein was detected in Western blots using an anti-plll antibody (Stengele et al., 1990 J. Mol. Biol. 212, 143-149). Approximately 10-20% of total P-III in phage preparations was present as fusion protein.
  • Phage displaying either set of fingers were capable of binding to specific DNA oligonucleotides, indicating that zinc fingers were expressed and correctly folded in both instances.
  • Paramagnetic heads coated with specific oligonucleotide were used as a medium on which to capture DNA-binding phage, and were consistently able to return between 100 and 500-fold more such phage, compared to free beads or beads coated with non-specific DNA.
  • phage displaying the three fingers of Zif268 were diluted 1:1.7 ⁇ 10 3 with Fd-Tet-SN phage not bearing zinc fingers, and the mixture incubated with beads coated with Zif268 operator DNA, one in three of the total phage eluted and transfected into E.
  • coli were shown by colony hybridisation to carry the Zif268 gene, indicating an enrichment factor of over 500 for the zinc finger phage.
  • zinc fingers displayed on fd phage are capable of preferential binding to DNA sequences with which they can form specific complexes, making possible the enrichment of wanted phage by factors of up to 500 in a single affinity purification step. Therefore, over multiple rounds of selection and amplification, very rare clones capable of sequence-specific DNA binding can be selected-from a large library.
  • a phage display library of zinc fingers from Zif268 The inventors have made a phage display library of the three fingers of Zif268 in which selected residues in the middle finger are randomised ( FIG. 2 ), and have isolated phage bearing zinc fingers with desired specificity using a modified Zif268 operator sequence (Christy & Nathan 1989 Proc. Natl. Acad. Sci. USA 86, 8737-8741) in which the middle DNA triplet is altered to the sequence of interest ( FIG. 3 ).
  • a modified Zif268 operator sequence Greek & Nathan 1989 Proc. Natl. Acad. Sci. USA 86, 8737-8741
  • FIG. 3 In order to be able to study both the primary and secondary putative base recognition positions which are suggested by database analysis (Jacobs 1992 EMBO J.
  • the inventors have designed the library of the middle finger so that, relative to the first residue in the ⁇ -helix (position +1), positions ⁇ 1 to +8, but excluding the conserved Leu and His, can be any amino acid except Phe, Tyr, Trp and Cys which occur only rarely at those positions (Jacobs 1993 Ph.D. thesis, University of Cambridge).
  • position +9 which might make an inter-finger contact with Ser at position ⁇ 2 (Pavletich & Pabo 1991)
  • Arg or Lys the two most frequently occurring residues at that position.
  • the size of the phage display library required, assuming full degeneracy of the 8 variable positions, is (16 7 ⁇ 2 1 ) 5.4 ⁇ 10 8 , but because of practical limitations in the efficiency of transformation with Fd-Tet-SN, the inventors were able to clone only 2.6 ⁇ 10 6 of these.
  • the library used is therefore some two hundred times smaller than the theoretical size necessary to cover all the possible variations of the ⁇ -helix. Despite this shortfall, it has been possible to isolate phage which bind with high affinity and specificity to given DNA sequences, demonstrating the remarkable versatility of the zinc finger motif.
  • guanine has a particularly important role in zinc finger-DNA interactions.
  • G selects fingers with Arg at position +6 or ⁇ 1 of the ⁇ -helix respectively.
  • G is present in the middle position of a triplet (e.g. Table 1b), the preferred amino acid at position +3 is His.
  • G at the 5′ end of a triplet selects Ser or Thr at +6 (e.g. Table 1p).
  • Adenine is also an important determinant of sequence specificity, recognised almost exclusively by Asn or Gln which again are able to make bidentate contacts (Seeman et al., 1976).
  • Gln is often selected at position ⁇ 1 of the ⁇ -helix, accompanied by small aliphatic residues at +2 (e.g. Table 1b).
  • Adenine in the middle of the triplet strongly selects Asn at +3 (e.g. Table 1c-e), except in the triplet CAG (Table 1a) which selected only two types of finger, both with His at +3 (one being the wild-type Zif268 which contaminated the library during this experiment).
  • cytosine and thymine cannot reliably be discriminated by a hydrogen bonding amino acid side chain in the major groove (Seeman et al., 1976). Nevertheless, C in the 3′ position of a triplet shows a marked preference for Asp or Glu at position ⁇ 1, together with Arg at +1 (e.g. Table 1e.g.). Asp is also sometimes selected at +3 and +6 when C is in the middle (e.g. Table 1o) and 5′ (e-g. Table 1a) position respectively.
  • Asp can accept a hydrogen bond from the amino group of C, one should note that the positive molecular charge of C in the major groove (Hunter 1993 J. Mol. Biol. 230, 1025-1054) will favour an interaction with Asp regardless of hydrogen bonding contacts.
  • C in the middle position most frequently selects Thr (e.g. Table 1i, Val or Leu (e.g. Table 1o) at +3.
  • T in the middle position most often selects Ser (e.g. Table 1i), Ala or Val (e.g. Table 1p) at +3.
  • the aliphatic amino acids are unable to make hydrogen bonds but Ala probably has a hydrophobic interaction with the methyl group of T, whereas a longer side chain such as Leu can exclude T and pack against the ring of C.
  • Ser and Thr are selected at +6 (as is occasionally the case for G at the 5′ end).
  • Thymine at the 3′ end of a triplet selects a variety of polar amino acids at ⁇ 1 (e.g. Table 1d), and occasionally returns fingers with Ser at +2 (e.g. Table 1a) which could make a contact as seen in the tramtrack crystal structure (Fairall et al., 1993).
  • affinity selection can favour those clones whose representation in the library is greatest even though their true affinity for DNA is less than that of other clones less abundant in the library. Phage display selection by affinity is therefore of limited value in distinguishing between permissive and specific interactions beyond those base contacts necessary to stabilise the complex. Thus in the absence of competition from fingers which are able to bind specifically to a given DNA, the tightest non-specific complexes will be selected from the phage library. Consequently, results obtained by phage display selection from a library must be confirmed by specificity assays, particularly when that library is of limited size.
  • amino acid sequence biases observed within a family of functionally equivalent zinc fingers indicate that, of the a-helical positions randomised in this study, only three primary ( ⁇ 1, +3 and +6) and one auxiliary (+2) positions are involved in recognition of DNA. Moreover, a limited set of amino acids are to be found at those positions, and it is presumed that these make contacts to bases. The indications therefore are that a code can be derived to describe zinc finger-DNA interactions.
  • sequence homologies are strongly suggestive of amino acid preferences for particular base-pairs, one cannot confidently deduce such rules until the specificity of individual fingers for DNA triplets is confirmed. The inventors therefore defer making a summary table of these preferences until the following example, in which is described how randomised DNA binding sites can be used to this end.
  • the original position or context of the randomised finger in the phage display library might bear on the efficacy of selected fingers when incorporated into a new DNA-binding domain.
  • Selections from a library of the outer fingers of a three finger peptide are capable of producing fingers which bind DNA in various different modes, while selections from a library of the middle finger should produce motifs which are more constrained. Accordingly, Rebar and Pabo do not assume that the first finger of Zif268 will always bind a triplet, and screened with a tetranucleotide binding site to allow for different binding modes.
  • motifs selected from libraries of the outer fingers might prove less amenable to the assembly of multifinger proteins, since binding of these fingers could be perturbed on constraining them to a particular binding mode, as would be the case for fingers which had to occupy the middle position of an assembled three-finger protein.
  • motifs selected from libraries of the middle finger, having been originally constrained will presumably be able to preserve their mode of binding even when placed in the outer positions of an assembled DNA-binding domain.
  • FIGS. 10A-10C shows different strategies for the design of tailored zinc finger proteins.
  • a three-finger DNA-binding motif is selected en bloc from a library of three randomised fingers.
  • B A three-finger DNA-binding motif is assembled out of independently selected fingers from a library of one randomised finger (e.g. the middle finger of Zif268).
  • C A three-finger DNA-binding motif is assembled out of independently selected fingers from three positionally specified libraries of randomised zinc fingers.
  • FIG. 11 illustrates the strategy of combinatorial assembly followed by en bloc selection.
  • Groups of triplet-specific zinc fingers (A) isolated by phage display selection are assembled in random combinations and re-displayed on phage (B).
  • a full-length target site (C) is used to select en bloc the most favourable combination of fingers (D).
  • This example describes a new technique to deal efficiently with the selection of a DNA binding site for a given zinc finger (essentially the converse of example 1). This is desirable as a safeguard against spurious selections based on the screening of display libraries. This may be done by screening against libraries of DNA triplet binding sites randomised in two positions but having one base fixed in the third position. The technique is applied here to determine the specificity of fingers previously selected by phage display. The inventors found that some of these fingers are able to specify a unique base in each position of the cognate triplet. This is further illustrated by examples of fingers which can discriminate between closely related triplets as measured by their respective equilibrium dissociation constants. Comparing the amino acid sequences of fingers which specify a particular base in a triplet, we infer that in most instances, sequence specific binding of zinc fingers to DNA can be achieved using a small set of amino acid-base contacts amenable to a code.
  • This example presents a convenient and rapid new method which can reveal the optimal binding site(s) of a DNA binding protein by single step selection from small libraries and use this to check the binding site preferences of those zinc fingers selected previously by phage display.
  • the inventors have used 12 different mini-libraries of the Zif268 binding site, each one with the central triplet having one position defined with a particular base pair and the other two positions randomised.
  • Each library therefore comprises 16 oligonucleotides and offers a number of potential binding sites to the middle finger, provided that the latter can tolerate the defined base pair.
  • Each zinc finger phage is screened against all 12 libraries individually immobilised in wells of a microtitre plate, and binding is detected by an enzyme immunoassay.
  • a pattern of acceptable bases at each position is disclosed, which the inventors term a “binding site signature”.
  • the information contained in a binding site signature encompasses the repertoire of binding sites recognised by a zinc finger.
  • the binding site signatures allow progress towards a specificity code for the interactions of zinc fingers with DNA.
  • FIG. 4 gives the binding site signatures of individual zinc finger phage.
  • the figure represents binding of zinc finger phage to randomised DNA immobilised in the wells of microtitre plates.
  • DNA libraries are applied to columns of wells (down the plate), while rows of wells (across the plate) contain equal volumes of a solution of a zinc finger phage.
  • the identity of each library is given as the middle triplet of the “bound” strand of Zif268 operator, where N represents a mixture of all 4 nucleotides.
  • the zinc finger phage is specified by the sequence of the variable region of the middle finger, numbered relative to the first helical residue (position 1), and the three primary recognition positions are highlighted. Bound phage are detected by an enzyme immunoassay. The approximate strength of binding is indicated by a grey scale proportional to the enzyme activity. From the pattern of binding to DNA libraries, called the “signature” of each clone, one or a small number of binding sites can be read off and these are written on the right of the figure.
  • Bound phage were detected using HRP-conjugated anti-M13 IgG (Pharmacia) and developed as described (Griffiths et al., 1994). Optical densities were quantitated using software package SOFTMAX 232 (Molecular Devices Corp).
  • FIGS. 5A-5F is a series of graphs of fractional saturation against concentration of DNA (nM).
  • the two outer fingers carry the native sequence, as do the the two cognate outer DNA triplets.
  • the sequence of amino acids occupying helical positions ⁇ 1 to +9 of the varied finger are shown in each case.
  • the graphs show that the middle finger can discriminate closely related triplets, usually by a factor of ten.
  • the graphs allowed the determination of apparent equilibrium dissociation constants, as below.
  • K d [DNA ].[P ]/[DNA.P], using the software package KALEIDAGRAPHTM Version 2.0 programme (Abelbeck Software). Owing to the sensitivity of the ELISA used to detect protein-DNA complex, the inventors were able to use zinc finger phage concentrations far below those of the DNA, as is required for accurate calculations of the K d .
  • the technique used here has the advantage that while the concentration of DNA (variable) must be known accurately, that of the zinc fingers (constant) need not be known (Choo & Klug 1993 Nucleic Acids Res. 21, 3341-3346).
  • the top row of FIG. 4 shows the signature of the second finger of wild type Zif268. From the pattern of strong signals indicating binding to oligonucleotide libraries having GNN, TNN, NGN and NNG as the middle triplet, it emerges that the optimal binding site for this finger is T/G,G,G, in accord with the published consensus sequence (Christy & Nathans 1989 Proc. Natl. Acad. Sci. USA 86, 8737-8741). This has implications for the interpretation of the X-ray crystal structure of Zif268 solved in complex with consensus operator having, TGG as the middle triplet (Pavletich & Pabo 1991).
  • binding site signatures can give insight into the details of zinc finger-DNA interactions.
  • the binding site signatures of other zinc fingers reveal that the phage selections performed in example 1 yielded highly sequence-specific DNA binding proteins. Some of these are able to specify a unique sequence for the middle triplet of a variant Zif268 binding site, and are therefore more specific than is Zif268 itself for its consensus site. Moreover, one can identify the fingers which recognise a particular oligonucleotide library, that is to say a specific base at a defined position, by looking down the columns of FIG. 4 . By comparing the amino acid sequences of these fingers one can identify any residues which have genuine preferences for particular bases on bound DNA. With a few exceptions, these are as previously predicted on the basis of phage display, and are summarised in Table 2.
  • Table 2 summarises frequently observed amino acid-base contacts in interactions of selected zinc fingers with DNA.
  • the given contacts comprise a “syllabic” recognition code for appropriate triplets.
  • Cognate amino acids and their positions in the ⁇ -helix are entered in a matrix relating each base to each position of a triplet.
  • Auxiliary amino acids from position +2 can enhance or modulate specificity of amino acids at position ⁇ 1 and these are listed as pairs Ser or Thr at position +6 permit Asp +2 of the following finger (denoted Asp ++2) to specify both G and T indirectly, and the pairs are listed.
  • the specificity of Ser +for T and Thr +3 for C may be interchangeable in rare instances while Val +3 appears to be consistently ambiguous.
  • the binding site signatures also reveal an important feature of the phage display library which is important to the interpretation of the selection results.
  • All the fingers in our panel, regardless of the amino acid present at position +6, are able to recognise G or both G and T at the 5′ end of a triplet.
  • the probable explanation for this is that the 5′ position of the middle triplet is fixed as either G or T by a contact from the invariant Asp at position +2 of finger 3 to the partner of either base on the complementary strand, analogous to those seen in the Zif268 (Pavletich & Pabo 1991 Science 252, 809-817) and tramtrack (Fairall et al., 1993) crystal structures (a contact to NH 2 of C or A respectively in the major groove).
  • position +2 is able to specify the base directly 3′ of the ‘cognate triplet’, and can thus work in conjunction with position +6 of the preceding finger.
  • the binding site signatures whilst pointing to amino acid-base contacts from the three primary positions, indicate that auxiliary positions can play other parts in base recognition.
  • a clear case in point is Gln at position ⁇ 1, which is specific for A at the 3′ end of a triplet when position +2 is a small non-polar amino acid such as Ala, though specific for T when polar residues such as Ser are at position +2.
  • binding site signature of a zinc finger reveals its differential base preferences at a given concentration of DNA. As the concentration of DNA is altered, one can expect the binding site signature of any clone to change, being more distinctive at low [DNA], and becoming less so at higher [DNA] 0 as the K d of less favourable sites is approached and further bases become acceptable at each position of the triplet. Furthermore, because two base positions are randomly occupied in any one library of oligonucleotides, binding site signatures are not formally able to exclude the possibility of context dependence for some interactions. Therefore to supplement binding site signatures, which are essentially comparative, quantitative determinations of the equilibrium dissociation constant of each phage for different DNA binding sites are required. After phage display selection and binding site signatures, these are the third and definitive stage in assessing the specificity of zinc fingers.
  • FIGS. 5A-5F Examples of such studies presented in FIGS. 5A-5F show reveal that zinc finger phages bind the operators indicated in their binding site signatures with K d s in the range of 10 ⁇ 8 ⁇ 10 ⁇ 9 M, and can discriminate against closely related binding sites by factors greater than an order of magnitude. Indeed FIGS. 5A-5F shows such differences in affinity for binding sites which differ in only one out of nine base pairs. Since the zinc fingers in our panel were selected from a library by non-competitive affinity purification, there is the possibility that fingers which are even more discriminatory can be isolated using a competitive selection process.
  • Measurements of dissociation constants allow different triplets to be ranked in order of preference according to the strength of binding.
  • the examples here indicate that the contacts from either position ⁇ 1 or +3 can contribute to discrimination.
  • the ambiguity in certain binding site signatures referred to above can be shown to have a basis in the equal affinity of certain figures fingers for closely related triplets. This is demonstrated by the K d s of the finger containing the amino acid sequence RGDALTSHER (Seq ID No. 100) for the triple triplets TUG and GTG.
  • a code for zinc ringer-DNA recognition One would expect that the versatility of the zinc finger motif will have allowed evolution to develop various modes or binding to DNA (and even to RNA), which will be too diverse to fall under the scope of a single code. However, although a code may not apply to all zinc finger-DNA interactions, there is now convincing evidence that a code applies to a substantial subset. This code will fall short of being able to predict unfailingly the DNA binding site preference of any given zinc finger from its amino acid sequence, but may yet be sufficiently comprehensive to allow the design of zinc fingers with specificity for a given DNA sequence.
  • DNA recognition involves four fixed principal (three primary and one auxiliary) positions on the ⁇ -helix, from where a limited and specific set of amino acid-base contacts result in recognition of a variety of DNA triplets.
  • a code can describe the interactions of zinc fingers with DNA. Towards this code, one can propose amino acid-base contacts for almost all the entries in a matrix relating each base to each position of a triplet (Table 2).
  • Arg in order that Ala may pick out T in the triplet GTG, Arg must not be used to recognise G from position +6, since this would distance the former too far from the DNA (see for example the finger containing the amino acid sequence RGDALTSHER) (Seq ID No. 100).
  • the pitch of the ⁇ -helix is 3.6 amino acids per turn, positions ⁇ 1, +3 and +6 are not an integral number of turns apart, so that position +3 is nearer to the DNA than are ⁇ 1 or +6.
  • short amino acids such as His and Asn, rather than the longer Arg and Gln, are used for the recognition of purines in the middle position of a triplet.
  • the versatility of the finger motif will likely allow other modes of binding to DNA.
  • one must take into account the malleability of nucleic acids such as is observed in Fairall et al., where a deformation of the double helix at a flexible base step allows a direct contact from Ser at position +2 of finger 1 to a T at the 3′ position of the cognate triplet.
  • water may be seen to play an important role, for example where short side chains such as Asp, Asn or Scr interact with bases from position ⁇ 1 (Qian et al., 1993 J. Am. Chem. Soc. 115, 1189-1190; Shakked et al., 1994 Nature (London) 368, 469-478).
  • the coded interactions of zinc fingers with DNA can be used to model the specificity of individual zinc fingers de novo, or more likely in conjunction with phage display selection of suitable candidates.
  • the additive effect of multiply repeated domains offers the opportunity to bind specifically and tightly to extended, and hence very rare, genomic loci.
  • zinc finger proteins might well be a good alternative to the use of antisense nucleic acids in suppressing or modifying the action of a given gene, whether normal or mutant.
  • extra functions could be introduced to these DNA binding domains by appending suitable natural or synthetic effectors.
  • murine cells made growth factor-independent by the action of the oncogene (Daley et al., 1988 Proc. Natl. Acad. Sci. U.S.A. 85, 9312-9316) are found to revert to factor dependence on transient transfection with a vector expressing the designed zinc finger polypeptide.
  • DNA-binding proteins designed to recognise specific DNA sequences could be incorporated in chimeric transcription factors, recombinases, nucleases etc. for a wide range of applications.
  • the inventors have shown that zinc finger mini-domains can discriminate between closely related DNA triplets, and have proposed that they can be linked together to form domains for the specific recognition of longer DNA sequences.
  • One interesting possibility for the use of such protein domains is to target selectively genetic differences in pathogens or transformed cells. Here one such application is described.
  • CML chronic myelogenous leukaemia
  • the breakpoints usually occur in the first intron of the c-ABL gene and in the breakpoint cluster region of the BCR gene (Shtivelman et al., 1985 Nature 315, 550-554), and give rise to a p210 BCR-ABL gene product (Konopka et al., 1984 Cell 37, 1035-1042).
  • ALL acute lymphoblastic leukaemia
  • the breakpoints usually occur in the first introns of both BCR and c-ABL (Hermans er al., 1987 Cell 51, 33-40), and result in a p190 BCR-ABL gene product ( FIG. 6 ) (Kurzrock et al., 1987 Nature 325, 631-635).
  • FIG. 6 shows the nucleotide sequences (Seq ID No.s 9-11) of the fusion point between BCR and ABL sequences in p190 cDNA, and of the corresponding exon boundaries in the BCR and c-ABL genes. Exon sequences are written in capital letters while introns are given in lowercase.
  • Line 1 shows p190 BCR-ABL cDNA
  • line 2 the BCR genomic sequence at junction of exon 1 and intron 1
  • line 3 the ABL genomic sequence at junction of intron 1 and exon 2 (Hermans et al 1987).
  • the 9 bp sequence in the p190 BCR-ABL cDNA used as a target is underlined, as are the homologous sequences in genomic BCR and c-ABL.
  • Facsimiles of these rearranged genes act as dominant transforming oncogenes in cell culture (Daley et al., 1988) and transgenic mice (Heisterkamp et al., 1990 Nature 344, 251-253).
  • the cDNAs bear a unique nucleotide sequence at the fusion point of the BCR and c-ABL genes, which can be recognised at the DNA level by a site-specific DNA-binding protein.
  • the present inventors have designed such a protein to recognise the unique fusion site in the p190 BCR-ABL c-DNA. This fusion is obviously distinct from the breakpoints in the spontaneous genomic translocations, which are thought to be variable among patients.
  • the design of such peptides has implications for cancer research, the primary aim here is to prove the principle of protein design, and to assess the feasibility of in vivo binding to chromosomal DNA in available model systems.
  • a nine base-pair target sequence (GCA, GAA, GCC) for a three zinc finger peptide was chosen which spanned the fusion point of the p190 BCR-ABL cDNA (Hermans et al., 1987).
  • the three triplets forming this binding site were each used to screen a zinc finger phage library over three rounds as described above in example 1.
  • the selected fingers were then analysed by binding site signatures to reveal their preferred triplet, and mutations to improve specificity were made to the finger selected for binding to GCA.
  • a phage display mini-library of putative BCR-ABL-binding three-finger proteins was cloned in fd phage, comprising six possible combinations of the six selected or designed fingers (1A, 1B; 2A; 3A, 3B and 3C) linked in the appropriate order. These fingers are illustrated in FIG. 7 (Seq ID Nos 12-17). In FIG. 7 regions of secondary structure are underlined below the list, while residue positions are given above, relative to the first position of the ⁇ -helix (position 1). Zinc finger phages were selected from a library of 2.6 ⁇ 10 6 variants, using three DNA binding sites each containing one of the triplets GCC, GAA or GCA.
  • Binding site signatures indicate that fingers 1A and 1B specify the triplet GCC, finger 2A specifics GAA, while the fingers selected using the triplet GCA all prefer binding to GCT.
  • finger 3A the specificity of which we believed, on the basis of recognition rules, could be changed by a point mutation.
  • Finger 3B based on the selected finger 3A, but in which Gln at helical position +2 was altered to Ala should be specific for GCA.
  • Finger 3C is an alternative version of finger 3A, in which the recognition of C is mediated by Asp +3 rather than by Thr +3.
  • the mini library was screened once with an oligonucleotide containing the 9 base-pair BCR-ABL target sequence to select for tight binding clones over weak binders and background vector phage. Because the library was small, the inventors did not include competitor DNA sequences for homologous regions of the genomic BCR and c-ABL genes but instead checked the selected clones for their ability to discriminate.
  • the peptide 1A-2A-3B henceforth referred to as the anti-BCR-ABL peptide, was used in further experiments.
  • the anti-BCR-ABL peptide has an apparent equilibrium dissociation constant (K d ) of 6.2 ⁇ 0.4 ⁇ 10 ⁇ 7 M for the p190 BCB-ABL cDNA sequence in vitro, and discriminates against the similar sequences found in genomic BCR and C-ABL DNA, by factors greater than an order of magnitude ( FIG. 8 ). Referring to FIG.
  • the graph shows binding (measured as an A 450-650 ) at various [DNA]. Binding reactions and complex detection by enzyme immunoassay were performed as described previously, and a full curve analysis was used in calculations of the K d (Choo & Klug 1993). The DNA used were oligonucleotides spanning 9 bp either side of the fusion point in the cDNA or the exon boundaries.
  • the measured dissociation constant is higher than that of three-finger peptides from naturally occurring proteins such as Spl (Kadonga et al., 1987 Cell 51, 1079-1090) or Zif268 (Christy et al., 1988), which have K d s in the range of 10 ⁇ 9 M, but rather is comparable to that of the two fingers from the tramtrack (ttk) protein (Fairall et al., 1992).
  • affinity of the anti-BCR-ABL peptide could be refined, if desired, by site-directed mutations or by “affinity maturation” of a phage display library (Hawkins et al., 1992 J. Mol. Biol. 226, 889-896).
  • the peptide was fused to the VP16 activation domain from herpes simplex virus (Fields 1993 Methods 5, 116-124) and used in transient transfection assays ( FIG. 9 ) to drive production of a CAT (chloramphenicol acetyl transferase) reporter gene from a binding site upstream of the TATA box (Gorman ct al., Mol. Cell. Biol. 2, 1044-1051).
  • CAT chloramphenicol acetyl transferase
  • reporter plasmids pMCAT6BA, pMCAT6A, and pMCAT6B were constructed by inserting 6 copies of the p190 BCR-ABL target site (CGCAGAAGCC) (Seq ID No. 121), the c-ABL second exon-intron junction sequence (TCCAGAAGCC) (Seq ID No. 122), or the BCR first exon-intron junction sequence (CGCAGGTGAG) (Seq ID No. 123) respectively, into pMCAT3 (Luscher et al., 1989 Genes Dev. 1507-1517).
  • CGCAGAAGCC p190 BCR-ABL target site
  • TCCAGAAGCC c-ABL second exon-intron junction sequence
  • CGCAGGTGAG BCR first exon-intron junction sequence
  • the anti-BCR-ABL/VP16 expression vector was generated by inserting the in-frame fusion between the activation domain of herpes simplex virus VP16 (Fields 1993) and the Zn finger peptide in the pEF-BOS vector (Mizushima & Shigezaku 1990 Nucl. Acids Res. 18, 5322).
  • C3H10T1/2 cells were transiently co-transfected with 10 mg of reporter plasmid and 10 mg of expression vector.
  • RSVL de Wet et al., 1987 Mol. Cell Biol. 7, 725-737
  • Rous sarcoma virus long terminal repeat linked to luciferase was used as an internal control to normalise for differences in transfection efficiency.
  • Plasmid pGSEC which has five consensus 17-mer GAL4-binding sites upstream from the minimal promoter of the adenovirus Elb TATA box
  • pMIVP16 vector which encodes an in-frame fusion between the DNA-binding domain of GAL4 and the activation domain of herpes simplex virus VP16, were used as a positive control (Sadowski et al., 1992 Gene 118, 137-141).
  • C3H10T1/2 cells were transiently cotransfected with a CAT reporter plasmid and an anti-BCR-ABL/VP 16 expression vector (pZN1A).
  • This construct was used to transiently transfect the IL-3-dependent murine cell line Ba/F3 (Palacios & Steinmetz 1985 Cell 41, 727-734), or alternatively Ba/F3+p190 and Ba/F3+p210 cell lines previously made IL-3-independent by integrated plasmid constructs expressing either p190 BCR-ABL or p210 BCR-ABL , respectively. Staining of the cells with the 9E10 antibody followed by a secondary fluorescent conjugate showed efficient nuclear localisation in those cells transfected with the anti-BCR-ABL peptide.
  • the anti-BCR-ABL expression vector was generated in the pEF-BOS vector (Mizushima & Shigezaku 1990), including an 11 amino acid c-myc epitope tag (EQKLISEEDLN) SEQ ID NO: 124 at the carboxy-terminal end, recognizable by the 9E10 antibody (Evan et al., 1985) and the nuclear localization signal PKKKRKV SEQ ID NO: 125 of the large T antigen of SV40 virus (Kalderon et al., 1984) at the amino-terminal end.
  • EQKLISEEDLN 11 amino acid c-myc epitope tag
  • PKKKRKV nuclear localization signal
  • Three glycine residues were introduced downstream of the nuclear localization signal as a spacer, to ensure exposure of the nuclear leader from the folded molecule.
  • Ba/F3 cells were transfected with 25 mg of the anti-BCR-ABL expression construct tagged with the 9E10 c-myc epitope as described (Sanchez-Garcia & Rabbitts 1994 Proc. Natl. Acad. Sci. U.S.A. in press) and protein production analyzed 48 h later by immunofluorescence-labelling as follows.
  • Cells were fixed in 4% (w/v) paraformaldehyde for 15 min, washed in phosphate-buffered saline (PBS), and permeabilized in methanol for 2 min. After blocking in 10% fetal calf serum in PBS for 30 min, the mouse 9E10 antibody was added.
  • PBS phosphate-buffered saline
  • FITC fluorescein isothiocyanate
  • SIGMA conjugated goat anti-mouse IgG
  • the blots were performed as follows: 10 mg of total cytoplasmic RNA, from the cells indicated, was glyoxylated and fractionated in 1.4% agarose gels in 10 mM NaPO 4 buffer, pH 7.0. After electrophoresis the gel was blotted onto HYBOND-N (Amersham), UV-cross linked and hybridized to an 32 P-labelled c-ABL probe. Autoradiography was for 14 h at ⁇ 70° C. Loading was monitored by reprobing the filters with a mouse b-acting cDNA.
  • a DNA-binding protein designed to recognise a specific DNA sequence in vitro is active in vivo where, directed to the nucleus by an appended localisation signal, it can bind its target sequence in chromosomal DNA. This is found on otherwise actively transcribing DNA, so presumably binding of the peptide blocks the path of the polymerase, causing stalling or abortion.
  • the use of a specific polypeptide in this case to target intragenic sequences is reminiscent of antisense oligonucleotide- or ribozyme- based approaches to inhibiting the expression of selected genes (Stein & Cheng 1993 Science 261, 1004-1012).
  • DNA-binding proteins can be tailored against genes altered by chromosomal translocations, or point mutations, as well as to regulatory sequences within genes. Also, like oligonucleotides which can be designed to repress transcription by triple helix formation in homopurine-homopyrimidine promoters (Cooney et al., 1988 Science 245, 725-730) DNA-binding proteins can bind to various unique regions outside genes, but in contrast they can direct gene expression by both up- or down-regulating, the initiation of transcription when fused to activation (Seipel er al., 1992 EMBO J.
  • phage display zinc finger library described in the preceding examples could be considered sub-optimal in a number of ways:
  • flanking fingers both recognised GCG triplets (in certain cases creating nearly symmetrical binding sites for the three zinc fingers, which enables the peptide to bind to the ‘bottom’ strand of DNA, thus evading the register of interactions we wished to set);
  • Asp++2 Asp+2 of finger three was dominant over the interactions of finger two (position+6) with the 5′ base of the middle triplet;
  • the middle finger is fully randomised in only four positions ( ⁇ 1, +2, +3 and +6) so that the library size is smaller and all codons are represented.
  • the library was cloned in the pCANTAB5E phagemid vector from Pharmacia, which allows higher transformation frequencies than the phage.
  • the first and third fingers recognise the triplets GAC and GCA, respectively, making for a highly asymmetric binding site.
  • Recognition of the 3′ A in the latter triplet by finger three is mediated by Gln-1/Ala+2, the significance of which is that the short Ala+2 should not make contacts to the DNA (in particular with the 5′ base of the middle triplet), thus alleviating the problem noted at (iii) above.
  • the human ras gene is susceptible to a number of different mutations, which can convert it into an oncogene.
  • a ras oncogene is found in a large number of human cancers.
  • One particular mutation is known as the G12V mutation (i.e. the polypeptide encoded by the mutant gene contains a substitution from glycine to valine). Because ras oncogenes are so common in human cancers, they are extremely significant targets for potential therapeutic methods.
  • a three finger protein has been designed which can recognise the G12V mutant of ras.
  • the protein was produced using rational design based on the known specificity rules.
  • a zinc finger framework (from one of the fingers selected to hind GCC) was modified by point mutations in position +3 to yield fingers recognising two additional different triplets.
  • the finger recognising GCC and the two derivatives were cloned in pCANTAB5E and expressed on the surface of phage.
  • the G12V-binding peptide “r-BP” was to be selected from a small library of related proteins. The reason a library was to be used is that while it was clear to us what 8/9 of the amino acid:base contacts should be, it was not clear whether the middle C of the GCC triplet should be recognised by +3 Asp, or Glu, or Ser, or Thr (see Table 2 above). Thus a three-finger peptide gene was assembled from 8 overlapping synthetic oligonucleotides which were annealed and ligated according to standard procedures and the ⁇ 300 bp product purified from a 2% agarose gel.
  • the gene for finger 1 contained a partial codon randomisation at position +3 which allowed for inclusion of each of the above amino acids (D, E, S & T) and also certain other residues which were in fact not predicted to be desirable (e.g. Asn).
  • the synthetic oligonucleotides were designed to have SfiI and NotI overhangs when annealed.
  • the ⁇ 300 bp fragment was ligated into SfiI/NotI -cut FdSN vector and the ligation mixture was electroporated into DH5 ⁇ cells. Phage were produced from these as previously described and a selection step carried out using the G12V sequence (also as described) to eliminate phage without insert and those phage of the library which bound poorly.
  • Asti at position +3 is unexpected and most probably due to the fact that proteins with a cytosine-specific residue at position +3 bind to some E. coli DNA sequence so tightly that they are lethal. Thus phage display selection is not always guaranteed to produce the tightest-binding clone, since passage through bacteria is essential to the technique, and the selected proteins may be those which do not bind to the genome of this host if such binding is deleterious.
  • the amino acid sequence (Seq ID No. 18) of the fingers is shown in FIG. 12 .
  • the numbers refer to the ⁇ -helical amino acid residues.
  • the fingers (F1, F2 & F3) bind to the G12V mutant nucleotide sequence:
  • the bold A shows the single point mutation by which the G12V sequence differs from the wild type sequence.
  • Assay of the protein in eukaryotes requires the use of a weak promoter.
  • the anti-RAS (G12V) protein When expression of the anti-RAS (G12V) protein is strong, the peptide presumably binds to the wild-type ras allele (which is required) leading to cell death.
  • a regulatable promoter e.g. for tetracycline
  • the G12V mutation is a naturally occurring genomic mutation (not only a cDNA mutation as was the p190 bcr-abl) human cell lines and other animal models can be used in research.
  • the protein can be used to diagnose the precise point mutation present in the genomic DNA, or more likely in PCR amplified genomic DNA, without sequencing. It should therefore be possible, without further inventive activity, to design diagnostic kits for detecting (e.g. point) mutations on DNA. ELISA-based methods should prove particularly suitable.
  • the transferrin receptor is thought particularly useful but, in theory, any receptor molecule (preferably of high affinity) expressed on the surface of a human target cell could act as a suitable ligand, either for a specific immunoglobulin or fragment, or for the receptor's natural ligand fused or coupled with the zinc finger polypeptide.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Plant Pathology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Virology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)
US10/400,017 1994-08-20 1995-08-17 Relating to binding proteins for recognition of DNA Expired - Lifetime USRE45721E1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US10/400,017 USRE45721E1 (en) 1994-08-20 1995-08-17 Relating to binding proteins for recognition of DNA

Applications Claiming Priority (9)

Application Number Priority Date Filing Date Title
GB9416880A GB9416880D0 (en) 1994-08-20 1994-08-20 Improvements in or relating to binding proteins for recognition of DNA
GB9416880.4 1994-08-20
GB9422534.9 1994-11-08
GB9422534A GB9422534D0 (en) 1994-11-08 1994-11-08 Improvements in or relating to binding proteins for recognition of DNA
GBGB9514698.1A GB9514698D0 (en) 1995-07-18 1995-07-18 Improvements in or relating to binding proteins for recognition of DNA
GB9514698.1 1995-07-18
PCT/GB1995/001949 WO1996006166A1 (fr) 1994-08-20 1995-08-17 Ameliorations concernant des proteines de liaison permettant de reconnaitre l'adn
US10/400,017 USRE45721E1 (en) 1994-08-20 1995-08-17 Relating to binding proteins for recognition of DNA
US3312901A 2001-12-27 2001-12-27

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US79340895A Reissue 1994-08-20 1995-08-17

Publications (1)

Publication Number Publication Date
USRE45721E1 true USRE45721E1 (en) 2015-10-06

Family

ID=27267339

Family Applications (7)

Application Number Title Priority Date Filing Date
US10/309,578 Expired - Lifetime USRE45795E1 (en) 1994-08-20 1995-08-17 Binding proteins for recognition of DNA
US08/793,408 Ceased US6007988A (en) 1994-08-20 1995-08-17 Binding proteins for recognition of DNA
US10/400,017 Expired - Lifetime USRE45721E1 (en) 1994-08-20 1995-08-17 Relating to binding proteins for recognition of DNA
US10/033,129 Expired - Lifetime USRE39229E1 (en) 1994-08-20 1995-08-17 Binding proteins for recognition of DNA
US09/139,762 Expired - Lifetime US6013453A (en) 1994-08-20 1998-08-25 Binding proteins for recognition of DNA
US10/397,930 Expired - Lifetime USRE42211E1 (en) 1994-08-20 2003-03-25 Relating to binding proteins for recognition of DNA
US11/500,162 Expired - Lifetime USRE42150E1 (en) 1994-08-20 2006-08-07 Binding proteins for recognition of DNA

Family Applications Before (2)

Application Number Title Priority Date Filing Date
US10/309,578 Expired - Lifetime USRE45795E1 (en) 1994-08-20 1995-08-17 Binding proteins for recognition of DNA
US08/793,408 Ceased US6007988A (en) 1994-08-20 1995-08-17 Binding proteins for recognition of DNA

Family Applications After (4)

Application Number Title Priority Date Filing Date
US10/033,129 Expired - Lifetime USRE39229E1 (en) 1994-08-20 1995-08-17 Binding proteins for recognition of DNA
US09/139,762 Expired - Lifetime US6013453A (en) 1994-08-20 1998-08-25 Binding proteins for recognition of DNA
US10/397,930 Expired - Lifetime USRE42211E1 (en) 1994-08-20 2003-03-25 Relating to binding proteins for recognition of DNA
US11/500,162 Expired - Lifetime USRE42150E1 (en) 1994-08-20 2006-08-07 Binding proteins for recognition of DNA

Country Status (8)

Country Link
US (7) USRE45795E1 (fr)
EP (2) EP0781331B1 (fr)
JP (2) JP4118327B2 (fr)
AT (2) ATE524545T1 (fr)
AU (1) AU698152B2 (fr)
CA (1) CA2196419C (fr)
DE (1) DE69535829D1 (fr)
WO (1) WO1996006166A1 (fr)

Families Citing this family (392)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
ES2330052T3 (es) 1991-03-01 2009-12-03 Dyax Corporation Proteina quimerica que comprende micro-proteinas que tienen dos o mas puentes disulfuro y relaizaciones de las mismas.
US6140466A (en) 1994-01-18 2000-10-31 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
US20050084885A1 (en) * 1994-01-18 2005-04-21 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
AU726759B2 (en) * 1994-08-20 2000-11-23 Gendaq Limited Improvements in or relating to binding proteins for recognition of DNA
GB9824544D0 (en) 1998-11-09 1999-01-06 Medical Res Council Screening system
US7262055B2 (en) 1998-08-25 2007-08-28 Gendaq Limited Regulated gene expression in plants
US7285416B2 (en) 2000-01-24 2007-10-23 Gendaq Limited Regulated gene expression in plants
USRE45795E1 (en) 1994-08-20 2015-11-10 Gendaq, Ltd. Binding proteins for recognition of DNA
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
WO1999021991A1 (fr) * 1997-10-29 1999-05-06 Shanghai Second Medical University Bmzf12: gene a doigt de zinc clone a partir de la moelle osseuse
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
AU746454B2 (en) 1998-03-02 2002-05-02 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
WO1999045127A2 (fr) * 1998-03-06 1999-09-10 Oxford Biomedica (Uk) Limited Activation amelioree de promedicaments
JP2002506640A (ja) * 1998-03-17 2002-03-05 ジェンダック・リミテッド 核酸結合タンパク質
EP1112355A1 (fr) * 1998-09-14 2001-07-04 Aston University Bibliotheques de genes et de proteines, et procedes s'y rapportant
US20050086718A1 (en) 1999-03-23 2005-04-21 Mendel Biotechnology, Inc. Plant transcriptional regulators of abiotic stress
US7897843B2 (en) 1999-03-23 2011-03-01 Mendel Biotechnology, Inc. Transcriptional regulation of plant biomass and abiotic stress tolerance
US6806062B1 (en) * 1998-10-05 2004-10-19 Novozymes A/S Fungal transcriptional activator useful in methods for producing polypeptides
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
US6599692B1 (en) 1999-09-14 2003-07-29 Sangamo Bioscience, Inc. Functional genomics using zinc finger proteins
US6534261B1 (en) 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US7070934B2 (en) 1999-01-12 2006-07-04 Sangamo Biosciences, Inc. Ligand-controlled regulation of endogenous gene expression
US7013219B2 (en) * 1999-01-12 2006-03-14 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6794136B1 (en) 2000-11-20 2004-09-21 Sangamo Biosciences, Inc. Iterative optimization in the design of binding proteins
US7030215B2 (en) 1999-03-24 2006-04-18 Sangamo Biosciences, Inc. Position dependent recognition of GNN nucleotide triplets by zinc fingers
US20030104526A1 (en) 1999-03-24 2003-06-05 Qiang Liu Position dependent recognition of GNN nucleotide triplets by zinc fingers
EP1181359A1 (fr) 1999-05-28 2002-02-27 Sangamo Biosciences Inc. Commutateurs genetiques
CA2369855A1 (fr) * 1999-05-28 2001-01-04 Sangamo Biosciences, Inc. Commutateurs moleculaires
GB9915126D0 (en) 1999-06-30 1999-09-01 Imp College Innovations Ltd Control of gene expression
WO2001002856A1 (fr) * 1999-07-02 2001-01-11 The Scripps Research Institute Procede de determination de l'interaction d'agents de liaison peptidique
EP1230355A2 (fr) * 1999-10-01 2002-08-14 Sangamo Biosciences Inc. Banque d'adn et son utilisation dans des procedes de selection et conception des polypeptides
EP1229781B1 (fr) 1999-11-17 2013-01-02 Mendel Biotechnology, Inc. Gènes de caractéristiques de semences de plantes
EP1950306A1 (fr) 1999-11-17 2008-07-30 Mendel Biotechnology, Inc. Gènes de tolérance au stress environnemental
ATE309536T1 (de) 1999-12-06 2005-11-15 Sangamo Biosciences Inc Methoden zur verwendung von randomisierten zinkfingerprotein-bibliotheken zur identifizierung von genfunktionen
CA2398155C (fr) * 2000-01-24 2011-07-19 Sangamo Biosciences, Inc. Polypeptides a liaison acide nucleique caracterises par des modules a liaison acide nucleique lies par une liaison flexible
WO2001059450A2 (fr) * 2000-02-08 2001-08-16 Sangamo Biosciences, Inc. Cellules destinees a la decouverte de medicaments
US20020061512A1 (en) * 2000-02-18 2002-05-23 Kim Jin-Soo Zinc finger domains and methods of identifying same
US20020064802A1 (en) * 2000-04-28 2002-05-30 Eva Raschke Methods for binding an exogenous molecule to cellular chromatin
WO2001083819A2 (fr) 2000-04-28 2001-11-08 Sangamo Biosciences, Inc. Procédés d'élaboration de molécules régulatrices exogènes
AU2001257421A1 (en) * 2000-04-28 2001-11-12 Sangamo Biosciences, Inc. Pharmacogenomics and identification of drug targets by reconstruction of signal transduction pathways based on sequences of accessible regions
EP1276860B1 (fr) * 2000-04-28 2007-09-26 Sangamo Biosciences Inc. Base de donnees de sequences regulatrices, leurs procedes d'elaboration et d'utilisation
US7923542B2 (en) 2000-04-28 2011-04-12 Sangamo Biosciences, Inc. Libraries of regulatory sequences, methods of making and using same
US20040039175A1 (en) * 2000-05-08 2004-02-26 Yen Choo Modulation of viral gene expression by engineered zinc finger proteins
US6492117B1 (en) * 2000-07-12 2002-12-10 Gendaq Limited Zinc finger polypeptides capable of binding DNA quadruplexes
US20030082561A1 (en) * 2000-07-21 2003-05-01 Takashi Sera Zinc finger domain recognition code and uses thereof
AU2001278496A1 (en) * 2000-07-21 2002-02-05 Syngenta Participations Ag Zinc finger domain recognition code and uses thereof
EP2410060A1 (fr) 2000-08-22 2012-01-25 Mendel Biotechnology, Inc. Gènes servant à modifier des caractéristiques de plantes IV
AU2001285252A1 (en) * 2000-08-25 2002-03-13 Gendaq, Ltd. Analysis of binding interactions
ATE448301T1 (de) 2000-09-08 2009-11-15 Univ Zuerich Sammlung von proteinen mit sich wiederholenden sequenzen (repeat proteins), die repetitive sequenzmodule enthalten
US6905816B2 (en) * 2000-11-27 2005-06-14 Intelligent Medical Devices, Inc. Clinically intelligent diagnostic devices and methods
US7026462B2 (en) 2000-12-07 2006-04-11 Sangamo Biosciences, Inc. Regulation of angiogenesis with zinc finger proteins
US7067317B2 (en) * 2000-12-07 2006-06-27 Sangamo Biosciences, Inc. Regulation of angiogenesis with zinc finger proteins
EP1353941B1 (fr) 2001-01-22 2013-03-13 Sangamo BioSciences, Inc. Proteines de liaison a doigt de zinc modifiees
US7947469B2 (en) 2001-01-22 2011-05-24 Gendaq, Ltd. Modulation of HIV infection
US6872548B2 (en) * 2001-01-31 2005-03-29 Human Genome Sciences, Inc. Scaffolded fusion polypeptides and compositions and methods for making the same
US7067617B2 (en) 2001-02-21 2006-06-27 The Scripps Research Institute Zinc finger binding domains for nucleotide sequence ANN
CA2457095A1 (fr) * 2001-08-17 2003-02-27 Toolgen, Inc. Bibliotheques de domaines a doigts de zinc
WO2003027247A2 (fr) 2001-09-24 2003-04-03 Sangamo Biosciences, Inc. Modulation de cellules souches au moyen de proteines a doigts de zinc (zfp)
US20040259258A1 (en) * 2001-12-07 2004-12-23 Kim Jin-Soo Regulation of prokaryotic gene expression with zinc finger proteins
WO2003048345A1 (fr) * 2001-12-07 2003-06-12 Toolgen, Inc. Balayage phenotypique de proteines chimeriques
US6899892B2 (en) * 2001-12-19 2005-05-31 Regents Of The University Of Minnesota Methods to reduce body fat
US7262054B2 (en) 2002-01-22 2007-08-28 Sangamo Biosciences, Inc. Zinc finger proteins for DNA binding and gene regulation in plants
CA2474486C (fr) 2002-01-23 2013-05-14 The University Of Utah Research Foundation Mutagenese chromosomique ciblee au moyen de nucleases en doigt a zinc
US20030232410A1 (en) * 2002-03-21 2003-12-18 Monika Liljedahl Methods and compositions for using zinc finger endonucleases to enhance homologous recombination
EP1532178A4 (fr) * 2002-06-11 2006-10-25 Scripps Research Inst Facteurs de transcription artificiels
DE50310378D1 (de) 2002-07-26 2008-10-02 Basf Plant Science Gmbh Neue selektionsverfahren
CA2494626A1 (fr) 2002-08-07 2004-03-04 Basf Plant Science Gmbh Sequences d'acide nucleique codant des proteines associees a une reaction de stress abiotique
US7361635B2 (en) 2002-08-29 2008-04-22 Sangamo Biosciences, Inc. Simultaneous modulation of multiple genes
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
ES2380017T3 (es) 2002-09-18 2012-05-07 Mendel Biotechnology, Inc. Polinucleótidos y polipéptidos en plantas
US20070178454A1 (en) * 2002-10-21 2007-08-02 Joung J K Context sensitive paralell optimization of zinc finger dna binding domains
US20060246440A1 (en) * 2002-10-23 2006-11-02 Joung J K Methods for producing zinc finger proteins that bind to extended dna target sequences
AU2003295692A1 (en) * 2002-11-15 2004-06-15 Sangamo Biosciences, Inc. Methods and compositions for analysis of regulatory sequences
US7696334B1 (en) * 2002-12-05 2010-04-13 Rosetta Genomics, Ltd. Bioinformatically detectable human herpesvirus 5 regulatory gene
CN100398652C (zh) * 2002-12-09 2008-07-02 图尔金株式会社 调节性锌指蛋白
AU2003303589B2 (en) 2002-12-26 2008-04-24 Syngenta Participations Ag Cell proliferation-related polypeptides and uses therefor
US20070042433A1 (en) * 2003-03-26 2007-02-22 Jianxin Bao Neuregulin protein regulation of synaptic proteins
EP1641930B1 (fr) 2003-04-15 2011-06-29 BASF Plant Science GmbH Sequence d'acides nucleiques codant pour des proteines associees a la reponse de stress abiotique et cellules vegetales et plantes a tolerance accrue au stress environnemental
US7888121B2 (en) 2003-08-08 2011-02-15 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US11311574B2 (en) 2003-08-08 2022-04-26 Sangamo Therapeutics, Inc. Methods and compositions for targeted cleavage and recombination
US8409861B2 (en) 2003-08-08 2013-04-02 Sangamo Biosciences, Inc. Targeted deletion of cellular DNA sequences
US20120196370A1 (en) 2010-12-03 2012-08-02 Fyodor Urnov Methods and compositions for targeted genomic deletion
EP3222715A1 (fr) 2003-08-08 2017-09-27 Sangamo BioSciences, Inc. Procédés et compositions pour clivage et recombinaison ciblés
US7216038B2 (en) 2003-09-11 2007-05-08 Franco Vitaliano Quantum information processing elements and quantum information processing platforms using such elements
US7219018B2 (en) * 2003-09-11 2007-05-15 Franco Vitaliano Quantum information processing elements and quantum information processing platforms using such elements
US7219017B2 (en) 2003-09-11 2007-05-15 Franco Vitaliano Quantum information processing elements and quantum information processing platforms using such elements
WO2005028630A2 (fr) 2003-09-19 2005-03-31 Sangamo Biosciences, Inc. Proteines a doigt de zinc issues du genie genetique pour la regulation de l'expression genique
US7972854B2 (en) 2004-02-05 2011-07-05 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
CA2561565C (fr) * 2004-04-08 2013-11-26 Sangamo Biosciences, Inc. Methodes de repression du gene phospholamban et de modulation de la contractilite cardiaque
CA2561714A1 (fr) * 2004-04-08 2005-10-27 Sangamo Biosciences, Inc. Procedes et compositions permettant de traiter les troubles neuropathiques et neurodegeneratifs
CA2579677A1 (fr) * 2004-09-16 2006-03-30 Sangamo Biosciences, Inc. Production de proteines: compositions et methodes
CN103289961A (zh) 2004-09-24 2013-09-11 巴斯福植物科学有限公司 编码与非生物性胁迫反应相关的蛋白质的核酸序列和环境胁迫抗性增加的植物细胞和植物
WO2006078431A2 (fr) 2004-12-22 2006-07-27 The University Of Iowa Research Foundation Compositions et procedes associes a des vecteurs retroviraux modifies pour une integration restreinte specifique au site
CA2594572A1 (fr) 2005-01-14 2006-07-20 University Of Guelph Gene et proteine de detection de sucre regules par l'azote et modulation associee
CA2599004C (fr) 2005-02-28 2015-05-26 Sangamo Biosciences, Inc. Methodes et compostions anti-angiogeniques
KR101419729B1 (ko) * 2005-07-26 2014-07-17 상가모 바이오사이언스 인코포레이티드 외래 핵산 서열의 표적화된 통합 및 발현
EP1915452A2 (fr) 2005-08-12 2008-04-30 BASF Plant Science GmbH Sequences d'acides nucleiques codant des proteines associees a la reponse au stress abiotique, cellules vegetales et vegetaux presentant une tolerance amelioree au stress environnemental
AU2006287553A1 (en) 2005-09-08 2007-03-15 Chromatin, Inc. Plants modified with mini-chromosomes
JP2009520463A (ja) 2005-11-28 2009-05-28 ザ スクリプス リサーチ インスティテュート Ｔｎｎのための亜鉛フィンガー結合ドメイン
US20090172834A1 (en) 2006-03-24 2009-07-02 Basf Plant Science Gmbh Proteins Associated With Abiotic Stress Response And Homologs
US20080015164A1 (en) * 2006-05-19 2008-01-17 Sangamo Biosciences, Inc. Methods and compositions for inactivation of dihydrofolate reductase
WO2007139982A2 (fr) 2006-05-25 2007-12-06 Sangamo Biosciences, Inc. Procédés et compositions pour l'inactivation de gènes
WO2007139898A2 (fr) * 2006-05-25 2007-12-06 Sangamo Biosciences, Inc. Demi-domaines résultant de clivage par génie génétique
ATE554096T1 (de) 2006-08-10 2012-05-15 Basf Plant Science Gmbh Verfahren zum erhöhen der resistenz gegen sojabohnenrost in transgenen pflanzen
PL2049663T3 (pl) 2006-08-11 2015-08-31 Dow Agrosciences Llc Homologiczna rekombinacja za pośrednictwem nukleazy z palcami cynkowymi
RU2428480C9 (ru) 2006-08-30 2012-03-20 Басф Плант Сайенс Гмбх Способ повышения резистентности трансгенных растений к патогенным воздействиям
CA2660861A1 (fr) 2006-10-12 2008-04-17 Basf Plant Science Gmbh Procede d'augmentation de la resistance aux pathogenes de plantes transgeniques
CN101589148B (zh) 2006-10-13 2014-07-02 巴斯福植物科学有限公司 产量提高的植物
US9217026B2 (en) * 2006-11-13 2015-12-22 Sangamo Biosciences, Inc. Method of inactivating a glucocorticoid receptor gene in an isolated cell
WO2008067840A1 (fr) 2006-12-08 2008-06-12 Swetree Technologies Ab Plantes ayant des caractéristiques de croissance améliorées et leur procédé de production
SI2415872T1 (sl) * 2006-12-14 2016-09-30 Dow Agrosciences Llc Optimizirani nekanonični proteini s cinkovim prstom
BRPI0807760A2 (pt) 2007-02-19 2014-06-17 Evonik Degussa Gmbh Bactérias corineformes com atividade de formato-thf-sintetase e/ou atividade de clivagem de glicina
CA2584934A1 (fr) 2007-04-17 2008-10-17 University Of Guelph Gene et proteine de detection de sucre regules par l'azote et modulation associee
CN101679977B (zh) 2007-04-26 2013-05-01 桑格摩生物科学股份有限公司 向ppp1r12c基因座的靶向整合
DE112008001452T5 (de) 2007-05-22 2010-12-16 Basf Plant Science Gmbh Pflanzen mit erhöhter Toleranz und/oder Resistenz gegen Umweltstress und erhöhter Biomasseproduktion
CN101772575B (zh) 2007-05-23 2015-03-11 先正达参股股份有限公司 多核苷酸标志物
KR101510778B1 (ko) 2007-07-12 2015-04-10 상가모 바이오사이언스 인코포레이티드 알파 1,6 당전이효소(ｆｕｔ8) 유전자 발현을 비활성화시키기 위한 방법 및 조성물
MX2010002931A (es) 2007-09-18 2010-06-01 Basf Plant Science Gmbh Plantas con rendimiento incrementado.
DE112008002456T5 (de) 2007-09-21 2011-01-13 Basf Plant Science Gmbh Pflanzen mit erhöhtem Ertrag
US11235026B2 (en) 2007-09-27 2022-02-01 Sangamo Therapeutics, Inc. Methods and compositions for modulating PD1
EP2205749B1 (fr) * 2007-09-27 2016-05-18 Dow AgroSciences LLC Protéines à doigt de zinc synthétisées ciblant des gènes de 5-énolpyruvyl shikimate-3-phosphate synthase
CA2700231C (fr) * 2007-09-27 2018-09-18 Sangamo Biosciences, Inc. Identification in vivo rapide de nucleases biologiquement actives
US8563314B2 (en) 2007-09-27 2013-10-22 Sangamo Biosciences, Inc. Methods and compositions for modulating PD1
EP2597155B1 (fr) 2007-10-25 2016-11-23 Sangamo BioSciences, Inc. Procédés et compositions pour une intégration ciblée
BRPI0821748A2 (pt) * 2007-12-19 2019-09-24 Basf Plant Science Gmbh método para produzir uma planta com rendimento aumentado, molécula isolada de ácido nucleico, construção de ácido nucleico, vetor, processo para produzir um polipeptídeo, polipeptídeo, anticorpo, núcleo de célula de planta, célula de planta, tecido de planta, material de propagação, semente, pólen, progênie, ou uma parte de planta, ou uma planta com rendimento aumento, processo para a indentificação de um composto, método para a produção de uma composição agrícola, composição, polipeptídeo ou molécula de ácido nucleico, uso dos ácidos nucléicos, e, método para a indentificação de uma planta com um rendimento aumentado
BR122017021070B1 (pt) 2007-12-28 2019-01-29 Swetree Technologies Ab método para produzir uma planta lenhosa perene transgênica apresentando um crescimento aumentado medido pela altura em comparação com seu tipo selvagem
MX2010009010A (es) * 2008-02-27 2010-09-09 Basf Plant Science Gmbh Plantas con mayor rendimiento.
EP2281050B1 (fr) 2008-04-14 2014-04-02 Sangamo BioSciences, Inc. Constructions donneuses linéaires pour une intégration ciblée
ES2594229T3 (es) 2008-04-30 2016-12-16 Sanbio, Inc. Células de regeneración nerviosa con alteraciones en la metilación del ADN
WO2009137872A1 (fr) * 2008-05-14 2009-11-19 Simons Haplomics Limited Méthodes et compositions permettant le traitement du cancer
JP2011521643A (ja) 2008-05-28 2011-07-28 サンガモ バイオサイエンシーズ， インコーポレイテッド Ｄｎａ結合ドメインおよび切断ドメインを連結するための組成物
KR101802393B1 (ko) * 2008-06-10 2017-11-28 상가모 테라퓨틱스, 인코포레이티드 Ｂａｘ- 및 ｂａｋ-결함 세포주들의 제조 방법 및 조성물
US8048307B2 (en) * 2008-08-14 2011-11-01 Brent Lee Dynamic filtration device using centrifugal force
CN103923892A (zh) 2008-08-19 2014-07-16 巴斯夫植物科学有限公司 通过在植物或其部分中提高或产生一种或更多活性具有提高产量的植物
CN102159722B (zh) 2008-08-22 2014-09-03 桑格摩生物科学股份有限公司 用于靶向单链切割和靶向整合的方法和组合物
US20110195843A1 (en) 2008-09-23 2011-08-11 Basf Plant Science Gmbh Plants with Increased Yield (LT)
AU2009306369A1 (en) 2008-10-23 2010-04-29 Basf Plant Science Gmbh A method for producing a transgenic cell with increased gamma-aminobutyric acid (GABA) content
EP2350289A1 (fr) 2008-10-23 2011-08-03 BASF Plant Science GmbH Plantes ayant un rendement augmenté (nue)
AU2009311697B2 (en) * 2008-10-29 2014-12-18 Sangamo Therapeutics, Inc. Methods and compositions for inactivating glutamine synthetase gene expression
EP2527454B1 (fr) 2008-11-03 2017-05-03 Swetree Technologies AB Matière végétale, plantes et procédé de production d'une plante dont les propriétés de la lignine sont modifiées
WO2010056808A2 (fr) * 2008-11-12 2010-05-20 The Regents Of The University Of California Compositions et procédés pour reprogrammer et redifférencier des cellules
US20110023152A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genome editing of cognition related genes in animals
US20110016543A1 (en) * 2008-12-04 2011-01-20 Sigma-Aldrich Co. Genomic editing of genes involved in inflammation
US20110023148A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genome editing of addiction-related genes in animals
US20110023145A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved in autism spectrum disorders
US20110023151A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genome editing of abc transporters
US20110016542A1 (en) * 2008-12-04 2011-01-20 Sigma-Aldrich Co. Canine genome editing with zinc finger nucleases
US20110016546A1 (en) * 2008-12-04 2011-01-20 Sigma-Aldrich Co. Porcine genome editing with zinc finger nucleases
US20110016540A1 (en) * 2008-12-04 2011-01-20 Sigma-Aldrich Co. Genome editing of genes associated with trinucleotide repeat expansion disorders in animals
US20110023149A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved in tumor suppression in animals
US20110023139A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved in cardiovascular disease
US20110023141A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved with parkinson's disease
US20110030072A1 (en) * 2008-12-04 2011-02-03 Sigma-Aldrich Co. Genome editing of immunodeficiency genes in animals
US20110023153A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved in alzheimer's disease
US20110016541A1 (en) * 2008-12-04 2011-01-20 Sigma-Aldrich Co. Genome editing of sensory-related genes in animals
US20110023150A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genome editing of genes associated with schizophrenia in animals
US20110016539A1 (en) * 2008-12-04 2011-01-20 Sigma-Aldrich Co. Genome editing of neurotransmission-related genes in animals
US20110023154A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Silkworm genome editing with zinc finger nucleases
US20110023147A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of prion disorder-related genes in animals
US20110023146A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of genes involved in secretase-associated disorders
US20110023158A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Bovine genome editing with zinc finger nucleases
US20110023140A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Rabbit 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
US20110023143A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of neurodevelopmental genes in animals
US20110023156A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Feline genome editing with zinc finger nucleases
KR101803737B1 (ko) 2008-12-04 2017-12-01 상가모 테라퓨틱스, 인코포레이티드 징크 핑거 뉴클레아제를 이용한 랫트의 유전체 편집
US20110023159A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Ovine genome editing with zinc finger nucleases
US20110023157A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Equine genome editing with zinc finger nucleases
EP2199399A1 (fr) 2008-12-17 2010-06-23 BASF Plant Science GmbH Production de cétocaroténoïdes dans les plantes
AR074783A1 (es) 2008-12-17 2011-02-09 Dow Agrosciences Llc Metodos y composiciones para expresar uno o mas productos de un acido nucleico exogeno integrado al locus zp15 de una celula vegetal
CA2934285C (fr) * 2009-02-04 2018-11-27 Sangamo Biosciences, Inc. Procedes et compositions pour traiter des neuropathies
US11235062B2 (en) * 2009-03-06 2022-02-01 Metaqor Llc Dynamic bio-nanoparticle elements
US11096901B2 (en) 2009-03-06 2021-08-24 Metaqor Llc Dynamic bio-nanoparticle platforms
CA2755192C (fr) 2009-03-20 2018-09-11 Sangamo Biosciences, Inc. Modification de cxcr4 en utilisant des proteines a doigt de zinc modifiees
CA2756833C (fr) * 2009-04-09 2019-11-19 Sangamo Biosciences, Inc. Integration ciblee dans des cellules souches
US8772008B2 (en) * 2009-05-18 2014-07-08 Sangamo Biosciences, Inc. Methods and compositions for increasing nuclease activity
AU2010266705B2 (en) 2009-06-30 2014-06-05 Sangamo Therapeutics, Inc. Rapid screening of biologically active nucleases and isolation of nuclease-modified cells
CA2766123A1 (fr) 2009-07-08 2011-01-13 Cellular Dynamics International, Inc. Cellules ips modifiees ayant une forme mutante du gene d?entree cellulaire du virus d?immunodeficience humaine (vih)
AU2010275363A1 (en) 2009-07-23 2012-02-02 Basf Plant Science Company Gmbh Plants with increased yield
US9234016B2 (en) * 2009-07-28 2016-01-12 Sangamo Biosciences, Inc. Engineered zinc finger proteins for treating trinucleotide repeat disorders
US20120178647A1 (en) * 2009-08-03 2012-07-12 The General Hospital Corporation Engineering of zinc finger arrays by context-dependent assembly
DK2462230T3 (en) * 2009-08-03 2015-10-19 Recombinetics Inc METHODS AND COMPOSITIONS FOR TARGETED RE-MODIFICATION
WO2011019385A1 (fr) 2009-08-11 2011-02-17 Sangamo Biosciences, Inc. Organismes homozygotes destinés à une modification ciblée
MY174390A (en) * 2009-10-22 2020-04-15 Sangamo Biosciences Inc Engineered zinc finger proteins targeting plant genes involved in fatty acid biosynthesis
US8956828B2 (en) 2009-11-10 2015-02-17 Sangamo Biosciences, Inc. Targeted disruption of T cell receptor genes using engineered zinc finger protein nucleases
EP2501816A4 (fr) 2009-11-17 2013-07-03 Basf Plant Science Co Gmbh Plantes à rendement accru
AU2010325549B2 (en) 2009-11-27 2017-04-20 Basf Plant Science Company Gmbh Optimized endonucleases and uses thereof
EA031322B1 (ru) 2010-01-22 2018-12-28 Дау Агросайенсиз Ллс Клетка или клеточная линия для экспрессии экзогенных нуклеотидных последовательностей и применение клетки или клеточной линии
CA2788560A1 (fr) 2010-02-08 2011-08-11 Sangamo Biosciences, Inc. Demi-domaines de clivage techniques
US9255259B2 (en) 2010-02-09 2016-02-09 Sangamo Biosciences, Inc. Targeted genomic modification with partially single-stranded donor molecules
CA2796600C (fr) 2010-04-26 2019-08-13 Sangamo Biosciences, Inc. Edition du genome d'un locus de rosa en utilisant des nucleases a doigt de zinc
WO2011139349A1 (fr) 2010-05-03 2011-11-10 Sangamo Biosciences, Inc. Compositions pour relier des modules en doigt de zinc
EP2571512B1 (fr) 2010-05-17 2017-08-23 Sangamo BioSciences, Inc. Nouvelles protéines se liant à l'adn et leurs utilisations
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
JP2013537410A (ja) 2010-07-23 2013-10-03 シグマ−アルドリッチ・カンパニー・リミテッド・ライアビリティ・カンパニー 標的化エンドヌクレアーゼおよび一本鎖核酸を用いたゲノム編集
WO2012047598A1 (fr) 2010-09-27 2012-04-12 Sangamo Biosciences, Inc. Procédés et compositions d'inhibition d'entrée de virus dans cellules
CA2814143C (fr) 2010-10-12 2020-09-08 The Children's Hospital Of Philadelphia Procedes et compositions pour traiter l'hemophilie b
EP2659404B1 (fr) 2010-12-29 2018-08-08 Sigma-Aldrich Co., LLC Cellules dont l'expression des protéines impliquées dans les processus adme et toxicologiques est perturbée
US9267123B2 (en) 2011-01-05 2016-02-23 Sangamo Biosciences, Inc. Methods and compositions for gene correction
EP2694554A1 (fr) 2011-04-08 2014-02-12 Gilead Biologics, Inc. Procédés et compositions pour normaliser le système vasculaire tumoral par inhibition de la loxl2
ES2671733T3 (es) 2011-06-30 2018-06-08 Sigma Aldrich Co. Llc Células deficientes en ácido CMP-N-acetilneuramínico hidroxilasa y/o glucoproteína alfa-1,3-galactosil transferasa
EP2732038B1 (fr) 2011-07-15 2018-09-05 The General Hospital Corporation Procédés d'assemblage d'effecteurs de type activateur de la transcription
JP6072788B2 (ja) 2011-07-25 2017-02-01 サンガモ バイオサイエンシーズ， インコーポレイテッド 嚢胞性線維症膜コンダクタンス制御因子（ｃｆｔｒ）遺伝子を改変するための方法および組成物
CN103917644A (zh) 2011-09-21 2014-07-09 桑格摩生物科学股份有限公司 调控转基因表达的方法和组合物
WO2013050318A1 (fr) 2011-10-07 2013-04-11 Basf Plant Science Company Gmbh Procédé de production de plantes ayant une résistance accrue à des pathogènes
MA36970A1 (fr) * 2011-10-11 2016-03-31 Aliophtha Ag Régulation de l'expression d'un récepteur par l'intermédiaire de l'administration de facteurs de transcription artificiels
EP2771457B1 (fr) 2011-10-27 2017-11-22 Sangamo Therapeutics, Inc. Procédés et compositions pour la modification du locus hprt
CA2854819C (fr) 2011-11-16 2022-07-19 Sangamo Biosciences, Inc. Proteines de liaison d'adn modifiees et utilisations de celles-ci
CN104245932A (zh) 2012-01-11 2014-12-24 西格马-奥尔德里奇有限责任公司 具有简单糖型的重组蛋白的产生
EP2806881A1 (fr) 2012-01-27 2014-12-03 SanBio, Inc. Procédés et compositions de modulation de l'angiogenèse et de la vasculogenèse
CN105658792B (zh) 2012-02-28 2020-11-10 西格马-奥尔德里奇有限责任公司 靶向组蛋白乙酰化
JP6490426B2 (ja) 2012-02-29 2019-03-27 サンガモ セラピューティクス， インコーポレイテッド ハンチントン病を治療するための方法および組成物
MX369788B (es) 2012-05-02 2019-11-21 Dow Agrosciences Llc Modificacion dirigida de deshidrogenasa de malato.
EP3646880A1 (fr) 2012-05-07 2020-05-06 Allergan, Inc. Procédé de traitement d'amd chez des patients souffrant d'un état réfractaire à la thérapie anti-vegf
RU2650819C2 (ru) 2012-05-07 2018-04-17 Сангамо Терапьютикс, Инк. Способы и композиции для опосредованной нуклеазой направленной интеграции трансгенов
US9890364B2 (en) 2012-05-29 2018-02-13 The General Hospital Corporation TAL-Tet1 fusion proteins and methods of use thereof
CA2878037C (fr) 2012-07-11 2021-08-31 Sangamo Biosciences, Inc. Procedes et compositions pour la delivrance d'agents biologiques
US10648001B2 (en) 2012-07-11 2020-05-12 Sangamo Therapeutics, Inc. Method of treating mucopolysaccharidosis type I or II
HUE041553T2 (hu) 2012-07-11 2019-05-28 Sangamo Therapeutics Inc Lizoszomális tárolási betegségek (LSD) kezelése és génterápia
ES2812599T3 (es) 2012-08-29 2021-03-17 Sangamo Therapeutics Inc Procedimientos y composiciones para el tratamiento de una afección genética
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
CN105264067B (zh) 2012-09-07 2020-11-10 美国陶氏益农公司 Fad3性能基因座及相应的能够诱导靶向断裂的靶位点特异性结合蛋白
UA119135C2 (uk) 2012-09-07 2019-05-10 ДАУ АГРОСАЙЄНСІЗ ЕлЕлСі Спосіб отримання трансгенної рослини
UA118090C2 (uk) 2012-09-07 2018-11-26 ДАУ АГРОСАЙЄНСІЗ ЕлЕлСі Спосіб інтегрування послідовності нуклеїнової кислоти, що представляє інтерес, у ген fad2 у клітині сої та специфічний для локусу fad2 білок, що зв'язується, здатний індукувати спрямований розрив
ES2824024T3 (es) 2012-10-10 2021-05-11 Sangamo Therapeutics Inc Compuestos modificadores de células T y usos de los mismos
EP2906602B1 (fr) 2012-10-12 2019-01-16 The General Hospital Corporation Protéines de fusion effecteur de type activateur de transcription (tale) - déméthylase 1 spécifique de la lysine (lsd1)
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
KR20150096696A (ko) 2012-12-13 2015-08-25 다우 아그로사이언시즈 엘엘씨 메이즈에서 특정 유전자좌로의 정밀 유전자 표적화
AU2014207618A1 (en) 2013-01-16 2015-08-06 Emory University Cas9-nucleic acid complexes and uses related thereto
EP3623463B1 (fr) 2013-02-07 2021-10-20 The General Hospital Corporation Activateurs transcriptionnels tale
PT2963113T (pt) 2013-02-14 2020-02-14 Univ Osaka Método para isolar região genómica específica utilizando molécula que se liga especificamente a sequência de adn endógena
AU2014218621B2 (en) 2013-02-25 2019-11-07 Sangamo Therapeutics, Inc. Methods and compositions for enhancing nuclease-mediated gene disruption
CA2906553C (fr) 2013-03-15 2022-08-02 The General Hospital Corporation Direction, par guidage arn, de proteines regulatrices genetiques et epigenomiques vers des loci genomiques specifiques
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
US9937207B2 (en) 2013-03-21 2018-04-10 Sangamo Therapeutics, Inc. Targeted disruption of T cell receptor genes using talens
CN105263312A (zh) 2013-04-05 2016-01-20 美国陶氏益农公司 用于在植物基因组内整合外源序列的方法和组合物
CA2910427C (fr) 2013-05-10 2024-02-20 Sangamo Biosciences, Inc. Procedes et compositions d'apport pour genie genomique medie par nuclease
CA2910489A1 (fr) 2013-05-15 2014-11-20 Sangamo Biosciences, Inc. Procedes et compositions pour le traitement d'une maladie genetique
US10011850B2 (en) 2013-06-21 2018-07-03 The General Hospital Corporation Using RNA-guided FokI Nucleases (RFNs) to increase specificity for RNA-Guided Genome Editing
CA2920899C (fr) 2013-08-28 2023-02-28 Sangamo Biosciences, Inc. Compositions de liaison de domaines de liaison a l'adn et de domaines de clivage
WO2015057976A1 (fr) 2013-10-17 2015-04-23 Sangamo Biosciences, Inc. Procédés d'apport et compositions pour ingénierie génomique à médiation par des nucléases dans des cellules souches hématopoïétiques
CN105899665B (zh) 2013-10-17 2019-10-22 桑格摩生物科学股份有限公司 用于核酸酶介导的基因组工程改造的递送方法和组合物
UA120503C2 (uk) 2013-11-04 2019-12-26 Дау Агросайєнсиз Елелсі Спосіб одержання трансгенної клітини рослини кукурудзи
UY35816A (es) 2013-11-04 2015-05-29 Dow Agrosciences Llc ?locus óptimos de la soja?.
WO2015066634A2 (fr) 2013-11-04 2015-05-07 Dow Agrosciences Llc Locus optimaux de soja
AU2014341929B2 (en) 2013-11-04 2017-11-30 Corteva Agriscience Llc Optimal maize loci
LT3066201T (lt) 2013-11-07 2018-08-10 Editas Medicine, Inc. Su crispr susiję būdai ir kompozicijos su valdančiomis grnr
BR112016010175A2 (pt) 2013-11-11 2017-10-03 Sangamo Biosciences Inc Repressor genético, seus usos, e composição farmacêutica
EP3960856A1 (fr) 2013-11-13 2022-03-02 Children's Medical Center Corporation Régulation de l'expression génique médiée par la nucléase
EP3757116A1 (fr) 2013-12-09 2020-12-30 Sangamo Therapeutics, Inc. Procédés et compositions pour l'ingénierie génomique
PL3102673T3 (pl) 2014-02-03 2020-11-02 Sangamo Therapeutics, Inc. Sposoby i kompozycje do leczenia talasemii beta
US20150232578A1 (en) 2014-02-14 2015-08-20 Symvivo Corporation Hybrid proteins and uses thereof
JP6606088B2 (ja) 2014-02-24 2019-11-13 サンガモ セラピューティクス， インコーポレイテッド ヌクレアーゼ媒介性標的化組み込みのための方法および組成物
EP3736325A1 (fr) 2014-03-04 2020-11-11 Sigma Aldrich Co. LLC Cellules résistantes aux virus et leurs utilisations
US9624498B2 (en) 2014-03-18 2017-04-18 Sangamo Biosciences, Inc. Methods and compositions for regulation of zinc finger protein expression
CA2946514C (fr) 2014-04-22 2023-08-29 Q-State Biosciences, Inc. Analyse optogenetique de composes
WO2015164748A1 (fr) 2014-04-24 2015-10-29 Sangamo Biosciences, Inc. Protéines effectrices de type activateur de transcription (tale) obtenues par génie génétique
MX2016014565A (es) 2014-05-08 2017-05-23 Sangamo Biosciences Inc Metodos y composiciones para tratar la enfermedad de huntington.
CA2947622A1 (fr) 2014-05-13 2015-11-19 Sangamo Biosciences, Inc. Methodes d'edition genomique et compositions pour la prevention ou le traitement d'une maladie
CA2949710A1 (fr) 2014-05-30 2015-12-03 The Board Of Trustees Of The Leland Stanford Junior University Compositions et methodes pour traiter des infections virales latentes
US9970001B2 (en) 2014-06-05 2018-05-15 Sangamo Therapeutics, Inc. Methods and compositions for nuclease design
WO2015193653A1 (fr) 2014-06-16 2015-12-23 Consejo Nacional De Investigaciones Cientificas Y Tecnicas Gènes et protéines chimériques de résistance à l'oxydation et plantes transgéniques les comprenant
IL286474B2 (en) 2014-06-23 2023-11-01 Massachusetts Gen Hospital Genome-wide random identification of DSBS assessed by sequencing (guide-sequence)
US20170159065A1 (en) 2014-07-08 2017-06-08 Vib Vzw Means and methods to increase plant yield
CA2955203C (fr) 2014-07-14 2022-01-04 Washington State University Knock-out nanos qui permet de pratiquer l'ablation des cellules de la lignee germinale
US11278572B2 (en) 2014-07-18 2022-03-22 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Reducing CXCR4 expression and/or function to enhance engraftment of hematopoietic stem cells
WO2016014837A1 (fr) 2014-07-25 2016-01-28 Sangamo Biosciences, Inc. Édition de gènes pour la thérapie génique du vih
US9816074B2 (en) 2014-07-25 2017-11-14 Sangamo Therapeutics, Inc. Methods and compositions for modulating nuclease-mediated genome engineering in hematopoietic stem cells
US9616090B2 (en) 2014-07-30 2017-04-11 Sangamo Biosciences, Inc. Gene correction of SCID-related genes in hematopoietic stem and progenitor cells
ES2886012T3 (es) 2014-09-16 2021-12-16 Sangamo Therapeutics Inc Métodos y composiciones para la ingeniería y corrección de genomas mediadas por nucleasas en células madre hematopoyéticas
CA2963080A1 (fr) 2014-10-01 2016-04-07 The General Hospital Corporation Procedes destines a augmenter l'efficacite de la reparation par homologie induite par nuclease
US10889834B2 (en) 2014-12-15 2021-01-12 Sangamo Therapeutics, Inc. Methods and compositions for enhancing targeted transgene integration
EP3247366A4 (fr) 2015-01-21 2018-10-31 Sangamo Therapeutics, Inc. Méthodes et compositions pour l'identification de nucléases à spécificité élevée
BR112017016789A2 (pt) 2015-02-11 2018-05-08 Basf Se métodos para produzir uma planta transgênica, para controlar a vegetação indesejada e para o cultivo da planta, molécula de ácido nucleico, construção de ácido nucleico, vetor, polipeptídeo hppd mutado, núcleo de célula vegetal, núcleo de célula vegetal transgênica, planta transgênica, uso do ácido nucleico, combinação útil, processo para a preparação de uma combinação útil e uso de uma combinação útil
US10048275B2 (en) 2015-03-13 2018-08-14 Q-State Biosciences, Inc. Cardiotoxicity screening methods
ES2959608T3 (es) 2015-04-03 2024-02-27 Dana Farber Cancer Inst Inc Composición y métodos de edición del genoma de células B
US10179918B2 (en) 2015-05-07 2019-01-15 Sangamo Therapeutics, Inc. Methods and compositions for increasing transgene activity
US10808020B2 (en) 2015-05-12 2020-10-20 Sangamo Therapeutics, Inc. Nuclease-mediated regulation of gene expression
EP3298448A4 (fr) 2015-05-21 2019-03-27 Q-State Biosciences, Inc. Microscope optogénétique
US10117911B2 (en) 2015-05-29 2018-11-06 Agenovir Corporation Compositions and methods to treat herpes simplex virus infections
GB2543873A (en) 2015-05-29 2017-05-03 Agenovir Corp Compositions and methods for cell targeted HPV treatment
EP3302525A2 (fr) 2015-06-05 2018-04-11 Novartis AG Méthodes et compositions permettant de diagnostiquer, traiter et surveiller le traitement de troubles associés à une déficience en shank3
US9957501B2 (en) 2015-06-18 2018-05-01 Sangamo Therapeutics, Inc. Nuclease-mediated regulation of gene expression
AU2016291778B2 (en) 2015-07-13 2021-05-06 Sangamo Therapeutics, Inc. Delivery methods and compositions for nuclease-mediated genome engineering
WO2017023570A1 (fr) 2015-08-06 2017-02-09 The Curators Of The University Of Missouri Animaux résistant aux pathogènes ayant des gènes cd163 modifiés
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
JP2018530536A (ja) 2015-09-11 2018-10-18 ザ ジェネラル ホスピタル コーポレイション ヌクレアーゼＤＳＢの完全照合およびシーケンシング（ＦＩＮＤ−ｓｅｑ）
EA037993B1 (ru) 2015-09-23 2021-06-21 Сангамо Терапьютикс, Инк. Репрессоры htt и их применение
JP2018529353A (ja) 2015-09-30 2018-10-11 ザ ジェネラル ホスピタル コーポレイション 配列決定法による切断事象の包括的生体外報告（ＣＩＲＣＬＥ−ｓｅｑ）
CA3004349A1 (fr) 2015-11-23 2017-06-01 Sangamo Therapeutics, Inc. Methodes et compositions pour modifier l'immunite
AU2016369490C1 (en) 2015-12-18 2021-12-23 Sangamo Therapeutics, Inc. Targeted disruption of the T cell receptor
CN108699132B (zh) 2015-12-18 2023-08-11 桑格摩生物治疗股份有限公司 Mhc细胞受体的靶向破坏
EP3402533B1 (fr) 2016-01-15 2021-08-04 Sangamo Therapeutics, Inc. Méthodes et compositions pour le traitement d'une maladie neurologique
CN109069568B (zh) 2016-02-02 2023-07-07 桑格摩生物治疗股份有限公司 用于连接dna结合结构域和切割结构域的组合物
AU2017238512B2 (en) 2016-03-23 2022-12-08 Dana-Farber Cancer Institute, Inc. Methods for enhancing the efficiency of gene editing
US11124805B2 (en) 2016-07-13 2021-09-21 Vertex Pharmaceuticals Incorporated Methods, compositions and kits for increasing genome editing efficiency
US20190247436A1 (en) 2016-07-21 2019-08-15 Maxcyte, Inc. Methods and compositions for modifying genomic dna
US11344511B2 (en) 2016-07-27 2022-05-31 Case Western Reserve University Compounds and methods of promoting myelination
US11193136B2 (en) 2016-08-09 2021-12-07 Vib Vzw Cellulose synthase inhibitors and mutant plants
AU2017315414B2 (en) 2016-08-24 2024-02-15 Sangamo Therapeutics, Inc. Engineered target specific nucleases
WO2018039440A1 (fr) 2016-08-24 2018-03-01 Sangamo Therapeutics, Inc. Régulation de l'expression génique à l'aide de nucléases modifiées
WO2018049009A2 (fr) 2016-09-07 2018-03-15 Sangamo Therapeutics, Inc. Modulation des gènes du foie
MX2019003768A (es) 2016-10-03 2019-06-24 Juno Therapeutics Inc Moleculas de enlace especificas de hpv.
EP3757120B8 (fr) 2016-10-04 2022-06-15 Precision Biosciences, Inc. Domaines co-stimulateurs destinés à être utilisés dans des cellules génétiquement modifiées
CN110290813A (zh) 2016-10-14 2019-09-27 通用医疗公司 表观遗传学调控的位点特异性核酸酶
CA3039673A1 (fr) 2016-10-20 2018-04-26 Sangamo Therapeutics, Inc. Methodes et compositions pour le traitement de la maladie de fabry
US11020492B2 (en) 2016-10-31 2021-06-01 Sangamo Therapeutics, Inc. Gene correction of SCID-related genes in hematopoietic stem and progenitor cells
IL266862B2 (en) 2016-12-01 2024-01-01 Sangamo Therapeutics Inc Tau modulators and methods and preparations for their administration
JP7416406B2 (ja) 2016-12-08 2024-01-17 ケース ウェスタン リザーブ ユニバーシティ 機能的ミエリン産生を増進させるための方法および組成物
US20190390241A1 (en) 2017-01-24 2019-12-26 Sigma-Aldrich Co. Llc Viral resistant cells and culture systems
SG11201906948UA (en) 2017-04-20 2019-08-27 Univ Oregon Health & Science Human gene correction
CA3061612A1 (fr) 2017-04-28 2018-11-01 Acuitas Therapeutics, Inc. Nouveaux lipides carbonyles et formulations nanoparticulaires lipidiques pour l'administration d'acides nucleiques
AU2018261366A1 (en) 2017-05-03 2019-10-31 Sangamo Therapeutics, Inc. Methods and compositions for modification of a cystic fibrosis transmembrane conductance regulator (CTFR) gene
JP7170666B2 (ja) 2017-05-08 2022-11-14 プレシジョン バイオサイエンシズ，インク． 操作された抗原受容体をコードする核酸分子及び阻害性核酸分子、並びにそれらの使用方法
WO2018218166A1 (fr) 2017-05-25 2018-11-29 The General Hospital Corporation Utilisation de désaminases clivées pour limiter la désamination hors cible non désirée d'édition de bases
US11512287B2 (en) 2017-06-16 2022-11-29 Sangamo Therapeutics, Inc. Targeted disruption of T cell and/or HLA receptors
EP3645038A1 (fr) 2017-06-30 2020-05-06 Precision Biosciences, Inc. Lymphocytes t génétiquement modifiés comprenant un intron modifié dans le gène alpha du récepteur des lymphocytes t
EP4215543A3 (fr) 2017-10-03 2023-10-11 Juno Therapeutics, Inc. Molécules de liaison spécifiques du vph
US20200239544A1 (en) 2017-10-03 2020-07-30 Precision Biosciences, Inc. Modified epidermal growth factor receptor peptides for use in genetically-modified cells
US20200299658A1 (en) 2017-11-01 2020-09-24 Precision Biosciences, Inc. Engineered nucleases that target human and canine factor viii genes as a treatment for hemophilia a
US11661611B2 (en) 2017-11-09 2023-05-30 Sangamo Therapeutics, Inc. Genetic modification of cytokine inducible SH2-containing protein (CISH) gene
AU2019209293B2 (en) 2018-01-17 2023-07-27 Vertex Pharmaceuticals Incorporated DNA-PK inhibitors
BR112020014140A2 (pt) 2018-01-17 2020-12-01 Vertex Pharmaceuticals Incorporated inibidores de dna-pk
WO2019143677A1 (fr) 2018-01-17 2019-07-25 Vertex Pharmaceuticals Incorporated Composés de quinoxalinone, compositions, procédés et kits pour augmenter l'efficacité d'édition du génome
BR112020015488A2 (pt) 2018-02-08 2020-12-08 Sangamo Therapeutics, Inc. Nucleases alvo-específicas modificadas
BR112020020245A2 (pt) 2018-04-05 2021-04-06 Editas Medicine, Inc. Métodos de produzir células expressando um receptor recombinante e composições relacionadas
EP3773908A1 (fr) 2018-04-05 2021-02-17 Juno Therapeutics, Inc. Récepteurs de lymphocytes t et cellules modifiées les exprimant
RU2020136054A (ru) 2018-04-05 2022-05-06 Джуно Терапьютикс, Инк. Т-клетки, экспрессирующие рекомбинантный рецептор, соответствующие полинуклеотиды и способы
EP3781587A4 (fr) 2018-04-18 2022-03-30 Sangamo Therapeutics, Inc. Compositions de protéines à doigts de zinc pour la modulation de la huntingtine (htt)
US11690921B2 (en) 2018-05-18 2023-07-04 Sangamo Therapeutics, Inc. Delivery of target specific nucleases
CN108802357B (zh) * 2018-06-27 2021-03-30 中南民族大学 一种用于免疫印迹分析的生物发光探针及其应用
SG11202101801RA (en) 2018-08-23 2021-03-30 Sangamo Therapeutics Inc Engineered target specific base editors
CN113015741A (zh) 2018-09-18 2021-06-22 桑格摩生物治疗股份有限公司 程序性细胞死亡1(pd1)特异性核酸酶
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
US11453639B2 (en) 2019-01-11 2022-09-27 Acuitas Therapeutics, Inc. Lipids for lipid nanoparticle delivery of active agents
WO2020159445A1 (fr) 2019-01-31 2020-08-06 Agency For Science, Technology And Research Inhibiteur cnx/erp57 destiné à être utilisé dans le traitement ou la prévention du cancer
US11946040B2 (en) 2019-02-04 2024-04-02 The General Hospital Corporation Adenine DNA base editor variants with reduced off-target RNA editing
EP3826664A4 (fr) 2019-02-06 2022-10-26 Sangamo Therapeutics, Inc. Procédé de traitement des mucopolysaccharidoses de type i
EP3946384A1 (fr) 2019-04-02 2022-02-09 Sangamo Therapeutics, Inc. Procédés pour le traitement de béta-thalassémie
WO2020206248A1 (fr) 2019-04-03 2020-10-08 Precision Biosciences, Inc. Cellules immunitaires génétiquement modifiées comprenant un arnsh adapté au microarn (shrnamir)
US20220204994A1 (en) 2019-04-05 2022-06-30 Precision Biosciences, Inc. Methods of preparing populations of genetically-modified immune cells
EP3958871A1 (fr) 2019-04-23 2022-03-02 Sangamo Therapeutics, Inc. Modulateurs de l'expression du gène cadre de lecture ouvert 72 sur le chromosome 9 et leurs utilisations
KR20220016474A (ko) 2019-05-01 2022-02-09 주노 쎄러퓨티크스 인코퍼레이티드 변형된 ｃｄ247 유전자 자리로부터 키메라 수용체를 발현하는 세포, 관련 폴리뉴클레오타이드 및 방법
AU2020265741A1 (en) 2019-05-01 2021-11-25 Editas Medicine, Inc. Cells expressing a recombinant receptor from a modified TGFBR2 Locus, related polynucleotides and methods
SG11202112553WA (en) 2019-06-13 2021-12-30 Allogene Therapeutics Inc Anti-talen antibodies and uses thereof
EP4004216A1 (fr) 2019-07-25 2022-06-01 Precision BioSciences, Inc. Compositions et procédés d'empilement séquentiel de séquences d'acides nucléiques dans un locus génomique
WO2021035054A1 (fr) 2019-08-20 2021-02-25 Precision Biosciences, Inc. Schémas posologiques de lymphodéplétion pour immunothérapies 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
WO2021067864A1 (fr) 2019-10-02 2021-04-08 Sangamo Therapeutics, Inc. Facteurs de transcription de protéine de doigt de zinc pour le traitement d'une maladie à prion
KR20220071248A (ko) 2019-10-02 2022-05-31 상가모 테라퓨틱스, 인코포레이티드 알파-시누클레인 발현을 억제시키기 위한 아연 핑거 단백질 전사 인자
DK3812472T3 (da) 2019-10-21 2023-02-20 Univ Freiburg Albert Ludwigs Virkelig uvildig in vitro-undersøgelse til profilering af off-target-aktivitet af en eller flere målspecifikke programmerbare nukleaser i celler (abnoba-seq)
US20220411479A1 (en) 2019-10-30 2022-12-29 Precision Biosciences, Inc. Cd20 chimeric antigen receptors and methods of use for immunotherapy
TW202132566A (zh) 2019-11-01 2021-09-01 美商聖加莫治療股份有限公司 用於治療或預防溶體貯積病之鋅指核酸酶變異體
WO2021087358A1 (fr) 2019-11-01 2021-05-06 Sangamo Therapeutics, Inc. Variants de la recombinase gin
EP4069285A1 (fr) 2019-12-06 2022-10-12 Precision BioSciences, Inc. Méthodes d'immunothérapie anticancéreuse utilisant des régimes de lymphodéplétion et des lymphocytes car-t allogéniques cd19, cd20 ou bcma
CN115210251A (zh) 2020-01-22 2022-10-18 桑格摩生物治疗股份有限公司 用于阻遏tau表达的锌指蛋白转录因子
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
US20230263121A1 (en) 2020-03-31 2023-08-24 Elo Life Systems Modulation of endogenous mogroside pathway genes in watermelon and other cucurbits
JP2023525513A (ja) 2020-05-06 2023-06-16 セレクティス ソシエテ アノニム 細胞ゲノムにおける外因性配列の標的化挿入のための方法
JP2023525510A (ja) 2020-05-06 2023-06-16 セレクティス ソシエテ アノニム 治療用タンパク質の送達のために細胞を遺伝子改変する方法
WO2021231259A1 (fr) 2020-05-11 2021-11-18 Precision Biosciences, Inc. Vecteurs viraux autolimités codant pour des nucléases
WO2021231661A2 (fr) 2020-05-13 2021-11-18 Juno Therapeutics, Inc. Procédé de production de lots de cellules donneuses exprimant un récepteur recombinant
EP4171616A1 (fr) 2020-06-26 2023-05-03 Juno Therapeutics GmbH Lymphocytes t modifiés exprimant un récepteur recombiné, polynucléotides et procédés associés
JP2023535365A (ja) 2020-07-16 2023-08-17 アクイタス セラピューティクス インコーポレイテッド 脂質ナノ粒子に使用するためのカチオン性脂質
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
WO2022046760A2 (fr) 2020-08-25 2022-03-03 Kite Pharma, Inc. Lymphocytes t à fonctionnalité améliorée
AU2021350099A1 (en) 2020-09-25 2023-04-27 Sangamo Therapeutics, Inc. Zinc finger fusion proteins for nucleobase editing
US20240018203A1 (en) 2020-10-02 2024-01-18 Sangamo Therapeutics, Inc. Novel zinc finger protein transcription factors for repressing alpha-synuclein expression
US20230365995A1 (en) 2020-10-07 2023-11-16 Precision Biosciences, Inc. Lipid nanoparticle compositions
US20240060079A1 (en) 2020-10-23 2024-02-22 Elo Life Systems Methods for producing vanilla plants with improved flavor and agronomic production
CN116802203A (zh) 2020-11-04 2023-09-22 朱诺治疗学股份有限公司 从经修饰的恒定cd3免疫球蛋白超家族链基因座表达嵌合受体的细胞、相关多核苷酸和方法
CA3200855A1 (fr) 2020-11-16 2022-05-19 Pig Improvement Company Uk Limited Animaux resistants a la grippe a ayant des genes anp32 modifies
WO2022165111A1 (fr) 2021-01-28 2022-08-04 Precision Biosciences, Inc. Modulation de la signalisation du tgf bêta dans des cellules eucaryotes génétiquement modifiées
WO2022226316A1 (fr) 2021-04-22 2022-10-27 Precision Biosciences, Inc. Compositions et procédés pour générer des plantes mâles stériles
WO2023064872A1 (fr) 2021-10-14 2023-04-20 Precision Biosciences, Inc. Associations de lymphocytes t car anti-bcma et d'inhibiteurs de gamma secrétase
WO2023070003A1 (fr) 2021-10-19 2023-04-27 Precision Biosciences, Inc. Procédés d'édition de gène permettant de traiter une déficience en alpha-1 antitrypsine (aat)
WO2023081900A1 (fr) 2021-11-08 2023-05-11 Juno Therapeutics, Inc. Lymphocytes t modifiés exprimant un récepteur recombiné de lymphocytes t (tcr) et systèmes et procédés apparentés
WO2023086790A1 (fr) 2021-11-09 2023-05-19 Amgen Inc. Procédé de fabrication d'un conjugué anticorps-peptides
WO2023091910A1 (fr) 2021-11-16 2023-05-25 Precision Biosciences, Inc. Méthodes d'immunothérapie anticancéreuse
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
WO2023122722A1 (fr) 2021-12-22 2023-06-29 Sangamo Therapeutics, Inc. Nouvelles protéines de fusion à doigt de zinc pour l'édition de nucléobases
WO2023131616A1 (fr) 2022-01-05 2023-07-13 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
WO2023168397A1 (fr) 2022-03-04 2023-09-07 Sigma-Aldrich Co. Llc Sélection métabolique par l'intermédiaire de la voie de biosynthèse de l'asparagine
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
WO2024073692A1 (fr) 2022-09-30 2024-04-04 Sigma-Aldrich Co. Llc Sélection métabolique par l'intermédiaire de la voie de biosynthèse de glycine-formate
WO2024073686A1 (fr) 2022-09-30 2024-04-04 Sigma-Aldrich Co. Llc Sélection métabolique par l'intermédiaire de la voie de biosynthèse de la sérine

Citations (58)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4990607A (en) 1989-03-14 1991-02-05 The Rockefeller University Alteration of gene expression in plants
WO1992002536A1 (fr) 1990-08-02 1992-02-20 The Regents Of The University Of Colorado Evolution polypeptidique systematique par traduction inverse
US5096814A (en) 1984-03-23 1992-03-17 Kernforschungsanlage Juelich Gmbh Macroporous and microporous inorganic carrier for immobilization of cells
US5096815A (en) 1989-01-06 1992-03-17 Protein Engineering Corporation Generation and selection of novel dna-binding proteins and polypeptides
US5198346A (en) 1989-01-06 1993-03-30 Protein Engineering Corp. Generation and selection of novel DNA-binding proteins and polypeptides
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5243041A (en) 1991-08-22 1993-09-07 Fernandez Pol Jose A DNA vector with isolated CDNA gene encoding metallopanstimulin
US5302519A (en) 1991-09-09 1994-04-12 Fred Hutchinson Cancer Research Center Method of producing a Mad polypeptide
US5324638A (en) 1992-05-13 1994-06-28 Sloan-Kettering Institute For Cancer Research Brain transcription factor, nucleic acids encoding same and uses thereof
US5324818A (en) 1991-08-21 1994-06-28 The Regents Of The University Of Michigan Proteins useful in the regulation of κB-containing genes
US5324819A (en) 1988-04-08 1994-06-28 Stryker Corporation Osteogenic proteins
US5340739A (en) 1988-07-13 1994-08-23 Brigham & Women's Hospital Hematopoietic cell specific transcriptional regulatory elements of serglycin and uses thereof
US5348864A (en) 1991-01-25 1994-09-20 E. R. Squibb & Sons, Inc. Mouse vav proto-oncogene DNA and protein sequences
US5350840A (en) 1989-11-13 1994-09-27 Massachusetts Institute Of Technology Localization and characterization of the Wilms' tumor gene
US5356802A (en) 1992-04-03 1994-10-18 The Johns Hopkins University Functional domains in flavobacterium okeanokoites (FokI) restriction endonuclease
US5376530A (en) 1987-12-16 1994-12-27 Institut Pasteur Steroid/thyroid hormone receptor-related gene, which is inappropriately expressed in human heptocellular carcinoma, and which is a retinoic acid receptor
WO1995011922A1 (fr) 1993-10-29 1995-05-04 Affymax Technologies N.V. Banques de presentation de peptides et d'anticorps in vitro
WO1995019431A1 (fr) 1994-01-18 1995-07-20 The Scripps Research Institute Derives de proteine a doigts zinciques et procedes associes
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
WO1996006166A1 (fr) 1994-08-20 1996-02-29 Medical Research Council Ameliorations concernant des proteines de liaison permettant de reconnaitre l'adn
WO1996006110A1 (fr) 1994-08-18 1996-02-29 Gilman, Michael, Z. Proteines composites se fixant a de l'adn et substances et procedes concernant ces proteines
US5498530A (en) 1991-10-16 1996-03-12 Affymax Technologies, N.V. Peptide library and screening method
WO1996011267A1 (fr) 1994-10-07 1996-04-18 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Adn et proteine a doigts de zinc et leur utilisation
WO1996020951A1 (fr) 1994-12-29 1996-07-11 Massachusetts Institute Of Technology Proteines chimeres de liaison d'adn
WO1996032475A2 (fr) 1995-04-12 1996-10-17 University Of Washington Procedes de preparation de proteines de liaison de l'adn
US5578483A (en) 1988-12-23 1996-11-26 The Salk Institute For Biological Studies Receptor transcription-repression activity compositions and methods
US5597693A (en) 1989-03-17 1997-01-28 The Salk Institute For Biological Studies Hormone response element compositions and assay
US5639592A (en) 1990-09-21 1997-06-17 The Salk Institute For Biological Studies Functional antagonism between proto-oncoprotein c-Jun and hormone receptors
WO1997027212A1 (fr) 1996-01-23 1997-07-31 Rigel Pharmaceuticals, Inc. Methodes de criblage de peptides effecteurs intracellulaires transdominants et de molecules d'arn
US5674738A (en) 1986-08-22 1997-10-07 Roche Molecular Systems, Inc. DNA encoding thermostable nucleic acid polymerase enzyme from thermus species Z05
US5686574A (en) * 1992-02-26 1997-11-11 The General Hospital Corporation Constitutive activator of retinoid acid response (car) receptor fusion protien
US5702914A (en) 1994-12-21 1997-12-30 The Salk Institute For Biological Studies Use of reporter genes for retinoid receptor screening assays having novel retinoid-associated response elements
US5789538A (en) 1995-02-03 1998-08-04 Massachusetts Institute Of Technology Zinc finger proteins with high affinity new DNA binding specificities
US5792640A (en) 1992-04-03 1998-08-11 The Johns Hopkins University General method to clone hybrid restriction endonucleases using lig gene
US5830707A (en) * 1994-05-23 1998-11-03 The Salk Institute For Biological Studies Method for site-specific integration of nucleic acids and related products
US5830721A (en) 1994-02-17 1998-11-03 Affymax Technologies N.V. DNA mutagenesis by random fragmentation and reassembly
EP0875567A2 (fr) 1997-04-30 1998-11-04 Basf Aktiengesellschaft Protéines à doigts de zinc se liant à l'oncogène mycm ainsi comment leur préparation et utilisation
WO1998053058A1 (fr) 1997-05-23 1998-11-26 Gendaq Limited Proteines de liaison d'acide nucleique
WO1998053059A1 (fr) 1997-05-23 1998-11-26 Medical Research Council Proteines de liaison d'acide nucleique
WO1998054311A1 (fr) 1997-05-27 1998-12-03 The Scripps Research Institute Derives de proteines a doigts de zinc et procedes associes
US5869618A (en) 1990-05-25 1999-02-09 Lippman; Marc E. Antibodies to ligand growth factors
US5871907A (en) 1991-05-15 1999-02-16 Medical Research Council Methods for producing members of specific binding pairs
US5871902A (en) 1994-12-09 1999-02-16 The Gene Pool, Inc. Sequence-specific detection of nucleic acid hybrids using a DNA-binding molecule or assembly capable of discriminating perfect hybrids from non-perfect hybrids
US5916794A (en) 1992-04-03 1999-06-29 Johns Hopkins University Methods for inactivating target DNA and for detecting conformational change in a nucleic acid
WO1999036553A2 (fr) 1998-01-15 1999-07-22 Ariad Gene Therapeutics, Inc. Regulation de phenomenes biologiques au moyen de proteines chimeres multimeres
US5939538A (en) 1996-10-25 1999-08-17 Immusol Incorporated Methods and compositions for inhibiting HIV infection of cells by cleaving HIV co-receptor RNA
WO1999041371A1 (fr) 1998-02-13 1999-08-19 Genetrace Systems, Inc. Utilisation de ribozymes afin d'identifier la fonction de genes
WO1999042474A2 (fr) 1998-02-20 1999-08-26 Genome Dynamics, Inc. Procédé permettant la conception de protéines à doigts de zinc se liant à l'adn
WO1999045132A1 (fr) 1998-03-02 1999-09-10 Massachusetts Institute Of Technology Proteines a poly-doigts de zinc a sequences de liaison ameliorees
WO1999047656A2 (fr) 1998-03-17 1999-09-23 Gendaq Limited Proteines de fixation a l'acide nucleique
WO1999048909A2 (fr) 1998-01-30 1999-09-30 Greisman Harvey A Strategie generale permettant de selectionner des proteines a doigts de zinc a haute affinite pour divers sites cibles de l'adn
US5972615A (en) 1998-01-21 1999-10-26 Urocor, Inc. Biomarkers and targets for diagnosis, prognosis and management of prostate disease
US6001885A (en) 1996-09-02 1999-12-14 Centre International De Recherches Dermatologiques Retinoid inhibition of expression of VEGF
WO2000023464A2 (fr) 1998-10-16 2000-04-27 Novartis Ag Domaines de liaison des doigts de zinc pour gnn
WO2000027878A1 (fr) 1998-11-09 2000-05-18 Gendaq Limited Systeme de criblage de polypeptides a motifs en doigt de zinc, destine a mettre en evidence une certaine capacite de liaison
WO2000042219A1 (fr) 1999-01-12 2000-07-20 Sangamo Biosciences, Inc. Selection de sites cibles pour proteines a doigts de zinc et methodes de fabrication de proteines a doigts de zinc destinees a se lier avec des sites preselectionnes
WO2000041566A1 (fr) 1999-01-12 2000-07-20 Sanagamo Biosciences, Inc. Regulation de l'expression genique endogene dans des cellules au moyen de proteines a doigts de zinc

Patent Citations (66)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5096814A (en) 1984-03-23 1992-03-17 Kernforschungsanlage Juelich Gmbh Macroporous and microporous inorganic carrier for immobilization of cells
US5674738A (en) 1986-08-22 1997-10-07 Roche Molecular Systems, Inc. DNA encoding thermostable nucleic acid polymerase enzyme from thermus species Z05
US5376530A (en) 1987-12-16 1994-12-27 Institut Pasteur Steroid/thyroid hormone receptor-related gene, which is inappropriately expressed in human heptocellular carcinoma, and which is a retinoic acid receptor
US5324819A (en) 1988-04-08 1994-06-28 Stryker Corporation Osteogenic proteins
US5340739A (en) 1988-07-13 1994-08-23 Brigham & Women's Hospital Hematopoietic cell specific transcriptional regulatory elements of serglycin and uses thereof
US5403484A (en) 1988-09-02 1995-04-04 Protein Engineering Corporation Viruses expressing chimeric binding proteins
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5578483A (en) 1988-12-23 1996-11-26 The Salk Institute For Biological Studies Receptor transcription-repression activity compositions and methods
US5198346A (en) 1989-01-06 1993-03-30 Protein Engineering Corp. Generation and selection of novel DNA-binding proteins and polypeptides
US5096815A (en) 1989-01-06 1992-03-17 Protein Engineering Corporation Generation and selection of novel dna-binding proteins and polypeptides
US4990607A (en) 1989-03-14 1991-02-05 The Rockefeller University Alteration of gene expression in plants
US5597693A (en) 1989-03-17 1997-01-28 The Salk Institute For Biological Studies Hormone response element compositions and assay
US5350840A (en) 1989-11-13 1994-09-27 Massachusetts Institute Of Technology Localization and characterization of the Wilms' tumor gene
US5869618A (en) 1990-05-25 1999-02-09 Lippman; Marc E. Antibodies to ligand growth factors
WO1992002536A1 (fr) 1990-08-02 1992-02-20 The Regents Of The University Of Colorado Evolution polypeptidique systematique par traduction inverse
US5639592A (en) 1990-09-21 1997-06-17 The Salk Institute For Biological Studies Functional antagonism between proto-oncoprotein c-Jun and hormone receptors
US5348864A (en) 1991-01-25 1994-09-20 E. R. Squibb & Sons, Inc. Mouse vav proto-oncogene DNA and protein sequences
US5871907A (en) 1991-05-15 1999-02-16 Medical Research Council Methods for producing members of specific binding pairs
US5324818A (en) 1991-08-21 1994-06-28 The Regents Of The University Of Michigan Proteins useful in the regulation of κB-containing genes
US5243041A (en) 1991-08-22 1993-09-07 Fernandez Pol Jose A DNA vector with isolated CDNA gene encoding metallopanstimulin
US5302519A (en) 1991-09-09 1994-04-12 Fred Hutchinson Cancer Research Center Method of producing a Mad polypeptide
US5498530A (en) 1991-10-16 1996-03-12 Affymax Technologies, N.V. Peptide library and screening method
US5686574A (en) * 1992-02-26 1997-11-11 The General Hospital Corporation Constitutive activator of retinoid acid response (car) receptor fusion protien
US5792640A (en) 1992-04-03 1998-08-11 The Johns Hopkins University General method to clone hybrid restriction endonucleases using lig gene
US5916794A (en) 1992-04-03 1999-06-29 Johns Hopkins University Methods for inactivating target DNA and for detecting conformational change in a nucleic acid
US5356802A (en) 1992-04-03 1994-10-18 The Johns Hopkins University Functional domains in flavobacterium okeanokoites (FokI) restriction endonuclease
US5487994A (en) 1992-04-03 1996-01-30 The Johns Hopkins University Insertion and deletion mutants of FokI restriction endonuclease
US5436150A (en) 1992-04-03 1995-07-25 The Johns Hopkins University Functional domains in flavobacterium okeanokoities (foki) restriction endonuclease
US5324638A (en) 1992-05-13 1994-06-28 Sloan-Kettering Institute For Cancer Research Brain transcription factor, nucleic acids encoding same and uses thereof
WO1995011922A1 (fr) 1993-10-29 1995-05-04 Affymax Technologies N.V. Banques de presentation de peptides et d'anticorps in vitro
WO1995019431A1 (fr) 1994-01-18 1995-07-20 The Scripps Research Institute Derives de proteine a doigts zinciques et procedes associes
US5830721A (en) 1994-02-17 1998-11-03 Affymax Technologies N.V. DNA mutagenesis by random fragmentation and reassembly
US5830707A (en) * 1994-05-23 1998-11-03 The Salk Institute For Biological Studies Method for site-specific integration of nucleic acids and related products
WO1996006110A1 (fr) 1994-08-18 1996-02-29 Gilman, Michael, Z. Proteines composites se fixant a de l'adn et substances et procedes concernant ces proteines
US6013453A (en) 1994-08-20 2000-01-11 Medical Research Council Binding proteins for recognition of DNA
US6007988A (en) 1994-08-20 1999-12-28 Medical Research Council Binding proteins for recognition of DNA
WO1996006166A1 (fr) 1994-08-20 1996-02-29 Medical Research Council Ameliorations concernant des proteines de liaison permettant de reconnaitre l'adn
WO1996011267A1 (fr) 1994-10-07 1996-04-18 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Adn et proteine a doigts de zinc et leur utilisation
US5871902A (en) 1994-12-09 1999-02-16 The Gene Pool, Inc. Sequence-specific detection of nucleic acid hybrids using a DNA-binding molecule or assembly capable of discriminating perfect hybrids from non-perfect hybrids
US5702914A (en) 1994-12-21 1997-12-30 The Salk Institute For Biological Studies Use of reporter genes for retinoid receptor screening assays having novel retinoid-associated response elements
WO1996020951A1 (fr) 1994-12-29 1996-07-11 Massachusetts Institute Of Technology Proteines chimeres de liaison d'adn
US5789538A (en) 1995-02-03 1998-08-04 Massachusetts Institute Of Technology Zinc finger proteins with high affinity new DNA binding specificities
WO1996032475A2 (fr) 1995-04-12 1996-10-17 University Of Washington Procedes de preparation de proteines de liaison de l'adn
WO1997027213A1 (fr) 1996-01-23 1997-07-31 The Board Of Trustees Of The Leland Stanford Junior University Procedes servant a rechercher par criblage des peptides effecteurs et des molecules d'arn transdominants
WO1997027212A1 (fr) 1996-01-23 1997-07-31 Rigel Pharmaceuticals, Inc. Methodes de criblage de peptides effecteurs intracellulaires transdominants et de molecules d'arn
US6001885A (en) 1996-09-02 1999-12-14 Centre International De Recherches Dermatologiques Retinoid inhibition of expression of VEGF
US5939538A (en) 1996-10-25 1999-08-17 Immusol Incorporated Methods and compositions for inhibiting HIV infection of cells by cleaving HIV co-receptor RNA
US6160091A (en) 1997-04-30 2000-12-12 Prolifix Limited Myc-binding zinc finger proteins, their preparation and their use
EP0875567A2 (fr) 1997-04-30 1998-11-04 Basf Aktiengesellschaft Protéines à doigts de zinc se liant à l'oncogène mycm ainsi comment leur préparation et utilisation
WO1998053059A1 (fr) 1997-05-23 1998-11-26 Medical Research Council Proteines de liaison d'acide nucleique
WO1998053058A1 (fr) 1997-05-23 1998-11-26 Gendaq Limited Proteines de liaison d'acide nucleique
WO1998053057A1 (fr) 1997-05-23 1998-11-26 Gendaq Limited Bibliotheque de polypeptides de fixation d'acide nucleique
WO1998053060A1 (fr) 1997-05-23 1998-11-26 Gendaq Limited Proteines de liaison d'acide nucleique
WO1998054311A1 (fr) 1997-05-27 1998-12-03 The Scripps Research Institute Derives de proteines a doigts de zinc et procedes associes
WO1999036553A2 (fr) 1998-01-15 1999-07-22 Ariad Gene Therapeutics, Inc. Regulation de phenomenes biologiques au moyen de proteines chimeres multimeres
US5972615A (en) 1998-01-21 1999-10-26 Urocor, Inc. Biomarkers and targets for diagnosis, prognosis and management of prostate disease
WO1999048909A2 (fr) 1998-01-30 1999-09-30 Greisman Harvey A Strategie generale permettant de selectionner des proteines a doigts de zinc a haute affinite pour divers sites cibles de l'adn
WO1999041371A1 (fr) 1998-02-13 1999-08-19 Genetrace Systems, Inc. Utilisation de ribozymes afin d'identifier la fonction de genes
WO1999042474A2 (fr) 1998-02-20 1999-08-26 Genome Dynamics, Inc. Procédé permettant la conception de protéines à doigts de zinc se liant à l'adn
WO1999045132A1 (fr) 1998-03-02 1999-09-10 Massachusetts Institute Of Technology Proteines a poly-doigts de zinc a sequences de liaison ameliorees
WO1999047656A2 (fr) 1998-03-17 1999-09-23 Gendaq Limited Proteines de fixation a l'acide nucleique
WO2000023464A2 (fr) 1998-10-16 2000-04-27 Novartis Ag Domaines de liaison des doigts de zinc pour gnn
WO2000027878A1 (fr) 1998-11-09 2000-05-18 Gendaq Limited Systeme de criblage de polypeptides a motifs en doigt de zinc, destine a mettre en evidence une certaine capacite de liaison
WO2000042219A1 (fr) 1999-01-12 2000-07-20 Sangamo Biosciences, Inc. Selection de sites cibles pour proteines a doigts de zinc et methodes de fabrication de proteines a doigts de zinc destinees a se lier avec des sites preselectionnes
WO2000041566A1 (fr) 1999-01-12 2000-07-20 Sanagamo Biosciences, Inc. Regulation de l'expression genique endogene dans des cellules au moyen de proteines a doigts de zinc
US6534261B1 (en) 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins

Non-Patent Citations (178)

* Cited by examiner, † Cited by third party
Title
Agarwal et al., "Stimulation of Transcript Elongation Requires Both the Zinc Finger and RNA Polymerase II Binding Domains of Human TFIS," Biochemistry 30(31):7842-7851 (1991).
Antao et al., "A Thermodynamic Study of Unusually Stable RNA and DNA Hairpins," Nuc. Acids. Res. 19(21):5901-5905 (1991).
Barbas et al., "Assembly of Combinatorial Antibody Libraries on Phage Surfaces: The Gene III Site," PNAS 88:7978-7982 (1991).
Barbas et al., "Semisynthetic Combinatorial Antibody Libraries: A Chemical Solution to the Diversity Problem," PNAS 89:4457-4461 (1992).
Barbas, C. F., "Recent Advances in Phage Display," Curr. Opin. Biotech. 4:526-530 (1993).
Beerli et al., "Positive and negative regulation of endogenous genes by designed transcription factors," Proc. Natl. Acad. Sci. U.S.A. 97:1495-1500 (2000).
Beerli et al., "Toward Controlling Gene Expression at Will: Specific Regulation of the erbB-2/HER-2 Promoter by Using Polydactyl Zinc Finger Proteins Constructed From Modular Building Blocks," Proc. Natl. Acad. Sci. U.S.A. 95:14628-14633 (1998).
Bellefroid et al., "Clustered Organization of Homologous KRAB Zinc-Finger Genes With Enhanced Expression in Human T Lymphoid Cells," EMBO J. 12(4):1363-1374 (1993).
Berg et al., "The Galvanization of Biology: A Growing Appreciation for the Roles of Zinc," Science 271:1081-1085 (1996).
Berg, J.M., "DNA Binding Specificity of Steroid Receptors," Cell 57:1065-1068 (1989).
Berg, J.M., "Letting Your Fingers do the Walking," Nature Biotechnology 15:323 (1997).
Berg, J.M., "Sp1 and the Subfamily of Zinc-Finger Proteins with Guanine-Rich Binding Sites," PNAS 89:11109-11110 (1992).
Bergqvist et al., "Loss of DNA-binding and new Transcriptional Trans-Activation Function in Polyomavirus Large T-Antigen with Mutation of Zinc Finger Motif," Nuc. Acids Res. 18(9):2715-2720 (1990).
Blaese et al. Vectors in cancer therapy: how will they deliver? Cancer Gene Therapy vol. 2(4):291-297, Oct. 1995.
Bonner, J.J. Vectors for the expression and analysis of DNA-binding proteins in yeast. Gene, vol. 104, pp. 113-118, 1991. *
Caponigro et al., "Transdominant Genetic Analysis of a Growth Control Pathway," PNAS 95:7508-7513 (1998).
Celenza et al., "A Yeast Gene That Is Essential for Release from Glucose Repression Encodes a Protein Kinase," Science 233:1175-1180 (1986).
Chavrier et al. A gene encoding a protein with zinc fingers is activated during G0/G1 transition in cultured cells. The EMBO Journal. vol. 7, No. 1, pp. 29-35, 1998. *
Chen et al., "PLZF-RARPlzf-RARα Fusion Proteins Generated From the Variant t(11;17)(q23;q21) Translocation in Acute Promyelocytic Leukemia Inhibit Ligand-Dependent Transactivation of Wild-Type Retinoic Acid Receptors," PNAS USA 91:1178-1182 (1994).
Cheng et al., "A Single Amino Acid Substitution in Zinc Finger 2 of Adrlp Changes its Binding Specificity at two Positions in UAS1," .J. Mol. Biol. 251:1-8 (1995).
Cheng et al., "Identification of Potential Target Genes for Adrlp through Characterization of Essential Nucleotides in UAS1," Mol. Cellular Biol. 14(6):3842-3852 (1994).
Choo et al., "All Wrapped Up," Nature Struct Biol 5(4):253-255 (1998).
Choo et al., "Designing DNA-Binding Proteins on the Surface of Filamentous Phage," Curr. Opin. Biotechnology 6:431-436 (1995).
Choo et al., "Promoter-Specific Activation of Gene Expression Directed By Bacteriophage-Selected Zinc Fingers," J. Mol. Biol. 273:525-532 (1997).
Choo et al., A Role in DNA-Binding for the Linker Sequences of the First Three Zinc Fingers of TFIIIA Nuc. Acids Res. 21(15):3341-3346 (1993).
Choo et al., Physical Basis of Protein-DNA Recognition Code, Curr. Opin. Struct. Biol. 7(1):117-125 (1997).
Choo et al: "In Vivo repression by a site-specific DNA-binding protein designed against an oncogenic sequence", Nature, vol. 372, Dec. 15, 1994, pp. 642-645.
Choo et al: "Selection of DNA binding sites for zinc fingers using rationally randomized DNA reveals coded interactions", Proc.Natl.Acad.Sci.USA, vol. 91, Nov. 1994, Biochemistry, pp. 11168-11172.
Choo et al: "Toward a code for the interactions of zinc fingers with DNA: Selection of randomized fingers displayed on phage", Proc.Natl.Acad.Sci.USA, vol. 91, Nov. 1994, Biochemistry, pp. 11163-11167.
Choo, Y., "End Effects in DNA Recognition Code," Nuc. Acids. Res. 26(2):554-557 (1998).
Choo, Y., "Recognition of DNA Methylation by Zinc Fingers," Nature Struct Biol. 5(4):264-265 (1998).
Christy et al., "A Gene Activated in Mouse 3T3 Cells by Serum Growth Factors Encodes A Protein With "Zinc Finger" Sequences," PNAS USA 85:7857-7861 (1988).
Clarke et al., "Zinc Fingers in Caenorhabditis elegans: Finding Families and Probing Pathways," Science 282:2018-2022 (1998).
Corbi et al., "Synthesis of a New Zinc Finger Peptide: Comparison of Its "Code" Deduced and "CASTing" Derived Binding Sites," FEBS Letters 417:71-74 (1997).
Courey et al. Synergistic activation by the glutamine-rich domains of human stranscription factor SP1. Cell, vol. 59, pp. 827-836, Dec. 1989. *
Crozatier et al., "Single Amino Acid Exchanges in Separate Domains of the Drosophila Serendipity Zinc Finger Protein Cause Embroyonic and Sex Biased Lethality," Genetics 131:905-916 (1992).
Debs et al., "Regulation of Gene Expression in Vivo by Liposome-Mediated Delivery of a Purified Transcription Factor," J. Biological Chemistry 265(18):10189-10192 (1990).
DesJardins et al., "Repeated CT Elements Bound by Zinc Finger Proteins Control the Absolute and Relative Activities of the Two Principal Human C-myc Promoters," Mol. Cell. Biol. 13(9):5710-5724 (1993).
Desjarlais et al., "Length-Encoded Multiplex binding Site Determination: Application to Zinc Finger Proteins," PNAS 91:11099-11103 (1994).
Desjarlais et al., "Redesigning the DNA-Binding Specificity of a Zinc Finger Protein: A Data Base-Guided Approach," Proteins: Structure, Function, and Genetics 12(2):101-104 (1992).
Desjarlais et al., "Redesigning the DNA-Binding Specificity of a Zinc Finger Protein: A Data Base-Guided Approach," Proteins: Structure, Function, and Genetics 3(3):272 (1992).
Desjarlais et al., "Use of a Zinc-Finger Consensus Sequence Framework and Specificity Rules to Design Specific DNA Binding, Proteins," PNAS 90:2256-2260 (1993).
Desjarlais et al: "Toward rules relating zinc finger protein sequences and DNA binding site preferences" Proc.Natl.Acad.Sci.USA, vol. 89, Aug. 1992, Biophysics, pp. 7345-7349.
Dibello et al., "The Drosophila Broad-Complex Encodes a Family of Related Proteins Containing Zinc Fingers," Genetics 129:385-397 (1991).
Donze et al., "Activation of delta-globin gene expression by erythroid Krupple-like factor: a potential approach for gene therapy of sickle cell disease," Blood 88:4051-4057 (1996).
Elrod-Erickson at al., "High-Resolution Structures of Variant Zif268-DNA Complexes: Implications for Understanding Zinc Finger-DNA Recognition," Structure 6(4):451-464 (1998).
Elrod-Erickson et al., "Zif268 Protein-DNA Complex Refined at 1.6: a Model System for Understanding Zinc Finger-DNA Interactions," Structure 4(10):1171-1180 (1996).
Fairall et al. Sequence-specific DNA binding by a two zinc-finger peptide from the Drosophila melanogaster Tramtrack protein. Journal of Molecular Biology, vol. 226, No. 2, pp. 349-366, 1992. *
Fairall et al., "The Crystal Structure of a Two Zinc-Finger Peptide Reveals an Extension to the Rules for Zinc-Finger /DNA Recognition," Nature 366:483-487 (1993).
Frankel et al., "Fingering Too Many Proteins," Cell 53:675 (1988).
Friesen et al., "Phage Display of RNA Binding Zinc Fingers from Transcription Factor IIA," J. Biological Chem. 272(17):10994-10997 (1997).
Friesen et al., "Specific RNA Binding Proteins Constructed from Zinc Fingers," Nature Structural Biology 5(7):543-546 (1998).
Gamsjaeger, et al., "Sticky Fingers: Zinc-Fingers As Protein-Recognition Motifs," Trends in Biochemical Sciences 32:63-70 (2007).
Gashler, et al., "A Novel Repression Module, and Extensive Activation Domain, and a Bipartite Nuclear Localization Signal Defined in the Immediate-Early Transcription Factor EGR-1," Molecular and Cellular Biology 13(8):4556-4571 (1993).
GenBank Accession No. AAC34714.1, Sep. 8, 1998. *
Ghosh "A relational database of transcription factors," Nucleic Acids Res 18:1749-1756 (1990).
Gillemans et al., "Altered DNA Binding Specificity Mutants of EKLF and Spl Show that EKLF is an Activator of the b-Globin Locus Control Region in vivo," Genes and Development 12:2863-2873 (1998).
Gogos et al., "Recognition of Diverse Sequences by Class I Zinc Fingers: Asymmetries and Indirect Effects on Specificity in the Interaction Between CF2II and A+T-Rich Sequences Elements," PNAS 93(5):2159-2164 (1996).
Gossen et al., "Tight Control of Gene Expression in Mammalian Cells by Tetracycline-Responsive Promoter," PNAS 89:5547-5551 (1992).
Greisman & Pabo, "A General Strategy for Selecting High-Affinity Zinc Finger Proteins for Diverse DNA Target Sites," Science 275:657-661 (1997).
Hall et al., "Functional Interaction Between the Two Zinc Finger Domains of the V-erbA Oncoprotein," Cell Growth & Differentiation 3:207-216 (1992).
Hamilton et al., "Comparison of the DNA Binding Characteristics of the Related Zinc Finger Proteins WT1 and EGR1," Biochemistry 37:2051-2058 (1998).
Hamilton et al., "High Affinity Binding Sites for the Wilms' Tumor Suppressor Protein WTl," Nuc. Acids. Res. 23(2):277-284 (1995).
Hanas et al., "Internal Deletion Mutants of Xenopus Transcription Factor IIIA," Nuc. Acids. Res. 17(23):9861-9870 (1989).
Hard et al, Science, Jul. 13, 1990, vol. 249, pp. 157-1160. *
Hayes et al., "Locations of Contacts Between Individual Zinc Fingers Xenopus laevis Transcription Factor IIIA and the Internal Control Region of a 5S RNA Gene," Biochemistry 31:11600-11605 (1992).
Heinzel et al., "A Complex containing N-CoR, MSin3 and Histone Deacetylese Medates Transcriptional Repression," Nature 387:43-48 (1997).
Hirst et al., "Discrimination of DNA Response Elements for Thyroid Hormone and Estrogen is Dependent on Dimerization of Receptor DNA Binding Domains," PNAS 89:5527-5531 (1992).
Hoffman et al., "Structures of DNA-Binding Mutant Zinc Finger Domains: Implications for DNA Binding," Protein Science 2:951-965 (1993).
Houbaviy et al. Cocrystal structure of YY1 bound to the adeno-associated virus P5 initiator. Proc Natl Acad Sci U S A. vol. 93, No. 24, pp. 13577-82, Nov. 1996. *
Hu et al. Targeting the Escherichia coli lac repressor to the mammalian cell nucleus. Gene, vol. 99, pp. 141-150, 1991. *
Imhof et al., "Transcriptional Regulation of the AP-2alpha Promoter by BTEB-1 and AP-2REP, a Novel WT-1/EGR-Related Zinc Finger Repressor," Molecular and Cellular Biology 19(1):194-204 (1999).
Inouye et al. Isolation of a cDNA Encoding a Metal Response Element Binding Protein Using a Novel Expression Cloning Procedure: The One Hybrid System. DNA and Cell Biology, vol. 13, No. 7, pp. 731-742, Jul. 1994. *
Isalan et al., "Comprehensive DNA Recognition Through Concerted Interactions from Adjacent Zinc Fingers," Biochemistry 37:12026-12033 (1998).
Isalan et al., "Synergy Between Adjacent Zinc Fingers in Sequence-Specific DNA Recognition," PNAS 94(11):5617-5621 (1997).
Jacobs, G.H., "Determination of the Base Recognition Positions of Zinc Fingers From Sequence Analysis," EMBO J. 11(12):4507-4517 (1992).
Jamieson et al. "A Zinc Finger Directory for High-Affinity DNA Recognition," PNAS 93:12834-12839 (1996).
Jamieson et al., "In Vitro Selection of Zinc Fingers with Altered DNA-Binding Specificity" Biochemistry 33:5689-5695 (1994).
Jones et al., "Replacing the complementarity-determining regions in a human antibody with those from a mouse" Nature 321:522-525 (1986).
Julian et al., "Replacement of His23 by Cys in a Zinc Finger of HIV-1NCp7 Led to a Change in 1HNMR-Derived 3D Structure and to a Loss of Biological Activity," FEBS Letters 331(1,2):43-48 (1993).
Kamiuchi et al., "New Multi Zinc Finger Protein: Biosynthetic Design and Characteristics of DNA Recognition," Nucleic Acids Symposium Series 37:153-154 (1997).
Kang et al., "Zinc Finger Proteins as Designer Transcription Factors," J. Biol. Chem. 275(12):8742-8748 (2000).
Kim et al. "Getting a handhold on DNA: design of poly-zinc finger proteins with femtomolar dissociation constants" Proc. Natl. Acad. Sci. USA 95:2812-2817 (1998).
Kim et al. "Transcriptional repression by zinc finger peptides. Exploring the potential for applications in gene herapy" J. Biol. Chem. 272:29795-29800 (1997).
Kim et al., "A 2.2 A° Resolution Crystal Structure of a Designed Zinc Finger Protein Bound to DNA," Nat. Struct. Biol. 3(11):940-945 (1996).
Kim et al., "Design of TATA Box-Binding Protein/Zinc Finger Fusions for Targeted Regulation of Gene Expression," PNAS 94:3616-3620 (1997).
Kim et al., "Hybrid Restriction Enzymes: Zinc Finger Fusions Fok l Cleavage Domain," PNAS 93:1156-1160 (1996).
Kim et al., "Serine at Position 2 in the DNA Recognition Helix of a Cys2-His2 Zinc Finger Peptide is Not, in General, Responsible for Base Recognition." J. Mol. Biol. 252:1-5 (1995).
Kim et al., "Site-Specific Cleavage of DNA-RNA Hybrids by Zinc Finger/Fokl Cleavage Domain Fusions," Gene 203:4349 (1997).
Kinzler et al., "The GLI Gene is Member of the Kruppel Family of Zinc Finger Proteins," Nature 332:371-374 (1988).
Klug et al., "Protein Motifs 5: Zinc Fingers," FASEB J. 9:597-604 (1995).
Klug et al: "Zinc Fingers", The FASEB Journal, May 1995, vol. 9, No. 8, pp. 597-604.
Klug, "Zinc Finger Peptides for the Regulation of Gene Expression," J. Mol. Biol. 293:215-218 (1999).
Klug, A., "Gene Regulatory Proteins and Their Interaction with DNA," Ann. NY Acad. Sci. 758:143-160 (1995).
Kothekar, "Computer Simulation of Zinc Finger Motif from Cellular Nucleic Acid Binding Proteins and Their Interaction with Consensus DNA Sequences," FEBS Letters 274(1,2):217-222 (1990).
Kriwacki et al. "Sequence-specific recognition of DNA by zinc finger peptides derived from the transcription factor Sp-1," Proc. Natl. Acad. Sci. USA 89:9759-9763 (1992).
Krizek et al. A consensus zinc finger peptide: Design, high-affinity metal binding, a pH-dependent structure, and a His to Cys sequence variant. Journal of the American Chemical Society, vol. 113, pp. 4518-1523, 1991. *
Krizek et al. A consensus zinc finger peptide: Design, high-affinity metal binding, a pH-dependent structure, and a His to Cys sequence variant. Journal of the American Chemical Society, vol. 113, pp. 4518-4523, 1991. *
Kudla et al., "The Regulatory Gene areA Mediating Nitrogen Metabolite R in Aspergillus nidulans Mutations Affecting Specificity of Gene Activation Alter a Loop Residue of Putative Zinc Finger," EMBO J. 9(5):1355-1364 (1990).
Laird-Offringa et al., "RNA-Binding Proteins Tamed," Nat. Structural Biol. 5(8):665-668 (1998).
Lee et al. Evidence for physical interaction between the zinc-finger transcription factors YY1 and Sp1. Proc Natl Acad Sci U S A., vol. 90, No. 13, pp. 6145-9, Jul. 1, 1993. *
Lee et al. The zinc finger motif: Conservation of chemical shifts and correlation with structure. FEBS, vol. 309, No. 1, pp. 29-32, Aug. 1992. *
Liu et al., "Design of Polydactyl Zinc-Finger Proteins for Unique Addressing Within Complex Genomes," Proc. Natl. Acad. Sci. U.S.A. 94:5525-5530 (1997).
Liu et al., "Regulation of an Endogenous Locus Using a Panel of Designed Zinc Finger Proteins Targeted to Accessible Chromatin Regions: Activation of Vascular Endothelial Growth Factor A," Journal of Biological Chemistry 276(14):11323-11334 (2001).
Liu et al., "Transcription Factor EGR-1 Suppresses the Growth and Transformation of Human HT-1080 Fibrosarcoma Cells by Induction of Transforming Growth Factor Beta 1," Proc. Natl. Acad. Sci. U.S.A. 93(21):11831-11836 (1996).
Mackay, et al., "Zinc Fingers Are Sticking Together," TIBS 23:1-4 (1998).
Mandel-Guffreund et al., "Quantitative Parameters for Amino Acid-Base Interaction: Implication for Predication of Protein-DNA Binding Sites," Nuc. Acids Res. 26(10):2306-2312 (1998).
Margolin et al., "Kruppel-Associated Boxes are Potent Transcriptional Repression Domains," PNAS 91:4509-4513 (1994).
Michael et al. Metal binding and folding properties of a minimalist Cys2His2 zinc finger peptide. Proceedings of the National Academy of Sciences, USA, vol. 89, pp. 4796-4800 Jun. 1992. *
Mizushima et al., "pEF-BOS, a Powerful Mammilian Expression Vector," Nuc. Acids. Res. 18(17):5322 (1990).
Mukhopadhyay et al. "The von Hippel-lindau Tumor Suppressor Gene Product Interacts with Sp1 to Repress Vascular Endothelial Growth Factor Promoter Activity" Mol. Cell. Biol. 17(9):5629-5639 (1997).
Nakagama et al., "Sequence and Structural Requirements for High-Affinity DNA Binding by the WT1 Gene Product," Molecular and Cellular Biology 15(3):1489-1498 (1995).
Narayan et al. Structures of zinc finger domains from transcription factor Sp1. The Journal of Biological Chemistry, vol. 272, No. 12, pp. 7801-7809, Mar. 1997. *
Nardelli et al., "Base Sequence Discrimination by Zinc-Finger DNA-Binding Domians," Nature 349:175-178 (1991).
Nardelli et al., "Zinc Finger-DNA Recognition: Analysis of Base Specificity by Site-Directed Mutagenesis," Nucleic Acids Research 20(16):4137-4144 (1992).
Nekludova et al., "Distinctive DNA Conformation With Enlarged Major Groove is Found in Zn-Finger-DNA and Other Protein-DNA Complexes," PNAS 91:6948-6952 (1994).
Orkin et al., "Report and Recommendations of the Panel to Assess the NIH Investment in Research on Gene Therapy," (Dec. 7, 1995).
Pabo et al., "Protein-DNA Recognition," Ann. Rev. Biochem. 53:293-321 (1984).
Pabo et al., "Systematic Analysis of Possible Hydrogen Bonds between Amino Acid Side Chains and B-form DNA," J. Biomolecular Struct. Dynamic 1:1039-1049 (1983).
Pabo, C. O., "Transcription Factors: Structural Families and Principles of DNA Recognition," Ann. Rev. Biochem. 61:1053-1095 (1992).
Pascual et al. Structure of the PHD Zinc Finger from Human Williams-Beuren Syndrome Transcription Factor. J. Mol. Biol. vol. 304, pp. 723-729, 2000. *
Pavletich et al., "Crystal Structure of a Five-Finger GLI-DNA Complex: New Perspectives on Zinc Fingers," Science, 261:1701-1707 (1993).
Pavletich et al., "Zinc Finger-DNA Recognition: Crystal Structure of a Zif268-DNA Complex at 2.1 A," Science 252:809-817 (991).
Pengue et al., "Kruppel-Associated Box-Mediated Repression of RNA Polymerase II Promoters is Influenced by the Arrangement of Basal Promoter Elements," PNAS 93:1015-1020 (1996).
Pengue et al., "Repression of Transcriptional Activity at a Distance by the Evolutionarily Conserved KRAB Domain Present in a Subfamily of Zinc Finger Proteins," Nuc. Acids Res. 22(15):2908-2914 (1994).
Pengue et al., "Transcriptional Silencing of Human Immunodeficiency Virus Type I Long Terminal Repeat-Driven Gene Expression by the Kruppel-Associated Box Repressor Domain Targeted to the Transactivating Response Element," J. Virology 69(10):6577-6580 (1995).
Pomerantz et al., "Analysis of Homeodomain Function by Structure-Based Design of a Transcription Factor." PNAS 92:9752-9756 (1995).
Pomerantz et al., "Structure-Based Design of a Dimeric Zinc Finger Protein," Biochemistry 37(4):965-970 (1998).
Pomerantz et al., "Structure-Based Design of Transcription Factors," Science 267:93-96 (1995).
Ptashne. How eukaryotic transcriptional activators work. Nature, vol. 335, pp. 683-689, Oct. 1988. *
Qian et al., "Two-Dimensional NMR Studies of the Zinc Finger Motif: Solution Structures and Dynamics of Mutant ZFY Domains Containing Aromatic Substitutions in the Hydrophobic Core," Biochemistry 31:7463-7476 (1992).
Quigley et al., "Complete Androgen Insensitivity Due to Deletion of Exon C of the Androgen Receptor Gene Highlights the Functional Importance of the Second Zinc Finger of the Androgen Receptor in Vivo," Molecular Endocrinology 6(7):1103-1112 (1992).
Rauscher et al., "Binding of the Wilms' Tumor Locus Zinc Finger Protein to the EGR- I Consensus Sequence," Science 250:1259-1262 (1990).
Ray et al., "Repressor to Activator Switch by Mutations in the First Zn Finger of the Glucocorticoid Receptor: Is Direct DNA Binding Necessary?," PNAS 88:7086-7090 (1991).
Rebar et al., "Phage Display Methods for Selecting Zinc Finger Proteins with Novel DNA-Binding Specificities," Methods in Enzymology 267:129-149 (1996).
Rebar et al: "Zinc Finger Phage: Affinity Selection of Fingers with New DNA-Binding Specificites", Science, vol. 263, Feb. 4, 1994, pp. 671-673.
Reith et al., "Cloning of the Major Histocompatibility Complex Class II Promoter Binding Protein Affected in a Hereditary Defect in Class II Gene Regulation," PNAS 86:4200-4204 (1989).
Rhodes et al., "Zinc Fingers: They Play a Key Part in Regulating the Activity of Genes in Many Species, From Yeast to Humans. Fewer Than 10 Years Ago No One Knew They Existed." Scientific American 268:56-65 (1993).
Rice et al., "Inhibitors of HIV Nucleocapsid Protein Zinc Fingers as Candidates for the Treatment of AIDS." Science. 270:1194-1197 (1995).
Rivera et al., "A Humanized System for Pharmacologic Control of Gene Expression," Nature Medicine 7(9):1028-1032 (1996).
Rollins et al., "Role of TFIIIA Zinc Fingers In vivo: Analysis of Single-Finger Function in Developing Xenopus Embryos," Molecular Cellular Biology 13(8):4776-4783 (1993).
Ronde et al., "Ontogeny of the v-erbA Oncoprotein from the Thyroid Hormone Receptor: An Alteration in the DNA Binding Domain Plays a Role Crucial for verbA Function," J. Virology 65(4):2037-2046 (1991).
Sadowski et al. GAL4-VP16 is an unusually potent transcriptional activator. Nature. vol. 335, pp. 563-564, Oct. 1988. *
Sadowski et al., "GAL4-VP16 is an unusually potent transcriptional activator," Nature 335:563-568 (1988).
Saleh et al., "A Novel Zinc Finger Gene on Human Chromosome 1 qter That is Alternatively Spliced in Human Tissues and Cell Lines," American Journal of Human Genetics 52:192-203 (1993).
Search of Swissprot. Data Base Performed CA Aug. 2000.
Shi et al., "A Direct Comparison of the Properties of Natural and Designed Finger Proteins," Chem. & Biol. 2(2):83-89 (1995).
Shi et al., "DNA Unwinding Induced by Zinc Finger Protein Binding." Biochemistry 35:3845-3848 (1996).
Shi et al., "Specific DNA-RNA Hybrid Binding by Zinc Finger Proteins," Science 268:282-284 (1995).
Singh et al., "Molecular Cloning of an Enhancer Binding Protein: Isolation by Screening of an Expression Library with a Recognition Site DNA," Cell 52:415-423 (1988).
Skerka et al., "Coordinate Expression and Distinct DNA-Binding Characteristics of the Four EGR-Zinc Finger Proteins in Jurkat T Lymphocytes," Immunobiology 198:179-191 (1997).
South et al., "The Nucleocapsid Protein Isolated from HIV-1 Particles Binds Zinc and Forms Retroviral-Type Zinc Fingers," Biochemistry 29:7786-7789 (1990).
Spengler et al., "Regulation of Apoptosis and Cell Cycle Arrest by ZZC1, A Novel Zinc finger Protein Expressed in the Pituitary Gland and the Brain," Embo J. 16(10):2814-2825 (1997).
Suzuki et al. "DNA Recognition Code of Transcription Factors in the Helix-turn-Helix, Probe Helix, Hormone Receptor, and Zinc Finger Families," PNAS 91:12357-12361 (1994).
Suzuki et al., "Stereochemical Basis of DNA Recognition by Zn Fingers," Nuc. Acids Res. 22(16):3397-3405 (1994).
Swimoff et al., "DNA-Binding Specificity of NGFI-A and Related Zinc Finger Transcription Factors," Mol. Cell. Biol. 15 (4):2275-2287 (1995).
Taylor et al., "Designing Zinc-Finger ADRI Mutants with Altered Specificity of DNA Binding to T in UASI Sequences," Biochemistry 34:3222-3230 (1995).
Thiel et al., "The Human Transcriptional Repressor Protein NABI: Expression and Biological Activity," Biochim Biophys Acta 1493:289-301 (2000).
Thiesen et al., "Amino Acid Substitutions in the SP1 Zinc Finger Domain Alter the DNA Binding Affinity to Cognate SP1 Target Site," Biochem. Biophys. Res. Communications 175(I):333-338 (1991).
Thiesen et al: "Dtermination of DNA binding specificities of mutated zinc finger domains", FEBS LETTERS, vol. 283, No. 1, May 1991, pp. 23-26.
Thiesen, H. J., "From Repression Domains to Designer Zinc Finger Proteins: A Novel Strategy for Intracellular Immunization Against HIV," Gene Expression 5:229-243 (1996).
Thukral et al., "Localization of a Minimal Binding Domain and Activation Regions in Yeast Regulatory Protein ADRI1," Molecular Cellular Biology 9(6):2360-2369 (1989).
Thukral et al., "Mutations in the Zinc Fingers of ADR1 That Change the Specificity of DNA Binding and Transactivation," Mol. Cell Biol. 12(6):2784-2792 (1992).
Thukral et al., "Two Monomers of Yeast Transcription Factor ADR1 Bind a Paldromic Sequence Symmetrically to Activate ADH2 Expression," Molecular Cellular Biol. 11(3):1566-1577 (1991).
Thurkral et al., "Alanine Scanning Site-Directed Mutagenesis of the Zinc Fingers of Transcription Factor ADR1: Residues that Contact DNA and that Transactivate," PNAS 88:9188-9192 (1991), + correction page.
Vortkamp et al., "Identification of Optimized Target Sequences for the GL13 Zinc Finger Protein," DNA Cell Biol. 14(7):629-634 (1995).
Wang et al., "Dimerization of Zinc Fingers Mediated by Peptides Evolved In Vitro From Random Sequences," Proc. Natl. Acad. Sci. U.S.A. 96:9568-9573 (1999).
Webster et al., "Conversion of the E1A Cys4 Zinc Finger to a Nonfunctional His2, Cys2 Zinc Finger by a Single Point Mutation," PNAS 88:9989-9993 (1991).
Whyatt et al., "The Two Zinc Finger-Like Domains of GATA-1 Have Different DNA Binding Specificities," EMBO J. 12(13):4993-5005 (1993).
Wilson et al., "In Vivo Mutational Analysis of the NGFI-A Zinc Fingers," J. Biol. Chem. 267(6):3718-3724 (1992).
Witzgall et al., "The Kruppel-Associated Box-A (KRAB-A) Domain of Zinc Finger Proteins Mediates Transcriptional Repression" PNAS 91:4514-4518 (1994).
Wolfe et al., Analysis of Zinc Fingers Optimized Via Phage Display: Evaluating the Utility of a Recognition Code, J. Mol. Biol. 285:1917-1934 (1999).
Wright et al., "Expression of a Zinc Finger Gene in HTLV-1 and HTLV-II Transformed Cells," Science 248:588-591 (1990).
Wu et al., "Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Reduces Reverse Transcriptase Pausing at a Secondary Structure near the Murine Leukemia Virus Polypurine Tract" J. Viral. 70(10):7132-7142 (1996).
Wu et al: "Binding zinc fingers by selection: Toward a therapeutic application", Proc.Natl.Acad.Sci.USA, vol. 92, Jan. 1995, Biochemistry, pp. 344-348.
Yang et al., "Surface Plasmon Resonance Based Kinetic Studies of Zinc Finger-DNA Interaction," J. Immunol. Methods 183:175-182 (1995).
Yu et al., "A Hairpin Ribozyme Inhibits Expression of Diverse Strains of Human Immunodeficiency Virus Type 1," PNAS 90:6340-6344 (1993).
Zhang et al., "Synthetic Zinc Finger Transcription Factor Action at an Endogenous Chromosomal Site. Activation of the Human Erythropoietin Gene," Journal of Biological Chemistry 275(43):33850-33860 (2000).

Also Published As

Publication number Publication date
EP2022856A3 (fr) 2009-04-01
JP4942637B2 (ja) 2012-05-30
ATE524545T1 (de) 2011-09-15
JP2008119007A (ja) 2008-05-29
CA2196419C (fr) 2007-08-21
USRE42150E1 (en) 2011-02-15
EP2022856A2 (fr) 2009-02-11
DE69535829D1 (de) 2008-10-16
USRE45795E1 (en) 2015-11-10
USRE39229E1 (en) 2006-08-08
US6007988A (en) 1999-12-28
EP0781331B1 (fr) 2008-09-03
EP0781331A1 (fr) 1997-07-02
US6013453A (en) 2000-01-11
JPH10504461A (ja) 1998-05-06
AU3229195A (en) 1996-03-14
AU698152B2 (en) 1998-10-22
ATE407205T1 (de) 2008-09-15
JP4118327B2 (ja) 2008-07-16
CA2196419A1 (fr) 1996-02-29
USRE42211E1 (en) 2011-03-08
EP2022856B1 (fr) 2011-09-14
WO1996006166A1 (fr) 1996-02-29

Similar Documents

Publication Publication Date Title
USRE45721E1 (en) Relating to binding proteins for recognition of DNA
US6977154B1 (en) Nucleic acid binding proteins
CA2290886C (fr) Proteines de liaison d'acide nucleique
WO1998053059A1 (fr) Proteines de liaison d'acide nucleique
EP1003853B1 (fr) Procedes d'analyse de proteines
AU2003215094B2 (en) Zinc finger libraries
AU726759B2 (en) Improvements in or relating to binding proteins for recognition of DNA
AU7539800A (en) Dna library
Cohen Selection of peptide aptamers that recognize and inhibit intracellular proteins
Greisman A strategy for selecting zinc finger proteins for desired DNA sequences

Legal Events

Date Code Title Description
AS Assignment

Owner name: UNITED KINGDOM RESEARCH AND INNOVATION, UNITED KINGDOM

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:MEDICAL RESEARCH COUNCIL;REEL/FRAME:046469/0108

Effective date: 20180401

Owner name: UNITED KINGDOM RESEARCH AND INNOVATION, UNITED KIN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:MEDICAL RESEARCH COUNCIL;REEL/FRAME:046469/0108

Effective date: 20180401