US20090263900A1 - Linear donor constructs for targeted integration - Google Patents

Linear donor constructs for targeted integration Download PDF

Info

Publication number
US20090263900A1
US20090263900A1 US12/386,059 US38605909A US2009263900A1 US 20090263900 A1 US20090263900 A1 US 20090263900A1 US 38605909 A US38605909 A US 38605909A US 2009263900 A1 US2009263900 A1 US 2009263900A1
Authority
US
United States
Prior art keywords
cell
sequence
nucleic acid
interest
domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US12/386,059
Other languages
English (en)
Inventor
Russell DeKelver
Philip D. Gregory
Michael C. Holmes
Fyodor Urnov
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sangamo Therapeutics Inc
Original Assignee
Sangamo Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=41095228&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20090263900(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Sangamo Therapeutics Inc filed Critical Sangamo Therapeutics Inc
Priority to US12/386,059 priority Critical patent/US20090263900A1/en
Assigned to SANGAMO BIOSCIENCES, INC. reassignment SANGAMO BIOSCIENCES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: URNOV, FYODOR, DEKELVER, RUSSELL, GREGORY, PHILIP D., HOLMES, MICHAEL C.
Publication of US20090263900A1 publication Critical patent/US20090263900A1/en
Priority to US13/134,766 priority patent/US9045763B2/en
Priority to US14/699,908 priority patent/US9376685B2/en
Priority to US15/146,276 priority patent/US9765360B2/en
Assigned to SANGAMO THERAPEUTICS, INC. reassignment SANGAMO THERAPEUTICS, INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: SANGAMO BIOSCIENCES, INC.
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination

Definitions

  • the present disclosure is in the field of genome engineering, particularly linear donor constructs for targeted integration into the genome of a cell.
  • targeted cleavage events can be used, for example, to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted recombination and targeted integration at a predetermined chromosomal locus.
  • targeted cleavage events can be used, for example, to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted recombination and targeted integration at a predetermined chromosomal locus.
  • United States Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; and 20060188987, and International Publication WO 2007/014275 the disclosures of which are incorporated by reference in their entireties for all purposes.
  • targeted integration using zinc finger nucleases has been demonstrated with circular (plasmid) DNAs having long ( ⁇ 750 base pair) homology arms. See, Moehle et al. (2007) Proc. Nat'l. Acad. Sci. USA 104(9):3055-3060.
  • compositions comprising shorter, linear exogenous polynucleotides that optionally can resist exonuclease degradation and use of these compositions in methods for targeted integration.
  • the present disclosure provides linear exogenous (donor) nucleic acids, compositions comprising these nucleic acids and methods of making and using these linear donor molecules.
  • the donor molecules described herein have two homology arms of between about 50 and 100 base pairs flanking a sequence of interest.
  • the donor sequences can be integrated in a targeted manner into the genome of a cell, for example using zinc finger nucleases (ZFNs) and/or meganucleases. Integration of the exogenous nucleic acid sequences into the genome is facilitated by targeted double-strand cleavage of the genome (chromosome) in the region of interest. Cleavage is preferably targeted to the region of interest through the use of fusion proteins comprising a zinc finger binding domain, which is engineered to bind a sequence within the region of interest, and a cleavage domain or a cleavage half-domain. Such cleavage stimulates integration of exogenous polynucleotide sequences at or near the cleavage site.
  • ZFNs zinc finger nucleases
  • meganucleases meganucleases
  • a linear nucleic acid molecule comprising homology arms of 50-100 base pairs flanking a sequence of interest.
  • the linear donor molecule stably persists in the cell into which it is introduced.
  • the linear donor molecule is modified to resist exonucleolytic cleavage, for example by placing one or more phosphorothioate phosphodiester bonds between one or more base pairs on the ends of the donor molecule.
  • the sequence of interest of the donor molecule may comprise one or more sequences encoding a functional polypeptide (e.g., a cDNA), with or without a promoter.
  • the nucleic acid sequence comprises a promoterless sequence encoding an antibody, an antigen, an enzyme, a growth factor, a receptor (cell surface or nuclear), a hormone, a lymphokine, a cytokine, a reporter, functional fragments of any of the above and combinations of the above. Expression of the integrated sequence is then ensured by transcription driven by an endogenous promoter or other control element in the region of interest.
  • a “tandem” cassette is integrated into the selected site in this manner, the first component of the cassette comprising a promotorless sequence as described above, followed by a transcription termination sequence, and a second sequence, encoding an autonomous expression cassette. Additional sequences (coding or non-coding sequences) may be included in the donor molecule between the homology arms, including but not limited to, sequences encoding a 2A peptide, SA site, IRES, etc.
  • the donor molecules of the disclosure can be inserted into a specified location in a genome following cleavage of the genome, for example using one or more fusion molecules comprising a DNA-binding domain targeted to the specified location in the genome and a cleavage domain (e.g., a zinc finger nuclease (ZFN) or naturally or non-naturally occurring meganuclease to a particular locus.
  • a cleavage domain e.g., a zinc finger nuclease (ZFN) or naturally or non-naturally occurring meganuclease to a particular locus.
  • a method for integrating an exogenous sequence as described herein into a region of interest in the genome of a cell comprising: (a) expressing a fusion protein in the cell, the fusion protein comprising a DNA-binding domain (e.g., zinc finger binding domain) and a cleavage domain or cleavage half-domain, wherein the DNA-binding domain (e.g., zinc finger binding domain) has been engineered to bind to a target site in the region of interest in the genome of the cell; and (b) contacting the cell with a donor polynucleotide as described herein, wherein binding of the fusion protein to the target site cleaves the genome of the cell in the region of interest, thereby resulting in integration of the exogenous sequence into the genome of the cell within the region of interest.
  • a DNA-binding domain e.g., zinc finger binding domain
  • a cleavage domain or cleavage half-domain wherein the DNA-binding domain (e.g., zinc
  • the methods comprise the steps of (a) expressing a first fusion protein in the cell, the first fusion protein comprising a first zinc finger binding domain and a first cleavage half-domain, wherein the first zinc finger binding domain has been engineered to bind to a first target site in the region of interest in the genome of the cell; (b) expressing a second fusion protein in the cell, the second fusion protein comprising a second zinc finger binding domain and a second cleavage half domain, wherein the second zinc finger binding domain binds to a second target site in the region of interest in the genome of the cell, wherein the second target site is different from the first target site; and (c) contacting the cell with a exogenous donor molecule as described herein, wherein binding of the first fusion protein to the first target site, and binding of the second fusion protein to the second target site, positions the cleavage half-domains such that the genome of the cell is cleaved in the region of interest, thereby resulting in integration of
  • the donor polynucleotide comprises a sequence encoding a functional polypeptide, which sequence is inserted into the genome of the cell.
  • the first and second cleavage half-domains are from a Type IIS restriction endonuclease, for example, FokI or StsI.
  • at least one of the fusion proteins may comprise an alteration in the amino acid sequence of the dimerization interface of the cleavage half-domain, for example such that obligate heterodimers of the cleavage half-domains are formed.
  • the cleavage domain may be a naturally or non-naturally occurring meganuclease.
  • the cell can be a mammalian cell, for example, a human, rat, mouse or rabbit cell, or a plant cell. Additionally, the cell may be derived from an insect, xenopus or nematode system. Furthermore, the cell may be arrested in the G2 phase of the cell cycle.
  • a linear donor nucleic acid molecule comprising homology arms of between 50 and 750 base pairs and a sequence of interest, wherein the homology arms flank the sequence of interest.
  • linear donor nucleic acid of any of 1 to 4 further comprising, between the homology arms, a sequence encoding a 2A peptide.
  • linear donor nucleic acid of any of 1 through 5 further comprising, between the homology arms, a sequence comprising an SA site.
  • polypeptide is selected from the group consisting of an antibody, an antigen, an enzyme, a growth factor, a receptor (cell surface or nuclear), a hormone, a lymphokine, a cytokine, a reporter gene, a selectable marker, a secreted factor, an epitope tag and functional fragments thereof and combinations thereof.
  • linear donor nucleic acid of any of 1 to 7 or 9, wherein the sequence contains a non-coding nucleic acid.
  • the linear donor nucleic acid according to claim 12 wherein the non-coding nucleic acid is selected from the group consisting of a miRNA, and SH-RNA, or siRNA.
  • a method for homology-dependent targeted integration of a sequence of interest into a region of interest in the genome of the cell comprising the steps of:
  • a fusion protein in the cell comprising a DNA-binding domain and cleavage domain or a cleavage half-domain, wherein the DNA-binding domain has been engineered to bind to a target site in the region of interest;
  • binding of the fusion protein to the target site cleaves the genome of the cell in the region of the interest, thereby resulting in homology-dependent targeted integration of the sequence of interest into the genome of the cell.
  • a method for homology-dependent targeted integration of a sequence of interest into a cell comprising:
  • binding of the first fusion protein to the first target site, and binding of the second fusion protein to the second target site positions the cleavage half-domains such that the genome of the cell is cleaved in the region of interest, thereby resulting in homology-dependent integration of the donor nucleic said into the genome of the cell.
  • sequence in interest from the integrated donor comprises a non-coding nucleic acid sequence.
  • Type IIS restriction endonuclease is selected from the group consisting of FokI and StsI.
  • FIG. 1 is a schematic diagram depicting construction of a linear donor polynucleotide as described herein.
  • the “x” denotes phosphorothioate phosphodiester bonds as the first and second bonds on the 5′ and 3′ ends of the polynucleotide.
  • FIG. 2 depicts the sequence of an exemplary linear donor (SEQ ID NO: 1) having homology arms of 100 base pairs.
  • the linear donor molecule comprises a left homology arm from nucleotides 1 to 100 (lowercase, underlined); a splice acceptor (SA) site, from nucleotides 107 to 132 (lowercase, bold); a sequence encoding a foot-in-mouth-disease virus (FMDV)-derived 2A self-processing sequence (2A peptide) from nucleotides 141 to 212 (uppercase, no underlining); a sequence encoding green fluorescent protein (GFP) poly(A) from nucleotides 219 to 1,215 (uppercase, underlined); and a right homology arm from nucleotides 1235 to 1334 (lowercase, underlined).
  • SA splice acceptor
  • FMDV foot-in-mouth-disease virus
  • FIG. 3 depicts the sequence of an exemplary linear donor (SEQ ID NO:2) having homology arms of 75 base pairs.
  • the linear donor molecule comprises a left homology arm from nucleotides 1 to 75 (lowercase, underlined); an SA site from nucleotides 82 to 107 (lowercase, bold); a sequence encoding a 2A peptide from nucleotides 116 to 187 (uppercase, no underlining); a sequence encoding GFP poly(A) from nucleotides 194 to 1,190 (uppercase, underlined); and a right homology arm from nucleotides 1210 to 1284 (lowercase, underlined).
  • FIG. 4 depicts the sequence of an exemplary linear donor (SEQ ID NO:3) having homology arms of 50 base pairs.
  • the linear donor molecule comprises a left homology arm from nucleotides 1 to 50 (lowercase, underlined); an SA site from nucleotides 57 to 82 (lowercase, bold); a sequence encoding a 2A peptide from nucleotides 91 to 162 (uppercase, no underlining); a sequence encoding GFP poly(A) from nucleotides 169 to 1,165 (uppercase, underlined); and a right homology arm from nucleotides 1,185 to 1,234 (lowercase, underlined).
  • FIG. 5 depicts the sequence of another exemplary linear donor (SEQ ID NO:4) having homology arms of 50 base pairs.
  • the linear donor molecule comprises a left homology arm from nucleotides 1 to 50 (lowercase, underlined); an hPGK promoter sequence from nucleotides 79 to 594 (lowercase, bold); a sequence encoding GFP poly(A) from nucleotides 615 to 1,611 (uppercase, underlined); and a right homology arm from nucleotides 1,639 to 1,688 (lowercase, underlined).
  • FIG. 6 depicts results of a PCR assay and shows modification of the PPP1R12C (AAVS1) locus when various donor molecules as described herein are introduced into K562 cells in the absence (lanes 2-7) or presence of AAVS1-targeted ZFNs (lanes 8-13).
  • FIG. 7 is a Southern blot showing modification of the PPP1R12C (AAVS1) locus when various donor molecules as described herein are introduced into K562 cells in the absence (lanes 3-7) or presence of AAVS1-targeted ZFNs (lanes 9-13). The percent of chromosomes modified by is listed below lanes 9-13.
  • FIG. 8 depicts the percentage of GFP-positive cells as evaluated by FACS.
  • the present disclosure relates to exogenous (donor) polynucleotides useful for homology-dependent targeted integration (TI) into a region of interest in a genome.
  • the donor polynucleotides described herein are linear molecules comprising homology arms (HA) of approximately 50-100 base pairs. The homology arms flank one or more sequences of interest to be inserted into the genome of a cell.
  • HA homology arms
  • These donor molecules are useful for targeted cleavage and recombination into a specified region of interest in a genome when used in combination with fusion proteins (zinc finger nucleases) comprising a cleavage domain (or a cleavage half-domain) and a zinc finger binding domain (and/or polynucleotides encoding these proteins).
  • a zinc finger binding domain can comprise one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers), and can be engineered to bind to any sequence within the region of interest.
  • the linear donor polynucleotides described are integrated at high rates into the cleavage site by homology-dependent methods.
  • linear donor molecules described herein include the rapid and efficient provision of donor molecules for use with ZFNs.
  • donor molecules used in combination with zinc finger nucleases (ZFNs) for targeted insertion into a specified locus of the genome are plasmid constructs containing long ( ⁇ 750 base pairs) homology arms flanking a transgene of interest. Construction of such plasmid donors is time-consuming, taking at least 2 weeks.
  • the linear donor molecules described herein can be constructed within hours and used immediately.
  • use of linear donors as described herein reduces or eliminates the phenomena of stable insertion of the plasmid donor into the host cell.
  • MOLECULAR CLONING A LABORATORY MANUAL , Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY , John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY , Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION , Third edition, Academic Press, San Diego, 1998 ; METHODS IN ENZYMOLOGY , Vol. 304, “Chromatin” (P. M. Wassarman and A. P.
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form.
  • polynucleotide refers to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form.
  • these terms are not to be construed as limiting with respect to the length of a polymer.
  • the terms can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones).
  • an analogue of a particular nucleotide has the same base-pairing specificity; i.e., an analogue of A will base-pair with T.
  • polypeptide “peptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues.
  • the term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of a corresponding naturally-occurring amino acids.
  • Binding refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific. Such interactions are generally characterized by a dissociation constant (K d ) of 10 ⁇ 6 M ⁇ 1 or lower. “Affinity” refers to the strength of binding: increased binding affinity being correlated with a lower K d .
  • a “binding protein” is a protein that is able to bind non-covalently to another molecule.
  • a binding protein can bind to, for example, a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein) and/or a protein molecule (a protein-binding protein).
  • a DNA-binding protein a DNA-binding protein
  • an RNA-binding protein an RNA-binding protein
  • a protein-binding protein it can bind to itself (to form homodimers, homotrimers, etc.) and/or it can bind to one or more molecules of a different protein or proteins.
  • a binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding and protein-binding activity.
  • a “zinc finger DNA binding protein” (or binding domain) is a protein, or a domain within a larger protein, that binds, DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion.
  • the term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.
  • Zinc finger binding domains can be “engineered” to bind to a predetermined nucleotide sequence.
  • methods for engineering zinc finger proteins are design and selection.
  • a designed zinc finger protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see, also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.
  • a “selected” zinc finger protein is a protein not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. See e.g., U.S. Pat. No. 5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No. 6,007,988; U.S. Pat. No. 6,013,453; U.S. Pat. No. 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197 and WO 02/099084.
  • sequence refers to a nucleotide sequence of any length, which can be DNA or RNA; can be linear, circular or branched and can be either single-stranded or double stranded.
  • donor sequence refers to a nucleotide sequence that is inserted into a genome.
  • a donor sequence can be of any length, for example between 2 and 10,000 nucleotides in length (or any integer value therebetween or thereabove), preferably between about 100 and 1,000 nucleotides in length (or any integer therebetween), more preferably between about 200 and 500 nucleotides in length.
  • a “homologous, non-identical sequence” refers to a first sequence which shares a degree of sequence identity with a second sequence, but whose sequence is not identical to that of the second sequence.
  • a polynucleotide comprising the wild-type sequence of a mutant gene is homologous and non-identical to the sequence of the mutant gene.
  • the degree of homology between the two sequences is sufficient to allow homologous recombination therebetween, utilizing normal cellular mechanisms.
  • Two homologous non-identical sequences can be any length and their degree of non-homology can be as small as a single nucleotide (e.g., for correction of a genomic point mutation by targeted homologous recombination) or as large as 10 or more kilobases (e.g., for insertion of a gene at a predetermined ectopic site in a chromosome).
  • Two polynucleotides comprising the homologous non-identical sequences need not be the same length.
  • an exogenous polynucleotide i.e., donor polynucleotide
  • an exogenous polynucleotide i.e., donor polynucleotide of between 20 and 10,000 nucleotides or nucleotide pairs can be used.
  • nucleic acid and amino acid sequence identity are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this fashion. In general, identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity.
  • the percent identity of two sequences is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100.
  • An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure , M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986).
  • the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects sequence identity.
  • Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters.
  • the degree of sequence similarity between polynucleotides can be determined by hybridization of polynucleotides under conditions that allow formation of stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
  • Two nucleic acid, or two polypeptide sequences are substantially homologous to each other when the sequences exhibit at least about 70%-75%, preferably 80%-82%, more preferably 85%-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity over a defined length of the molecules, as determined using the methods above.
  • substantially homologous also refers to sequences showing complete identity to a specified DNA or polypeptide sequence.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; Nucleic Acid Hybridization: A Practical Approach , editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).
  • Selective hybridization of two nucleic acid fragments can be determined as follows. The degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit the hybridization of a completely identical sequence to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern (DNA) blot, Northern (RNA) blot, solution hybridization, or the like, see Sambrook, et al., Molecular Cloning: A Laboratory Manual , Second Edition, (1989) Cold Spring Harbor, N.Y.).
  • hybridization assays that are well known in the art (e.g., Southern (DNA) blot, Northern (RNA) blot, solution hybridization, or the like, see Sambrook, et al., Molecular Cloning: A Laboratory Manual , Second Edition, (1989) Cold Spring Harbor, N.Y.).
  • Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency. If conditions of low stringency are employed, the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.
  • a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.
  • a nucleic acid probe When utilizing a hybridization-based detection system, a nucleic acid probe is chosen that is complementary to a reference nucleic acid sequence, and then by selection of appropriate conditions the probe and the reference sequence selectively hybridize, or bind, to each other to form a duplex molecule.
  • a nucleic acid molecule that is capable of hybridizing selectively to a reference sequence under moderately stringent hybridization conditions typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe.
  • Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence of the selected nucleic acid probe.
  • Hybridization conditions useful for probe/reference sequence hybridization where the probe and reference sequence have a specific degree of sequence identity, can be determined as is known in the art (see, for example, Nucleic Acid Hybridization: A Practical Approach , editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).
  • Hybridization stringency refers to the degree to which hybridization conditions disfavor the formation of hybrids containing mismatched nucleotides, with higher stringency correlated with a lower tolerance for mismatched hybrids.
  • Factors that affect the stringency of hybridization include, but are not limited to, temperature, pH, ionic strength, and concentration of organic solvents such as, for example, formamide and dimethylsulfoxide. As is known to those of skill in the art, hybridization stringency is increased by higher temperatures, lower ionic strength and lower solvent concentrations.
  • stringency conditions for hybridization it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of the sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions.
  • the selection of a particular set of hybridization conditions is selected following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual , Second Edition, (1989) Cold Spring Harbor, N.Y.).
  • “Recombination” refers to a process of exchange of genetic information between two polynucleotides.
  • “homologous recombination (HR)” refers to the specialized form of such exchange that takes place, for example, during repair of double-strand breaks in cells. This process requires nucleotide sequence homology, uses a “donor” molecule to template repair of a “target” molecule (i.e., the one that experienced the double-strand break), and is variously known as “non-crossover gene conversion” or “short tract gene conversion,” because it leads to the transfer of genetic information from the donor to the target.
  • such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and/or “synthesis-dependent strand annealing,” in which the donor is used to resynthesize genetic information that will become part of the target, and/or related processes.
  • Such specialized HR often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.
  • “Cleavage” refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, fusion polypeptides are used for targeted double-stranded DNA cleavage.
  • a “cleavage domain” comprises one or more polypeptide sequences which possesses catalytic activity for DNA cleavage.
  • a cleavage domain can be contained in a single polypeptide chain or cleavage activity can result from the association of two (or more) polypeptides.
  • a “cleavage half-domain” is a polypeptide sequence which, in conjunction with a second polypeptide (either identical or different) forms a complex having cleavage activity (preferably double-strand cleavage activity).
  • Chromatin is the nucleoprotein structure comprising the cellular genome.
  • Cellular chromatin comprises nucleic acid, primarily DNA, and protein, including histones and non-histone chromosomal proteins.
  • the majority of eukaryotic cellular chromatin exists in the form of nucleosomes, wherein a nucleosome core comprises approximately 150 base pairs of DNA associated with an octamer comprising two each of histones H2A, H2B, H3 and H4; and linker DNA (of variable length depending on the organism) extends between nucleosome cores.
  • a molecule of histone H1 is generally associated with the linker DNA.
  • chromatin is meant to encompass all types of cellular nucleoprotein, both prokaryotic and eukaryotic.
  • Cellular chromatin includes both chromosomal and episomal chromatin.
  • a “chromosome,” is a chromatin complex comprising all or a portion of the genome of a cell.
  • the genome of a cell is often characterized by its karyotype, which is the collection of all the chromosomes that comprise the genome of the cell.
  • the genome of a cell can comprise one or more chromosomes.
  • an “episome” is a replicating nucleic acid, nucleoprotein complex or other structure comprising a nucleic acid that is not part of the chromosomal karyotype of a cell.
  • Examples of episomes include plasmids and certain viral genomes.
  • an “accessible region” is a site in cellular chromatin in which a target site present in the nucleic acid can be bound by an exogenous molecule which recognizes the target site. Without wishing to be bound by any particular theory, it is believed that an accessible region is one that is not packaged into a nucleosomal structure. The distinct structure of an accessible region can often be detected by its sensitivity to chemical and enzymatic probes, for example, nucleases.
  • a “target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist.
  • the sequence 5′-GAATTC-3′ is a target site for the Eco RI restriction endonuclease.
  • exogenous molecule is a molecule that is not normally present in a cell, but can be introduced into a cell by one or more genetic, biochemical or other methods. “Normal presence in the cell” is determined with respect to the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic development of muscle is an exogenous molecule with respect to an adult muscle cell. Similarly, a molecule induced by heat shock is an exogenous molecule with respect to a non-heat-shocked cell.
  • An exogenous molecule can comprise, for example, a coding sequence for any polypeptide or fragment thereof, a functioning version of a malfunctioning endogenous molecule or a malfunctioning version of a normally-functioning endogenous molecule.
  • An exogenous molecule can also be the same type of molecule as an endogenous molecule but be derived from a different species than the species the endogenous molecule is derived from.
  • a human nucleic acid sequence may be introduced into a cell line originating from a hamster or mouse.
  • An exogenbus molecule can be, among other things, a small molecule, such as is generated by a combinatorial chemistry process, or a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules.
  • Nucleic acids include DNA and RNA, can be single- or double-stranded; can be linear, branched or circular; and can be of any length. Nucleic acids include those capable of forming duplexes, as well as triplex-forming nucleic acids. See, for example, U.S. Pat. Nos. 5,176,996 and 5,422,251.
  • Exogenous nucleic acid molecules that can be targeted for insertion into a genome are also referred to as “donor” polynucleotides.
  • Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases and helicases.
  • exogenous molecule can be the same type of molecule as an endogenous molecule, e.g., an exogenous protein or nucleic acid.
  • an exogenous nucleic acid can comprise an infecting viral genome, a plasmid or episome introduced into a cell, or a chromosome that is not normally present in the cell.
  • Methods for the introduction of exogenous molecules into cells include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer.
  • an “endogenous” molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions.
  • an endogenous nucleic acid can comprise a chromosome, the genome of a mitochondrion, chloroplast or other organelle, or a naturally-occurring episomal nucleic acid.
  • Additional endogenous molecules can include proteins, for example, transcription factors and enzymes.
  • a “fusion” molecule is a molecule in which two or more subunit molecules are linked, preferably covalently.
  • the subunit molecules can be the same chemical type of molecule, or can be different chemical types of molecules.
  • Examples of the first type of fusion molecule include, but are not limited to, fusion proteins (for example, a fusion between a ZFP DNA-binding domain and a cleavage domain) and fusion nucleic acids (for example, a nucleic acid encoding the fusion protein described supra).
  • Examples of the second type of fusion molecule include, but are not limited to, a fusion between a triplex-forming nucleic acid and a polypeptide, and a fusion between a minor groove binder and a nucleic acid.
  • Fusion protein in a cell can result from delivery of the fusion protein to the cell or by delivery of a polynucleotide encoding the fusion protein to a cell, wherein the polynucleotide is transcribed, and the transcript is translated, to generate the fusion protein.
  • Trans-splicing, polypeptide cleavage and polypeptide ligation can also be involved in expression of a protein in a cell. Methods for polynucleotide and polypeptide delivery to cells are presented elsewhere in this disclosure.
  • Gene expression refers to the conversion of the information, contained in a gene, into a gene product.
  • a gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA.
  • Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.
  • Modulation of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression.
  • Eukaryotic cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells.
  • Plant cells include, but are not limited to, cells of monocotyledonous (monocots) or dicotyledonous (dicots) plants.
  • monocots include cereal plants such as maize, rice, barley, oats, wheat, sorghum, rye, sugarcane, pineapple, onion, banana, and coconut.
  • dicots include tobacco, tomato, sunflower, cotton, sugarbeet, potato, lettuce, melon, soybean, canola (rapeseed), and alfalfa.
  • Plant cells may be from any part of the plant and/or from any stage of plant development.
  • a “region of interest” is any region of cellular chromatin, such as, for example, a gene or a non-coding sequence within or adjacent to a gene, in which it is desirable to bind an exogenous molecule. Binding can be for the purposes of targeted DNA cleavage and/or targeted recombination.
  • a region of interest can be present in a chromosome, an episome, an organellar genome (e.g., mitochondrial, chloroplast), or an infecting viral genome, for example.
  • a region of interest can be within the coding region of a gene, within transcribed non-coding regions such as, for example, leader sequences, trailer sequences or introns, or within non-transcribed-regions, either upstream or downstream of the coding region.
  • a region of interest can be as small as a single nucleotide pair or up to 2,000 nucleotide pairs in length, or any integral value of nucleotide pairs.
  • operative linkage and “operatively linked” (or “operably linked”) are used interchangeably with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components.
  • a transcriptional regulatory sequence such as a promoter
  • a transcriptional regulatory sequence is generally operatively linked in cis with a coding sequence, but need not be directly adjacent to it.
  • an enhancer is a transcriptional regulatory sequence that is operatively linked to a coding sequence, even though they are not contiguous.
  • the term “operatively linked” can refer to the fact that each of the components performs the same function in linkage to the other component as it would if it were not so linked.
  • the ZFP DNA-binding domain and the cleavage domain are in operative linkage if, in the fusion polypeptide, the ZFP DNA-binding domain portion is able to bind its target site and/or its binding site, while the cleavage domain is able to cleave DNA in the vicinity of the target site.
  • a “functional fragment” of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, yet retains the same function as the full-length protein, polypeptide or nucleic acid.
  • a functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one or more amino acid or nucleotide substitutions.
  • DNA-binding function of a polypeptide can be determined, for example, by filter-binding, electrophoretic mobility-shift, or immunoprecipitation assays. DNA cleavage can be assayed by gel electrophoresis. See Ausubel et al., supra.
  • the ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays or complementation, both genetic and biochemical. See, for example, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No. 5,585,245 and PCT WO 98/44350.
  • polynucleotides for insertion into the genome also referred to as “exogenous” polynucleotides or “donor” polynucleotides. It has been shown that plasmid donors carrying 750 bp homology arms flanking a transgene of interest, in combination with designed zinc finger nucleases (ZFNs) can be used for targeted gene alteration. See, e.g., Moehle et al. (2007) Proc. Nat'l. Acad. Sci. USA 104(9):3055-3060 and U.S. Patent Publication No. 20050064474.
  • ZFNs zinc finger nucleases
  • linear donor sequences of the disclosure comprising short homology arms of approximately 50-100 base pairs can be effectively integrated into the genome of cell.
  • the linear donor sequences described herein take only hours to construct.
  • the linear donor sequences described herein are 25 to 50 base pairs in length (or any value therebetween, including 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides).
  • the sequences are between 50 and 75 nucleotides in length (including 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75 nucleotides in length).
  • the sequences are between 75 and 100 nucleotides in length (including 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length).
  • the donor polynucleotides are between 100 and 150 nucleotides in length (or any value therebetween).
  • the donor polynucleotides are between 50 and 750 nucleotides in length (e.g., 50 and 100, 50 and 150, 50 and 200, 50 and 250, 50 and 300, 50 and 350, 50 and 400, 50 and 450, 50 and 500, 50 and 550, 50 and 600, 50 and 650, 50 and 700).
  • the donor sequences described herein may be isolated from plasmids, cells or other sources using standard techniques known in the art such as PCR. Alternatively, they may be chemically synthesized using standard oligonucleotide synthesis techniques. Typically, the donor polynucleotides are made by PCR using a primer with a 50-100 bp 5′ portion homologous to the genomic target, and a 15-18 bp portion identical to the ORF of interest ( FIG. 1 ).
  • the linear donor polynucleotides described herein may include one or more phosphorothioate phosphodiester bonds between terminal base pairs to protect the linear donor polynucleotide from exonucleolytic degradation. These bonds may be in two or more positions at the 5′ and/or 3′ ends of the molecule and may be added during isolation or synthesis using standard methodology. See, e.g., Ciafre et al. (1995) Nucleic Acids Res. 23(20):4134-42; Johansson et al. (2002) Vaccine 20(27-28):3379-88. In embodiments in which the donor polynucleotide is isolated by PCR using primers ( FIG.
  • the 5′ ends of the primer (and donor polynucleotide) are typically phosphorothioate phosphodiester bonds.
  • the linear donor polynucleotides may include one or more 5′ deoxynucleotides, biotin and/one or more amine groups, all of which have been shown to reduce exonucleolytic degradation.
  • exogenous (donor) polynucleotide may comprise any sequence of interest (exogenous sequence).
  • exogenous sequences include, but are not limited to any polypeptide coding sequence (e.g., cDNAs), promoter sequences, enhancer sequences, epitope tags, marker genes, cleavage enzyme recognition sites, epitope tags and various types of expression constructs.
  • Marker genes include, but are not limited to, sequences encoding proteins that mediate antibiotic resistance (e.g., ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), sequences encoding colored or fluorescent or luminescent proteins (e.g., green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase), and proteins which mediate enhanced cell growth and/or gene amplification (e.g., dihydrofolate reductase).
  • Epitope tags include, for example, one or more copies of FLAG, His, myc, Tap, HA or any detectable amino acid sequence.
  • the exogenous (donor) polynucleotide may also comprise sequences which do not encode polypeptides but rather any type of noncoding sequence, as well as one or more control elements (e.g., promoters).
  • the exogenous nucleic acid sequence may produce one or more RNA molecules (e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs (miRNAs), etc.).
  • a donor molecule can contain several, discontinuous regions of homology to cellular chromatin.
  • the regions of homology can flank two or more regions containing the desired alterations.
  • the exogenous sequence comprises a polynucleotide encoding any polypeptide of which expression in the cell is desired, including, but not limited to antibodies, antigens, enzymes, receptors (cell surface or nuclear), hormones, lymphokines, cytokines, reporter polypeptides, growth factors, and functional fragments of any of the above.
  • the coding sequences may be, for example, cDNAs.
  • a donor molecule can be a linear molecule following linearization, as a result of ZFN directed cleavage, of a plasmid taken up by a cell.
  • the linear donor molecule can reside in the genome of the cell wherein the donor molecule becomes available for homology directed targeted integration following ZFN directed cleavage and release of the donor from the genome.
  • the exogenous sequence may comprise a sequence encoding a polypeptide that is lacking or non-functional in the subject having a genetic disease, including but not limited to any of the following genetic diseases: achondroplasia, achromatopsia, acid maltase deficiency, adenosine deaminase deficiency (OMIM No.
  • adrenoleukodystrophy aicardi syndrome, alpha-1 antitrypsin deficiency, alpha-thalassemia, androgen insensitivity syndrome, apert syndrome, arrhythmogenic right ventricular, dysplasia, ataxia telangictasia, barth syndrome, beta-thalassemia, blue rubber bleb nevus syndrome, canavan disease, chronic granulomatous diseases (CGD), cri du chat syndrome, cystic fibrosis, dercum's disease, ectodermal dysplasia, fanconi anemia, fibrodysplasia ossificans progressive, fragile X syndrome, galactosemis, Gaucher's disease, generalized gangliosidoses (e.g., GM1), hemochromatosis, the hemoglobin C mutation in the 6 th codon of beta-globin (HbC), hemophilia, Huntington's disease, Hurler Syndrome, hypophosphatasia, Klinefleter
  • leukodystrophy long QT syndrome, Marfan syndrome, Moebius syndrome, mucopolysaccharidosis (MPS), nail patella syndrome, nephrogenic diabetes insipdius, neurofibromatosis, Neimann-Pick disease, osteogenesis imperfecta, porphyria, Prader-Willi syndrome, progeria, Proteus syndrome, retinoblastoma, Rett syndrome, Rubinstein-Taybi syndrome, Sanfilippo syndrome, severe combined immunodeficiency (SCID), Shwachman syndrome, sickle cell disease (sickle cell anemia), Smith-Magenis syndrome, Stickler syndrome, Tay-Sachs disease, Thrombocytopenia Absent Radius (TAR) syndrome, Treacher Collins syndrome, trisomy, tuberous sclerosis, Turner's syndrome, urea cycle disorder, von Hippel-Landau disease, Waardenburg syndrome, Williams syndrome, Wilson's disease, Wiskott-Aldrich syndrome
  • Additional exemplary diseases that can be treated by targeted integration include acquired immunodeficiencies, lysosomal storage diseases (e.g., Gaucher's disease, GM1, Fabry disease and Tay-Sachs disease), mucopolysaccahidosis (e.g. Hunter's disease, Hurler's disease), hemoglobinopathies (e.g., sickle cell diseases, HbC, ⁇ -thalassemia, ⁇ -thalassemia) and hemophilias.
  • lysosomal storage diseases e.g., Gaucher's disease, GM1, Fabry disease and Tay-Sachs disease
  • mucopolysaccahidosis e.g. Hunter's disease, Hurler's disease
  • hemoglobinopathies e.g., sickle cell diseases, HbC, ⁇ -thalassemia, ⁇ -thalassemia
  • hemophilias e.g., sickle cell diseases, HbC, ⁇ -thalassemia
  • the exogenous sequences can comprise a marker gene (described above), allowing selection of cells that have undergone targeted integration, and a linked sequence encoding an additional functionality.
  • marker genes include GFP, drug selection marker(s) and the like.
  • exogenous sequences may also transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.
  • the disclosed methods and compositions include fusion proteins comprising a cleavage domain (or a cleavage half-domain) and a zinc finger domain, in which the zinc finger domain, by binding to a sequence a region of interest in the genome of a cell directs the activity of the cleavage domain (or cleavage half-domain) to the vicinity of the sequence and, hence, induces cleavage (e.g., a double stranded break) in the region of interest.
  • a zinc finger domain can be engineered to bind to virtually any desired sequence. Accordingly, one or more zinc finger binding domains can be engineered to bind to one or more sequences in the region of interest.
  • a fusion protein comprising a zinc finger binding domain and a cleavage domain (or of two fusion proteins, each comprising a zinc finger binding domain and a cleavage half-domain), in a cell, effects cleavage in the region of interest.
  • Selection of a sequence in a region of interest for binding by a zinc finger domain can be accomplished, for example, according to the methods disclosed in co-owned U.S. Pat. No. 6,453,242 (Sep. 17, 2002), which also discloses methods for designing ZFPs to bind to a selected sequence. It will be clear to those skilled in the art that simple visual inspection of a nucleotide sequence can also be used for selection of a target site. Accordingly, any means for target site selection can be used in the methods described herein.
  • Target sites are generally composed of a plurality of adjacent target subsites.
  • a target subsite refers to the sequence (usually either a nucleotide triplet, or a nucleotide quadruplet that can overlap by one nucleotide with an adjacent quadruplet) bound by an individual zinc finger. See, for example, WO 02/077227. If the strand with which a zinc finger protein makes most contacts is designated the target strand “primary recognition strand,” or “primary contact strand,” some zinc finger proteins bind to a three base triplet in the target strand and a fourth base on the non-target strand.
  • a target site generally has a length of at least 9 nucleotides and, accordingly, is bound by a zinc finger binding domain comprising at least three zinc fingers.
  • a zinc finger binding domain comprising at least three zinc fingers.
  • binding of, for example, a 4-finger binding domain to a 12-nucleotide target site, a 5-finger binding domain to a 15-nucleotide target site or a 6-finger binding domain to an 18-nucleotide target site is also possible.
  • binding of larger binding domains e.g., 7-, 8-, 9-finger and more
  • a target site it is not necessary for a target site to be a multiple of three nucleotides.
  • a target site it is not necessary for a target site to be a multiple of three nucleotides.
  • one or more of the individual zinc fingers of a multi-finger binding domain can bind to overlapping quadruplet subsites.
  • a three-finger protein can bind a 10-nucleotide sequence, wherein the tenth nucleotide is part of a quadruplet bound by a terminal finger
  • a four-finger protein can bind a 13-nucleotide sequence, wherein the thirteenth nucleotide is part of a quadruplet bound by a terminal finger, etc.
  • the length and nature of amino acid linker sequences between individual zinc fingers in a multi-finger binding domain also affects binding to a target sequence.
  • a so-called “non-canonical linker,” “long linker” or “structured linker” between adjacent zinc fingers in a multi-finger binding domain can allow those fingers to bind subsites which are not immediately adjacent.
  • Non-limiting examples of such linkers are described, for example, in U.S. Pat. No. 6,479,626 and WO 01/53480. Accordingly, one or more subsites, in a target site for a zinc finger binding domain, can be separated from each other by 1, 2, 3, 4, 5 or more nucleotides.
  • a four-finger binding domain can bind to a 13-nucleotide target site comprising, in sequence, two contiguous 3-nucleotide subsites, an intervening nucleotide, and two contiguous triplet subsites.
  • Distance between sequences refers to the number of nucleotides or nucleotide pairs intervening between two sequences, as measured from the edges of the sequences nearest each other.
  • the two target sites can be on opposite.
  • DNA strands (Example 1). In other embodiments, both target sites are on the same DNA strand.
  • the DNA binding domain comprises a zinc finger protein.
  • a zinc finger binding domain comprises one or more zinc fingers. Miller et al. (1985) EMBO J. 4:1609-1614; Rhodes (1993) Scientific American Feb.:56-65; U.S. Pat. No. 6,453,242.
  • the zinc finger binding domains described herein generally include 2, 3, 4, 5, 6 or even more zinc fingers.
  • each zinc finger domain contains two beta sheets (held in a beta turn which contains the two invariant cysteine residues) and an alpha helix (containing the two invariant histidine residues), which are held in a particular conformation through coordination of a zinc atom by the two cysteines and the two histidines.
  • Zinc fingers include both canonical C 2 H 2 zinc fingers (i.e., those in which the zinc ion is coordinated by two cysteine and two histidine residues) and non-canonical zinc fingers such as, for example, C 3 H zinc fingers (those in which the zinc ion is coordinated by three cysteine residues and one histidine residue) and C 4 zinc fingers (those in which the zinc ion is coordinated by four cysteine residues). See also WO 02/057293.
  • Zinc finger binding domains can be engineered to bind to a target site (see above) using standard techniques. See, Example 1; co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261, including references cited therein.
  • An engineered zinc finger binding domain can have a novel binding specificity, compared to a naturally-occurring zinc finger protein.
  • Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence.
  • Exemplary selection methods including phage display and two-hybrid systems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB 2,338,237.
  • an individual zinc finger binds to a three-nucleotide (i.e., triplet) sequence (or a four-nucleotide sequence which can overlap, by one nucleotide, with the four-nucleotide binding site of an adjacent zinc finger)
  • the length of a sequence to which a zinc finger binding domain is engineered to bind e.g., a target sequence
  • binding sites for individual zinc fingers (i.e., subsites) in a target site need not be contiguous, but can be separated by one or several nucleotides, depending on the length and nature of the amino acids sequences between the zinc fingers (i.e., the inter-finger linkers) in a multi-finger binding domain.
  • adjacent zinc fingers can be separated by amino acid linker sequences of approximately 5 amino acids (so-called “canonical” inter-finger linkers) or, alternatively, by one or more non-canonical linkers.
  • canonical inter-finger linkers amino acid linker sequences of approximately 5 amino acids
  • non-canonical linkers amino acid linker sequences of approximately 5 amino acids
  • non-canonical linkers amino acid linker sequences of approximately 5 amino acids
  • non-canonical linkers e.g., co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261.
  • insertion of longer (“non-canonical”) inter-finger linkers between certain of the zinc fingers may be preferred as it may increase the affinity and/or specificity of binding by the binding domain. See, for example, U.S. Pat. No. 6,479,626 and WO 01/53480.
  • multi-finger zinc finger binding domains can also be characterized with respect to the presence and location of non-canonical inter-finger linkers.
  • a six-finger zinc finger binding domain comprising three fingers (joined by two canonical inter-finger linkers), a long linker and three additional fingers (joined by two canonical inter-finger linkers) is denoted a 2 ⁇ 3 configuration.
  • a binding domain comprising two fingers (with a canonical linker therebetween), a long linker and two additional fingers (joined by a canonical linker) is denoted a 2 ⁇ 2 protein.
  • a protein comprising three two-finger units (in each of which the two fingers are joined by a canonical linker), and in which each two-finger unit is joined to the adjacent two finger unit by a long linker, is referred to as a 3 ⁇ 2 protein.
  • a long or non-canonical inter-finger linker between two adjacent zinc fingers in a multi-finger binding domain often allows the two fingers to bind to subsites which are not immediately contiguous in the target sequence. Accordingly, there can be gaps of one or more nucleotides between subsites in a target site; i.e., a target site can contain one or more nucleotides that are not contacted by a zinc finger.
  • a 2 ⁇ 2 zinc finger binding domain can bind to two six-nucleotide sequences separated by one nucleotide, i.e., it binds to a 13-nucleotide target site. See also Moore et al. (2001a) Proc. Natl. Acad. Sci. USA 98:1432-1436; Moore et al. (2001b) Proc. Natl. Acad. Sci. USA 98:1437-1441 and WO 01/53480.
  • a target subsite is a three- or four-nucleotide sequence that is bound by a single zinc finger.
  • a two-finger unit is denoted a binding module.
  • a binding module can be obtained by, for example, selecting for two adjacent fingers in the context of a multi-finger protein (generally three fingers) which bind a particular six-nucleotide target sequence.
  • modules can be constructed by assembly of individual zinc fingers. See also WO 98/53057 and WO 01/53480.
  • the DNA-binding domain may be derived from a nuclease.
  • the recognition sequences of homing endonucleases and meganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII are known. See also U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res.
  • the cleavage domain portion of the fusion proteins disclosed herein can be obtained from any endonuclease or exonuclease.
  • Exemplary endonucleases from which a cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388.
  • Non limiting examples of homing endonucleases and meganucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevII are known. See also U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al.
  • Restriction endonucleases are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding.
  • Certain restriction enzymes e.g., Type IIS
  • Fok I catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as well as Li et al.
  • fusion proteins comprise the cleavage domain (or cleavage half-domain) from at least one Type IS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered.
  • Fok I An exemplary Type IIS restriction enzyme, whose cleavage domain is separable from the binding domain, is Fok I.
  • This particular enzyme is active as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575. Accordingly, for the purposes of the present disclosure, the portion of the Fok I enzyme used in the disclosed fusion proteins is considered a cleavage half-domain.
  • two fusion proteins, each comprising a FokI cleavage half-domain can be used to reconstitute a catalytically active cleavage domain.
  • a single polypeptide molecule containing a zinc finger binding domain and two Fok I cleavage half-domains can also be used. Parameters for targeted cleavage and targeted sequence alteration using zinc finger-Fok I fusions are provided elsewhere in this disclosure.
  • a cleavage domain or cleavage half-domain can be any portion of a protein that retains cleavage activity, or that retains the ability to multimerize (e.g., dimerize) to form a functional cleavage domain.
  • Type IIS restriction enzymes are described in co-owned International Publication WO 2007/014275, incorporated by reference herein in its entirety.
  • cleavage domains may also be modified.
  • variants of the cleavage half-domain are employed, which variants that minimize or prevent homodimerization of the cleavage half-domains.
  • Non-limiting examples of such modified cleavage half-domains are described in detail in WO 2007/014275, incorporated by reference in its entirety herein. See, also, Examples.
  • the cleavage domain comprises an engineered cleavage half-domain (also referred to as dimerization domain mutants) that minimize or prevent homodimerization are known to those of skill the art and described for example in U.S. Patent Publication Nos.
  • Additional engineered cleavage half-domains of Fok I form an obligate heterodimers can also be used in the ZFNs described herein.
  • the first cleavage half-domain includes mutations at amino acid residues at positions 490 and 538 of Fok I and the second cleavage half-domain includes mutations at amino acid residues 486 and 499.
  • the cleavage domain comprises two cleavage half-domains, both of which are part of a single polypeptide comprising a binding domain, a first cleavage half-domain and a second cleavage half-domain.
  • the cleavage half-domains can have the same amino acid sequence or different amino acid sequences, so long as they function to cleave the DNA.
  • two fusion proteins are required for cleavage if the fusion proteins comprise cleavage half-domains.
  • a single protein comprising two cleavage half-domains can be used.
  • the two cleavage half-domains can be derived from the same endonuclease (or functional fragments thereof), or each cleavage half-domain can be derived from a different endonuclease (or functional fragments thereof).
  • two cleavage half-domains are used wherein one of the half domains is enzymatically inactive, such that a single-stranded nick is introduced at the target site (see for example co-owned U.S. provisional application 61/189,800).
  • the target sites for the two fusion proteins are preferably disposed, with respect to each other, such that binding of the two fusion proteins to their respective target sites places the cleavage half-domains in a spatial orientation to each other that allows the cleavage half-domains to form a functional cleavage domain, e.g., by dimerizing.
  • the near edges of the target sites are separated by 5-8 nucleotides or by 15-18 nucleotides.
  • any integral number of nucleotides or nucleotide pairs can intervene between two target sites (e.g., from 2 to 50 nucleotides or more). In general, the point of cleavage lies between the target sites.
  • fusion proteins and polynucleotides encoding same are known to those of skill in the art. For example, methods for the design and construction of fusion protein comprising zinc finger proteins (and polynucleotides encoding same) are described in co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261; and International Publication WO 2007/014275.
  • polynucleotides encoding such fusion proteins are constructed. These polynucleotides can be inserted into a vector and the vector can be introduced into a cell (see below for additional disclosure regarding vectors and methods for introducing polynucleotides into cells).
  • a fusion protein comprises a zinc finger binding domain and a cleavage half-domain from the Fok I restriction enzyme, and two such fusion proteins are expressed in a cell.
  • Expression of two fusion proteins in a cell can result from delivery of the two proteins to the cell; delivery of one protein and one nucleic acid encoding one of the proteins to the cell; delivery of two nucleic acids, each encoding one of the proteins, to the cell; or by delivery of a single nucleic acid, encoding both proteins, to the cell.
  • a fusion protein comprises a single polypeptide chain comprising two cleavage half domains and a zinc finger binding domain. In this case, a single fusion protein is expressed in a cell and, without wishing to be bound by theory, is believed to cleave DNA as a result of formation of an intramolecular dimer of the cleavage half-domains.
  • Two fusion proteins each comprising a zinc finger binding domain and a cleavage half-domain, may be expressed in a cell, and bind to target sites which are juxtaposed in such a way that a functional cleavage domain is reconstituted and DNA is cleaved in the vicinity of the target sites.
  • cleavage occurs between the target sites of the two zinc finger binding domains.
  • One or both of the zinc finger binding domains and/or cleavage domains can be engineered.
  • the components of the fusion proteins may be arranged such that the zinc finger domain is nearest the amino terminus of the fusion protein, and the cleavage half-domain is nearest the carboxy-terminus. Dimerization of the cleavage half-domains to form a functional nuclease is brought about by binding of the fusion proteins to sites on opposite DNA strands, with the 5′ ends of the binding sites being proximal to each other.
  • the components of the fusion proteins may be arranged such that the cleavage half-domain is nearest the amino terminus of the fusion protein, and the zinc finger domain is nearest the carboxy-terminus.
  • dimerization of the cleavage half-domains to form a functional nuclease is brought about by binding of the fusion proteins to sites on opposite DNA strands, with the 3′ ends of the binding sites being proximal to each other.
  • a first fusion protein contains the cleavage half-domain nearest the amino terminus of the fusion protein, and the zinc finger domain nearest the carboxy-terminus
  • a second fusion protein is arranged such that the zinc finger domain is nearest the amino terminus of the fusion protein, and the cleavage half-domain is nearest the carboxy-terminus.
  • both fusion proteins bind to the same DNA strand, with the binding site of the first fusion protein containing the zinc finger domain nearest the carboxy terminus located to the 5′ side of the binding site of the second fusion protein containing the zinc finger domain nearest the amino terminus.
  • the two fusion proteins can bind in the region of interest in the same or opposite polarity, and their binding sites (i.e., target sites) can be separated by any number of nucleotides, e.g., from 0 to 200 nucleotides or any integral value therebetween.
  • the binding sites for two fusion proteins, each comprising a zinc finger binding domain and a cleavage half-domain can be located between 5 and 18 nucleotides apart, for example, 5-8 nucleotides apart, or 15-18 nucleotides apart, or 6 nucleotides apart, or 16 nucleotides apart, as measured from the edge of each binding site nearest the other binding site, and cleavage occurs between the binding sites.
  • the site at which the DNA is cleaved generally lies between the binding sites for the two fusion proteins. Double-strand breakage of DNA often results from two single-strand breaks, or “nicks,” offset by 1, 2, 3, 4, 5, 6 or more nucleotides, (for example, cleavage of double-stranded DNA by native Fok I results from single-strand breaks offset by 4 nucleotides). Thus, cleavage does not necessarily occur at exactly opposite sites on each DNA strand.
  • the structure of the fusion proteins and the distance between the target sites can influence whether cleavage occurs adjacent a single nucleotide pair, or whether cleavage occurs at several sites. However, for targeted integration, cleavage within a range of nucleotides is generally sufficient, and cleavage between particular base pairs is not required.
  • the amino acid sequence between the zinc finger domain and the cleavage domain is denoted the “ZC linker.”
  • the ZC linker is to be distinguished from the inter-finger linkers discussed above. ZC linkers are described in detail, for example, in WO 2007/014275.
  • the fusion protein As discussed in detail below, the fusion protein (ZFN), or a polynucleotide encoding same, is introduced into a cell. Once introduced into, or expressed in, the cell, the fusion protein binds to the target sequence in PPP1R12C and cleaves within this gene locus.
  • the disclosed methods and compositions can be used to cleave DNA in cellular chromatin, which facilitates targeted integration of an exogenous sequence (donor polynucleotide) as described herein.
  • exogenous sequence donor polynucleotide
  • integration is meant both physical insertion (e.g., into the genome of a host cell) and, in addition, integration by copying of the donor sequence into the host cell genome via the nucleic acid replication processes.
  • one or more zinc finger binding domains are engineered to bind a target site at or near the predetermined cleavage site, and a fusion protein comprising the engineered zinc finger binding domain and a cleavage domain is expressed in a cell.
  • the DNA is cleaved, preferably via a double stranded break, near the target site by the cleavage domain.
  • the presence of a double-stranded break facilitates integration of exogenous sequences as described herein via homologous recombination.
  • Targeted integration of exogenous sequences can be used to generate cells and cell lines for protein expression. See, for example, co-owned U.S. Patent Application Publication No. 2006/0063231 (the disclosure of which is hereby incorporated by reference herein, in its entirety, for all purposes).
  • the chromosomal integration site should be compatible with high-level transcription of the integrated sequences, preferably in a wide range of cell types and developmental states.
  • transcription of integrated sequences varies depending on the integration site due to, among other things, the chromatin structure of the genome at the integration site. Accordingly, genomic target sites that support high-level transcription of integrated sequences are desirable.
  • exogenous sequences not result in ectopic activation of one or more cellular genes (e.g., oncogenes).
  • ectopic expression may be desired.
  • exogenous (donor) sequence can be introduced into the cell prior to, concurrently with, or subsequent to, expression of the fusion protein(s).
  • compositions are also provided that may enhance levels of targeted recombination including, but not limited to, the use of additional ZFP-functional domain fusions. See, WO 2007/014275.
  • Exemplary molecules of this type include, but are not limited to, compounds which affect microtubule polymerization (e.g., vinblastine, nocodazole, Taxol), compounds that interact with DNA (e.g., cis-platinum(II) diamine dichloride, Cisplatin, doxorubicin) and/or compounds that affect DNA synthesis (e.g., thymidine, hydroxyurea, L-mimosine, etoposide, 5-fluorouracil).
  • compounds which affect microtubule polymerization e.g., vinblastine, nocodazole, Taxol
  • compounds that interact with DNA e.g., cis-platinum(II) diamine dichloride, Cisplatin, doxorubicin
  • compounds that affect DNA synthesis e.g., thymidine, hydroxyurea, L-mimosine, etoposide, 5-fluorouracil.
  • HDAC histone deacetylase
  • Additional methods for cell-cycle arrest include overexpression of proteins which inhibit the activity of the CDK cell-cycle kinases, for example, by introducing a cDNA encoding the protein into the cell or by introducing into the cell an engineered ZFP which activates expression of the gene encoding the protein.
  • Cell-cycle arrest is also achieved by inhibiting the activity of cyclins and CDKs, for example, using RNAi methods (e.g., U.S. Pat. No. 6,506,559) or by introducing into the cell an engineered ZFP which represses expression of one or more genes involved in cell-cycle progression such as, for example, cyclin and/or CDK genes. See, e.g., co-owned U.S. Pat. No. 6,534,261 for methods for the synthesis of engineered zinc finger proteins for regulation of gene expression.
  • targeted cleavage is conducted in the absence of a donor polynucleotide (preferably in S or G 2 phase), and recombination occurs between homologous chromosomes.
  • a donor polynucleotide preferably in S or G 2 phase
  • nucleic acids as described herein may be introduced into a cell using any suitable method.
  • DNA constructs may be introduced into (e.g., into the genome of) a desired plant host by a variety of conventional techniques.
  • Conventional techniques see, for example, Weissbach & Weissbach Methods for Plant Molecular Biology (1988, Academic Press, N.Y.) Section VIII, pp. 421-463; and Grierson & Corey, Plant Molecular Biology (1988, 2d Ed.), Blackie, London, Ch. 7-9.
  • the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using biolistic methods, such as DNA particle bombardment (see, e.g., Klein et al (1987) Nature 327:70-73).
  • the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector.
  • Agrobacterium tumefaciens -mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et al (1984) Science 233:496-498, and Fraley et al (1983) Proc. Nat'l. Acad. Sci. USA 80:4803.
  • gene transfer may be achieved using non-Agrobacterium bacteria or viruses such as Rhizobium sp. NGR234 , Sinorhizoboium meliloti, Mesorhizobium loti , potato virus X, cauliflower mosaic virus and cassaya vein mosaic virus and/or tobacco mosaic virus, See, e.g., Chung et al. (2006) Trends Plant Sci. 11(1):1-4.
  • non-Agrobacterium bacteria or viruses such as Rhizobium sp. NGR234 , Sinorhizoboium meliloti, Mesorhizobium loti , potato virus X, cauliflower mosaic virus and cassaya vein mosaic virus and/or tobacco mosaic virus, See, e.g., Chung et al. (2006) Trends Plant Sci. 11(1):1-4.
  • the virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria using binary T DNA vector (Bevan (1984) Nuc. Acid Res. 12:8711-8721) or the co-cultivation procedure (Horsch et al (1985) Science 227:1229-1231).
  • binary T DNA vector Bevan (1984) Nuc. Acid Res. 12:8711-8721
  • the co-cultivation procedure Horsch et al (1985) Science 227:1229-1231.
  • the Agrobacterium transformation system is used to engineer dicotyledonous plants (Bevan et al (1982) Ann. Rev. Genet 16:357-384; Rogers et al (1986) Methods Enzymol. 118:627-641).
  • the Agrobacterium transformation system may also be used to transform, as well as transfer, DNA to monocotyledonous plants and plant cells. See U.S. Pat. No. 5,591,616; Hemalsteen et al (1984) EMBO J 3:3039-3041; Hooykass-Van Slogteren et al (1984) Nature 311:763-764; Grimsley et al (1987) Nature 325:1677-179; Boulton et al (1989) Plant Mol. Biol. 12:31-40.; and Gould et al (1991) Plant Physiol. 95:426-434.
  • Alternative gene transfer and transformation methods include, but are not limited to, protoplast transformation through calcium-, polyethylene glycol (PEG)- or electroporation-mediated uptake of naked DNA (see Paszkowski et al. (1984) EMBO J 3:2717-2722, Potrykus et al. (1985) Molec. Gen. Genet. 199:169-177; Fromm et al. (1985) Proc. Nat. Acad. Sci. USA 82:5824-5828; and Shimamoto (1989) Nature 338:274-276) and electroporation of plant tissues (D'Halluin et al. (1992) Plant Cell 4:1495-1505).
  • PEG polyethylene glycol
  • Additional methods for plant cell transformation include microinjection, silicon carbide mediated DNA uptake (Kaeppler et al. (1990) Plant Cell Reporter 9:415-418), and microprojectile bombardment (see Klein et al. (1988) Proc. Nat. Acad. Sci. USA 85:4305-4309; and Gordon-Kamm et al. (1990) Plant Cell 2:603-618).
  • the fusion protein(s) can be introduced as polypeptides and/or polynucleotides.
  • two polynucleotides each comprising sequences encoding one of the aforementioned polypeptides, can be introduced into a cell, and when the polypeptides are expressed and each binds to its target sequence, cleavage occurs at or near the target sequence.
  • a single polynucleotide comprising sequences encoding both fusion polypeptides is introduced into a cell.
  • Polynucleotides can be DNA, RNA or any modified forms or analogues or DNA and/or RNA.
  • one or more ZFPs or ZFP fusion proteins can be cloned into a vector for transformation into prokaryotic or eukaryotic cells for replication and/or expression.
  • Vectors can be prokaryotic vectors, e.g., plasmids, or shuttle vectors, insect vectors, or eukaryotic vectors.
  • ZFNs can also be cloned into an expression vector, for administration to a plant cell, animal cell, preferably a mammalian cell or a human cell, fungal cell, bacterial cell, or protozoal cell using standard techniques described for example in Sambrook et al., supra and United States Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; and 20060188987, and International Publication WO 2007/014275.
  • the ZFNs and donor sequences are delivered in vivo or ex vivo for gene therapy uses.
  • Non-viral vector delivery systems for delivering polynucleotides to cells include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
  • Viral vector delivery systems for delivery of the ZFNs include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • Methods of non-viral delivery of nucleic acids in vivo or ex vivo include electroporation, lipofection (see, U.S. Pat. Nos. 5,049,386; 4,946,787 and commercially available reagents such as TransfectamTM and LipofectinTM), microinjection, biolistics, virosomes, liposomes (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al. Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem.
  • nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.) and BTX Molecular Delivery Systems (Holliston, Mass.).
  • adenoviral based systems can be used.
  • Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and high levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system.
  • Adeno-associated virus (“AAV”) vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S.
  • At least six viral vector approaches are currently available for gene transfer in clinical trials, which utilize approaches that involve complementation of defective vectors by genes inserted into helper cell lines to generate the transducing agent.
  • pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn et al., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138 (1997)).
  • PA317/pLASN was the first therapeutic vector used in a gene therapy trial. (Blaese et al., Science 270:475-480 (1995)). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors. (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoff et al., Hum. Gene Ther. 1:111-2 (1997).
  • Recombinant adeno-associated virus vectors are a promising alternative gene delivery systems based on the defective and nonpathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from a plasmid that retains only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system. (Wagner et al.; Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther. 9:748-55 (1996)).
  • Ad Replication-deficient recombinant adenoviral vectors
  • Ad can be produced at high titer and readily infect a number of different cell types.
  • Most adenovirus vectors are engineered such that a transgene replaces the Ad E1a, E1b, and/or E3 genes; subsequently the replication defective vector is propagated in human 293 cells that supply deleted gene function in trans.
  • Ad vectors can transduce multiple types of tissues in vivo, including nondividing, differentiated cells such as those found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity.
  • Ad vector An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et al., Infection 24:1 5-10 (1996); Sterman et al. Hum. Gene Ther. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).
  • Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and ⁇ 2 cells or PA317 cells, which package retrovirus.
  • Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host (if applicable), other viral sequences being replaced by an expression cassette encoding the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line.
  • AAV vectors used in gene therapy typically only possess inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and integration into the host genome.
  • ITR inverted terminal repeat
  • Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
  • the cell line is also infected with adenovirus as a helper.
  • the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
  • the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.
  • a viral vector can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus.
  • the ligand is chosen to have affinity for a receptor known to be present on the cell type of interest. For example, Han et al., Proc. Natl. Acad. Sci.
  • Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor.
  • This principle can be extended to other virus-target cell pairs, in which the target cell expresses a receptor and the virus expresses a fusion protein comprising a ligand for the cell-surface receptor.
  • filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor.
  • Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below.
  • vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
  • Ex vivo cell transfection for diagnostics, research, or for gene therapy is well known to those of skill in the art.
  • cells are isolated from the subject organism, transfected with a ZFP nucleic acid (gene or cDNA) and exogenous sequence, and re-infused back into the subject organism (e.g., patient).
  • a ZFP nucleic acid gene or cDNA
  • Various cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et al., Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) and the references cited therein for a discussion of how to isolate and culture cells from patients).
  • stem cells are used in ex vivo procedures for cell transfection and gene therapy.
  • the advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow.
  • Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such a GM-CSF, IFN- ⁇ and TNF- ⁇ are known (see Inaba et al., J. Exp. Med. 176:1693-1702 (1992)).
  • Stem cells are isolated for transduction and differentiation using known methods. For example, stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD8+ (T cells), CD45+ (panb cells), GR-1 (granulocytes), and lad (differentiated antigen presenting cells) (see Inaba et al., J. Exp. Med. 176:1693-1702 (1992)).
  • T cells CD4+ and CD8+
  • CD45+ panb cells
  • GR-1 granulocytes
  • lad differentiated antigen presenting cells
  • the cell to be used is an oocyte.
  • cells derived from model organisms may be used. These can include cells derived from xenopus, insect cells (e.g., drosophilia) and nematode cells.
  • Vectors comprising nucleic acids as described herein can also be administered directly to an organism for transduction of cells in vivo.
  • naked DNA can be administered.
  • Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • Vectors useful for introduction of transgenes into hematopoietic stem cells include adenovirus Type 35.
  • Vectors suitable for introduction of transgenes into immune cells include non-integrating lentivirus vectors. See, for example, Ory et al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al. (1998) J. Virol. 72:8463-8471; Zuffery et al. (1998) J. Virol. 72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions available, as described below (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).
  • one or more of the ZFN fusion proteins can be also be introduced into the cell as polypeptides using methods described for example in WO 2007/014275.
  • protein delivery vehicles include, “membrane translocation polypeptides,” for example peptide have amphiphilic or hydrophobic amino acid subsequences that have the ability to act as membrane-translocating carriers, toxin molecules, liposomes and liposome derivatives such as immunoliposomes (including targeted liposomes).
  • ZFPs and expression vectors encoding ZFPs can be administered directly to the patient for targeted cleavage integration into the PPP1R12C locus for therapeutic or prophylactic applications, for example, cancer, ischemia, diabetic retinopathy, macular degeneration, rheumatoid arthritis, psoriasis, HIV infection, sickle cell anemia, Alzheimer's disease, muscular dystrophy, neurodegenerative diseases, vascular disease, cystic fibrosis, stroke, and the like.
  • Administration of therapeutically effective amounts is by any of the routes normally used for introducing ZFP into ultimate contact with the tissue to be treated.
  • the ZFPs are administered in any suitable manner, preferably with pharmaceutically acceptable carriers. Suitable methods of administering such modulators are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions that are available (see, e.g., Remington's Pharmaceutical Sciences, 17 th ed. 1985)).
  • the ZFPs can be made into aerosol formulations (i.e., they can be “nebulized”) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the disclosed compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally.
  • the formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Transformed plant cells which are produced by any of the above plant cell transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype.
  • Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences.
  • Plant regeneration from cultured protoplasts is described in Evans, et al., “Protoplasts Isolation and Culture” in Handbook of Plant Cell Culture , pp. 124-176, Macmillian Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts , pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, pollens, embryos or parts thereof. Such regeneration techniques are described generally in Klee et al (1987) Ann. Rev. of Plant Phys. 38:467-486.
  • Nucleic acids introduced into a plant cell can be used to confer desired traits on essentially any plant.
  • a wide variety of plants and plant cell systems may be engineered for the desired physiological and agronomic characteristics described herein using the nucleic acid constructs of the present disclosure and the various transformation methods mentioned above.
  • target plants and plant cells for engineering include, but are not limited to, those monocotyledonous and dicotyledonous plants, such as crops including grain crops (e.g., wheat, maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear, strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops (e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g., lettuce, spinach); flowering plants (e.g., petunia, rose, chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plants used in phytoremediation (e.g., heavy metal accumulating plants); oil crops (e.g., sunflower, rape seed) and plants used for experimental purposes (e.g., Arabidopsis).
  • crops including grain crops e.g., wheat, maize, rice, millet, barley
  • the disclosed methods and compositions have use over a broad range of plants, including, but not limited to, species from the genera Asparagus, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucurbita, Daucus, Erigeron, Glycine, Gossypium, Hordeum, Lactuca, Lolium, Lycopersicon, Malus, Manihot, Nicotiana, Orychophragmus, Oryza, Persea, Phaseolus, Pisum, Pyrus, Prunus, Raphanus, Secale, Solanum, Sorghum, Triticum, Vitis, Vigna, and Zea.
  • the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
  • a transformed plant cell, callus, tissue or plant may be identified and isolated by selecting or screening the engineered plant material for traits encoded by the marker genes present on the transforming DNA. For instance, selection may be performed by growing the engineered plant material on media containing an inhibitory amount of the antibiotic or herbicide to which the transforming gene construct confers resistance. Further, transformed plants and plant cells may also be identified by screening for the activities of any visible marker genes (e.g., the ⁇ -glucuronidase, luciferase, B or C1 genes) that may be present on the recombinant nucleic acid constructs. Such selection and screening methodologies are well known to those skilled in the art.
  • any visible marker genes e.g., the ⁇ -glucuronidase, luciferase, B or C1 genes
  • Physical and biochemical methods also may be used to identify plant or plant cell transformants containing inserted gene constructs. These methods include but are not limited to: 1) Southern analysis or PCR amplification for detecting and determining the structure of the recombinant DNA insert; 2) Northern blot, S1 RNase protection, primer-extension or reverse transcriptase-PCR amplification for detecting and examining RNA transcripts of the gene constructs; 3) enzymatic assays for detecting enzyme or ribozyme activity, where such gene products are encoded by the gene construct; 4) protein gel electrophoresis, Western blot techniques, immunoprecipitation, or enzyme-linked immunoassays, where the gene construct products are proteins.
  • the present disclosure also encompasses seeds of the transgenic plants described above wherein the seed has the transgene or gene construct.
  • the present disclosure further encompasses the progeny, clones, cell lines or cells of the transgenic plants described above wherein said progeny, clone, cell line or cell has the transgene or gene construct.
  • Linear donor constructs with homology arms of 50, 75 or 100 base pairs flanking a sequence encoding a protein of interest were designed and constructed as follows.
  • Donor constructs included homology arms contained within the PPP1R12C locus (also referred to as AAVS1 or p84 site) or within the endogenous IL2R ⁇ locus. See, U.S. Provisional Application No. 60/926,322, filed Apr. 26, 2007 for a description of the PPP1R12C locus, incorporated by reference in its entirety herein.
  • Donor constructs were prepared by PCR using primers with 50, 75 or 100 base pairs of homology to the genomic target (PPP1R12C or IL2R ⁇ ).
  • the templates used for these PCRs were plasmid molecules containing two long (approx. 750 bp) fragments homologous to the genomic target, flanking a GFP construct (GFP constructs are elaborated upon in sections 0139 for AAVS1 and 0140 for IL2R ⁇ ).
  • the primers were constructed to include phosphorothioate phosphodiester bonds at the first and second phosphodiester bonds of the 5′ ends of the primers to protect the linear donor from exonucleolytic degradation.
  • Phosphorothioate phosphodiester bonds were introduced using standard techniques, for example as described in Ciafre et al. (1995) Nucleic Acids Res. 23(20):4134-42 and Johansson et al. (2002) Vaccine 20(27-28):3379-88.
  • donor constructs can be prepared by PCR as shown schematically in FIG. 1 .
  • the donors can be made by PCR using a primer with a 50, 75 or 100 base pair 5′ portion homologous to the genomic target (PPP1R12C or IL2R ⁇ ) and a 15-30 base pair portion identical to the open reading frame (ORF) of interest.
  • the primers can be constructed to include phosphorothioate phosphodiester bonds at the first and second phosphodiester bonds of the 5′ ends of the primers to protect the linear donor from exonucleolytic degradation. Phosphorothioate phosphodiester bonds can be introduced using standard techniques, for example as described in Ciafre et al. (1995) Nucleic Acids Res. 23(20):4134-42 and Johansson et al. (2002) Vaccine 20(27-28):3379-88.
  • PCR primers for constructs containing 50, 75 and 100 base pair homology arms to PPP1R12C are shown in Table 1 and PCR primers for constructs containing 50 base pair homology arms to IL2R ⁇ are shown in Table 2.
  • FIGS. 2 , 3 and 4 show donor molecules targeted to PPP1R12C (AAVS1).
  • FIG. 2 shows a linear donor molecule (SEQ ID NO:1) targeted to AAVS1 and having homology arms of 100 base pairs and referred to as AAVS1 100 bp HA donor.
  • the left homology arm of AAVS1 100 bp HA extends from nucleotides 1 to 100 (lowercase, underlined); an SA site extends from nucleotides 107 to 132 (lowercase, bold); a sequence encoding a 2A peptide from nucleotides 141 to 212 (uppercase, no underlining); a sequence encoding green fluorescent protein (GFP) poly(A) extends from nucleotides 219 to 1,215 (uppercase, underlined); and a right homology arm extends from nucleotides 1235 to 1334 (lowercase, underlined).
  • GFP green fluorescent protein
  • FIG. 3 shows a linear donor molecule (SEQ ID NO:2) having homology arms of 75 base pairs and designated AAVS1 75 bp HA.
  • the left homology arm extends from nucleotides 1 to 75 (lowercase, underlined); an SA site extends from nucleotides 82 to 107 (lowercase, bold); a sequence encoding a 2A peptide from nucleotides 116 to 187 (uppercase, no underlining); a sequence encoding GFP poly(A) extends from nucleotides 194 to 1,190 (uppercase, underlined); and a right homology arm extends from nucleotides 1210 to 1284 (lowercase, underlined).
  • FIG. 4 shows a linear donor molecule (SEQ ID NO:3) having homology arms of 50 base pairs and designated AAVS1 50 bp HA.
  • AAVS1 50 bp HA comprises a left homology arm from nucleotides 1 to 50 (lowercase, underlined); an SA site from nucleotides 57 to 82 (lowercase, bold); a sequence encoding a 2A peptide from nucleotides 91 to 162 (uppercase, no underlining); a sequence encoding GFP poly(A) from nucleotides 169 to 1,165 (uppercase, underlined); and a right homology arm from nucleotides 1,185 to 1,234 (lowercase, underlined).
  • the sequence of a donor molecule for IL2R ⁇ is shown in FIG. 5 (SEQ ID NO:4).
  • This molecule comprises homology arms of 50 base pairs (left homology arm from nucleotides 1 to 50 (lowercase, underlined) and right homology arm from nucleotides 1,639 to 1,688 (lower, underlined)).
  • the IL2R ⁇ 50 bp HA donor molecule also comprises an hPGK promoter sequence from nucleotides 79 to 594 (lowercase, bold) and a sequence encoding GFP poly(A) from nucleotides 615 to 1,611 (uppercase, underlined).
  • linear donor constructs having short (50-100 base pair) homology arms To evaluate targeted integration of linear donor constructs having short (50-100 base pair) homology arms, the linear donors and a pair of fusion proteins comprising a zinc finger protein nuclease (ZFNs) as described in U.S. Provisional Application No. 60/926,322, filed Apr. 26, 2007 and shown in Table 3 (DNA target sites indicated in uppercase letters; non-contacted nucleotides indicated in lowercase), were transfected into K562 cells using the AmaxaTM Nucleofection kit as shown in Table 4.
  • ZFNs zinc finger protein nuclease
  • the SA-2A-GFP-pA donor refers to the 1,647 bp circular donor fragment described in U.S. Provisional Application No. 60/926,322, corresponding to positions 60318104-60319750 of PPP1R12C.
  • TI rate of targeted integration
  • Results of PCR and Southern blotting are shown in FIG. 6 and FIG. 7 , respectively.
  • the top of each lane is marked with the sample number (left column, Table 4) and the percent of chromosomes modified by is listed below each lane.
  • Results are shown in Table 5 and FIG. 8 and confirm that the GFP ORF of the linear donor sequences was integrated into the genome.
  • linear donor constructs with short homology arms can be used to efficiently transfer a sequence encoding a polypeptide of interest to a specified genomic location.
  • the linear donor constructs described herein are rapidly generated by PCR using a plasmid template and can be protected from exonucleolytic degradation using phosphorothioate modification.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Saccharide Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
US12/386,059 2005-07-26 2009-04-13 Linear donor constructs for targeted integration Abandoned US20090263900A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US12/386,059 US20090263900A1 (en) 2008-04-14 2009-04-13 Linear donor constructs for targeted integration
US13/134,766 US9045763B2 (en) 2005-07-26 2011-06-16 Linear donor constructs for targeted integration
US14/699,908 US9376685B2 (en) 2005-07-26 2015-04-29 Linear donor constructs for targeted integration
US15/146,276 US9765360B2 (en) 2005-07-26 2016-05-04 Linear donor constructs for targeted integration

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US12404708P 2008-04-14 2008-04-14
US12/386,059 US20090263900A1 (en) 2008-04-14 2009-04-13 Linear donor constructs for targeted integration

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
US11/493,423 Continuation-In-Part US20070134796A1 (en) 2003-08-08 2006-07-26 Targeted integration and expression of exogenous nucleic acid sequences
US13/134,766 Continuation US9045763B2 (en) 2005-07-26 2011-06-16 Linear donor constructs for targeted integration

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US13/134,766 Continuation US9045763B2 (en) 2005-07-26 2011-06-16 Linear donor constructs for targeted integration

Publications (1)

Publication Number Publication Date
US20090263900A1 true US20090263900A1 (en) 2009-10-22

Family

ID=41095228

Family Applications (4)

Application Number Title Priority Date Filing Date
US12/386,059 Abandoned US20090263900A1 (en) 2005-07-26 2009-04-13 Linear donor constructs for targeted integration
US13/134,766 Active 2027-11-26 US9045763B2 (en) 2005-07-26 2011-06-16 Linear donor constructs for targeted integration
US14/699,908 Active US9376685B2 (en) 2005-07-26 2015-04-29 Linear donor constructs for targeted integration
US15/146,276 Active US9765360B2 (en) 2005-07-26 2016-05-04 Linear donor constructs for targeted integration

Family Applications After (3)

Application Number Title Priority Date Filing Date
US13/134,766 Active 2027-11-26 US9045763B2 (en) 2005-07-26 2011-06-16 Linear donor constructs for targeted integration
US14/699,908 Active US9376685B2 (en) 2005-07-26 2015-04-29 Linear donor constructs for targeted integration
US15/146,276 Active US9765360B2 (en) 2005-07-26 2016-05-04 Linear donor constructs for targeted integration

Country Status (8)

Country Link
US (4) US20090263900A1 (ja)
EP (1) EP2281050B1 (ja)
JP (3) JP2011518555A (ja)
AU (1) AU2009238629C1 (ja)
CA (1) CA2720903C (ja)
ES (1) ES2459880T3 (ja)
HK (1) HK1151065A1 (ja)
WO (1) WO2009131632A1 (ja)

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100047805A1 (en) * 2008-08-22 2010-02-25 Sangamo Biosciences, Inc. Methods and compositions for targeted single-stranded cleavage and targeted integration
US20100199389A1 (en) * 2008-12-17 2010-08-05 Dow Agrosciences Llc Targeted integration into the Zp15 locus
US20110041195A1 (en) * 2009-08-11 2011-02-17 Sangamo Biosciences, Inc. Organisms homozygous for targeted modification
US20110189775A1 (en) * 2010-01-22 2011-08-04 Dow Agrosciences Llc Targeted genomic alteration
WO2011100058A1 (en) 2010-02-09 2011-08-18 Sangamo Biosciences, Inc. Targeted genomic modification with partially single-stranded donor molecules
WO2012047598A1 (en) 2010-09-27 2012-04-12 Sangamo Biosciences, Inc. Methods and compositions for inhibiting viral entry into cells
WO2013016446A2 (en) 2011-07-25 2013-01-31 Sangamo Biosciences, Inc. Methods and compositions for alteration of a cystic fibrosis transmembrane conductance regulator (cftr) gene
WO2013166315A1 (en) 2012-05-02 2013-11-07 Dow Agrosciences Llc Targeted modification of malate dehydrogenase
WO2013169802A1 (en) 2012-05-07 2013-11-14 Sangamo Biosciences, Inc. Methods and compositions for nuclease-mediated targeted integration of transgenes
WO2014039702A2 (en) 2012-09-07 2014-03-13 Dow Agrosciences Llc Fad2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
WO2014165612A2 (en) 2013-04-05 2014-10-09 Dow Agrosciences Llc Methods and compositions for integration of an exogenous sequence within the genome of plants
WO2015017866A1 (en) 2013-08-02 2015-02-05 Enevolv, Inc. Processes and host cells for genome, pathway, and biomolecular engineering
US9175280B2 (en) 2010-10-12 2015-11-03 Sangamo Biosciences, Inc. Methods and compositions for treating hemophilia B
WO2018035158A1 (en) 2016-08-15 2018-02-22 Enevolv, Inc. Cell-free sensor systems
US9909131B2 (en) 2013-11-04 2018-03-06 Dow Agrosciences Llc Optimal soybean loci
US9914930B2 (en) 2012-09-07 2018-03-13 Dow Agrosciences Llc FAD3 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
US10072062B2 (en) * 2010-07-21 2018-09-11 Sangamo Therapeutics, Inc. Methods and compositions for modification of a HLA locus
US10093940B2 (en) 2013-11-04 2018-10-09 Dow Agrosciences Llc Optimal maize loci
US10233465B2 (en) 2013-11-04 2019-03-19 Dow Agrosciences Llc Optimal soybean loci
US10273493B2 (en) 2013-11-04 2019-04-30 Dow Agrosciences Llc Optimal maize loci
US10415046B2 (en) 2012-12-13 2019-09-17 Dow Agrosciences Llc Precision gene targeting to a particular locus in maize
WO2019178613A1 (en) 2018-03-16 2019-09-19 Immusoft Corporation B cells genetically engineered to secrete follistatin and methods of using the same to treat follistatin-related diseases, conditions, disorders and to enhance muscle growth and strength
US10612041B2 (en) 2014-03-21 2020-04-07 The Board Of Trustees Of The Leland Stanford Junior University Genome editing without nucleases
US10774338B2 (en) 2014-01-16 2020-09-15 The Regents Of The University Of California Generation of heritable chimeric plant traits
EP3741852A2 (en) 2014-05-02 2020-11-25 Iontas Ltd Preparation of libraries of protein variants expressed in eukaryotic cells and use for selecting binding molecules
US11352603B2 (en) 2013-03-14 2022-06-07 Immusoft Corporation Methods for in vitro memory B cell differentiation and transduction with VSV-G pseudotyped viral vectors
US11655275B2 (en) 2017-05-03 2023-05-23 Sangamo Therapeutics, Inc. Methods and compositions for modification of a cystic fibrosis transmembrane conductance regulator (CFTR) gene
US12031146B2 (en) 2021-05-17 2024-07-09 The Board Of Trustees Of The Leland Stanford Junior University Genome editing without nucleases

Families Citing this family (95)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120196370A1 (en) 2010-12-03 2012-08-02 Fyodor Urnov Methods and compositions for targeted genomic deletion
EP2281050B1 (en) * 2008-04-14 2014-04-02 Sangamo BioSciences, Inc. Linear donor constructs for targeted integration
WO2011064736A1 (en) 2009-11-27 2011-06-03 Basf Plant Science Company Gmbh Optimized endonucleases and uses thereof
AU2010325563B2 (en) * 2009-11-27 2017-02-02 Basf Plant Science Company Gmbh Chimeric endonucleases and uses thereof
ES2565216T3 (es) * 2010-04-13 2016-04-01 Sigma-Aldrich Co. Llc Métodos de generación de proteínas marcadas endógenamente
BR112013001685B1 (pt) 2010-07-23 2021-10-13 Sigma-Aldrich Co. Llc Método para editar pelo menos uma sequência cromossômica endógena em uma célula pela inserção de uma sequência na sequência cromossômica
WO2012165270A1 (ja) * 2011-05-27 2012-12-06 公立大学法人横浜市立大学 遺伝子ターゲティングベクター、その作製方法及び利用方法
US10648001B2 (en) 2012-07-11 2020-05-12 Sangamo Therapeutics, Inc. Method of treating mucopolysaccharidosis type I or II
EP2872154B1 (en) 2012-07-11 2017-05-31 Sangamo BioSciences, Inc. Methods and compositions for delivery of biologics
HUE030799T2 (en) 2012-07-11 2017-06-28 Sangamo Biosciences Inc Methods and preparations for the treatment of lysosomal storage diseases
AU2013308770B2 (en) 2012-08-29 2019-01-17 Sangamo Therapeutics, Inc. Methods and compositions for treatment of a genetic condition
EP2906684B8 (en) 2012-10-10 2020-09-02 Sangamo Therapeutics, Inc. T cell modifying compounds and uses thereof
KR102596302B1 (ko) * 2012-11-01 2023-11-01 팩터 바이오사이언스 인크. 세포에서 단백질을 발현시키는 방법들과 생성물들
MX2015006220A (es) * 2012-11-20 2016-07-20 Simplot Co J R Insercion de adn de tranferencia mediada por efectores tipo activadores de la transcripción (tal).
AU2013355327A1 (en) 2012-12-05 2015-06-11 Sangamo Therapeutics, Inc. Methods and compositions for regulation of metabolic disorders
WO2014130955A1 (en) 2013-02-25 2014-08-28 Sangamo Biosciences, Inc. Methods and compositions for enhancing nuclease-mediated gene disruption
RS63188B1 (sr) * 2013-03-15 2022-06-30 Cibus Us Llc Ciljana genska modifikacija primenom popravke gena posredovane oligonukleotidom
EP2975942B1 (en) 2013-03-21 2018-08-08 Sangamo Therapeutics, Inc. Targeted disruption of t cell receptor genes using engineered zinc finger protein nucleases
US9771403B2 (en) 2013-12-09 2017-09-26 Sangamo Therapeutics, Inc. Methods and compositions for treating hemophilia
US10072066B2 (en) 2014-02-03 2018-09-11 Sangamo Therapeutics, Inc. Methods and compositions for treatment of a beta thalessemia
US10370680B2 (en) 2014-02-24 2019-08-06 Sangamo Therapeutics, Inc. Method of treating factor IX deficiency using nuclease-mediated targeted integration
US9624498B2 (en) 2014-03-18 2017-04-18 Sangamo Biosciences, Inc. Methods and compositions for regulation of zinc finger protein expression
WO2015175642A2 (en) 2014-05-13 2015-11-19 Sangamo Biosciences, Inc. Methods and compositions for prevention or treatment of a disease
NZ728159A (en) 2014-07-14 2018-08-31 Univ Washington State Nanos2 knock-out that ablates germline cells
WO2016014837A1 (en) 2014-07-25 2016-01-28 Sangamo Biosciences, Inc. Gene editing for hiv gene therapy
WO2016019144A2 (en) 2014-07-30 2016-02-04 Sangamo Biosciences, Inc. Gene correction of scid-related genes in hematopoietic stem and progenitor cells
WO2016025759A1 (en) * 2014-08-14 2016-02-18 Shen Yuelei Dna knock-in system
EP3878948A1 (en) 2014-09-16 2021-09-15 Sangamo Therapeutics, Inc. Methods and compositions for nuclease-mediated genome engineering and correction in hematopoietic stem cells
US10889834B2 (en) 2014-12-15 2021-01-12 Sangamo Therapeutics, Inc. Methods and compositions for enhancing targeted transgene integration
WO2016118726A2 (en) 2015-01-21 2016-07-28 Sangamo Biosciences, Inc. Methods and compositions for identification of highly specific nucleases
US10179918B2 (en) 2015-05-07 2019-01-15 Sangamo Therapeutics, Inc. Methods and compositions for increasing transgene activity
IL284707B2 (en) 2015-05-12 2024-01-01 Sangamo Therapeutics Inc Regulation of nuclease-mediated gene expression
US9957501B2 (en) 2015-06-18 2018-05-01 Sangamo Therapeutics, Inc. Nuclease-mediated regulation of gene expression
EP3322297A4 (en) 2015-07-13 2019-04-17 Sangamo Therapeutics, Inc. RELEASE METHOD AND COMPOSITIONS FOR NUCLEASE-DERIVED GENOMINE ENGINEERING
CN108472314A (zh) 2015-07-31 2018-08-31 明尼苏达大学董事会 修饰的细胞和治疗方法
CN109475107A (zh) 2015-08-06 2019-03-15 密苏里大学管理者 具有修饰的cd163基因的抗病原体动物
WO2017029833A1 (ja) * 2015-08-20 2017-02-23 大学共同利用機関法人情報・システム研究機構 動物細胞ゲノム部位特異的外来ｄｎａ挿入方法及び前記挿入方法を用いて得られる細胞
CN108348576B (zh) 2015-09-23 2022-01-11 桑格摩生物治疗股份有限公司 Htt阻抑物及其用途
EP4144844A1 (en) * 2015-10-12 2023-03-08 DuPont US Holding, LLC Protected dna templates for gene modification and increased homologous recombination in cells and methods of use
PE20181206A1 (es) 2015-10-28 2018-07-23 Sangamo Therapeutics Inc Construcciones especificas del higado, casetes de expresion del factor viii y metodos de uso de estos
WO2017083092A1 (en) 2015-11-10 2017-05-18 Dow Agrosciences Llc Methods and systems for predicting the risk of transgene silencing
EP3380622A4 (en) 2015-11-23 2019-08-07 Sangamo Therapeutics, Inc. METHODS AND COMPOSITIONS FOR MODIFYING IMMUNITY
EA201891212A1 (ru) 2015-12-18 2019-01-31 Сангамо Терапьютикс, Инк. Адресная дезорганизация клеточного рецептора гкгс
CA3008413A1 (en) 2015-12-18 2017-06-22 Sangamo Therapeutics, Inc. Targeted disruption of the t cell receptor
SG11201805680SA (en) 2016-01-15 2018-07-30 Sangamo Therapeutics Inc Methods and compositions for the treatment of neurologic disease
EP3411056A4 (en) 2016-02-02 2019-10-02 Sangamo Therapeutics, Inc. COMPOUNDS FOR NETWORKING DNA BINDING DOMAINS AND SPLITTING DOMAINS
ES2634802B1 (es) * 2016-03-28 2018-07-03 Victor Manuel GALVEZ JEREZ Molécula de ácido nucleico y método para modificar de forma bialélica un gen diana o locus presente en el material genético de una célula
EP3872177A1 (en) * 2016-05-27 2021-09-01 Life Technologies Corporation Compositions and methods for enhancing homologous recombination
PL3504229T3 (pl) 2016-08-24 2022-04-19 Sangamo Therapeutics, Inc. Regulacja ekspresji genów przy użyciu nukleaz poddanych inżynierii
EP3504327B1 (en) 2016-08-24 2021-10-06 Sangamo Therapeutics, Inc. Engineered target specific nucleases
AU2017324462B2 (en) 2016-09-07 2024-03-21 Sangamo Therapeutics, Inc. Modulation of liver genes
AU2017336094A1 (en) 2016-09-29 2019-04-18 Immunitybio, Inc. HLA class I-deficient NK-92 cells with decreased immunogenicity
BR112019007210A2 (pt) 2016-10-20 2019-08-13 Sangamo Therapeutics Inc métodos e composições para o tratamento da doença de fabry
WO2018081775A1 (en) 2016-10-31 2018-05-03 Sangamo Therapeutics, Inc. Gene correction of scid-related genes in hematopoietic stem and progenitor cells
EP3546575A4 (en) * 2016-11-28 2019-12-11 Osaka University GENOMEDITIERUNGSVERFAHREN
CN110168078B (zh) 2017-01-06 2024-05-14 免疫生物公司 具有降低的cd96/tigit表达的遗传修饰的nk-92细胞
KR102637590B1 (ko) 2017-02-17 2024-02-15 데날리 테라퓨틱스 인크. 트란스페린 수용체 유전자삽입 모델
IL301115A (en) 2017-04-28 2023-05-01 Acuitas Therapeutics Inc New lipid carbonyl and lipid nanoparticle formulations for delivery of nucleic acids
US11512287B2 (en) 2017-06-16 2022-11-29 Sangamo Therapeutics, Inc. Targeted disruption of T cell and/or HLA receptors
CA3079748A1 (en) 2017-11-09 2019-05-16 Sangamo Therapeutics, Inc. Genetic modification of cytokine inducible sh2-containing protein (cish) gene
CA3089587A1 (en) 2018-02-08 2019-08-15 Sangamo Therapeutics, Inc. Engineered target specific nucleases
MX2020010461A (es) 2018-04-05 2021-01-15 Juno Therapeutics Inc Linfocitos t que expresan un receptor recombinante, polinucleotidos y metodos relacionados.
BR112020020245A2 (pt) 2018-04-05 2021-04-06 Editas Medicine, Inc. Métodos de produzir células expressando um receptor recombinante e composições relacionadas
WO2019204457A1 (en) 2018-04-18 2019-10-24 Sangamo Therapeutics, Inc. Zinc finger protein compositions for modulation of huntingtin (htt)
US11690921B2 (en) 2018-05-18 2023-07-04 Sangamo Therapeutics, Inc. Delivery of target specific nucleases
IL280951B1 (en) 2018-08-23 2024-04-01 Sangamo Therapeutics Inc Engineered target-specific base editors
CA3111711A1 (en) 2018-09-18 2020-03-26 Sangamo Therapeutics, Inc. Programmed cell death 1 (pd1) specific nucleases
WO2020069029A1 (en) 2018-09-26 2020-04-02 Emendobio Inc. Novel crispr nucleases
MX2021004276A (es) 2018-10-18 2021-09-08 Intellia Therapeutics Inc Composiciones y metodos para tratar deficiencia de alfa-1 antitripsina.
BR112021007301A2 (pt) 2018-10-18 2021-07-27 Intellia Therapeutics, Inc. composições e métodos para expressar fator ix
CN114207130A (zh) 2018-10-18 2022-03-18 英特利亚治疗股份有限公司 用于从白蛋白基因座进行转基因表达的组合物和方法
BR112021007323A2 (pt) 2018-10-18 2021-07-27 Intellia Therapeutics, Inc. construtos de ácido nucleico e métodos para uso
CN113474328A (zh) 2019-01-11 2021-10-01 爱康泰生治疗公司 用于脂质纳米颗粒递送活性剂的脂质
WO2020149395A1 (ja) * 2019-01-18 2020-07-23 国立大学法人大阪大学 栄養障害型表皮水疱症治療薬
US20220098621A1 (en) 2019-02-05 2022-03-31 Emendobio Inc. Crispr compositions and methods for promoting gene editing of ribosomal protein s19 (rps19) gene
WO2020163307A1 (en) 2019-02-06 2020-08-13 Emendobio Inc. New engineered high fidelity cas9
WO2020223571A1 (en) 2019-05-01 2020-11-05 Juno Therapeutics, Inc. Cells expressing a chimeric receptor from a modified cd247 locus, related polynucleotides and methods
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
WO2021028359A1 (en) 2019-08-09 2021-02-18 Sangamo Therapeutics France Controlled expression of chimeric antigen receptors in t cells
WO2021231661A2 (en) 2020-05-13 2021-11-18 Juno Therapeutics, Inc. Process for producing donor-batched cells expressing a recombinant receptor
KR20230042283A (ko) 2020-06-26 2023-03-28 주노 테라퓨틱스 게엠베하 재조합 수용체를 조건부로 발현하는 조작된 ｔ 세포, 관련된 폴리뉴클레오티드 및 방법
WO2022016070A1 (en) 2020-07-16 2022-01-20 Acuitas Therapeutics, Inc. Cationic lipids for use in lipid nanoparticles
WO2022098787A1 (en) 2020-11-04 2022-05-12 Juno Therapeutics, Inc. Cells expressing a chimeric receptor from a modified invariant cd3 immunoglobulin superfamily chain locus and related polynucleotides and methods
WO2022101641A1 (en) 2020-11-16 2022-05-19 Pig Improvement Company Uk Limited Influenza a-resistant animals having edited anp32 genes
TW202305118A (zh) 2021-03-23 2023-02-01 美商艾歐凡斯生物治療公司 腫瘤浸潤淋巴球之ｃｉｓｈ基因編輯及其在免疫療法中之用途
CA3225985A1 (en) 2021-07-01 2023-01-05 Indapta Therapeutics, Inc. Engineered natural killer (nk) cells and related methods
AU2022379633A1 (en) 2021-10-27 2024-04-11 Regeneron Pharmaceuticals, Inc. Compositions and methods for expressing factor ix for hemophilia b therapy
WO2023081900A1 (en) 2021-11-08 2023-05-11 Juno Therapeutics, Inc. Engineered t cells expressing a recombinant t cell receptor (tcr) and related systems and methods
WO2023105244A1 (en) 2021-12-10 2023-06-15 Pig Improvement Company Uk Limited Editing tmprss2/4 for disease resistance in livestock
WO2023114515A2 (en) 2021-12-17 2023-06-22 Denali Therapeutics Inc. Anti-pilra antibodies, uses thereof, and related methods and reagents
TW202332767A (zh) 2022-02-02 2023-08-16 美商雷傑納榮製藥公司 用於治療龐貝氏症之抗ＴｆＲ：ＧＡＡ及抗ＣＤ６３：ＧＡＡ插入
WO2023212677A2 (en) 2022-04-29 2023-11-02 Regeneron Pharmaceuticals, Inc. Identification of tissue-specific extragenic safe harbors for gene therapy approaches
WO2024007020A1 (en) 2022-06-30 2024-01-04 Indapta Therapeutics, Inc. Combination of engineered natural killer (nk) cells and antibody therapy and related methods
WO2024013514A2 (en) 2022-07-15 2024-01-18 Pig Improvement Company Uk Limited Gene edited livestock animals having coronavirus resistance
WO2024100604A1 (en) 2022-11-09 2024-05-16 Juno Therapeutics Gmbh Methods for manufacturing engineered immune cells

Citations (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5356802A (en) * 1992-04-03 1994-10-18 The Johns Hopkins University Functional domains in flavobacterium okeanokoites (FokI) restriction endonuclease
US5410248A (en) * 1993-12-29 1995-04-25 General Electric Company Method for the simultaneous detection of velocity and acceleration distribution in moving fluids
US5420032A (en) * 1991-12-23 1995-05-30 Universitge Laval Homing endonuclease which originates from chlamydomonas eugametos and recognizes and cleaves a 15, 17 or 19 degenerate double stranded nucleotide sequence
US5436150A (en) * 1992-04-03 1995-07-25 The Johns Hopkins University Functional domains in flavobacterium okeanokoities (foki) restriction endonuclease
US5487994A (en) * 1992-04-03 1996-01-30 The Johns Hopkins University Insertion and deletion mutants of FokI restriction endonuclease
US5789538A (en) * 1995-02-03 1998-08-04 Massachusetts Institute Of Technology Zinc finger proteins with high affinity new DNA binding specificities
US5925523A (en) * 1996-08-23 1999-07-20 President & Fellows Of Harvard College Intraction trap assay, reagents and uses thereof
US6007988A (en) * 1994-08-20 1999-12-28 Medical Research Council Binding proteins for recognition of DNA
US6140466A (en) * 1994-01-18 2000-10-31 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
US6242568B1 (en) * 1994-01-18 2001-06-05 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
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
US6479626B1 (en) * 1998-03-02 2002-11-12 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
US6506559B1 (en) * 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
US6528313B1 (en) * 1989-03-20 2003-03-04 Institut Pasteur Procedure for specific replacement of a copy of a gene present in the recipient genome by the integration of a gene different from that where the integration is made
US6534261B1 (en) * 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US20030232410A1 (en) * 2002-03-21 2003-12-18 Monika Liljedahl Methods and compositions for using zinc finger endonucleases to enhance homologous recombination
US6833252B1 (en) * 1992-05-05 2004-12-21 Institut Pasteur Nucleotide sequence encoding the enzyme I-SecI and the uses thereof
US20050208489A1 (en) * 2002-01-23 2005-09-22 Dana Carroll Targeted chromosomal mutagenasis using zinc finger nucleases
US20060063231A1 (en) * 2004-09-16 2006-03-23 Sangamo Biosciences, Inc. Compositions and methods for protein production
US20060188987A1 (en) * 2003-08-08 2006-08-24 Dmitry Guschin Targeted deletion of cellular DNA sequences
US20070117128A1 (en) * 2005-10-18 2007-05-24 Smith James J Rationally-designed meganucleases with altered sequence specificity and DNA-binding affinity
US20080299580A1 (en) * 2007-04-26 2008-12-04 Sangamo Biosciences, Inc. Targeted integration into the PPP1R12C locus

Family Cites Families (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2681922C (en) 1994-01-18 2012-05-15 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
GB9824544D0 (en) 1998-11-09 1999-01-06 Medical Res Council Screening system
GB9703369D0 (en) 1997-02-18 1997-04-09 Lindqvist Bjorn H Process
GB2338237B (en) 1997-02-18 2001-02-28 Actinova Ltd In vitro peptide or protein expression library
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
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
NZ507451A (en) * 1998-05-07 2003-12-19 Transkaryotic Therapies Inc Genomic sequences upstream of the coding region of the g-csf gene for protein production and delivery and enhancement of g-csf transcription and translation trasfection and infection into mamalian cell
US6794136B1 (en) 2000-11-20 2004-09-21 Sangamo Biosciences, Inc. Iterative optimization in the design of binding proteins
AU2693501A (en) 2000-01-24 2001-07-31 Gendaq Ltd Nucleic acid binding polypeptides characterized by flexible linkers connected nucleic acid binding modules
US20020061512A1 (en) 2000-02-18 2002-05-23 Kim Jin-Soo Zinc finger domains and methods of identifying same
AU2001263155A1 (en) 2000-05-16 2001-11-26 Massachusetts Institute Of Technology Methods and compositions for interaction trap assays
AU2001283377B2 (en) * 2000-08-14 2007-09-13 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Enhanced homologous recombination mediated by lambda recombination proteins
JP2002060786A (ja) 2000-08-23 2002-02-26 Kao Corp 硬質表面用殺菌防汚剤
US7105348B2 (en) * 2000-10-31 2006-09-12 Regeneron Pharmaceuticals, Inc. Methods of modifying eukaryotic cells
US6586251B2 (en) * 2000-10-31 2003-07-01 Regeneron Pharmaceuticals, Inc. Methods of modifying eukaryotic cells
CA2435394C (en) 2001-01-22 2018-01-09 Sangamo Biosciences, Inc. Modified zinc finger binding proteins
GB0108491D0 (en) 2001-04-04 2001-05-23 Gendaq Ltd Engineering zinc fingers
US20040224385A1 (en) 2001-08-20 2004-11-11 Barbas Carlos F Zinc finger binding domains for cnn
US7888121B2 (en) 2003-08-08 2011-02-15 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
EP3222715A1 (en) 2003-08-08 2017-09-27 Sangamo BioSciences, Inc. Methods and compositions for targeted cleavage and recombination
WO2007014275A2 (en) * 2005-07-26 2007-02-01 Sangamo Biosciences, Inc. Targeted integration and expression of exogenous nucleic acid sequences
CN105296527B (zh) * 2006-08-11 2020-11-27 陶氏益农公司 锌指核酸酶介导的同源重组
EP2281050B1 (en) * 2008-04-14 2014-04-02 Sangamo BioSciences, Inc. Linear donor constructs for targeted integration

Patent Citations (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6528313B1 (en) * 1989-03-20 2003-03-04 Institut Pasteur Procedure for specific replacement of a copy of a gene present in the recipient genome by the integration of a gene different from that where the integration is made
US6528314B1 (en) * 1989-03-20 2003-03-04 Institut Pasteur Procedure for specific replacement of a copy of a gene present in the recipient genome by the integration of a gene different from that where the integration is made
US5420032A (en) * 1991-12-23 1995-05-30 Universitge Laval Homing endonuclease which originates from chlamydomonas eugametos and recognizes and cleaves a 15, 17 or 19 degenerate double stranded nucleotide sequence
US5356802A (en) * 1992-04-03 1994-10-18 The Johns Hopkins University Functional domains in flavobacterium okeanokoites (FokI) restriction endonuclease
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
US6833252B1 (en) * 1992-05-05 2004-12-21 Institut Pasteur Nucleotide sequence encoding the enzyme I-SecI and the uses thereof
US5410248A (en) * 1993-12-29 1995-04-25 General Electric Company Method for the simultaneous detection of velocity and acceleration distribution in moving fluids
US6140466A (en) * 1994-01-18 2000-10-31 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
US6242568B1 (en) * 1994-01-18 2001-06-05 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
US6007988A (en) * 1994-08-20 1999-12-28 Medical Research Council Binding proteins for recognition of DNA
US6013453A (en) * 1994-08-20 2000-01-11 Medical Research Council Binding proteins for recognition of DNA
US5789538A (en) * 1995-02-03 1998-08-04 Massachusetts Institute Of Technology Zinc finger proteins with high affinity new DNA binding specificities
US6200759B1 (en) * 1996-08-23 2001-03-13 President And Fellows Of Harvard College Interaction trap assay, reagents and uses thereof
US5925523A (en) * 1996-08-23 1999-07-20 President & Fellows Of Harvard College Intraction trap assay, reagents and uses thereof
US6506559B1 (en) * 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
US6479626B1 (en) * 1998-03-02 2002-11-12 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
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
US6534261B1 (en) * 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US20050208489A1 (en) * 2002-01-23 2005-09-22 Dana Carroll Targeted chromosomal mutagenasis using zinc finger nucleases
US20030232410A1 (en) * 2002-03-21 2003-12-18 Monika Liljedahl Methods and compositions for using zinc finger endonucleases to enhance homologous recombination
US20060188987A1 (en) * 2003-08-08 2006-08-24 Dmitry Guschin Targeted deletion of cellular DNA sequences
US20060063231A1 (en) * 2004-09-16 2006-03-23 Sangamo Biosciences, Inc. Compositions and methods for protein production
US20070117128A1 (en) * 2005-10-18 2007-05-24 Smith James J Rationally-designed meganucleases with altered sequence specificity and DNA-binding affinity
US20080299580A1 (en) * 2007-04-26 2008-12-04 Sangamo Biosciences, Inc. Targeted integration into the PPP1R12C locus

Cited By (74)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9631186B2 (en) 2008-08-22 2017-04-25 Sangamo Biosciences, Inc. Methods and compositions for targeted single-stranded cleavage and targeted integration
US11149321B2 (en) 2008-08-22 2021-10-19 Sangamo Therapeutics, Inc. Methods and compositions for targeted single-stranded cleavage and targeted integration
US8703489B2 (en) * 2008-08-22 2014-04-22 Sangamo Biosciences, Inc. Methods and compositions for targeted single-stranded cleavage and targeted integration
US9200266B2 (en) 2008-08-22 2015-12-01 Sangamo Biosciences, Inc. Methods and compositions for targeted single-stranded cleavage and targeted integration
US10689717B2 (en) 2008-08-22 2020-06-23 Sangamo Therapeutics, Inc. Methods and compositions for targeted single-stranded cleavage and targeted integration
US20100047805A1 (en) * 2008-08-22 2010-02-25 Sangamo Biosciences, Inc. Methods and compositions for targeted single-stranded cleavage and targeted integration
US10113207B2 (en) 2008-08-22 2018-10-30 Sangamo Therapeutics, Inc. Methods and compositions for targeted single-stranded cleavage and targeted integration
US20100199389A1 (en) * 2008-12-17 2010-08-05 Dow Agrosciences Llc Targeted integration into the Zp15 locus
US8329986B2 (en) 2008-12-17 2012-12-11 Dow Agrosciences, Llc Targeted integration into the Zp15 locus
US10827731B2 (en) 2009-08-11 2020-11-10 Sangamo Therapeutics, Inc. Method of inactivating the IPK1 gene in corn
US20110041195A1 (en) * 2009-08-11 2011-02-17 Sangamo Biosciences, Inc. Organisms homozygous for targeted modification
US20110189775A1 (en) * 2010-01-22 2011-08-04 Dow Agrosciences Llc Targeted genomic alteration
US10260062B2 (en) 2010-01-22 2019-04-16 Sangamo Therapeutics, Inc. Targeted genomic alteration
US9970028B2 (en) 2010-02-09 2018-05-15 Sangamo Therapeutics, Inc. Targeted genomic modification with partially single-stranded donor molecules
US9255259B2 (en) 2010-02-09 2016-02-09 Sangamo Biosciences, Inc. Targeted genomic modification with partially single-stranded donor molecules
EP2660318A1 (en) 2010-02-09 2013-11-06 Sangamo BioSciences, Inc. Targeted genomic modification with partially single-stranded donor molecules
US9005973B2 (en) 2010-02-09 2015-04-14 Sangamo Biosciences, Inc. Targeted genomic modification with partially single-stranded donor molecules
US20110207221A1 (en) * 2010-02-09 2011-08-25 Sangamo Biosciences, Inc. Targeted genomic modification with partially single-stranded donor molecules
WO2011100058A1 (en) 2010-02-09 2011-08-18 Sangamo Biosciences, Inc. Targeted genomic modification with partially single-stranded donor molecules
US10858416B2 (en) 2010-07-21 2020-12-08 Sangamo Therapeutics, Inc. Methods and compositions for modification of a HLA locus
US10072062B2 (en) * 2010-07-21 2018-09-11 Sangamo Therapeutics, Inc. Methods and compositions for modification of a HLA locus
US9566352B2 (en) 2010-09-27 2017-02-14 Sangamo Biosciences, Inc. Methods and compositions for inhibiting viral entry into cells
EP3511420A1 (en) 2010-09-27 2019-07-17 Sangamo Therapeutics, Inc. Methods and compositions for inhibiting viral entry into cells
WO2012047598A1 (en) 2010-09-27 2012-04-12 Sangamo Biosciences, Inc. Methods and compositions for inhibiting viral entry into cells
US9175280B2 (en) 2010-10-12 2015-11-03 Sangamo Biosciences, Inc. Methods and compositions for treating hemophilia B
US9629930B2 (en) 2010-10-12 2017-04-25 Sangamo Biosciences, Inc. Methods and compositions for treating hemophilia B
US9161995B2 (en) 2011-07-25 2015-10-20 Sangamo Biosciences, Inc. Methods and compositions for alteration of a cystic fibrosis transmembrane conductance regulator (CFTR) gene
WO2013016446A2 (en) 2011-07-25 2013-01-31 Sangamo Biosciences, Inc. Methods and compositions for alteration of a cystic fibrosis transmembrane conductance regulator (cftr) gene
US9523098B2 (en) 2012-05-02 2016-12-20 Dow Agrosciences Llc Targeted modification of malate dehydrogenase
US11085092B2 (en) 2012-05-02 2021-08-10 Corteva Agriscience Llc Targeted modification of malate dehydrogenase
US10358684B2 (en) 2012-05-02 2019-07-23 Dow Agrosciences Llc Targeted modification of malate dehydrogenase
WO2013166315A1 (en) 2012-05-02 2013-11-07 Dow Agrosciences Llc Targeted modification of malate dehydrogenase
WO2013169802A1 (en) 2012-05-07 2013-11-14 Sangamo Biosciences, Inc. Methods and compositions for nuclease-mediated targeted integration of transgenes
US10174331B2 (en) 2012-05-07 2019-01-08 Sangamo Therapeutics, Inc. Methods and compositions for nuclease-mediated targeted integration of transgenes
EP3404099A1 (en) 2012-09-07 2018-11-21 Dow AgroSciences LLC Fad2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
US10287595B2 (en) 2012-09-07 2019-05-14 Dow Agrosciences Llc Fad2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
US9493779B2 (en) 2012-09-07 2016-11-15 Dow Agrosciences Llc FAD2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
EP3406715A1 (en) 2012-09-07 2018-11-28 Dow AgroSciences LLC Fad3 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
US9963711B2 (en) 2012-09-07 2018-05-08 Sangamo Therapeutics, Inc. FAD2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
EP3431600A1 (en) 2012-09-07 2019-01-23 Dow AgroSciences LLC Fad2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
US10844389B2 (en) 2012-09-07 2020-11-24 Dow Agrosciences Llc FAD2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
US9914930B2 (en) 2012-09-07 2018-03-13 Dow Agrosciences Llc FAD3 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
WO2014039702A2 (en) 2012-09-07 2014-03-13 Dow Agrosciences Llc Fad2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
US10577616B2 (en) 2012-09-07 2020-03-03 Dow Agrosciences Llc FAD2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
WO2014039692A2 (en) 2012-09-07 2014-03-13 Dow Agrosciences Llc Fad2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
US10961540B2 (en) 2012-09-07 2021-03-30 Dow Agrosciences Llc FAD3 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
US10526610B2 (en) 2012-09-07 2020-01-07 Dow Agrosciences Llc FAD3 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
US10415046B2 (en) 2012-12-13 2019-09-17 Dow Agrosciences Llc Precision gene targeting to a particular locus in maize
US11352603B2 (en) 2013-03-14 2022-06-07 Immusoft Corporation Methods for in vitro memory B cell differentiation and transduction with VSV-G pseudotyped viral vectors
WO2014165612A2 (en) 2013-04-05 2014-10-09 Dow Agrosciences Llc Methods and compositions for integration of an exogenous sequence within the genome of plants
US10501748B2 (en) 2013-04-05 2019-12-10 Dow Agrosciences Llc Methods and compositions for integration of an exogenous sequence within the genome of plants
US11198883B2 (en) 2013-04-05 2021-12-14 Dow Agrosciences Llc Methods and compositions for integration of an exogenous sequence within the genome of plants
EP3679785A2 (en) 2013-04-05 2020-07-15 Dow AgroSciences LLC Methods and compositions for integration of an exogenous sequence within the genome of plants
WO2015017866A1 (en) 2013-08-02 2015-02-05 Enevolv, Inc. Processes and host cells for genome, pathway, and biomolecular engineering
US9909131B2 (en) 2013-11-04 2018-03-06 Dow Agrosciences Llc Optimal soybean loci
US10233465B2 (en) 2013-11-04 2019-03-19 Dow Agrosciences Llc Optimal soybean loci
US10093940B2 (en) 2013-11-04 2018-10-09 Dow Agrosciences Llc Optimal maize loci
US11198882B2 (en) 2013-11-04 2021-12-14 Corteva Agriscience Llc Optimal maize loci
US10106804B2 (en) 2013-11-04 2018-10-23 Dow Agrosciences Llc Optimal soybean loci
US11098317B2 (en) 2013-11-04 2021-08-24 Corteva Agriscience Llc Optimal maize loci
US11098316B2 (en) 2013-11-04 2021-08-24 Corteva Agriscience Llc Optimal soybean loci
US11149287B2 (en) 2013-11-04 2021-10-19 Corteva Agriscience Llc Optimal soybean loci
US10273493B2 (en) 2013-11-04 2019-04-30 Dow Agrosciences Llc Optimal maize loci
US10774338B2 (en) 2014-01-16 2020-09-15 The Regents Of The University Of California Generation of heritable chimeric plant traits
US10612041B2 (en) 2014-03-21 2020-04-07 The Board Of Trustees Of The Leland Stanford Junior University Genome editing without nucleases
US11286477B2 (en) 2014-05-02 2022-03-29 Iontas Limited Preparation of libraries of protein variants expressed in eukaryotic cells and use for selecting binding molecules
EP3741852A2 (en) 2014-05-02 2020-11-25 Iontas Ltd Preparation of libraries of protein variants expressed in eukaryotic cells and use for selecting binding molecules
US11912984B2 (en) 2014-05-02 2024-02-27 Iontas Limited Preparation of libraries of protein variants expressed in eukaryotic cells and use for selecting binding molecules
US11926818B2 (en) 2014-05-02 2024-03-12 Iontas Limited Preparation of libraries of protein variants expressed in eukaryotic cells and use for selecting binding molecules
WO2018035158A1 (en) 2016-08-15 2018-02-22 Enevolv, Inc. Cell-free sensor systems
EP4053146A2 (en) 2016-08-15 2022-09-07 enEvolv, Inc. Molecule sensor systems
US11655275B2 (en) 2017-05-03 2023-05-23 Sangamo Therapeutics, Inc. Methods and compositions for modification of a cystic fibrosis transmembrane conductance regulator (CFTR) gene
WO2019178613A1 (en) 2018-03-16 2019-09-19 Immusoft Corporation B cells genetically engineered to secrete follistatin and methods of using the same to treat follistatin-related diseases, conditions, disorders and to enhance muscle growth and strength
US12031146B2 (en) 2021-05-17 2024-07-09 The Board Of Trustees Of The Leland Stanford Junior University Genome editing without nucleases

Also Published As

Publication number Publication date
CA2720903A1 (en) 2009-10-29
JP2016208980A (ja) 2016-12-15
US20160237457A1 (en) 2016-08-18
AU2009238629C1 (en) 2015-04-30
EP2281050A1 (en) 2011-02-09
WO2009131632A1 (en) 2009-10-29
US20150225727A1 (en) 2015-08-13
JP2014221053A (ja) 2014-11-27
US9376685B2 (en) 2016-06-28
HK1151065A1 (en) 2012-01-20
AU2009238629A1 (en) 2009-10-29
CA2720903C (en) 2019-01-15
US9045763B2 (en) 2015-06-02
JP2011518555A (ja) 2011-06-30
JP6081963B2 (ja) 2017-02-15
US9765360B2 (en) 2017-09-19
AU2009238629B2 (en) 2014-10-30
ES2459880T3 (es) 2014-05-12
EP2281050B1 (en) 2014-04-02
US20110281361A1 (en) 2011-11-17

Similar Documents

Publication Publication Date Title
US9765360B2 (en) Linear donor constructs for targeted integration
US11649468B2 (en) Targeted integration into the PPP1R12C locus
US9970028B2 (en) Targeted genomic modification with partially single-stranded donor molecules
CA2910427C (en) Delivery methods and compositions for nuclease-mediated genome engineering
US20060188987A1 (en) Targeted deletion of cellular DNA sequences
AU2015200431A1 (en) Linear Donor Constructs For Targeted Integration

Legal Events

Date Code Title Description
AS Assignment

Owner name: SANGAMO BIOSCIENCES, INC., CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:DEKELVER, RUSSELL;GREGORY, PHILIP D.;HOLMES, MICHAEL C.;AND OTHERS;REEL/FRAME:022834/0353;SIGNING DATES FROM 20090507 TO 20090512

STCB Information on status: application discontinuation

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

AS Assignment

Owner name: SANGAMO THERAPEUTICS, INC., CALIFORNIA

Free format text: CHANGE OF NAME;ASSIGNOR:SANGAMO BIOSCIENCES, INC.;REEL/FRAME:043297/0470

Effective date: 20170105