US20200291424A1 - Targeted deletion of cellular dna sequences - Google Patents
Targeted deletion of cellular dna sequences Download PDFInfo
- Publication number
- US20200291424A1 US20200291424A1 US16/859,049 US202016859049A US2020291424A1 US 20200291424 A1 US20200291424 A1 US 20200291424A1 US 202016859049 A US202016859049 A US 202016859049A US 2020291424 A1 US2020291424 A1 US 2020291424A1
- Authority
- US
- United States
- Prior art keywords
- cleavage
- domain
- cell
- sequence
- dna
- 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.)
- Pending
Links
- 238000012217 deletion Methods 0.000 title claims abstract description 72
- 230000037430 deletion Effects 0.000 title claims abstract description 72
- 108091092356 cellular DNA Proteins 0.000 title description 4
- 238000003776 cleavage reaction Methods 0.000 claims abstract description 278
- 230000007017 scission Effects 0.000 claims abstract description 272
- 238000000034 method Methods 0.000 claims abstract description 162
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 110
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 110
- 108090000623 proteins and genes Proteins 0.000 claims description 198
- 239000002773 nucleotide Substances 0.000 claims description 154
- 125000003729 nucleotide group Chemical group 0.000 claims description 133
- 241000196324 Embryophyta Species 0.000 claims description 65
- 101710163270 Nuclease Proteins 0.000 claims description 31
- 108010017070 Zinc Finger Nucleases Proteins 0.000 claims description 30
- 101710185494 Zinc finger protein Proteins 0.000 claims description 15
- 102100023597 Zinc finger protein 816 Human genes 0.000 claims description 15
- 230000004568 DNA-binding Effects 0.000 claims description 11
- 235000013311 vegetables Nutrition 0.000 claims description 3
- 241000218922 Magnoliophyta Species 0.000 claims description 2
- 235000013399 edible fruits Nutrition 0.000 claims description 2
- 239000004459 forage Substances 0.000 claims description 2
- 244000038559 crop plants Species 0.000 claims 1
- 229910052725 zinc Inorganic materials 0.000 abstract description 127
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 abstract description 126
- 239000011701 zinc Substances 0.000 abstract description 126
- 108020004414 DNA Proteins 0.000 abstract description 122
- 102000040430 polynucleotide Human genes 0.000 abstract description 53
- 108091033319 polynucleotide Proteins 0.000 abstract description 53
- 239000002157 polynucleotide Substances 0.000 abstract description 53
- 239000000203 mixture Substances 0.000 abstract description 38
- 230000001413 cellular effect Effects 0.000 abstract description 34
- 102000053602 DNA Human genes 0.000 abstract description 17
- 230000008263 repair mechanism Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 238
- 230000027455 binding Effects 0.000 description 141
- 102000004169 proteins and genes Human genes 0.000 description 84
- 150000007523 nucleic acids Chemical class 0.000 description 82
- 235000018102 proteins Nutrition 0.000 description 82
- 102000039446 nucleic acids Human genes 0.000 description 74
- 108020004707 nucleic acids Proteins 0.000 description 74
- 230000014509 gene expression Effects 0.000 description 63
- 108090000765 processed proteins & peptides Proteins 0.000 description 60
- 239000013598 vector Substances 0.000 description 60
- 230000004927 fusion Effects 0.000 description 55
- 102000004196 processed proteins & peptides Human genes 0.000 description 50
- 229920001184 polypeptide Polymers 0.000 description 48
- 239000000047 product Substances 0.000 description 43
- 239000013612 plasmid Substances 0.000 description 41
- 238000002744 homologous recombination Methods 0.000 description 35
- 230000006801 homologous recombination Effects 0.000 description 35
- 239000002502 liposome Substances 0.000 description 34
- 108010077544 Chromatin Proteins 0.000 description 32
- 230000003321 amplification Effects 0.000 description 32
- 210000003483 chromatin Anatomy 0.000 description 32
- 238000003199 nucleic acid amplification method Methods 0.000 description 32
- 238000005215 recombination Methods 0.000 description 27
- 230000006798 recombination Effects 0.000 description 27
- 210000000349 chromosome Anatomy 0.000 description 25
- 238000009396 hybridization Methods 0.000 description 24
- 210000001519 tissue Anatomy 0.000 description 24
- 101100438883 Homo sapiens CCR5 gene Proteins 0.000 description 23
- 125000003275 alpha amino acid group Chemical group 0.000 description 22
- 230000000694 effects Effects 0.000 description 21
- 108091008146 restriction endonucleases Proteins 0.000 description 21
- 241000700605 Viruses Species 0.000 description 20
- 230000006870 function Effects 0.000 description 20
- 235000001014 amino acid Nutrition 0.000 description 19
- 230000003612 virological effect Effects 0.000 description 19
- 230000004075 alteration Effects 0.000 description 18
- -1 episomes Chemical class 0.000 description 18
- 230000008569 process Effects 0.000 description 17
- 101710149870 C-C chemokine receptor type 5 Proteins 0.000 description 16
- 150000001413 amino acids Chemical class 0.000 description 16
- 238000001415 gene therapy Methods 0.000 description 16
- 238000012546 transfer Methods 0.000 description 16
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 15
- 108091028043 Nucleic acid sequence Proteins 0.000 description 15
- 238000001890 transfection Methods 0.000 description 15
- 108010042407 Endonucleases Proteins 0.000 description 14
- 238000006471 dimerization reaction Methods 0.000 description 14
- 150000001875 compounds Chemical class 0.000 description 13
- 239000013604 expression vector Substances 0.000 description 13
- 239000012634 fragment Substances 0.000 description 13
- 230000001105 regulatory effect Effects 0.000 description 13
- 239000000539 dimer Substances 0.000 description 12
- 230000035772 mutation Effects 0.000 description 12
- 230000000875 corresponding effect Effects 0.000 description 11
- 238000003780 insertion Methods 0.000 description 11
- 230000037431 insertion Effects 0.000 description 11
- 102000005962 receptors Human genes 0.000 description 11
- 108020003175 receptors Proteins 0.000 description 11
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 10
- 101000721661 Homo sapiens Cellular tumor antigen p53 Proteins 0.000 description 10
- 108700020796 Oncogene Proteins 0.000 description 10
- 108700019146 Transgenes Proteins 0.000 description 10
- 239000000523 sample Substances 0.000 description 10
- 241000701161 unidentified adenovirus Species 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 9
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 9
- 210000001744 T-lymphocyte Anatomy 0.000 description 9
- 125000000539 amino acid group Chemical group 0.000 description 9
- 239000000427 antigen Substances 0.000 description 9
- 108091007433 antigens Proteins 0.000 description 9
- 102000036639 antigens Human genes 0.000 description 9
- 230000002759 chromosomal effect Effects 0.000 description 9
- 238000013461 design Methods 0.000 description 9
- 238000004520 electroporation Methods 0.000 description 9
- 239000005090 green fluorescent protein Substances 0.000 description 9
- 238000001727 in vivo Methods 0.000 description 9
- 230000001404 mediated effect Effects 0.000 description 9
- 239000012528 membrane Substances 0.000 description 9
- 210000004940 nucleus Anatomy 0.000 description 9
- 210000000130 stem cell Anatomy 0.000 description 9
- 230000005945 translocation Effects 0.000 description 9
- 239000013603 viral vector Substances 0.000 description 9
- 108091026890 Coding region Proteins 0.000 description 8
- 102000004533 Endonucleases Human genes 0.000 description 8
- 241000725303 Human immunodeficiency virus Species 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 201000010099 disease Diseases 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 150000002632 lipids Chemical class 0.000 description 8
- 230000008685 targeting Effects 0.000 description 8
- 230000001225 therapeutic effect Effects 0.000 description 8
- 238000013459 approach Methods 0.000 description 7
- 238000003556 assay Methods 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 210000000170 cell membrane Anatomy 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 7
- 238000009472 formulation Methods 0.000 description 7
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 7
- 108020004999 messenger RNA Proteins 0.000 description 7
- 231100000350 mutagenesis Toxicity 0.000 description 7
- 230000010076 replication Effects 0.000 description 7
- 230000002103 transcriptional effect Effects 0.000 description 7
- 230000009466 transformation Effects 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 102100035875 C-C chemokine receptor type 5 Human genes 0.000 description 6
- 101150091887 Ctla4 gene Proteins 0.000 description 6
- 230000007018 DNA scission Effects 0.000 description 6
- 241000702421 Dependoparvovirus Species 0.000 description 6
- 102100031780 Endonuclease Human genes 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 6
- 241000700584 Simplexvirus Species 0.000 description 6
- 108091081024 Start codon Proteins 0.000 description 6
- 230000002068 genetic effect Effects 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 230000003993 interaction Effects 0.000 description 6
- 210000004962 mammalian cell Anatomy 0.000 description 6
- 238000002703 mutagenesis Methods 0.000 description 6
- 230000002018 overexpression Effects 0.000 description 6
- 238000004806 packaging method and process Methods 0.000 description 6
- 210000001938 protoplast Anatomy 0.000 description 6
- 230000001177 retroviral effect Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 230000010337 G2 phase Effects 0.000 description 5
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 5
- 108070000030 Viral receptors Proteins 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 210000004102 animal cell Anatomy 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 238000004422 calculation algorithm Methods 0.000 description 5
- 238000012937 correction Methods 0.000 description 5
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 5
- 230000002950 deficient Effects 0.000 description 5
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 5
- 230000010354 integration Effects 0.000 description 5
- 239000003446 ligand Substances 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 5
- 230000008929 regeneration Effects 0.000 description 5
- 238000011069 regeneration method Methods 0.000 description 5
- 230000008439 repair process Effects 0.000 description 5
- 208000007056 sickle cell anemia Diseases 0.000 description 5
- 238000010561 standard procedure Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 239000003053 toxin Substances 0.000 description 5
- 231100000765 toxin Toxicity 0.000 description 5
- 108700012359 toxins Proteins 0.000 description 5
- 238000010361 transduction Methods 0.000 description 5
- 230000026683 transduction Effects 0.000 description 5
- 230000014621 translational initiation Effects 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 239000013607 AAV vector Substances 0.000 description 4
- 208000030507 AIDS Diseases 0.000 description 4
- 241000238631 Hexapoda Species 0.000 description 4
- 108010033040 Histones Proteins 0.000 description 4
- 241000713666 Lentivirus Species 0.000 description 4
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 4
- 108091061960 Naked DNA Proteins 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 241000713311 Simian immunodeficiency virus Species 0.000 description 4
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 230000003115 biocidal effect Effects 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 230000003197 catalytic effect Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000003623 enhancer Substances 0.000 description 4
- 210000003527 eukaryotic cell Anatomy 0.000 description 4
- 210000005260 human cell Anatomy 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 238000001802 infusion Methods 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000001638 lipofection Methods 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 238000000520 microinjection Methods 0.000 description 4
- 238000010369 molecular cloning Methods 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 4
- 208000011580 syndromic disease Diseases 0.000 description 4
- 238000013519 translation Methods 0.000 description 4
- 241001430294 unidentified retrovirus Species 0.000 description 4
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 3
- 108091007914 CDKs Proteins 0.000 description 3
- 102000014914 Carrier Proteins Human genes 0.000 description 3
- 102000052510 DNA-Binding Proteins Human genes 0.000 description 3
- 102000006947 Histones Human genes 0.000 description 3
- 102000009331 Homeodomain Proteins Human genes 0.000 description 3
- 108010048671 Homeodomain Proteins Proteins 0.000 description 3
- 101100061678 Homo sapiens CTLA4 gene Proteins 0.000 description 3
- 241000701085 Human alphaherpesvirus 3 Species 0.000 description 3
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 3
- 102000011931 Nucleoproteins Human genes 0.000 description 3
- 108010061100 Nucleoproteins Proteins 0.000 description 3
- 108010047956 Nucleosomes Proteins 0.000 description 3
- 241001631646 Papillomaviridae Species 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- 108091027981 Response element Proteins 0.000 description 3
- 238000002105 Southern blotting Methods 0.000 description 3
- 208000036142 Viral infection Diseases 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 230000000735 allogeneic effect Effects 0.000 description 3
- 108091008324 binding proteins Proteins 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 230000022131 cell cycle Effects 0.000 description 3
- 210000003763 chloroplast Anatomy 0.000 description 3
- 239000013611 chromosomal DNA Substances 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 230000000447 dimerizing effect Effects 0.000 description 3
- 230000005782 double-strand break Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000002538 fungal effect Effects 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 238000001502 gel electrophoresis Methods 0.000 description 3
- 230000000415 inactivating effect Effects 0.000 description 3
- 238000001990 intravenous administration Methods 0.000 description 3
- 229920002521 macromolecule Polymers 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 239000002853 nucleic acid probe Substances 0.000 description 3
- 210000001623 nucleosome Anatomy 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 239000008194 pharmaceutical composition Substances 0.000 description 3
- 229920001983 poloxamer Polymers 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 210000001236 prokaryotic cell Anatomy 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 230000009870 specific binding Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 230000009261 transgenic effect Effects 0.000 description 3
- 230000032258 transport Effects 0.000 description 3
- 241000701447 unidentified baculovirus Species 0.000 description 3
- 241000712461 unidentified influenza virus Species 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- 230000009385 viral infection Effects 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 241000589158 Agrobacterium Species 0.000 description 2
- 108700031308 Antennapedia Homeodomain Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000193738 Bacillus anthracis Species 0.000 description 2
- 102100022548 Beta-hexosaminidase subunit alpha Human genes 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 101150017501 CCR5 gene Proteins 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 2
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 2
- 108090000994 Catalytic RNA Proteins 0.000 description 2
- 102000053642 Catalytic RNA Human genes 0.000 description 2
- 102000000844 Cell Surface Receptors Human genes 0.000 description 2
- 108010001857 Cell Surface Receptors Proteins 0.000 description 2
- 206010010099 Combined immunodeficiency Diseases 0.000 description 2
- 241000711573 Coronaviridae Species 0.000 description 2
- 108050006400 Cyclin Proteins 0.000 description 2
- 102000016736 Cyclin Human genes 0.000 description 2
- 201000003883 Cystic fibrosis Diseases 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- 108700020911 DNA-Binding Proteins Proteins 0.000 description 2
- 241000725619 Dengue virus Species 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 108010053187 Diphtheria Toxin Proteins 0.000 description 2
- 102000016607 Diphtheria Toxin Human genes 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000991587 Enterovirus C Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 241000531123 GB virus C Species 0.000 description 2
- 208000015872 Gaucher disease Diseases 0.000 description 2
- 241000713813 Gibbon ape leukemia virus Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 2
- 208000031220 Hemophilia Diseases 0.000 description 2
- 208000009292 Hemophilia A Diseases 0.000 description 2
- 241000711549 Hepacivirus C Species 0.000 description 2
- 241000724675 Hepatitis E virus Species 0.000 description 2
- 241000709721 Hepatovirus A Species 0.000 description 2
- 102000003964 Histone deacetylase Human genes 0.000 description 2
- 108090000353 Histone deacetylase Proteins 0.000 description 2
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 2
- 101000687346 Homo sapiens PR domain zinc finger protein 2 Proteins 0.000 description 2
- 241000700588 Human alphaherpesvirus 1 Species 0.000 description 2
- 241000701806 Human papillomavirus Species 0.000 description 2
- 206010020649 Hyperkeratosis Diseases 0.000 description 2
- 208000026350 Inborn Genetic disease Diseases 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- WZNJWVWKTVETCG-YFKPBYRVSA-N L-mimosine Chemical compound OC(=O)[C@@H](N)CN1C=CC(=O)C(O)=C1 WZNJWVWKTVETCG-YFKPBYRVSA-N 0.000 description 2
- 201000001779 Leukocyte adhesion deficiency Diseases 0.000 description 2
- 108091036060 Linker DNA Proteins 0.000 description 2
- 241000227653 Lycopersicon Species 0.000 description 2
- 241000220225 Malus Species 0.000 description 2
- 241000712079 Measles morbillivirus Species 0.000 description 2
- 208000002678 Mucopolysaccharidoses Diseases 0.000 description 2
- 206010056886 Mucopolysaccharidosis I Diseases 0.000 description 2
- 241000711386 Mumps virus Species 0.000 description 2
- 241000714177 Murine leukemia virus Species 0.000 description 2
- KYRVNWMVYQXFEU-UHFFFAOYSA-N Nocodazole Chemical compound C1=C2NC(NC(=O)OC)=NC2=CC=C1C(=O)C1=CC=CS1 KYRVNWMVYQXFEU-UHFFFAOYSA-N 0.000 description 2
- 238000000636 Northern blotting Methods 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 102000043276 Oncogene Human genes 0.000 description 2
- 241000209094 Oryza Species 0.000 description 2
- 102100024885 PR domain zinc finger protein 2 Human genes 0.000 description 2
- 108010081690 Pertussis Toxin Proteins 0.000 description 2
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 2
- 241000018646 Pinus brutia Species 0.000 description 2
- 235000011613 Pinus brutia Nutrition 0.000 description 2
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 description 2
- 101710182846 Polyhedrin Proteins 0.000 description 2
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N Propane Chemical compound CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 description 2
- 102000001253 Protein Kinase Human genes 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 241000125945 Protoparvovirus Species 0.000 description 2
- 108700033844 Pseudomonas aeruginosa toxA Proteins 0.000 description 2
- 241000220324 Pyrus Species 0.000 description 2
- 108091030071 RNAI Proteins 0.000 description 2
- 241000711798 Rabies lyssavirus Species 0.000 description 2
- 102000053062 Rad52 DNA Repair and Recombination Human genes 0.000 description 2
- 108700031762 Rad52 DNA Repair and Recombination Proteins 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 241000725643 Respiratory syncytial virus Species 0.000 description 2
- 241000710799 Rubella virus Species 0.000 description 2
- 241000607142 Salmonella Species 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 108020004459 Small interfering RNA Proteins 0.000 description 2
- 244000062793 Sorghum vulgare Species 0.000 description 2
- 208000022292 Tay-Sachs disease Diseases 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- 241000700618 Vaccinia virus Species 0.000 description 2
- JXLYSJRDGCGARV-WWYNWVTFSA-N Vinblastine Natural products O=C(O[C@H]1[C@](O)(C(=O)OC)[C@@H]2N(C)c3c(cc(c(OC)c3)[C@]3(C(=O)OC)c4[nH]c5c(c4CCN4C[C@](O)(CC)C[C@H](C3)C4)cccc5)[C@@]32[C@H]2[C@@]1(CC)C=CCN2CC3)C JXLYSJRDGCGARV-WWYNWVTFSA-N 0.000 description 2
- 206010068348 X-linked lymphoproliferative syndrome Diseases 0.000 description 2
- 230000001594 aberrant effect Effects 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000000443 aerosol Substances 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 201000006288 alpha thalassemia Diseases 0.000 description 2
- 102000013529 alpha-Fetoproteins Human genes 0.000 description 2
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 238000000211 autoradiogram Methods 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 108010081355 beta 2-Microglobulin Proteins 0.000 description 2
- 208000005980 beta thalassemia Diseases 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 210000002798 bone marrow cell Anatomy 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000025084 cell cycle arrest Effects 0.000 description 2
- 230000006369 cell cycle progression Effects 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 230000007248 cellular mechanism Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 208000016532 chronic granulomatous disease Diseases 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 210000000172 cytosol Anatomy 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- MWRBNPKJOOWZPW-CLFAGFIQSA-N dioleoyl phosphatidylethanolamine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-CLFAGFIQSA-N 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 238000009510 drug design Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000007824 enzymatic assay Methods 0.000 description 2
- 230000001036 exonucleolytic effect Effects 0.000 description 2
- 230000009368 gene silencing by RNA Effects 0.000 description 2
- 208000016361 genetic disease Diseases 0.000 description 2
- 208000006454 hepatitis Diseases 0.000 description 2
- 231100000283 hepatitis Toxicity 0.000 description 2
- 208000029570 hepatitis D virus infection Diseases 0.000 description 2
- 230000002363 herbicidal effect Effects 0.000 description 2
- 239000004009 herbicide Substances 0.000 description 2
- 238000005734 heterodimerization reaction Methods 0.000 description 2
- 235000014304 histidine Nutrition 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 150000002634 lipophilic molecules Chemical class 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000011987 methylation Effects 0.000 description 2
- 238000007069 methylation reaction Methods 0.000 description 2
- 210000003470 mitochondria Anatomy 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 206010028093 mucopolysaccharidosis Diseases 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 229950006344 nocodazole Drugs 0.000 description 2
- 230000006780 non-homologous end joining Effects 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 238000007899 nucleic acid hybridization Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 238000002823 phage display Methods 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 229960000502 poloxamer Drugs 0.000 description 2
- 230000008488 polyadenylation Effects 0.000 description 2
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102000021127 protein binding proteins Human genes 0.000 description 2
- 108091011138 protein binding proteins Proteins 0.000 description 2
- 108060006633 protein kinase Proteins 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000008707 rearrangement Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000014493 regulation of gene expression Effects 0.000 description 2
- 238000007634 remodeling Methods 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 108091092562 ribozyme Proteins 0.000 description 2
- 230000005783 single-strand break Effects 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000000699 topical effect Effects 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- 210000003956 transport vesicle Anatomy 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- 229960003048 vinblastine Drugs 0.000 description 2
- JXLYSJRDGCGARV-XQKSVPLYSA-N vincaleukoblastine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-XQKSVPLYSA-N 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 description 1
- ALNDFFUAQIVVPG-NGJCXOISSA-N (2r,3r,4r)-3,4,5-trihydroxy-2-methoxypentanal Chemical compound CO[C@@H](C=O)[C@H](O)[C@H](O)CO ALNDFFUAQIVVPG-NGJCXOISSA-N 0.000 description 1
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- BRCNMMGLEUILLG-NTSWFWBYSA-N (4s,5r)-4,5,6-trihydroxyhexan-2-one Chemical group CC(=O)C[C@H](O)[C@H](O)CO BRCNMMGLEUILLG-NTSWFWBYSA-N 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- VSNHCAURESNICA-NJFSPNSNSA-N 1-oxidanylurea Chemical compound N[14C](=O)NO VSNHCAURESNICA-NJFSPNSNSA-N 0.000 description 1
- 230000005730 ADP ribosylation Effects 0.000 description 1
- 102100024643 ATP-binding cassette sub-family D member 1 Human genes 0.000 description 1
- 108010013043 Acetylesterase Proteins 0.000 description 1
- 101710159080 Aconitate hydratase A Proteins 0.000 description 1
- 101710159078 Aconitate hydratase B Proteins 0.000 description 1
- 208000029483 Acquired immunodeficiency Diseases 0.000 description 1
- 201000010028 Acrocephalosyndactylia Diseases 0.000 description 1
- 208000002485 Adiposis dolorosa Diseases 0.000 description 1
- 201000011452 Adrenoleukodystrophy Diseases 0.000 description 1
- 208000024341 Aicardi syndrome Diseases 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 244000296825 Amygdalus nana Species 0.000 description 1
- 235000003840 Amygdalus nana Nutrition 0.000 description 1
- 206010056292 Androgen-Insensitivity Syndrome Diseases 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 108020005544 Antisense RNA Proteins 0.000 description 1
- 208000025490 Apert syndrome Diseases 0.000 description 1
- 241000219194 Arabidopsis Species 0.000 description 1
- 208000006400 Arbovirus Encephalitis Diseases 0.000 description 1
- 241000712892 Arenaviridae Species 0.000 description 1
- 235000005340 Asparagus officinalis Nutrition 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 206010003591 Ataxia Diseases 0.000 description 1
- 235000005781 Avena Nutrition 0.000 description 1
- 244000075850 Avena orientalis Species 0.000 description 1
- 108700020463 BRCA1 Proteins 0.000 description 1
- 102000036365 BRCA1 Human genes 0.000 description 1
- 101150072950 BRCA1 gene Proteins 0.000 description 1
- 108700020462 BRCA2 Proteins 0.000 description 1
- 102000052609 BRCA2 Human genes 0.000 description 1
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 231100000699 Bacterial toxin Toxicity 0.000 description 1
- 201000005943 Barth syndrome Diseases 0.000 description 1
- 241000219310 Beta vulgaris subsp. vulgaris Species 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 241000702628 Birnaviridae Species 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- 208000015885 Blue rubber bleb nevus Diseases 0.000 description 1
- 208000003508 Botulism Diseases 0.000 description 1
- 241000219198 Brassica Species 0.000 description 1
- 235000011331 Brassica Nutrition 0.000 description 1
- 240000002791 Brassica napus Species 0.000 description 1
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 1
- 101150008921 Brca2 gene Proteins 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 101710082513 C-X-C chemokine receptor type 4 Proteins 0.000 description 1
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 description 1
- 108700011778 CCR5 Proteins 0.000 description 1
- 108010017088 CCR5 Receptors Proteins 0.000 description 1
- 102000004274 CCR5 Receptors Human genes 0.000 description 1
- 108010061299 CXCR4 Receptors Proteins 0.000 description 1
- 102000012000 CXCR4 Receptors Human genes 0.000 description 1
- 241000714198 Caliciviridae Species 0.000 description 1
- 208000022526 Canavan disease Diseases 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- 240000008574 Capsicum frutescens Species 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- 229940123587 Cell cycle inhibitor Drugs 0.000 description 1
- 102000009410 Chemokine receptor Human genes 0.000 description 1
- 108050000299 Chemokine receptor Proteins 0.000 description 1
- 241000606161 Chlamydia Species 0.000 description 1
- 206010008631 Cholera Diseases 0.000 description 1
- 206010008723 Chondrodystrophy Diseases 0.000 description 1
- 235000007516 Chrysanthemum Nutrition 0.000 description 1
- 244000189548 Chrysanthemum x morifolium Species 0.000 description 1
- 241000219109 Citrullus Species 0.000 description 1
- 241000207199 Citrus Species 0.000 description 1
- 108010021408 Clostridium perfringens iota toxin Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 240000004270 Colocasia esculenta var. antiquorum Species 0.000 description 1
- 208000006992 Color Vision Defects Diseases 0.000 description 1
- 206010053138 Congenital aplastic anaemia Diseases 0.000 description 1
- 241000218631 Coniferophyta Species 0.000 description 1
- 241000709687 Coxsackievirus Species 0.000 description 1
- 206010011385 Cri-du-chat syndrome Diseases 0.000 description 1
- 241000724252 Cucumber mosaic virus Species 0.000 description 1
- 241000219122 Cucurbita Species 0.000 description 1
- 101710095468 Cyclase Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 1
- 230000007035 DNA breakage Effects 0.000 description 1
- 102100033195 DNA ligase 4 Human genes 0.000 description 1
- 108050004671 DNA ligase 4 Proteins 0.000 description 1
- 102100033934 DNA repair protein RAD51 homolog 2 Human genes 0.000 description 1
- 102100027830 DNA repair protein XRCC2 Human genes 0.000 description 1
- 102100027829 DNA repair protein XRCC3 Human genes 0.000 description 1
- 102100027828 DNA repair protein XRCC4 Human genes 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 241000450599 DNA viruses Species 0.000 description 1
- 101710096438 DNA-binding protein Proteins 0.000 description 1
- 241000208175 Daucus Species 0.000 description 1
- 244000000626 Daucus carota Species 0.000 description 1
- 235000002767 Daucus carota Nutrition 0.000 description 1
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 206010012689 Diabetic retinopathy Diseases 0.000 description 1
- 239000004338 Dichlorodifluoromethane Substances 0.000 description 1
- 235000002723 Dioscorea alata Nutrition 0.000 description 1
- 235000007056 Dioscorea composita Nutrition 0.000 description 1
- 235000009723 Dioscorea convolvulacea Nutrition 0.000 description 1
- 235000005362 Dioscorea floribunda Nutrition 0.000 description 1
- 235000004868 Dioscorea macrostachya Nutrition 0.000 description 1
- 235000005361 Dioscorea nummularia Nutrition 0.000 description 1
- 235000005360 Dioscorea spiculiflora Nutrition 0.000 description 1
- 101100300807 Drosophila melanogaster spn-A gene Proteins 0.000 description 1
- 206010058314 Dysplasia Diseases 0.000 description 1
- 108010024212 E-Selectin Proteins 0.000 description 1
- 102100023471 E-selectin Human genes 0.000 description 1
- 201000011001 Ebola Hemorrhagic Fever Diseases 0.000 description 1
- 241001466953 Echovirus Species 0.000 description 1
- 241000224431 Entamoeba Species 0.000 description 1
- 241000709661 Enterovirus Species 0.000 description 1
- 101800001467 Envelope glycoprotein E2 Proteins 0.000 description 1
- 101710091045 Envelope protein Proteins 0.000 description 1
- 208000031637 Erythroblastic Acute Leukemia Diseases 0.000 description 1
- 208000036566 Erythroleukaemia Diseases 0.000 description 1
- 101001091269 Escherichia coli Hygromycin-B 4-O-kinase Proteins 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 208000024720 Fabry Disease Diseases 0.000 description 1
- 201000004939 Fanconi anemia Diseases 0.000 description 1
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 1
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 1
- 241000724791 Filamentous phage Species 0.000 description 1
- 241000711950 Filoviridae Species 0.000 description 1
- 241000710781 Flaviviridae Species 0.000 description 1
- 241000710831 Flavivirus Species 0.000 description 1
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 1
- 235000016623 Fragaria vesca Nutrition 0.000 description 1
- 240000009088 Fragaria x ananassa Species 0.000 description 1
- 235000011363 Fragaria x ananassa Nutrition 0.000 description 1
- 208000001914 Fragile X syndrome Diseases 0.000 description 1
- 201000011240 Frontotemporal dementia Diseases 0.000 description 1
- 108010001515 Galectin 4 Proteins 0.000 description 1
- 102100039556 Galectin-4 Human genes 0.000 description 1
- 208000009796 Gangliosidoses Diseases 0.000 description 1
- 208000034951 Genetic Translocation Diseases 0.000 description 1
- 241000224466 Giardia Species 0.000 description 1
- 208000010055 Globoid Cell Leukodystrophy Diseases 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- 102000053187 Glucuronidase Human genes 0.000 description 1
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 206010053185 Glycogen storage disease type II Diseases 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 208000037357 HIV infectious disease Diseases 0.000 description 1
- 101100028493 Haloferax volcanii (strain ATCC 29605 / DSM 3757 / JCM 8879 / NBRC 14742 / NCIMB 2012 / VKM B-1768 / DS2) pan2 gene Proteins 0.000 description 1
- 244000020551 Helianthus annuus Species 0.000 description 1
- 235000003222 Helianthus annuus Nutrition 0.000 description 1
- 208000018565 Hemochromatosis Diseases 0.000 description 1
- 108010085686 Hemoglobin C Proteins 0.000 description 1
- 102100021519 Hemoglobin subunit beta Human genes 0.000 description 1
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 208000005176 Hepatitis C Diseases 0.000 description 1
- 208000005331 Hepatitis D Diseases 0.000 description 1
- 208000002972 Hepatolenticular Degeneration Diseases 0.000 description 1
- 108091027305 Heteroduplex Proteins 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 108010036115 Histone Methyltransferases Proteins 0.000 description 1
- 102000011787 Histone Methyltransferases Human genes 0.000 description 1
- 102100022893 Histone acetyltransferase KAT5 Human genes 0.000 description 1
- 101710116149 Histone acetyltransferase KAT5 Proteins 0.000 description 1
- 102000003893 Histone acetyltransferases Human genes 0.000 description 1
- 108090000246 Histone acetyltransferases Proteins 0.000 description 1
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 1
- 101000649306 Homo sapiens DNA repair protein XRCC2 Proteins 0.000 description 1
- 101000649315 Homo sapiens DNA repair protein XRCC4 Proteins 0.000 description 1
- 101000851181 Homo sapiens Epidermal growth factor receptor Proteins 0.000 description 1
- 101000615488 Homo sapiens Methyl-CpG-binding domain protein 2 Proteins 0.000 description 1
- 101001128138 Homo sapiens NACHT, LRR and PYD domains-containing protein 2 Proteins 0.000 description 1
- 101000981336 Homo sapiens Nibrin Proteins 0.000 description 1
- 101001109800 Homo sapiens Pro-neuregulin-1, membrane-bound isoform Proteins 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 241000209219 Hordeum Species 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 241000598436 Human T-cell lymphotropic virus Species 0.000 description 1
- 206010020460 Human T-cell lymphotropic virus type I infection Diseases 0.000 description 1
- 241000714260 Human T-lymphotropic virus 1 Species 0.000 description 1
- 241000714259 Human T-lymphotropic virus 2 Species 0.000 description 1
- 241000701074 Human alphaherpesvirus 2 Species 0.000 description 1
- 241000701027 Human herpesvirus 6 Species 0.000 description 1
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 1
- 208000023105 Huntington disease Diseases 0.000 description 1
- 208000015178 Hurler syndrome Diseases 0.000 description 1
- 208000025500 Hutchinson-Gilford progeria syndrome Diseases 0.000 description 1
- 206010049933 Hypophosphatasia Diseases 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000029462 Immunodeficiency disease Diseases 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 208000028547 Inborn Urea Cycle disease Diseases 0.000 description 1
- 102100034349 Integrase Human genes 0.000 description 1
- 102000012330 Integrases Human genes 0.000 description 1
- 108010061833 Integrases Proteins 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000018682 Interleukin Receptor Common gamma Subunit Human genes 0.000 description 1
- 108010066719 Interleukin Receptor Common gamma Subunit Proteins 0.000 description 1
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 1
- 235000006350 Ipomoea batatas var. batatas Nutrition 0.000 description 1
- 241000588748 Klebsiella Species 0.000 description 1
- 208000028226 Krabbe disease Diseases 0.000 description 1
- 108010054278 Lac Repressors Proteins 0.000 description 1
- 241000208822 Lactuca Species 0.000 description 1
- 240000008415 Lactuca sativa Species 0.000 description 1
- 235000003228 Lactuca sativa Nutrition 0.000 description 1
- 206010050638 Langer-Giedion syndrome Diseases 0.000 description 1
- 241000589248 Legionella Species 0.000 description 1
- 208000007764 Legionnaires' Disease Diseases 0.000 description 1
- 241000222722 Leishmania <genus> Species 0.000 description 1
- 206010024238 Leptospirosis Diseases 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 235000002262 Lycopersicon Nutrition 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 208000016604 Lyme disease Diseases 0.000 description 1
- 208000030289 Lymphoproliferative disease Diseases 0.000 description 1
- 208000015439 Lysosomal storage disease Diseases 0.000 description 1
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 1
- 235000011430 Malus pumila Nutrition 0.000 description 1
- 235000015103 Malus silvestris Nutrition 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 208000000916 Mandibulofacial dysostosis Diseases 0.000 description 1
- 240000003183 Manihot esculenta Species 0.000 description 1
- 208000001826 Marfan syndrome Diseases 0.000 description 1
- 240000004658 Medicago sativa Species 0.000 description 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 1
- 108010049137 Member 1 Subfamily D ATP Binding Cassette Transporter Proteins 0.000 description 1
- 102000003792 Metallothionein Human genes 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102100021299 Methyl-CpG-binding domain protein 2 Human genes 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 108010059724 Micrococcal Nuclease Proteins 0.000 description 1
- 201000002983 Mobius syndrome Diseases 0.000 description 1
- 208000034167 Moebius syndrome Diseases 0.000 description 1
- 241000713869 Moloney murine leukemia virus Species 0.000 description 1
- 208000001804 Monosomy 5p Diseases 0.000 description 1
- 108010086093 Mung Bean Nuclease Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 208000000175 Nail-Patella Syndrome Diseases 0.000 description 1
- 108700019961 Neoplasm Genes Proteins 0.000 description 1
- 102000048850 Neoplasm Genes Human genes 0.000 description 1
- 208000009905 Neurofibromatoses Diseases 0.000 description 1
- 101100355599 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) mus-11 gene Proteins 0.000 description 1
- 102100024403 Nibrin Human genes 0.000 description 1
- 241000208125 Nicotiana Species 0.000 description 1
- 108010008964 Non-Histone Chromosomal Proteins Proteins 0.000 description 1
- 102000006570 Non-Histone Chromosomal Proteins Human genes 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 241000712464 Orthomyxoviridae Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 206010031243 Osteogenesis imperfecta Diseases 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 241000711504 Paramyxoviridae Species 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 241000150350 Peribunyaviridae Species 0.000 description 1
- 241000218196 Persea Species 0.000 description 1
- 201000005702 Pertussis Diseases 0.000 description 1
- 240000007377 Petunia x hybrida Species 0.000 description 1
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 1
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 241000218657 Picea Species 0.000 description 1
- 208000000609 Pick Disease of the Brain Diseases 0.000 description 1
- 208000024571 Pick disease Diseases 0.000 description 1
- 241000709664 Picornaviridae Species 0.000 description 1
- 241000219843 Pisum Species 0.000 description 1
- 206010035148 Plague Diseases 0.000 description 1
- 102000012338 Poly(ADP-ribose) Polymerases Human genes 0.000 description 1
- 108010061844 Poly(ADP-ribose) Polymerases Proteins 0.000 description 1
- 229920000776 Poly(Adenosine diphosphate-ribose) polymerase Polymers 0.000 description 1
- 241000097929 Porphyria Species 0.000 description 1
- 208000010642 Porphyrias Diseases 0.000 description 1
- 201000010769 Prader-Willi syndrome Diseases 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 208000007932 Progeria Diseases 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 108010043400 Protamine Kinase Proteins 0.000 description 1
- 108700040121 Protein Methyltransferases Proteins 0.000 description 1
- 102000055027 Protein Methyltransferases Human genes 0.000 description 1
- 101710149951 Protein Tat Proteins 0.000 description 1
- 101710188315 Protein X Proteins 0.000 description 1
- 241000588769 Proteus <enterobacteria> Species 0.000 description 1
- 208000007531 Proteus syndrome Diseases 0.000 description 1
- 235000011432 Prunus Nutrition 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 201000004681 Psoriasis Diseases 0.000 description 1
- 235000014443 Pyrus communis Nutrition 0.000 description 1
- 101150006234 RAD52 gene Proteins 0.000 description 1
- 102000044126 RNA-Binding Proteins Human genes 0.000 description 1
- 230000004570 RNA-binding Effects 0.000 description 1
- 101710105008 RNA-binding protein Proteins 0.000 description 1
- 239000012979 RPMI medium Substances 0.000 description 1
- 241000220259 Raphanus Species 0.000 description 1
- 101100425557 Rattus norvegicus Tle3 gene Proteins 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 241000702247 Reoviridae Species 0.000 description 1
- 201000000582 Retinoblastoma Diseases 0.000 description 1
- 208000006289 Rett Syndrome Diseases 0.000 description 1
- 241000220010 Rhode Species 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 241000606651 Rickettsiales Species 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 241000702670 Rotavirus Species 0.000 description 1
- 241000714474 Rous sarcoma virus Species 0.000 description 1
- 206010039281 Rubinstein-Taybi syndrome Diseases 0.000 description 1
- 230000018199 S phase Effects 0.000 description 1
- 101001025539 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) Homothallic switching endonuclease Proteins 0.000 description 1
- 241000209056 Secale Species 0.000 description 1
- 108090000184 Selectins Proteins 0.000 description 1
- 102000003800 Selectins Human genes 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 201000004283 Shwachman-Diamond syndrome Diseases 0.000 description 1
- 201000001388 Smith-Magenis syndrome Diseases 0.000 description 1
- 241000207763 Solanum Species 0.000 description 1
- 235000002634 Solanum Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 235000009337 Spinacia oleracea Nutrition 0.000 description 1
- 244000300264 Spinacia oleracea Species 0.000 description 1
- 241000295644 Staphylococcaceae Species 0.000 description 1
- 208000027077 Stickler syndrome Diseases 0.000 description 1
- 101001091268 Streptomyces hygroscopicus Hygromycin-B 7''-O-kinase Proteins 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 235000021536 Sugar beet Nutrition 0.000 description 1
- 101800001271 Surface protein Proteins 0.000 description 1
- 101710192266 Tegument protein VP22 Proteins 0.000 description 1
- 206010043376 Tetanus Diseases 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 241000710924 Togaviridae Species 0.000 description 1
- 101710183280 Topoisomerase Proteins 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- 201000003199 Treacher Collins syndrome Diseases 0.000 description 1
- 241000224526 Trichomonas Species 0.000 description 1
- 208000035378 Trichorhinophalangeal syndrome type 2 Diseases 0.000 description 1
- RTKIYFITIVXBLE-UHFFFAOYSA-N Trichostatin A Natural products ONC(=O)C=CC(C)=CC(C)C(=O)C1=CC=C(N(C)C)C=C1 RTKIYFITIVXBLE-UHFFFAOYSA-N 0.000 description 1
- 208000037280 Trisomy Diseases 0.000 description 1
- 241000223104 Trypanosoma Species 0.000 description 1
- 208000026911 Tuberous sclerosis complex Diseases 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 102100033254 Tumor suppressor ARF Human genes 0.000 description 1
- 208000026928 Turner syndrome Diseases 0.000 description 1
- 206010046865 Vaccinia virus infection Diseases 0.000 description 1
- 108010000134 Vascular Cell Adhesion Molecule-1 Proteins 0.000 description 1
- 102100023543 Vascular cell adhesion protein 1 Human genes 0.000 description 1
- 241000219977 Vigna Species 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 235000009392 Vitis Nutrition 0.000 description 1
- 241000219095 Vitis Species 0.000 description 1
- 208000026724 Waardenburg syndrome Diseases 0.000 description 1
- 206010049644 Williams syndrome Diseases 0.000 description 1
- 208000018839 Wilson disease Diseases 0.000 description 1
- 208000006110 Wiskott-Aldrich syndrome Diseases 0.000 description 1
- 210000001766 X chromosome Anatomy 0.000 description 1
- 108010074310 X-ray repair cross complementing protein 3 Proteins 0.000 description 1
- 102100036976 X-ray repair cross-complementing protein 6 Human genes 0.000 description 1
- 101710124907 X-ray repair cross-complementing protein 6 Proteins 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 241000209149 Zea Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 108091007916 Zinc finger transcription factors Proteins 0.000 description 1
- 102000038627 Zinc finger transcription factors Human genes 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 208000008919 achondroplasia Diseases 0.000 description 1
- 201000000761 achromatopsia Diseases 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 208000021841 acute erythroid leukemia Diseases 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 201000009628 adenosine deaminase deficiency Diseases 0.000 description 1
- 102000030621 adenylate cyclase Human genes 0.000 description 1
- 108060000200 adenylate cyclase Proteins 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 244000193174 agave Species 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
- 208000006682 alpha 1-Antitrypsin Deficiency Diseases 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 230000003126 arrythmogenic effect Effects 0.000 description 1
- 210000004507 artificial chromosome Anatomy 0.000 description 1
- 208000036556 autosomal recessive T cell-negative B cell-negative NK cell-negative due to adenosine deaminase deficiency severe combined immunodeficiency Diseases 0.000 description 1
- 229940065181 bacillus anthracis Drugs 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 239000000688 bacterial toxin Substances 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 102000015736 beta 2-Microglobulin Human genes 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 239000003139 biocide Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 229960001561 bleomycin Drugs 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000001390 capsicum minimum Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000012820 cell cycle checkpoint Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000010307 cell transformation Effects 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 230000007073 chemical hydrolysis Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 238000000749 co-immunoprecipitation Methods 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 201000007254 color blindness Diseases 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000012786 cultivation procedure Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 229940124447 delivery agent Drugs 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 230000001687 destabilization Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 229960002086 dextran Drugs 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- PXBRQCKWGAHEHS-UHFFFAOYSA-N dichlorodifluoromethane Chemical compound FC(F)(Cl)Cl PXBRQCKWGAHEHS-UHFFFAOYSA-N 0.000 description 1
- 235000019404 dichlorodifluoromethane Nutrition 0.000 description 1
- 239000005546 dideoxynucleotide Substances 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- 235000004879 dioscorea Nutrition 0.000 description 1
- 206010013023 diphtheria Diseases 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 208000002169 ectodermal dysplasia Diseases 0.000 description 1
- 208000031068 ectodermal dysplasia syndrome Diseases 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 238000012407 engineering method Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 description 1
- 229960005420 etoposide Drugs 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 229940126864 fibroblast growth factor Drugs 0.000 description 1
- 229960002949 fluorouracil Drugs 0.000 description 1
- 230000000799 fusogenic effect Effects 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 102000054766 genetic haplotypes Human genes 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 201000004502 glycogen storage disease II Diseases 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 208000034737 hemoglobinopathy Diseases 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 102000055650 human NRG1 Human genes 0.000 description 1
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 208000018337 inherited hemoglobinopathy Diseases 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 239000012212 insulator Substances 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- WZNJWVWKTVETCG-UHFFFAOYSA-N kojic acid Natural products OC(=O)C(N)CN1C=CC(=O)C(O)=C1 WZNJWVWKTVETCG-UHFFFAOYSA-N 0.000 description 1
- 208000036546 leukodystrophy Diseases 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 208000004731 long QT syndrome Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 208000002780 macular degeneration Diseases 0.000 description 1
- 235000009973 maize Nutrition 0.000 description 1
- 235000005739 manihot Nutrition 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 238000012737 microarray-based gene expression Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000029115 microtubule polymerization Effects 0.000 description 1
- 235000019713 millet Nutrition 0.000 description 1
- 229950002289 mimosine Drugs 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 238000009126 molecular therapy Methods 0.000 description 1
- 101150071637 mre11 gene Proteins 0.000 description 1
- 208000005340 mucopolysaccharidosis III Diseases 0.000 description 1
- 208000011045 mucopolysaccharidosis type 3 Diseases 0.000 description 1
- 238000012243 multiplex automated genomic engineering Methods 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 201000006938 muscular dystrophy Diseases 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
- 230000002988 nephrogenic effect Effects 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 201000004931 neurofibromatosis Diseases 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000007500 overflow downdraw method Methods 0.000 description 1
- 108700025694 p53 Genes Proteins 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 101150081585 panB gene Proteins 0.000 description 1
- 238000004091 panning Methods 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 210000000680 phagosome Anatomy 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 150000008298 phosphoramidates Chemical class 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 229930195732 phytohormone Natural products 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 239000001294 propane Substances 0.000 description 1
- 239000003380 propellant Substances 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 239000013636 protein dimer Substances 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 235000014774 prunus Nutrition 0.000 description 1
- 101150010682 rad50 gene Proteins 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 102000037983 regulatory factors Human genes 0.000 description 1
- 108091008025 regulatory factors Proteins 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 230000000754 repressing effect Effects 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 238000012340 reverse transcriptase PCR Methods 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 208000002491 severe combined immunodeficiency Diseases 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000013605 shuttle vector Substances 0.000 description 1
- HBMJWWWQQXIZIP-UHFFFAOYSA-N silicon carbide Chemical compound [Si+]#[C-] HBMJWWWQQXIZIP-UHFFFAOYSA-N 0.000 description 1
- 229910010271 silicon carbide Inorganic materials 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- MFBOGIVSZKQAPD-UHFFFAOYSA-M sodium butyrate Chemical compound [Na+].CCCC([O-])=O MFBOGIVSZKQAPD-UHFFFAOYSA-M 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000004960 subcellular localization Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000004797 therapeutic response Effects 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical group [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 206010043554 thrombocytopenia Diseases 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 241001147422 tick-borne encephalitis virus group Species 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 201000006532 trichorhinophalangeal syndrome type II Diseases 0.000 description 1
- RTKIYFITIVXBLE-QEQCGCAPSA-N trichostatin A Chemical compound ONC(=O)/C=C/C(/C)=C/[C@@H](C)C(=O)C1=CC=C(N(C)C)C=C1 RTKIYFITIVXBLE-QEQCGCAPSA-N 0.000 description 1
- 230000010415 tropism Effects 0.000 description 1
- 208000009999 tuberous sclerosis Diseases 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000003160 two-hybrid assay Methods 0.000 description 1
- 238000010396 two-hybrid screening Methods 0.000 description 1
- 230000034512 ubiquitination Effects 0.000 description 1
- 238000010798 ubiquitination Methods 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 208000030954 urea cycle disease Diseases 0.000 description 1
- 208000007089 vaccinia Diseases 0.000 description 1
- 208000019553 vascular disease Diseases 0.000 description 1
- 230000002861 ventricular Effects 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 239000000277 virosome Substances 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
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
- C12N15/63—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
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
Definitions
- the present disclosure is in the field of genome engineering and targeted deletion (i.e. “knock-out” technology).
- One such alteration is deletion, i.e., removal of sequences from a genome.
- Deletions can be as small as a single nucleotide pair, or can encompass hundreds, thousands or even millions of nucleotide pairs.
- the ability to reproducibly induce targeted deletions is useful in the identification of gene function (e.g., by gene “knock-out” studies) and can also be useful for inactivating genes (e.g., viral receptors) whose function is required for pathological processes.
- ZFNs zinc finger/nuclease fusion proteins
- the process of ZFN-mediated mutagenesis as currently implemented using a single ZFN dimer has a number of limitations.
- the sizes of the deletions introduced by this method are generally quite small. Although deletions in excess of 100 bp are occasionally seen, the vast majority of deletions (probably more than 90%) are of fewer than about 20 bp. Therefore the method is unsuitable for generating large deletions at high efficiency. The ability to generate large deletions at high frequency would be required if, for example, it were necessary to eliminate entire regulatory region of a gene.
- a second shortcoming of existing methods for ZFN-mediated mutagenesis is that the heterogeneity of the deletions, coupled with their small sizes, makes it extremely difficult to monitor or quantify the mutagenesis process using conventional approaches such as PCR.
- larger deletions are much more readily detected and quantified in a background of excess unmodified gene sequence using a standard method such as PCR followed by agarose gel electrophoresis.
- a method for deleting sequences in a region of interest in double-stranded DNA comprising expressing first, second, third and fourth fusion proteins in a cell, wherein each of the fusion proteins comprises (i) a zinc finger DNA-binding domain that binds to a target site in the DNA, and (ii) a cleavage half-domain; further wherein (a) the first and second fusion proteins bind to first and second target sites respectively, wherein a first cleavage site lies between the first and second target sites (i.e., the first and second target sites straddle the first cleavage site) and (b) the third and fourth fusion proteins bind to third and fourth target sites respectively, wherein a second cleavage site lies between the third and fourth target sites (i.e., the third and fourth target sites straddle the second clea
- Also provided is a method for deleting sequences in a region of interest in double-stranded DNA comprising expressing first and second nucleases in a cell, wherein the first nuclease cleaves a first cleavage site and the second nuclease cleaves a second cleavage site; and DNA ends are rejoined such that sequences between the first and second cleavage sites are deleted.
- At least one of the nucleases is a fusion protein comprising (i) a zinc finger DNA-binding domain that binds to a target site in the DNA, wherein the target site is at or adjacent to the first or second cleavage site; and (ii) a cleavage domain.
- First and second DNA ends are generated by cleavage at the first cleavage site; while third and fourth DNA ends are generated by cleavage at the second cleavage site.
- the first and second cleavage sites are present on the same DNA molecule (e.g., on the same chromosome). In these cases, if the second and third ends, as defined above, are considered to be part of a DNA fragment containing sequences that lie between the first and second cleavage sites (i.e.
- first and second cleavage sites are located on different chromosomes, chromosomal translocations and/or chromosomal fusions can result.
- targeted cleavages and resultant deletion can also occur on extrachromosomal nucleic acids, such as episomes, intracellular vectors, organellar genomes, etc.
- ends generated directly by the cleavage event may be rejoined to cause a deletion.
- the ends generated by cleavage may be further processed (e.g., by exonucleolytic resection) and these ends resulting from cleavage can be rejoined. Rejoining can occur by cellular repair mechanisms such as those collectively denoted “non-homologous end-joining.”
- sequences in a region of interest are deleted, wherein the region of interest is in cellular chromatin.
- the first and second cleavage sites can be on the same chromosomes, on different chromosomes, on an extrachromosomal nucleic acid, or the first cleavage sit can be present on a chromosome and the second cleavage site can be present on an extrachromosomal nucleic acid.
- the target sites bound by the fusion proteins are present in pairs wherein, for each pair of target sites, a cleavage site lies therebetween.
- the first and second target sites straddle a first cleavage site and the third and fourth target sites straddle a second cleavage site.
- the target sites can be separated by any number of nucleotide pairs, commensurate with dimerization of the fusion proteins to regenerate a functional cleavage domain.
- maximal cleavage efficiency varies with both the distance between target sites and the length of the linker sequences between the zinc finger portion and the nuclease half-domain portion of the fusion proteins.
- the first and second target sites can be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide pairs.
- the third and fourth target sites can be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide pairs.
- this distance is expressed as the number of nucleotide pairs intervening between the near edges of the target sites, and does not include any nucleotide pair that is present in either of the target sites.
- the size of a deletion induced by the disclosed methods and compositions is determined by the distance between the first and second cleavage sites. Accordingly, deletions of any size, in any region of interest, can be obtained. Deletions of 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 nucleotide pairs, or any integral value of nucleotide pairs within this range, can be obtained. In addition deletions of a sequence of any integral value of nucleotide pairs greater than 1,000 nucleotide pairs can be obtained using the methods and compositions disclosed herein.
- the region of interest, in which deletion is induced, can be in a gene.
- the gene can be a gene involved in a disease or pathological condition.
- the gene can be a viral receptor.
- Certain chemokine receptors also function as viral receptors; for example, the chemokine receptor CCR-5 also functions a receptor for human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS).
- HIV human immunodeficiency virus
- AIDS acquired immune deficiency syndrome
- the present disclosure provides methods for inducing targeted deletions in a CCR-5 gene, optionally a human CCR-5 gene, for treatment of AIDS.
- deleted CCR-5 gene sequences and cells comprising deleted CCR-5 genes comprising deleted CCR-5 genes; optionally, human cells.
- the cells are primary cells obtained from an individual, which may optionally be returned to the same individual or a different individual.
- the primary cells are T-cells or dendritic cells.
- Cells can also include cultured cells, cells in an organism and cells that have been removed from an organism for treatment in cases where the cells and/or their descendants will be returned to the organism after treatment.
- a region of interest in cellular chromatin can be, for example, a genomic sequence or portion thereof.
- Compositions include fusion polypeptides comprising an engineered zinc finger binding domain (e.g., a zinc finger binding domain having a novel specificity) and a cleavage domain, and fusion polypeptides comprising an engineered zinc finger binding domain and a cleavage half-domain.
- Cleavage domains and cleavage half domains can be obtained, for example, from various restriction endonucleases and/or homing endonucleases.
- Cellular chromatin can be present in any type of cell including, but not limited to, prokaryotic and eukaryotic cells, fungal cells, plant cells, animal cells, mammalian cells, primate cells and human cells.
- a protein e.g., a fusion protein can be expressed in a cell, e.g., by delivering the fusion protein to the cell or by delivering a polynucleotide encoding the fusion protein to a cell, wherein the polynucleotide, if DNA, is transcribed, and an RNA molecule delivered to the cell or a transcript of a DNA molecule delivered to the cell is translated, to generate the protein.
- Methods for polynucleotide and polypeptide delivery to cells are presented elsewhere in this disclosure.
- the cleavage half-domains can be derived from the same endonuclease or from different endonucleases.
- Endonucleases include, but are not limited to, homing endonucleases and restriction endonucleases.
- Exemplary restriction endonucleases are Type IIS restriction endonucleases; an exemplary Type IIS restriction endonuclease is FokI.
- a cleavage domain can comprise two cleavage half-domains that are covalently linked in the same polypeptide.
- the two cleavage half-domains can be derived from the same endonuclease or from different endonucleases.
- the cleavage half domain can be derived from, for example, a homing endonuclease or a restriction endonuclease, for example, a Type IIS restriction endonuclease.
- An exemplary Type IIS restriction endonuclease is FokI.
- cleavage specificity it is possible to obtain increased cleavage specificity by utilizing fusion proteins in which one or both cleavage half-domains contains an alteration in the amino acid sequence of the dimerization interface.
- the target sites for the fusion proteins can comprise any number of nucleotides. Preferably, they are at least nine nucleotides in length, but they can also be larger (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18 and up to 100 nucleotides, including any integral value between 9 and 100 nucleotides); moreover, different target sequences need not necessarily be the same length.
- the distance between the nearest edges of the target sites can be any integral number of nucleotide pairs between 1 and 50, (e.g., 5 or 6 base pairs) as measured from the near end of one binding site to the closest end of the other binding site.
- cellular chromatin can be cleaved at a site located between the target sites of two fusion proteins.
- the target sites are on opposite DNA strands.
- expression of the fusion proteins in the cell can be accomplished either by introduction of the proteins into the cell or by introduction of one or more polynucleotides into the cell, which are optionally transcribed (if the polynucleotide is DNA), and the transcript(s) translated, to produce the fusion proteins.
- two polynucleotides, each comprising sequences encoding one pair of fusion proteins can be introduced into a cell.
- a single polynucleotide comprising sequences encoding both pairs of fusion proteins can be introduced into the cell.
- a zinc finger binding domain can be engineered, for example designed and/or selected. See, for example, U.S. Pat. Nos. 5,789,538; 6,007,988; 6,013,453; 6,140,466; 6,242,568; 6,410,248; 6,453,242; 6,534,261; 6,733,970; 6,746,838; 6,785,613; 6,790,941; 6,794,136; 6,866,997; and 6,933,113, as well as U.S. Patent Publication No. 2005/0064477. See also International Patent Publication No. WO 02/42459.
- Polynucleotides encoding fusions between a zinc finger binding domain and a cleavage domain or cleavage half-domain can be DNA or RNA, can be linear or circular, and can be single-stranded or double-stranded. They can be delivered to the cell as naked nucleic acid, as a complex with one or more delivery agents (e.g., liposomes, poloxamers) or contained in a viral delivery vehicle, such as, for example, an adenovirus, adeno-associated virus (AAV) or lentivirus.
- a polynucleotide can encode one or more fusion proteins.
- FIG. 1 is a photograph of an agarose gel in which amplification products of the human CCR-5 gene were analyzed.
- Lane 1 shows amplification products of DNA from K562 cells transfected with a plasmid encoding green fluorescent protein.
- Lane 2 shows amplification products of DNA from K562 cells transfected with a plasmid encoding a pair of ZFNs designed to cleave between +162 and +168 of the human CCR-5 gene.
- Lane 3 shows amplification products of DNA from K562 cells transfected with a plasmid encoding a pair of ZFNs designed to cleave between +627 and +633 of the human CCR-5 gene.
- Lane 4 shows amplification products of DNA from K562 cells transfected with both of the aforementioned ZFN pairs.
- FIG. 2 shows nucleotide sequences of amplification products of the CCR-5 gene obtained from K562 cells that had been transfected with two plasmids: one encoding a pair of ZFNs designed to cleave between +162 and +168 of the human CCR-5 gene and the other encoding a pair of ZFNs designed to cleave between +627 and +633 of the human CCR-5 gene (i.e., corresponding to lane 4 of FIG. 1 ).
- the topmost line shows a partial nucleotide sequence of the wild-type human CCR-5 gene from +146 to +185 (SEQ ID NO:45) and from +616 to +646 (SEQ ID NO:46), with the ZFN target sites underlined. Coordinates are with respect to the first nucleotide pair of the translation initiation codon.
- Each of the remaining lines represents a contiguous nucleotide sequence obtained from an amplification product of DNA obtained from cells that had been transfected as described in the preceding paragraph.
- portions of each contiguous sequence have been separated for purposes of alignment with the wild-type sequence.
- the bottom-most two lines provide descriptions of two additional sequences that were obtained.
- FIG. 3 shows an autoradiogram of a Southern blot of genomic DNA purified from transfected K562 cells and digested with XhoI and NdeI.
- Lane 1 shows DNA from cells transfected with two plasmids: one encoding a pair of ZFNs designed to cleave between +162 and +168 of the human CCR-5 gene and the other encoding a pair of ZFNs designed to cleave between +627 and +633 of the human CCR-5 gene.
- Lane 2 shows DNA from cells transfected with a plasmid encoding GFP.
- the upper arrow indicates a band derived from amplification of wild-type sequences; the lower arrow identifies a band obtained from amplification of deleted CCR-5 loci.
- FIG. 4 is a photograph of an agarose gel in which amplification products of the human CCR-5 gene were analyzed.
- Lane 1 shows size markers.
- Lane 2 shows amplification products of DNA from human T-cells transfected with a plasmid encoding a pair of ZFNs designed to cleave between +162 and +168 of the human CCR-5 gene and a plasmid encoding a pair of ZFNs designed to cleave between +627 and +633 of the human CCR-5 gene.
- Lane 3 shows amplification products of DNA from human T-cells transfected with a plasmid encoding green fluorescent protein. The arrow indicates a band representing amplification products of deleted CCR-5 loci.
- FIG. 5 shows nucleotide sequences of amplification products of the CCR-5 gene obtained from primary human T-cells that had been transfected with two plasmids: one encoding a pair of ZFNs designed to cleave between +162 and +168 of the human CCR-5 gene and the other encoding a pair of ZFNs designed to cleave between +627 and +633 of the human CCR-5 gene.
- the topmost line shows a partial nucleotide sequence of the wild-type human CCR-5 gene from +146 to +185 (SEQ ID NO: 63) and from +616 to +645 (SEQ ID NO: 64).
- the target sites for the ZFNs are underlined. Coordinates are with respect to the first nucleotide pair of the translation initiation codon.
- Each of the remaining lines represents a contiguous nucleotide sequence obtained from an amplification product of DNA obtained from cells that had been transfected as described in the preceding paragraph. In the written representation shown in the Figure, portions of each contiguous sequence have been separated for purposes of alignment with the wild-type sequence.
- Double-stranded DNA includes that present in chromosomes, episomes, organellar genomes (e.g., mitochondria, chloroplasts), artificial chromosomes and any other type of nucleic acid present in a cell such as, for example, amplified sequences, double minute chromosomes and the genomes of endogenous or infecting bacteria and viruses.
- Chromosomal sequences can be normal (i.e., wild-type) or mutant; mutant sequences can comprise, for example, insertions, deletions, translocations, rearrangements, and/or point mutations.
- a chromosomal sequence can also comprise one of a number of different alleles.
- compositions useful for targeted deletion include pairs of fusion proteins, each fusion protein comprising a cleavage domain (or a cleavage half-domain) and a zinc finger binding domain, polynucleotides encoding these proteins and combinations of polypeptides and polypeptide-encoding polynucleotides.
- a zinc finger binding domain can comprise one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers), and can be engineered to bind to any sequence.
- fusion proteins comprising a cleavage domain (or cleavage half-domain) and a zinc finger domain engineered to recognize a target sequence in said genomic region.
- the presence of such fusion proteins in a cell results in binding of the fusion proteins to their target sites, cleavage at two cleavage sites, and deletion of sequences therebetween.
- ZFN dimer refers to a pair of zinc finger/cleavage half domain fusion proteins, each of which binds to a distinct target site such that DNA is cleaved at a cleavage site which lies between the target sites.
- the process can be used for highly efficient deletion of large DNA sequences (e.g., several hundred base pairs). This enables disruption of DNA elements (e.g., exons, introns, regulatory sequences) that may not be completely removable via introduction of small ( ⁇ 20 bp) deletions.
- DNA elements e.g., exons, introns, regulatory sequences
- 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; Wolfe, 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 International Patent Publication Nos. 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. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; and 6,200,759; and International Patent Publication Nos. 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 (1981) Advances in Applied Mathematics 2:482-489. 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 (1986) Nucl. Acids Res. 14(6):6745-6763.
- 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. Details of these programs can be found online. With respect to sequences described herein, the range of desired degrees of sequence identity is approximately 80% to 100% and any integer value therebetween. Typically the percent identities between sequences are at least 70-75%, preferably 80-82%, more preferably 85-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity.
- 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.
- FIR homologous recombination
- This process requires nucleotide sequence homology, uses a “donor” molecule to template repair of a “target” molecule (i.e., the one that experienced the double-strand break), and is variously known as “non-crossover gene conversion” or “short tract gene conversion,” because it leads to the transfer of genetic information from the donor to the target.
- such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and/or “synthesis-dependent strand annealing,” in which the donor is used to resynthesize genetic information that will become part of the target, and/or related processes.
- Such specialized HR often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.
- “Cleavage” refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, fusion polypeptides are used for targeted double-stranded DNA cleavage.
- a “cleavage domain” comprises one or more polypeptide sequences which possesses catalytic activity for DNA cleavage.
- a cleavage domain can be contained in a single polypeptide chain or cleavage activity can result from the association of two (or more) polypeptides.
- a “cleavage half-domain” is a polypeptide sequence which, in conjunction with a second polypeptide (either identical or different) forms a complex having cleavage activity (preferably double-strand cleavage activity).
- Chromatin is the nucleoprotein structure comprising the cellular genome.
- Cellular chromatin comprises nucleic acid, primarily DNA, and protein, including histones and non-histone chromosomal proteins.
- the majority of eukaryotic cellular chromatin exists in the form of nucleosomes, wherein a nucleosome core comprises approximately 150 base pairs of DNA associated with an octamer comprising two each of histones H2A, H2B, H3 and H4; and linker DNA (of variable length depending on the organism) extends between nucleosome cores.
- a molecule of histone H1 is generally associated with the linker DNA.
- chromatin is meant to encompass all types of cellular nucleoprotein, both prokaryotic and eukaryotic.
- Cellular chromatin includes both chromosomal and episomal chromatin.
- a “chromosome” is a chromatin complex comprising all or a portion of the genome of a cell.
- the genome of a cell is often characterized by its karyotype, which is the collection of all the chromosomes that comprise the genome of the cell.
- the genome of a cell can comprise one or more chromosomes.
- an “episome” is a replicating nucleic acid, nucleoprotein complex or other structure comprising a nucleic acid that is not part of the chromosomal karyotype of a cell.
- Examples of episomes include plasmids and certain viral genomes.
- an “accessible region” is a site in cellular chromatin in which a target site present in the nucleic acid can be bound by an exogenous molecule which recognizes the target site. Without wishing to be bound by any particular theory, it is believed that an accessible region is one that is not packaged into a nucleosomal structure. The distinct structure of an accessible region can often be detected by its sensitivity to chemical and enzymatic probes, for example, nucleases.
- a “target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist.
- the sequence 5′-GAATTC-3′ is a target site for the Eco RI restriction endonuclease.
- exogenous molecule is a molecule that is not normally present in a cell, but can be introduced into a cell by one or more genetic, biochemical or other methods. “Normal presence in the cell” is determined with respect to the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic development of muscle is an exogenous molecule with respect to an adult muscle cell. Similarly, a molecule induced by heat shock is an exogenous molecule with respect to a non-heat-shocked cell.
- An exogenous molecule can comprise, for example, a functioning version of a malfunctioning endogenous molecule or a malfunctioning version of a normally-functioning endogenous molecule.
- An exogenous molecule can be, among other things, a small molecule, such as is generated by a combinatorial chemistry process, or a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules.
- Nucleic acids include DNA and RNA, can be single- or double-stranded; can be linear, branched or circular; and can be of any length. Nucleic acids include those capable of forming duplexes, as well as triplex-forming nucleic acids. See, for example, U.S. Pat. Nos. 5,176,996 and 5,422,251.
- Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases and helicases.
- exogenous molecule can be the same type of molecule as an endogenous molecule, e.g., an exogenous protein or nucleic acid.
- an exogenous nucleic acid can comprise an infecting viral genome, a plasmid or episome introduced into a cell, or a chromosome that is not normally present in the cell.
- Methods for the introduction of exogenous molecules into cells include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer.
- an “endogenous” molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions.
- an endogenous nucleic acid can comprise a chromosome, the genome of a mitochondrion, chloroplast or other organelle, or a naturally-occurring episomal nucleic acid.
- Additional endogenous molecules can include proteins, for example, transcription factors and enzymes.
- a “fusion” molecule is a molecule in which two or more subunit molecules are linked, preferably covalently.
- the subunit molecules can be the same chemical type of molecule, or can be different chemical types of molecules.
- Examples of the first type of fusion molecule include, but are not limited to, fusion proteins (for example, a fusion between a ZFP DNA-binding domain and a cleavage domain) and fusion nucleic acids (for example, a nucleic acid encoding the fusion protein described supra).
- Examples of the second type of fusion molecule include, but are not limited to, a fusion between a triplex-forming nucleic acid and a polypeptide, and a fusion between a minor groove binder and a nucleic acid.
- Fusion protein in a cell can result from delivery of the fusion protein to the cell or by delivery of a polynucleotide encoding the fusion protein to a cell, wherein the polynucleotide is transcribed, and the transcript is translated, to generate the fusion protein.
- Trans-splicing, polypeptide cleavage and polypeptide ligation can also be involved in expression of a protein in a cell. Methods for polynucleotide and polypeptide delivery to cells are presented elsewhere in this disclosure.
- Gene expression refers to the conversion of the information, contained in a gene, into a gene product.
- a gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA.
- Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.
- Modulation of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression.
- Eucaryotic cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells.
- a “region of interest” is any region of cellular chromatin, such as, for example, a gene or a non-coding sequence within or adjacent to a gene, in which it is desirable to bind an exogenous molecule. Binding can be for the purposes of targeted DNA cleavage and/or targeted recombination.
- a region of interest can be present in a chromosome, an episome, an organellar genome (e.g., mitochondrial, chloroplast), or an infecting viral genome, for example.
- a region of interest can be within the coding region of a gene, within transcribed non-coding regions such as, for example, leader sequences, trailer sequences or introns, or within non-transcribed regions, either upstream or downstream of the coding region.
- a region of interest can be as small as a single nucleotide pair or up to 2,000 nucleotide pairs in length, or any integral value of nucleotide pairs.
- operative linkage and “operatively linked” (or “operably linked”) are used interchangeably with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components.
- a transcriptional regulatory sequence such as a promoter
- a transcriptional regulatory sequence is generally operatively linked in cis with a coding sequence, but need not be directly adjacent to it.
- an enhancer is a transcriptional regulatory sequence that is operatively linked to a coding sequence, even though they are not contiguous.
- the term “operatively linked” can refer to the fact that each of the components performs the same function in linkage to the other component as it would if it were not so linked.
- the ZFP DNA-binding domain and the cleavage domain are in operative linkage if, in the fusion polypeptide, the ZFP DNA-binding domain portion is able to bind its target site and/or its binding site, while the cleavage domain is able to cleave DNA in the vicinity of the target site.
- a “functional fragment” of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, yet retains the same function as the full-length protein, polypeptide or nucleic acid.
- a functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one ore more amino acid or nucleotide substitutions.
- DNA-binding function of a polypeptide can be determined, for example, by filter-binding, electrophoretic mobility-shift, or immunoprecipitation assays. DNA cleavage can be assayed by gel electrophoresis. See Ausubel et al., supra.
- the ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays or complementation, both genetic and biochemical. See, for example, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No. 5,585,245 and International Patent Publication No. WO 98/44350.
- the disclosed methods and compositions include fusion proteins comprising a cleavage domain (or a cleavage half-domain) and a zinc finger domain, in which the zinc finger domain, by binding to a sequence in cellular chromatin (e.g., a target site or a binding site), directs the activity of the cleavage domain (or cleavage half-domain) to the vicinity of the sequence and, hence, induces cleavage in the vicinity of the target sequence.
- a zinc finger domain can be engineered to bind to virtually any desired sequence.
- one or more zinc finger binding domains can be engineered to bind to one or more sequences in the region of interest.
- Expression of a fusion protein comprising a zinc finger binding domain and a cleavage domain (or of two fusion proteins, each comprising a zinc finger binding domain and a cleavage half-domain), in a cell effects cleavage in the region of interest.
- Selection of a sequence in cellular chromatin for binding by a zinc finger domain can be accomplished, for example, according to the methods disclosed in co-owned U.S. Pat. No. 6,453,242 (Sep. 17, 2002), which also discloses methods for designing ZFPs to bind to a selected sequence. It will be clear to those skilled in the art that simple visual inspection of a nucleotide sequence can also be used for selection of a target site. Accordingly, any means for target site selection can be used in the claimed methods.
- Target sites are generally composed of a plurality of adjacent target subsites.
- a target subsite refers to the sequence (usually either a nucleotide triplet, or a nucleotide quadruplet that can overlap by one nucleotide with an adjacent quadruplet) bound by an individual zinc finger. See, for example, International Patent Publication No. 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 International Patent Publication No. WO 01/53480. Accordingly, one or more subsites, in a target site for a zinc finger binding domain, can be separated from each other by 1, 2, 3, 4, 5 or more nucleotides.
- a four-finger binding domain can bind to a 13-nucleotide target site comprising, in sequence, two contiguous 3-nucleotide subsites, an intervening nucleotide, and two contiguous triplet subsites.
- Distance between sequences refers to the number of nucleotides or nucleotide pairs intervening between two sequences, as measured from the edges of the sequences nearest each other.
- the two target sites can be on opposite DNA strands. In other embodiments, both target sites are on the same DNA strand.
- a zinc finger binding domain comprises one or more zinc fingers. Miller et al. (1985) EMBO J. 4:1609-1614; Rhodes (1993) Scientific American February: 56-65; U.S. Pat. No. 6,453,242. Typically, a single zinc finger domain is about 30 amino acids in length. Structural studies have demonstrated that each zinc finger domain (motif) contains two beta sheets (held in a beta turn which contains the two invariant cysteine residues) and an alpha helix (containing the two invariant histidine residues), which are held in a particular conformation through coordination of a zinc atom by the two cysteines and the two histidines.
- Zinc fingers include both canonical C 2 H 2 zinc fingers (i.e., those in which the zinc ion is coordinated by two cysteine and two histidine residues) and non-canonical zinc fingers such as, for example, C 3 H zinc fingers (those in which the zinc ion is coordinated by three cysteine residues and one histidine residue) and C 4 zinc fingers (those in which the zinc ion is coordinated by four cysteine residues). See also International Patent Publication No. WO 02/057293.
- Zinc finger binding domains can be engineered to bind to a sequence of choice. See, for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416.
- An engineered zinc finger binding domain can have a novel binding specificity, compared to a naturally-occurring zinc finger protein.
- Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261.
- Exemplary selection methods including phage display and two-hybrid systems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as International Patent Publication Nos. WO 98/37186; WO 98/53057; WO 00/27878; and 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 International Patent Publication No. 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 International Patent Publication No. 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 International Patent Publication Nos. WO 98/53057 and WO 01/53480.
- the cleavage domain portion of the fusion proteins disclosed herein can be obtained from any endo- or exonuclease.
- Exemplary endonucleases from which a cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388.
- Additional enzymes which cleave DNA are known (e.g., Si Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993).
- these enzymes or functional fragments thereof can be used as a source of cleavage domains and cleavage half-domains.
- a cleavage half-domain e.g., fusion proteins comprising a zinc finger binding domain and a cleavage half-domain
- a cleavage half-domain can be derived from any nuclease or portion thereof, as set forth above, that requires dimerization for cleavage activity.
- two fusion proteins are required for cleavage if the fusion proteins comprise cleavage half-domains.
- the two cleavage half-domains can be derived from the same endonuclease (or functional fragments thereof), or each cleavage half-domain can be derived from a different endonuclease (or functional fragments thereof).
- the target sites for the two fusion proteins are preferably disposed, with respect to each other, such that binding of the two fusion proteins 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.
- each fusion protein comprising a cleavage half-domain
- the primary contact strand for the zinc finger portion of each fusion protein will be on a different DNA strands and in opposite orientation. That is, for a pair of ZFP/cleavage half-domain fusions, the target sequences are on opposite strands and the two proteins bind in opposite orientation.
- 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
- FokI catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as well as Li et al.
- fusion proteins comprise the cleavage domain (or cleavage half-domain) from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered.
- FokI An exemplary Type IIS restriction enzyme, whose cleavage domain is separable from the binding domain, is FokI.
- This particular enzyme is active as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95:10,570-10,575. Accordingly, for the purposes of the present disclosure, the portion of the FokI 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 FokI cleavage half-domains can also be used. Parameters for targeted cleavage and targeted sequence alteration using zinc finger-Fold fusions are provided elsewhere in this disclosure.
- Type IIS restriction enzymes are listed in Table 1. Additional restriction enzymes also contain separable binding and cleavage domains, and these are contemplated by the present disclosure. See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.
- fusion proteins and polynucleotides encoding same are known to those of skill in the art. For example, methods for the design and construction of fusion protein comprising zinc finger proteins (and polynucleotides encoding same) are described in co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261. In certain embodiments, polynucleotides encoding such fusion proteins are constructed. These polynucleotides can be inserted into a vector and the vector can be introduced into a cell (see below for additional disclosure regarding vectors and methods for introducing polynucleotides into cells).
- a fusion protein comprises a zinc finger binding domain and a cleavage half-domain from the FokI restriction enzyme, and two such fusion proteins are expressed in a cell.
- Expression of two fusion proteins in a cell can result from delivery of the two proteins to the cell; delivery of one protein and one nucleic acid encoding one of the proteins to the cell; delivery of two nucleic acids, each encoding one of the proteins, to the cell; or by delivery of a single nucleic acid, encoding both proteins, to the cell.
- a fusion protein comprises a single polypeptide chain comprising two cleavage half domains and a zinc finger binding domain. In this case, a single fusion protein is expressed in a cell and, without wishing to be bound by theory, is believed to cleave DNA as a result of formation of an intramolecular dimer of the cleavage half-domains.
- the components of the fusion proteins are arranged such that the zinc finger domain is nearest the amino terminus of the fusion protein, and the cleavage half-domain is nearest the carboxy-terminus.
- This mirrors the relative orientation of the cleavage domain in naturally-occurring dimerizing cleavage domains such as those derived from the FokI enzyme, in which the DNA-binding domain is nearest the amino terminus and the cleavage half-domain is nearest the carboxy terminus.
- the amino acid sequence between the zinc finger binding domain (which is delimited by the N-terminal most of the two conserved cysteine residues and the C-terminal-most of the two conserved histidine residues) and the cleavage domain (or half-domain) is denoted the “ZC linker.”
- the ZC linker is to be distinguished from the inter-finger linkers discussed above.
- the ZC linker is located between the second histidine residue of the C-terminal-most zinc finger and the N-terminal-most amino acid residue of the cleavage half-domain (which is generally glutamine (Q) in the sequence QLV).
- the ZC linker can be any amino acid sequence.
- the length of the linker and the distance between the target sites (binding sites) are interrelated. See, for example, Smith et al. (2000) Nucleic Acids Res. 28:3361-3369; Bibikova et al. (2001) Mol.
- the disclosed methods and compositions can be used to cleave DNA at a region of interest in cellular chromatin (e.g., at a desired or predetermined site in a genome, for example, in a gene, either mutant or wild-type).
- a zinc finger binding domain is engineered to bind a target site at or near the predetermined cleavage site, and a fusion protein comprising the engineered zinc finger binding domain and a cleavage domain is expressed in a cell.
- the DNA is cleaved near the target site by the cleavage domain.
- the exact site of cleavage can depend on the length of the ZC linker.
- two fusion proteins each comprising a zinc finger binding domain and a cleavage half-domain, are expressed in a cell, and bind to target sites which are juxtaposed in such a way that a functional cleavage domain is reconstituted and DNA is cleaved in the vicinity of the target sites.
- cleavage occurs between the target sites of the two zinc finger binding domains.
- One or both of the zinc finger binding domains can be engineered.
- the binding site can encompass the cleavage site, or the near edge of the binding site can be 1, 2, 3, 4, 5, 6, 10, 25, 50 or more nucleotides (or any integral value between 1 and 50 nucleotides) from the cleavage site.
- the exact location of the binding site, with respect to the cleavage site, will depend upon the particular cleavage domain, and the length of the ZC linker.
- the binding sites generally straddle the cleavage site.
- the near edge of the first binding site can be 1, 2, 3, 4, 5, 6, 10, 25 or more nucleotides (or any integral value between 1 and 50 nucleotides) on one side of the cleavage site
- the near edge of the second binding site can be 1, 2, 3, 4, 5, 6, 10, 25 or more nucleotides (or any integral value between 1 and 50 nucleotides) on the other side of the cleavage site.
- the methods described herein can employ an engineered zinc finger binding domain fused to a cleavage domain.
- the binding domain is engineered to bind to a target sequence, at or near which cleavage is desired.
- the fusion protein, or a polynucleotide encoding same is introduced into a cell. Once introduced into, or expressed in, the cell, the fusion protein binds to the target sequence and cleaves at or near the target sequence. The exact site of cleavage depends on the nature of the cleavage domain and/or the presence and/or nature of linker sequences between the binding and cleavage domains.
- the distance between the near edges of the binding sites can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25 or more nucleotides (or any integral value between 1 and 50 nucleotides).
- Optimal levels of cleavage can also depend on both the distance between the binding sites of the two fusion proteins (See, for example, Smith et al. (2000) Nucleic Acids Res. 28:3361-3369; Bibikova et al. (2001) Mol. Cell. Biol. 21:289-297) and the length of the ZC linker in each fusion protein.
- the length of the linker between the ZFP and the FokI cleavage half-domain can influence cleavage efficiency.
- the ZC linker In one experimental system utilizing a ZFP-FokI fusion with a ZC linker of 4 amino acid residues, optimal cleavage was obtained when the near edges of the binding sites for two ZFP-FokI nucleases were separated by 6 base pairs.
- This particular fusion nuclease comprised the following amino acid sequence between the zinc finger portion and the nuclease half-domain:
- the cleavage domain comprises two cleavage half-domains, both of which are part of a single polypeptide comprising a binding domain, a first cleavage half-domain and a second cleavage half-domain.
- the cleavage half-domains can have the same amino acid sequence or different amino acid sequences, so long as they function to cleave the DNA.
- Cleavage half-domains may also be provided in separate molecules.
- two fusion polypeptides may be introduced into a cell, wherein each polypeptide comprises a binding domain and a cleavage half-domain.
- the cleavage half-domains can have the same amino acid sequence or different amino acid sequences, so long as they function to cleave the DNA.
- the binding domains bind to target sequences which are typically disposed in such a way that, upon binding of the fusion polypeptides, the two cleavage half-domains are presented in a spatial orientation to each other that allows reconstitution of a cleavage domain (e.g., by dimerization of the half-domains), thereby positioning the half-domains relative to each other to form a functional cleavage domain, resulting in cleavage of cellular chromatin in a region of interest.
- cleavage by the reconstituted cleavage domain occurs at a site located between the two target sequences.
- One or both of the proteins can be engineered to bind to its target site.
- the two fusion proteins can bind in the region of interest in the same or opposite polarity, and their binding sites (i.e., target sites) can be separated by any number of nucleotides, e.g., from 0 to 200 nucleotides or any integral value therebetween.
- the binding sites for two fusion proteins, each comprising a zinc finger binding domain and a cleavage half-domain can be located between 5 and 18 nucleotides apart, for example, 5-8 nucleotides apart, or 15-18 nucleotides apart, or 6 nucleotides apart, or 16 nucleotides apart, as measured from the edge of each binding site nearest the other binding site, and cleavage occurs between the binding sites.
- the site at which the DNA is cleaved generally lies between the binding sites for the two fusion proteins. Double-strand breakage of DNA often results from two single-strand breaks, or “nicks,” offset by 1, 2, 3, 4, 5, 6 or more nucleotides, (for example, cleavage of double-stranded DNA by native FokI results from single-strand breaks offset by 4 nucleotides). Thus, cleavage does not necessarily occur at exactly opposite sites on each DNA strand.
- the structure of the fusion proteins and the distance between the target sites can influence whether cleavage occurs adjacent a single nucleotide pair, or whether cleavage occurs at several sites. However, for many applications, including targeted recombination (see infra) cleavage within a range of nucleotides is generally sufficient, and cleavage between particular base pairs is not required.
- the fusion protein(s) can be introduced as polypeptides and/or polynucleotides.
- two polynucleotides, each comprising sequences encoding one of the aforementioned polypeptides, can be introduced into a cell, and when the polypeptides are expressed and each binds to its target sequence, cleavage occurs at or near the target sequence.
- a single polynucleotide comprising sequences encoding both fusion polypeptides is introduced into a cell.
- Polynucleotides can be DNA, RNA or any modified forms or analogues or DNA and/or RNA.
- single cleavage half-domains can exhibit limited double-stranded cleavage activity.
- either protein specifies an approximately 9-nucleotide target site.
- the aggregate target sequence of 18 nucleotides is likely to be unique in a mammalian genome, any given 9-nucleotide target site occurs, on average, approximately 23,000 times in the human genome.
- non-specific cleavage due to the site-specific binding of a single half-domain, may occur.
- the methods described herein contemplate the use of a dominant-negative mutant of a cleavage half-domain such as FokI (or a nucleic acid encoding same) that is expressed in a cell along with the two fusion proteins.
- the dominant-negative mutant is capable of dimerizing but is unable to cleave, and also blocks the cleavage activity of a half-domain to which it is dimerized.
- By providing the dominant-negative mutant in molar excess to the fusion proteins only regions in which both fusion proteins are bound will have a high enough local concentration of functional cleavage half-domains for dimerization and cleavage to occur. At sites where only one of the two fusion proteins are bound, its cleavage half-domain forms a dimer with the dominant negative mutant half-domain, and undesirable, non-specific cleavage does not occur.
- Methods for targeted cleavage which involve the use of fusions between a ZFP and a cleavage half-domain (such as, e.g., a ZFP/FokI fusion) require the use of two such fusion molecules, each generally directed to a distinct target sequence.
- Target sequences for the two fusion proteins can be chosen so that targeted cleavage is directed to a unique site in a genome, as discussed above.
- a potential source of reduced cleavage specificity could result from homodimerization of one of the two ZFP/cleavage half-domain fusions.
- One approach for reducing the probability of this type of aberrant cleavage at sequences other than the intended target site involves generating variants of the cleavage half-domain that minimize or prevent homodimerization. Preferably, one or more amino acids in the region of the half-domain involved in its dimerization are altered.
- the structure of the cleavage half-domains is reported to be similar to the arrangement of the cleavage half-domains during cleavage of DNA by FokI. Wah et al. (1998) Proc. Natl. Acad. Sci. USA 95:10564-10569.
- This structure indicates that amino acid residues at positions 483 and 487 play a key role in the dimerization of the FokI cleavage half-domains.
- the structure also indicates that amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 are all close enough to the dimerization interface to influence dimerization. Accordingly, amino acid sequence alterations at one or more of the aforementioned positions will likely alter the dimerization properties of the cleavage half-domain.
- Such changes can be introduced, for example, by constructing a library containing (or encoding) different amino acid residues at these positions and selecting variants with the desired properties, or by rationally designing individual mutants. In addition to preventing homodimerization, it is also possible that some of these mutations may increase the cleavage efficiency above that obtained with two wild-type cleavage half-domains.
- alteration of a FokI cleavage half-domain at any amino acid residue which affects dimerization can be used to prevent one of a pair of ZFP/FokI fusions from undergoing homodimerization which can lead to cleavage at undesired sequences.
- one or both of the fusion proteins can comprise one or more amino acid alterations that inhibit self-dimerization, but allow heterodimerization of the two fusion proteins to occur such that cleavage occurs at the desired target site.
- alterations are present in both fusion proteins, and the alterations have additive effects; i.e., homodimerization of either fusion, leading to aberrant cleavage, is minimized or abolished, while heterodimerization of the two fusion proteins is facilitated compared to that obtained with wild-type cleavage half-domains. See Example 5.
- a genomic sequence e.g., a region of interest in cellular chromatin
- a homologous non-identical sequence i.e., targeted recombination.
- Previous attempts to replace particular sequences have involved contacting a cell with a polynucleotide comprising sequences bearing homology to a chromosomal region (i.e., a donor DNA), followed by selection of cells in which the donor DNA molecule had undergone homologous recombination into the genome.
- the success rate of these methods is low, due to poor efficiency of homologous recombination and a high frequency of non-specific insertion of the donor DNA into regions of the genome other than the target site.
- the present disclosure provides methods of targeted sequence alteration characterized by a greater efficiency of targeted recombination and a lower frequency of non-specific insertion events.
- the methods involve making and using engineered zinc finger binding domains fused to cleavage domains (or cleavage half-domains) to make one or more targeted double-stranded breaks in cellular DNA. Because double-stranded breaks in cellular DNA stimulate homologous recombination several thousand-fold in the vicinity of the cleavage site, such targeted cleavage allows for the alteration or replacement (via homologous recombination) of sequences at virtually any site in the genome.
- targeted replacement of a selected genomic sequence also requires the introduction of the replacement (or donor) sequence.
- the donor sequence can be introduced into the cell prior to, concurrently with, or subsequent to, expression of the fusion protein(s).
- the donor polynucleotide contains sufficient homology to a genomic sequence to support homologous recombination between it and the genomic sequence to which it bears homology. Approximately 25, 50 100 or 200 nucleotides or more of sequence homology between a donor and a genomic sequence (or any integral value between 10 and 200 nucleotides, or more) will support homologous recombination therebetween.
- Donor sequences can range in length from 10 to 5,000 nucleotides (or any integral value of nucleotides therebetween) or longer. It will be readily apparent that the donor sequence is typically not identical to the genomic sequence that it replaces.
- the sequence of the donor polynucleotide can contain one or more single base changes, insertions, deletions, inversions or rearrangements with respect to the genomic sequence, so long as sufficient homology is present to support homologous recombination.
- a donor sequence can contain a non-homologous sequence flanked by two regions of homology.
- donor sequences can comprise a vector molecule containing sequences that are not homologous to the region of interest in cellular chromatin.
- the homologous region(s) of a donor sequence will have at least 50% sequence identity to a genomic sequence with which recombination is desired. In certain embodiments, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity is present. Any value between 1% and 100% sequence identity can be present, depending upon the length of the donor polynucleotide.
- a donor molecule can contain several, discontinuous regions of homology to cellular chromatin. For example, for targeted insertion of sequences not normally present in a region of interest, said sequences can be present in a donor nucleic acid molecule and flanked by regions of homology to sequence in the region of interest.
- certain sequence differences may be present in the donor sequence as compared to the genomic sequence.
- such nucleotide sequence differences will not change the amino acid sequence, or will make silent amino acid changes (i.e., changes which do not affect the structure or function of the protein).
- the donor polynucleotide can optionally contain changes in sequences corresponding to the zinc finger domain binding sites in the region of interest, to prevent cleavage of donor sequences that have been introduced into cellular chromatin by homologous recombination.
- the donor polynucleotide can be DNA or RNA, single-stranded or double-stranded and can be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3′ terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls et al.
- Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.
- a polynucleotide can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance.
- donor polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV).
- viruses e.g., adenovirus, AAV
- a double-stranded break in a cellular sequence activates cellular mechanisms which repair the break by transfer of sequence information from the donor molecule into the cellular (e.g., genomic or chromosomal) sequence; i.e., by a processes of homologous recombination.
- Applicants' methods advantageously combine the powerful targeting capabilities of engineered ZFPs with a cleavage domain (or cleavage half-domain) to specifically target a double-stranded break to the region of the genome at which recombination is desired.
- the efficiency of insertion of donor sequences by homologous recombination is inversely related to the distance, in the cellular DNA, between the double-stranded break and the site at which recombination is desired. In other words, higher homologous recombination efficiencies are observed when the double-stranded break is closer to the site at which recombination is desired.
- a precise site of recombination is not predetermined (e.g., the desired recombination event can occur over an interval of genomic sequence)
- the length and sequence of the donor nucleic acid, together with the site(s) of cleavage are selected to obtain the desired recombination event.
- cellular chromatin is cleaved within 10,000 nucleotides on either side of that nucleotide pair. In certain embodiments, cleavage occurs within 500, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, or 2 nucleotides, or any integral value between 2 and 1,000 nucleotides, on either side of the nucleotide pair whose sequence is to be changed.
- the binding sites for two fusion proteins each comprising a zinc finger binding domain and a cleavage half-domain, can be located 5-8 or 15-18 nucleotides apart, as measured from the edge of each binding site nearest the other binding site, and cleavage occurs between the binding sites. Whether cleavage occurs at a single site or at multiple sites between the binding sites is immaterial, since the cleaved genomic sequences are replaced by the donor sequences.
- the midpoint of the region between the binding sites is within 10,000 nucleotides of that nucleotide pair, preferably within 1,000 nucleotides, or 500 nucleotides, or 200 nucleotides, or 100 nucleotides, or 50 nucleotides, or 20 nucleotides, or 10 nucleotides, or 5 nucleotide, or 2 nucleotides, or one nucleotide, or at the nucleotide pair of interest.
- a homologous chromosome can serve as the donor polynucleotide.
- correction of a mutation in a heterozygote can be achieved by engineering fusion proteins which bind to and cleave the mutant sequence on one chromosome, but do not cleave the wild-type sequence on the homologous chromosome.
- the double-stranded break on the mutation-bearing chromosome stimulates a homology-based “gene conversion” process in which the wild-type sequence from the homologous chromosome is copied into the cleaved chromosome, thus restoring two copies of the wild-type sequence.
- Methods and compositions are also provided that may enhance levels of targeted recombination including, but not limited to, the use of additional ZFP-functional domain fusions to activate expression of genes involved in homologous recombination, such as, for example, members of the RAD52 epistasis group (e.g., Rad50, Rad51, Rad51B, Rad51C, Rad51D, Rad52, Rad54, Rad54B, Mre11, XRCC2, XRCC3), genes whose products interact with the aforementioned gene products (e.g., BRCA1, BRCA2) and/or genes in the NBS1 complex.
- members of the RAD52 epistasis group e.g., Rad50, Rad51, Rad51B, Rad51C, Rad51D, Rad52, Rad54, Rad54B, Mre11, XRCC2, XRCC3
- genes whose products interact with the aforementioned gene products e.g., BRCA1, BRCA2
- genes in the NBS1 complex e.
- ZFP-functional domain fusions can be used, in combination with the methods and compositions disclosed herein, to repress expression of genes involved in non-homologous end joining (e.g., Ku70/80, XRCC4, poly(ADP ribose) polymerase, DNA ligase 4).
- non-homologous end joining e.g., Ku70/80, XRCC4, poly(ADP ribose) polymerase, DNA ligase 4
- Non-homologous end joining e.g., Ku70/80, XRCC4, poly(ADP ribose) polymerase, DNA ligase 4
- Non-homologous end joining e.g., Ku70/80, XRCC4, poly(ADP ribose) polymerase, DNA ligase 4
- Yanez et al. 1998 Gene Therapy 5:149-159; Hoeijmakers (2001) Nature 411:366-374; Johnson et
- Additional repression methods include the use of antisense oligonucleotides and/or small interfering RNA (siRNA or RNAi) targeted to the sequence of the gene to be repressed.
- siRNA or RNAi small interfering RNA
- fusions of these protein (or functional fragments thereof) with a zinc finger binding domain targeted to the region of interest can be used to recruit these proteins (recombination proteins) to the region of interest, thereby increasing their local concentration and further stimulating homologous recombination processes.
- a polypeptide involved in homologous recombination as described above (or a functional fragment thereof) can be part of a triple fusion protein comprising a zinc finger binding domain, a cleavage domain (or cleavage half-domain) and the recombination protein (or functional fragment thereof).
- Additional proteins involved in gene conversion and recombination-related chromatin remodeling include histone acetyltransferases (e.g., Esalp, Tip60), histone methyltransferases (e.g., Dotlp), histone kinases and histone phosphatases.
- the p53 protein has been reported to play a central role in repressing homologous recombination (HR). See, for example, Valerie et al., (2003) Oncogene 22:5792-5812; Janz et al. (2002) Oncogene 21:5929-5933.
- HR homologous recombination
- the rate of HR in p53-deficient human tumor lines is 10,000-fold greater than in primary human fibroblasts, and there is a 100-fold increase in HR in tumor cells with a non-functional p53 compared to those with functional p53.
- Mekeel et al. (1997) Oncogene 14:1847-1857.
- Any method for downregulation of p53 activity can be used, including but not limited to cotransfection and overexpression of a p53 dominant negative mutant or targeted repression of p53 gene expression according to methods disclosed, e.g., in co-owned U.S. Pat. No. 6,534,261.
- Exemplary molecules of this type include, but are not limited to, compounds which affect microtubule polymerization (e.g., vinblastine, nocodazole, Taxol), compounds that interact with DNA (e.g., cis-platinum(II) diamine dichloride, Cisplatin, doxorubicin) and/or compounds that affect DNA synthesis (e.g., thymidine, hydroxyurea, L-mimosine, etoposide, 5-fluorouracil).
- compounds which affect microtubule polymerization e.g., vinblastine, nocodazole, Taxol
- compounds that interact with DNA e.g., cis-platinum(II) diamine dichloride, Cisplatin, doxorubicin
- compounds that affect DNA synthesis e.g., thymidine, hydroxyurea, L-mimosine, etoposide, 5-fluorouracil.
- HDAC histone deacetylase
- Additional methods for cell-cycle arrest include overexpression of proteins which inhibit the activity of the CDK cell-cycle kinases, for example, by introducing a cDNA encoding the protein into the cell or by introducing into the cell an engineered ZFP which activates expression of the gene encoding the protein.
- Cell-cycle arrest is also achieved by inhibiting the activity of cyclins and CDKs, for example, using RNAi methods (e.g., U.S. Pat. No. 6,506,559) or by introducing into the cell an engineered ZFP which represses expression of one or more genes involved in cell-cycle progression such as, for example, cyclin and/or CDK genes. See, e.g., co-owned U.S. Pat. No. 6,534,261 for methods for the synthesis of engineered zinc finger proteins for regulation of gene expression.
- targeted cleavage is conducted in the absence of a donor polynucleotide (preferably in S or G 2 phase), and recombination occurs between homologous chromosomes.
- a donor polynucleotide preferably in S or G 2 phase
- homologous recombination is a multi-step process requiring the modification of DNA ends and the recruitment of several cellular factors into a protein complex
- addition of one or more exogenous factors, along with donor DNA and vectors encoding zinc finger-cleavage domain fusions, can be used to facilitate targeted homologous recombination.
- An exemplary method for identifying such a factor or factors employs analyses of gene expression using microarrays (e.g., Affymetrix Gene Chip® arrays) to compare the mRNA expression patterns of different cells.
- cells that exhibit a higher capacity to stimulate double strand break-driven homologous recombination in the presence of donor DNA and zinc finger-cleavage domain fusions can be analyzed for their gene expression patterns compared to cells that lack such capacity.
- Genes that are upregulated or downregulated in a manner that directly correlates with increased levels of homologous recombination are thereby identified and can be cloned into any one of a number of expression vectors.
- These expression constructs can be co-transfected along with zinc finger-cleavage domain fusions and donor constructs to yield improved methods for achieving high-efficiency homologous recombination.
- expression of such genes can be appropriately regulated using engineered zinc finger proteins which modulate expression (either activation or repression) of one or more these genes. See, e.g., co-owned U.S. Pat. No. 6,534,261 for methods for the synthesis of engineered zinc finger proteins for regulation of gene expression.
- Candidate genes that are differentially expressed in cells that carry out homologous recombination at a higher rate, either unaided or in response to compounds that arrest the cells in G2, are identified, cloned, and re-introduced into cells to determine whether their expression is sufficient to re-capitulate the improved rates.
- expression of said candidate genes is activated using engineered zinc finger transcription factors as described, for example, in co-owned U.S. Pat. No. 6,534,261.
- a nucleic acid encoding one or more ZFPs or ZFP fusion proteins can be cloned into a vector for transformation into prokaryotic or eukaryotic cells for replication and/or expression.
- Vectors can be prokaryotic vectors, e.g., plasmids, or shuttle vectors, insect vectors, or eukaryotic vectors.
- a nucleic acid encoding a ZFP can also be cloned into an expression vector, for administration to a plant cell, animal cell, preferably a mammalian cell or a human cell, fungal cell, bacterial cell, or protozoal cell.
- sequences encoding a ZFP or ZFP fusion protein are typically subcloned into an expression vector that contains a promoter to direct transcription.
- Suitable bacterial and eukaryotic promoters are well known in the art and described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989; 3 rd ed., 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., supra.
- Bacterial expression systems for expressing the ZFP are available in, e.g., E.
- Kits for such expression systems are commercially available.
- Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known by those of skill in the art and are also commercially available.
- the promoter used to direct expression of a ZFP-encoding nucleic acid depends on the particular application. For example, a strong constitutive promoter is typically used for expression and purification of ZFP. In contrast, when a ZFP is administered in vivo for gene regulation, either a constitutive or an inducible promoter is used, depending on the particular use of the ZFP.
- a preferred promoter for administration of a ZFP can be a weak promoter, such as HSV TK or a promoter having similar activity.
- the promoter typically can also include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tet-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard (1992) PNAS 89:5547; Oligino et al. (1998) Gene Ther. 5:491-496; Wang et al. (1997) Gene Ther. 4:432-441; Neering et al. (1996) Blood 88:1147-1155; and Rendahl et al. (1998) Nat. Biotechnol. 16:757-761).
- the MNDU3 promoter can also be used, and is preferentially active in CD34 + hematopoietic stem cells.
- the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in host cells, either prokaryotic or eukaryotic.
- a typical expression cassette thus contains a promoter operably linked, e.g., to a nucleic acid sequence encoding the ZFP, and signals required, e.g., for efficient polyadenylation of the transcript, transcriptional termination, ribosome binding sites, or translation termination. Additional elements of the cassette may include, e.g., enhancers, and heterologous splicing signals.
- the particular expression vector used to transport the genetic information into the cell is selected with regard to the intended use of the ZFP, e.g., expression in plants, animals, bacteria, fungus, protozoa, etc. (see expression vectors described below).
- Standard bacterial expression vectors include plasmids such as pBR322-based plasmids, pSKF, pET23D, and commercially available fusion expression systems such as GST and LacZ.
- An exemplary fusion protein is the maltose binding protein, “MBP.” Such fusion proteins are used for purification of the ZFP.
- Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, for monitoring expression, and for monitoring cellular and subcellular localization, e.g., c-myc or FLAG.
- Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus.
- eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
- Some expression systems have markers for selection of stably transfected cell lines such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase.
- High yield expression systems are also suitable, such as using a baculovirus vector in insect cells, with a ZFP encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.
- the elements that are typically included in expression vectors also include a replicon that functions in E. coli , a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of recombinant sequences.
- Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, which are then purified using standard techniques (see, e.g., Colley et al. (1989) J. Biol. Chem. 264:17619-17622 ; Guide to Protein Purification, in Methods in Enzymology , vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison (1977) J. Bact. 132:349-351; Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).
- Any of the well known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, ultrasonic methods (e.g., sonoporation), liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the protein of choice.
- Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
- Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
- Methods of non-viral delivery of nucleic acids encoding engineered ZFPs include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids.
- nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.) and BTX Molecular Delivery Systems (Holliston, Mass.).
- Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM).
- Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, International Patent Publication Nos. WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration).
- lipid:nucleic acid complexes including targeted liposomes such as immunolipid complexes
- the preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes is well known to one of skill in the art (see, e.g., Crystal (1995) Science 270:404-410; Blaese et al. (1995) Cancer Gene Ther. 2:291-297; Behr et al. (1994) Bioconjugate Chem. 5:382-389; Remy et al. (1994) Bioconjugate Chem. 5:647-654; Gao et al. (1995) Gene Therapy 2:710-722; Ahmad et al. (1992) Cancer Res. 52:4817-4820; U.S. Pat. Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028; and
- RNA or DNA viral based systems for the delivery of nucleic acids encoding engineered ZFPs take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus.
- Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo).
- Conventional viral based systems for the delivery of ZFPs include, but are not limited to, retroviral, lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
- Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system depends on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression.
- Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al. (1992) J. Virol. 66:2731-2739; Johann et al. (1992) J. Virol. 66:1635-1640; Sommerfelt et al. (1990) Virol. 176:58-59; Wilson et al. (1989) J. Virol. 63:2374-2378; Miller et al. (1991) J. Virol. 65:2220-2224; International Patent Publication No. WO 94/26877).
- MiLV murine leukemia virus
- GaLV gibbon ape leukemia virus
- SIV Simian Immunodeficiency virus
- HAV human immunodeficiency virus
- Adenoviral based systems can be used.
- Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and high levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system.
- Adeno-associated virus (“AAV”) vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al. (1987) Virology 160:38-47; U.S. Pat. No.
- Adeno-associated virus vectors include AAV serotypes 1, 2, 5, 6, 7, 8 and 9; as well as chimeric AAV serotypes, e.g., AAV 2/1 and AAV 2/5. Both single-stranded and double-stranded (e.g., self-complementary) AAV vectors can be used.
- 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. (1995) Blood 85:3048-305; Kohn et al. (1995) Nat. Med. 1:1017-102; Malech et al. (1997) PNAS 94(22):12133-12138).
- PA317/pLASN was the first therapeutic vector used in a gene therapy trial. (Blaese et al. (1995) Science 270:475-480). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors. (Ellem et al. (1997) Immunol Immunother 44(1):10-20; Dranoff et al. (1997) Hum. Gene Ther. 1:111-2.
- 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. (1998) Lancet 351(9117):1702-3, Kearns et al. (1996) Gene Ther. 9:748-55).
- Ad vectors 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. 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.
- 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 w2 cells or PA317 cells, which package retrovirus.
- Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host (if applicable), other viral sequences being replaced by an expression cassette encoding the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line.
- AAV vectors used in gene therapy typically only possess inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and integration into the host genome.
- ITR inverted terminal repeat
- Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
- the cell line is also infected with adenovirus as a helper.
- the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
- the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.
- a viral vector can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus.
- the ligand is chosen to have affinity for a receptor known to be present on the cell type of interest.
- Han et al. (1995) Proc. Natl. Acad. Sci. USA 92:9747-9751 reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor.
- filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor.
- Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below.
- vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
- Ex vivo cell transfection for diagnostics, research, or for gene therapy is well known to those of skill in the art.
- cells are isolated from the subject organism, transfected with a ZFP nucleic acid (gene or cDNA), and re-infused back into the subject organism (e.g., patient).
- a ZFP nucleic acid gene or cDNA
- Various cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et al., Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) and the references cited therein for a discussion of how to isolate and culture cells from patients).
- stem cells are used in ex vivo procedures for cell transfection and gene therapy.
- the advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow.
- Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such a GM-CSF, IFN- ⁇ and TNF- ⁇ are known (see Inaba et al. (1992) J. Exp. Med. 176:1693-1702).
- 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 Tad (differentiated antigen presenting cells) (see Inaba et al. (1992) J. Exp. Med. 176:1693-1702).
- unwanted cells such as CD4+ and CD8+(T cells), CD45+(panB cells), GR-1 (granulocytes), and Tad (differentiated antigen presenting cells) (see Inaba et al. (1992) J. Exp. Med. 176:1693-1702).
- Vectors e.g., retroviruses, adenoviruses, liposomes, etc.
- therapeutic ZFP nucleic acids can also be administered directly to an organism for transduction of cells in vivo.
- naked DNA can be administered.
- Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
- Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions available, as described below (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).
- DNA constructs may be introduced into the genome of a desired plant host by a variety of conventional techniques. For reviews of such techniques see, for example, Weissbach & Weissbach Methods for Plant Molecular Biology (1988, Academic Press, N.Y.) Section VIII, pp. 421-463; and Grierson & Corey, Plant Molecular Biology (1988, 2d Ed.), Blackie, London, Ch. 7-9.
- the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using biolistic methods, such as DNA particle bombardment (see, e.g., Klein et al. (1987) Nature 327:70-73).
- the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector.
- Agrobacterium tumefaciens -mediated transformation techniques including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et al. (1984) Science 233:496-498, and Fraley et al. (1983) Proc. Nat'l. Acad. Sci. USA 80:4803.
- the virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria using binary T DNA vector (Bevan (1984) Nuc. Acid Res.
- Agrobacterium transformation system is used to engineer dicotyledonous plants (Bevan et al. (1982) Ann. Rev. Genet 16:357-384; Rogers et al. (1986) Methods Enzymol. 118:627-641).
- the Agrobacterium transformation system may also be used to transform, as well as transfer, DNA to monocotyledonous plants and plant cells. See Hernalsteen et al. (1984) EMBO J 3:3039-3041; Hooykass-Van Slogteren et al.
- Alternative gene transfer and transformation methods include, but are not limited to, protoplast transformation through calcium-, polyethylene glycol (PEG)- or electroporation-mediated uptake of naked DNA (see Paszkowski et al. (1984) EMBO J 3:2717-2722, Potrykus et al. (1985) Molec. Gen. Genet. 199:169-177; Fromm et al. (1985) Proc. Nat. Acad. Sci. USA 82:5824-5828; and Shimamoto (1989) Nature 338:274-276) and electroporation of plant tissues (D'Halluin et al. (1992) Plant Cell 4:1495-1505).
- PEG polyethylene glycol
- Additional methods for plant cell transformation include microinjection, silicon carbide mediated DNA uptake (Kaeppler et al. (1990) Plant Cell Reporter 9:415-418), and microprojectile bombardment (see Klein et al. (1988) Proc. Nat. Acad. Sci. USA 85:4305-4309; and Gordon-Kamm et al. (1990) Plant Cell 2:603-618).
- the disclosed methods and compositions can be used to insert exogenous sequences into a predetermined location in a plant cell genome. This is useful inasmuch as expression of an introduced transgene into a plant genome depends critically on its integration site. Accordingly, genes encoding, e.g., nutrients, antibiotics or therapeutic molecules can be inserted, by targeted recombination, into regions of a plant genome favorable to their expression.
- Transformed plant cells which are produced by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype.
- Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences.
- Plant regeneration from cultured protoplasts is described in Evans, et al., “Protoplasts Isolation and Culture” in Handbook of Plant Cell Culture , pp. 124-176, Macmillian Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, pollens, embryos or parts thereof. Such regeneration techniques are described generally in Klee et al. (1987) Ann. Rev. of Plant Phys. 38:467-486.
- Nucleic acids introduced into a plant cell can be used to confer desired traits on essentially any plant.
- a wide variety of plants and plant cell systems may be engineered for the desired physiological and agronomic characteristics described herein using the nucleic acid constructs of the present disclosure and the various transformation methods mentioned above.
- target plants and plant cells for engineering include, but are not limited to, those monocotyledonous and dicotyledonous plants, such as crops including grain crops (e.g., wheat, maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear, strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops (e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g., lettuce, spinach); flowering plants (e.g., petunia, rose, chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plants used in phytoremediation (e.g., heavy metal accumulating plants); oil crops (e.g., sunflower, rape seed) and plants used for experimental purposes (e.g., Arabidopsis ).
- crops including grain crops (e.g., wheat, maize, rice, millet, barley), fruit
- the disclosed methods and compositions have use over a broad range of plants, including, but not limited to, species from the genera Asparagus, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucurbita, Daucus, Glycine, Hordeum, Lactuca, Lycopersicon, Malus, Manihot, Nicotiana, Oryza, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Solanum, Sorghum, Triticum, Vitis, Vigna , and Zea.
- the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
- a transformed plant cell, callus, tissue or plant may be identified and isolated by selecting or screening the engineered plant material for traits encoded by the marker genes present on the transforming DNA. For instance, selection may be performed by growing the engineered plant material on media containing an inhibitory amount of the antibiotic or herbicide to which the transforming gene construct confers resistance. Further, transformed plants and plant cells may also be identified by screening for the activities of any visible marker genes (e.g., the ⁇ -glucuronidase, luciferase, B or C1 genes) that may be present on the recombinant nucleic acid constructs. Such selection and screening methodologies are well known to those skilled in the art.
- any visible marker genes e.g., the ⁇ -glucuronidase, luciferase, B or C1 genes
- Physical and biochemical methods also may be used to identify plant or plant cell transformants containing inserted gene constructs. These methods include but are not limited to: 1) Southern analysis or PCR amplification for detecting and determining the structure of the recombinant DNA insert; 2) Northern blot, 51 RNase protection, primer-extension or reverse transcriptase-PCR amplification for detecting and examining RNA transcripts of the gene constructs; 3) enzymatic assays for detecting enzyme or ribozyme activity, where such gene products are encoded by the gene construct; 4) protein gel electrophoresis, Western blot techniques, immunoprecipitation, or enzyme-linked immunoassays, where the gene construct products are proteins.
- RNA e.g., mRNA
- Effects of gene manipulation using the methods disclosed herein can be observed by, for example, northern blots of the RNA (e.g., mRNA) isolated from the tissues of interest. Typically, if the amount of mRNA has increased, it can be assumed that the corresponding endogenous gene is being expressed at a greater rate than before. Other methods of measuring gene and/or CYP74B activity can be used. Different types of enzymatic assays can be used, depending on the substrate used and the method of detecting the increase or decrease of a reaction product or by-product.
- the levels of and/or CYP74B protein expressed can be measured immunochemically, i.e., ELISA, RIA, EIA and other antibody based assays well known to those of skill in the art, such as by electrophoretic detection assays (either with staining or western blotting).
- the transgene may be selectively expressed in some tissues of the plant or at some developmental stages, or the transgene may be expressed in substantially all plant tissues, substantially along its entire life cycle. However, any combinatorial expression mode is also applicable.
- the present disclosure also encompasses seeds of the transgenic plants described above wherein the seed has the transgene or gene construct.
- the present disclosure further encompasses the progeny, clones, cell lines or cells of the transgenic plants described above wherein said progeny, clone, cell line or cell has the transgene or gene construct.
- polypeptide compounds such as ZFP fusion proteins
- ZFP fusion proteins An important factor in the administration of polypeptide compounds, such as ZFP fusion proteins, is ensuring that the polypeptide has the ability to traverse the plasma membrane of a cell, or the membrane of an intra-cellular compartment such as the nucleus.
- Cellular membranes are composed of lipid-protein bilayers that are freely permeable to small, nonionic lipophilic compounds and are inherently impermeable to polar compounds, macromolecules, and therapeutic or diagnostic agents.
- proteins and other compounds such as liposomes have been described, which have the ability to translocate polypeptides such as ZFPs across a cell membrane.
- membrane translocation polypeptides have amphiphilic or hydrophobic amino acid subsequences that have the ability to act as membrane-translocating carriers.
- homeodomain proteins have the ability to translocate across cell membranes.
- the shortest internalizable peptide of a homeodomain protein, Antennapedia was found to be the third helix of the protein, from amino acid position 43 to 58 (see, e.g., Prochiantz (1996) Current Opinion in Neurobiology 6:629-634).
- Another subsequence, the h (hydrophobic) domain of signal peptides was found to have similar cell membrane translocation characteristics (see, e.g., Lin et al. (1995) J. Biol. Chem. 270(1):4255-14258).
- Examples of peptide sequences which can be linked to a protein, for facilitating uptake of the protein into cells include, but are not limited to: an 11 amino acid peptide of the tat protein of HIV; a 20 residue peptide sequence which corresponds to amino acids 84-103 of the p16 protein (see Fahraeus et al. (1996) Current Biology 6:84); the third helix of the 60-amino acid long homeodomain of Antennapedia (Derossi et al. (1994) J. Biol. Chem.
- a signal peptide such as the Kaposi fibroblast growth factor (K-FGF) h region (Lin et al., supra); or the VP22 translocation domain from HSV (Elliot & O'Hare (1997) Cell 88:223-233).
- K-FGF Kaposi fibroblast growth factor
- VP22 translocation domain from HSV
- Other suitable chemical moieties that provide enhanced cellular uptake may also be chemically linked to ZFPs.
- Membrane translocation domains i.e., internalization domains
- Toxin molecules also have the ability to transport polypeptides across cell membranes. Often, such molecules (called “binary toxins”) are composed of at least two parts: a translocation/binding domain or polypeptide and a separate toxin domain or polypeptide. Typically, the translocation domain or polypeptide binds to a cellular receptor, and then the toxin is transported into the cell.
- binary toxins are composed of at least two parts: a translocation/binding domain or polypeptide and a separate toxin domain or polypeptide. Typically, the translocation domain or polypeptide binds to a cellular receptor, and then the toxin is transported into the cell.
- Clostridium perfringens iota toxin diphtheria toxin (DT), Pseudomonas exotoxin A (PE), pertussis toxin (PT), Bacillus anthracis toxin, and pertussis adenylate cyclase (CYA)
- DT diphtheria toxin
- PE Pseudomonas exotoxin A
- PT pertussis toxin
- Bacillus anthracis toxin Bacillus anthracis toxin
- pertussis adenylate cyclase CYA
- Such peptide sequences can be used to translocate ZFPs across a cell membrane.
- ZFPs can be conveniently fused to or derivatized with such sequences.
- the translocation sequence is provided as part of a fusion protein.
- a linker can be used to link the ZFP and the translocation sequence. Any suitable linker can be used, e.g., a peptide linker.
- the ZFP can also be introduced into an animal cell, preferably a mammalian cell, via a liposomes and liposome derivatives such as immunoliposomes.
- liposome refers to vesicles comprised of one or more concentrically ordered lipid bilayers, which encapsulate an aqueous phase.
- the aqueous phase typically contains the compound to be delivered to the cell, i.e., a ZFP.
- the liposome fuses with the plasma membrane, thereby releasing the drug into the cytosol.
- the liposome is phagocytosed or taken up by the cell in a transport vesicle. Once in the endosome or phagosome, the liposome either degrades or fuses with the membrane of the transport vesicle and releases its contents.
- the liposome In current methods of drug delivery via liposomes, the liposome ultimately becomes permeable and releases the encapsulated compound (in this case, a ZFP) at the target tissue or cell.
- the encapsulated compound in this case, a ZFP
- this can be accomplished, for example, in a passive manner wherein the liposome bilayer degrades over time through the action of various agents in the body.
- active drug release involves using an agent to induce a permeability change in the liposome vesicle.
- Liposome membranes can be constructed so that they become destabilized when the environment becomes acidic near the liposome membrane (see, e.g., Wang et al. (1987) PNAS 84(22):7851-5 ; Biochemistry 28:908 (1989)).
- DOPE Dioleoylphosphatidylethanolamine
- Such liposomes typically comprise a ZFP and a lipid component, e.g., a neutral and/or cationic lipid, optionally including a receptor-recognition molecule such as an antibody that binds to a predetermined cell surface receptor or ligand (e.g., an antigen).
- a lipid component e.g., a neutral and/or cationic lipid, optionally including a receptor-recognition molecule such as an antibody that binds to a predetermined cell surface receptor or ligand (e.g., an antigen).
- Suitable methods include, for example, sonication, extrusion, high pressure/homogenization, microfluidization, detergent dialysis, calcium-induced fusion of small liposome vesicles and ether-fusion methods, all of which are known to those of skill in the art.
- targeting moieties that are specific to a particular cell type, tissue, and the like.
- targeting moieties e.g., ligands, receptors, and monoclonal antibodies
- targeting moieties include monoclonal antibodies specific to antigens associated with neoplasms, such as prostate cancer specific antigen and MAGE. Tumors can also be diagnosed by detecting gene products resulting from the activation or over-expression of oncogenes, such as ras or c-erbB2. In addition, many tumors express antigens normally expressed by fetal tissue, such as the alphafetoprotein (AFP) and carcinoembryonic antigen (CEA).
- AFP alphafetoprotein
- CEA carcinoembryonic antigen
- Sites of viral infection can be diagnosed using various viral antigens such as hepatitis B core and surface antigens (HBVc, HBVs) hepatitis C antigens, Epstein-Barr virus antigens, human immunodeficiency type-1 virus (HIV1) and papilloma virus antigens.
- Inflammation can be detected using molecules specifically recognized by surface molecules which are expressed at sites of inflammation such as integrins (e.g., VCAM-1), selectin receptors (e.g., ELAM-1) and the like.
- Standard methods for coupling targeting agents to liposomes can be used. These methods generally involve incorporation into liposomes of lipid components, e.g., phosphatidylethanolamine, which can be activated for attachment of targeting agents, or derivatized lipophilic compounds, such as lipid derivatized bleomycin.
- lipid components e.g., phosphatidylethanolamine
- derivatized lipophilic compounds such as lipid derivatized bleomycin.
- Antibody targeted liposomes can be constructed using, for instance, liposomes which incorporate protein A (see Renneisen et al. (1990) J. Biol. Chem. 265:16337-16342 and Leonetti et al. (1990) PNAS 87:2448-2451.
- the dose administered to a patient, or to a cell which will be introduced into a patient, in the context of the present disclosure, should be sufficient to effect a beneficial therapeutic response in the patient over time.
- particular dosage regimens can be useful for determining phenotypic changes in an experimental setting, e.g., in functional genomics studies, and in cell or animal models.
- the dose will be determined by the efficacy and K d of the particular ZFP employed, the nuclear volume of the target cell, and the condition of the patient, as well as the body weight or surface area of the patient to be treated.
- the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound or vector in a particular patient.
- the maximum therapeutically effective dosage of ZFP for approximately 99% binding to target sites is calculated to be in the range of less than about 1.5 ⁇ 10 5 to 1.5 ⁇ 10 6 copies of the specific ZFP molecule per cell.
- the appropriate dose of an expression vector encoding a ZFP can also be calculated by taking into account the average rate of ZFP expression from the promoter and the average rate of ZFP degradation in the cell.
- a weak promoter such as a wild-type or mutant HSV TK promoter is used, as described above.
- the dose of ZFP in micrograms is calculated by taking into account the molecular weight of the particular ZFP being employed.
- the physician evaluates circulating plasma levels of the ZFP or nucleic acid encoding the ZFP, potential ZFP toxicities, progression of the disease, and the production of anti-ZFP antibodies. Administration can be accomplished via single or divided doses.
- ZFPs and expression vectors encoding ZFPs can be administered directly to the patient for targeted cleavage and/or recombination, and for therapeutic or prophylactic applications, for example, cancer, ischemia, diabetic retinopathy, macular degeneration, rheumatoid arthritis, psoriasis, HIV infection, sickle cell anemia, Alzheimer's disease, muscular dystrophy, neurodegenerative diseases, vascular disease, cystic fibrosis, stroke, and the like.
- Administration of therapeutically effective amounts is by any of the routes normally used for introducing ZFP into ultimate contact with the tissue to be treated.
- the ZFPs are administered in any suitable manner, preferably with pharmaceutically acceptable carriers. Suitable methods of administering such modulators are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
- Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions that are available (see, e.g., Remington's Pharmaceutical Sciences, 17 th ed. 1985)).
- the ZFPs can be made into aerosol formulations (i.e., they can be “nebulized”) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
- Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
- the disclosed compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally.
- the formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
- the disclosed methods and compositions for targeted cleavage can be used to induce mutations in a genomic sequence, e.g., large deletion mutations.
- Generation of targeted deletions, as disclosed herein, can be used to create gene knock-outs (e.g., for functional genomics or target validation) and for purposes of cell engineering or protein overexpression.
- ZFNs zinc finger/nuclease half-domain fusion proteins
- Targeted deletion of infecting or integrated viral genomes can be used to treat viral infections in a host. Additionally, targeted deletion of genes encoding receptors for viruses can be used to block expression of such receptors, thereby preventing viral infection and/or viral spread in a host organism. Targeted deletion of genes encoding viral receptors (e.g., the CCR5 and CXCR4 receptors for HIV) can be used to render the receptors unable to bind to virus, thereby preventing new infection and blocking the spread of existing infections.
- targeted deletion of genes encoding viral receptors e.g., the CCR5 and CXCR4 receptors for HIV
- viruses or viral receptors that may be targeted include herpes simplex virus (HSV), such as HSV-1 and HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV), HHV6 and HHV7.
- HSV herpes simplex virus
- VZV varicella zoster virus
- EBV Epstein-Barr virus
- CMV cytomegalovirus
- HHV6 and HHV7 herpes simplex virus
- the hepatitis family of viruses includes hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitis virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV).
- viruses or their receptors may be targeted, including, but not limited to, Picornaviridae (e.g., polioviruses, etc.); Caliciviridae; Togaviridae (e.g., rubella virus, dengue virus, etc.); Flaviviridae; Coronaviridae; Reoviridae; Birnaviridae; Rhabodoviridae (e.g., rabies virus, etc.); Filoviridae; Paramyxoviridae (e.g., mumps virus, measles virus, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g., influenza virus types A, B and C, etc.); Bunyaviridae; Arenaviridae; Retroviradae; lentiviruses (e.g., HTLV-I; HTLV-II; HIV-1 (also known as HTLV-III, LAV, ARV, hTLR, etc.) HIV-II
- Receptors for HIV include CCR-5 and CXCR-4.
- the genome of an infecting bacterium can be mutagenized by targeted deletion, to block or ameliorate bacterial infections.
- Certain genetic diseases result from expression of a mutant gene product. In such cases, inactivation of the mutant gene product by targeted deletion of its gene may ameliorate or cure the disease.
- Exemplary genetic diseases include, but are not limited to, 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
- acquired immunodeficiencies e.g., Gaucher's disease, GM1, Fabry disease and Tay-Sachs disease
- mucopolysaccahidosis e.g. Hunter's disease, Hurler's disease
- hemoglobinopathies e.g., sickle cell diseases, HbC, ⁇ -thalassemia, ⁇ -thalassemia
- hemophilias e.g., sickle cell diseases, HbC, ⁇ -thalassemia, ⁇ -thalassemia
- a pluripotent cell e.g., a hematopoietic stem cell
- Methods for mobilization, enrichment and culture of hematopoietic stem cells are known in the art. See for example, U.S. Pat. Nos. 5,061,620; 5,681,559; 6,335,195; 6,645,489; and 6,667,064.
- Treated stem cells can be returned to a patient for treatment of various diseases including, but not limited to, AIDS, SCID and sickle-cell anemia.
- overexpression of an oncogene may be reversed either by mutating the gene or by inactivating its control sequences by deletion. Any pathology dependent upon expression of a particular genomic sequence can be corrected or alleviated by targeted deletion of part or all of the sequence.
- Targeted deletion can also be used to alter non-coding sequences (e.g., regulatory sequences such as promoters, enhancers, initiators, terminators, splice sites) to alter the levels of expression of a gene product.
- non-coding sequences e.g., regulatory sequences such as promoters, enhancers, initiators, terminators, splice sites
- Such methods can be used, for example, for therapeutic purposes, functional genomics and/or target validation studies.
- compositions and methods described herein also allow for novel approaches and systems to address immune reactions of a host to allogeneic grafts.
- a major problem faced when allogeneic stem cells (or any type of allogeneic cell) are grafted into a host recipient is the high risk of rejection by the host's immune system, primarily mediated through recognition of the Major Histocompatibility Complex (MHC) on the surface of the engrafted cells.
- MHC Major Histocompatibility Complex
- the MHC comprises the HLA class I protein(s) that function as heterodimers that are comprised of a common ⁇ subunit and variable a subunits. It has been demonstrated that tissue grafts derived from stem cells that are devoid of HLA escape the host's immune response.
- genes encoding HLA proteins involved in graft rejection can be mutagenized or altered by deletion, in either their coding or regulatory sequences, so that their expression is blocked or they express a non-functional product.
- HLA class I can be removed from the cells to rapidly and reliably generate HLA class I null stem cells from any donor, thereby reducing the need for closely matched donor/recipient MHC haplotypes during stem cell grafting.
- any gene e.g., the ⁇ 2 microglobulin gene, the CTLA4 gene
- inactivation of any gene can be achieved, for example, by cleavage at two sites followed by joining so as to delete the sequence between the two cleavage sites.
- Targeted modification of chromatin structure can be used to facilitate the binding of fusion proteins to cellular chromatin.
- Targeted cleavage is accomplished, in certain embodiments, using fusion proteins (ZFNs) comprising a zinc finger DNA-binding domain and a nuclease half-domain.
- ZFNs fusion proteins
- a number of zinc finger proteins were designed to bind to sites in the human CCR5 gene (GenBank® Accession Number AY221093).
- the proteins were designed in pairs such that, for each pair, target sites occurred on opposite DNA strands and the near edges of the target sites were separated by 5 nucleotide pairs.
- Table 2 shows the nucleotide sequences of the target sites for these zinc finger domains, and the locations of the target sites within the human CCR-5 gene. The amino acid sequences of the recognition regions of their zinc finger portions are also shown.
- Polynucleotides encoding fusions of the zinc finger domains shown in Table 2 to the FokI cleavage half-domain were constructed, in which sequences encoding the zinc finger domain are upstream of sequences encoding the cleavage half-domain, such that, in the encoded proteins, the zinc finger domain is nearest the N-terminus, and the cleavage half-domain is nearest the C-terminus, of the fusion protein.
- K562 erythroleukemia cells were cultured in RPMI medium with 10% bovine serum. At a density of approximately 1 ⁇ 10 6 cells/ml, cells were transfected with two DNA constructs, each encoding a pair of zinc finger nucleases (ZFNs), with the ZFN coding sequences separated by a 2A peptide sequence.
- ZFNs zinc finger nucleases
- the first plasmid (denoted 004) encoded the r162b2 and 168c4 ZFNS (see Table 2 above) which were designed to cleave between +162 and +168 (with respect to the translation start) of the human CCR-5 gene; the second plasmid (denoted 043) encoded the r627s1 and 633b5 ZFNs (Table 2) which were designed to cleave between +627 and +633. Control transfections used only one of these two plasmids, or used a plasmid encoding green fluorescent protein (GFP).
- GFP green fluorescent protein
- CCR5longF (SEQ ID NO: 26) GATGGTGCTTTCATGAATTCC and CCR5longR: (SEQ ID NO: 27) GTGTCACAAGCCCACAGATA.
- Amplification products were analyzed by electrophoresis on 2% agarose e-gels (Invitrogen).
- Results are shown in FIG. 1 .
- a lower molecular weight amplification product is obtained from cells transfected with plasmids encoding the two ZFN pairs (lane 4).
- the size of this low molecular-weight band is consistent with removal of approximately 465 nucleotide pairs from the CCR-5 sequences, which corresponds to the distance between the two targeted cleavage sites.
- amplification products were cloned into the Topo-4® vector (Invitrogen, Carlsbad, Calif.) following the manufacturer's protocol. Two classes of insert size were obtained. Plasmids containing inserts of the smaller size class were analyzed to determine the nucleotide sequence of their inserts. The results are shown in FIG. 2 , in which a representative number of sequences in the region around and between the two targeted cleavage sites are shown. It can be seen, from the sequences obtained, that sequence alterations induced by cleavage at two cleavage sites can include deletion of some or all of the sequence between the two cleavage sites, and can also include deletion of additional sequences on one or both sides.
- Genomic DNA was isolated from K562 cells that had been transfected with plasmids encoding the two nuclease pairs described earlier in this example, or from control K562 cells that had been transfected with a plasmid encoding green fluorescent protein.
- Ten micrograms of DNA was digested with XhoI and NdeI. The digestion products were fractionated on an agarose gel and transferred to a nylon membrane. The membrane was incubated with a labeled probe comprising sequences corresponding to nucleotides ⁇ 246 through +9, with respect to the first base pair of the translation initiation codon of the human CCR-5 gene.
- the probe used in this experiment identifies a 2.8 kbp XhoI-NdeI fragment in DNA from cells transfected with the GFP-encoding plasmid, corresponding to wild-type CCR-5 sequences ( FIG. 3 , lane 2).
- a band at approximately 2.3 kbp, corresponding to deleted molecules is also present ( FIG. 3 , lane 1). Quantitation of this lower molecular weight band indicated a deletion frequency of approximately 10%.
- Human T-cells were obtained by leukopheresis from a healthy donor, the T cells were depleted in CD8 cells, then activated for two days with PHA+IL2. Transfection was performed by electroporation, using a Maxcyte electroporation device. Cells were transfected with two DNA constructs, each encoding a pair of zinc finger nucleases (ZFNs), with the ZFN coding sequences separated by a 2A peptide sequence.
- ZFNs zinc finger nucleases
- the first plasmid (denoted 149) encoded the r162p11 and 168i13 ZFNS (see Table 2 above) which were designed to cleave between +162 and +168 (with respect to the translation start) of the human CCR-5 gene;
- the second plasmid (denoted 141) encoded the r627s1 and 633a10 ZFNs (Table 2) which were designed to cleave between +627 and +633.
- Control transfections used a plasmid encoding green fluorescent protein (GFP).
- CCR5longF (SEQ ID NO: 26) GATGGTGCTTTCATGAATTCC and CCR5longR: (SEQ ID NO: 27) GTGTCACAAGCCCACAGATA Amplification products were analyzed by electrophoresis on 2% agarose e-gels (Invitrogen).
- Results are shown in FIG. 4 .
- a lower molecular weight amplification product is obtained from cells transfected with plasmids encoding the two ZFN pairs (lane 2 of FIG. 4 ).
- the size of this low molecular-weight band is consistent with removal of approximately 465 nucleotide pairs from the CCR-5 sequences, which corresponds to the distance between the two targeted cleavage sites.
- the lower molecular weight band was excised from the gel, DNA was eluted from the band and cloned into the Topo-4® vector (Invitrogen, Carlsbad, Calif.) following the manufacturer's protocol. Resulting plasmids were analyzed to determine the nucleotide sequence of their inserts. The results are shown in FIG. 5 , in which a representative number of sequences in the region around and between the two targeted cleavage sites are shown. It can be seen, from the sequences obtained, that the amplification products present in the lower band contained deletions of approximately 430 nucleotide pairs, whose endpoints lay at or near the targeted cleavage sites.
- a number of zinc finger proteins were designed to bind to sites in the human CTLA4 gene (GenBank® Accession Number NM_005214). The proteins were designed in pairs such that, for each pair, target sites occurred on opposite DNA strands. For one pair, the near edges of the target sites were separated by 5 nucleotide pairs and, for the other pair, the near edges of the target sites were separated by 6 nucleotide pairs.
- Table 3 shows the nucleotide sequences of the target sites for these zinc finger domains, and the locations of the target sites within the human CTLA4 gene. The amino acid sequences of the recognition regions of their zinc finger portions are also shown.
- Polynucleotides encoding fusions of the zinc finger domains shown in Table 3 to the FokI cleavage half-domain were constructed, in which sequences encoding the zinc finger domain are upstream of sequences encoding the cleavage half-domain, such that, in the encoded proteins, the zinc finger domain is nearest the N-terminus, and the cleavage half-domain is nearest the C-terminus, of the fusion protein.
- K562 cells were cultured and transfected as described in Example 2. Cells were transfected with four DNA constructs, each encoding one of the zinc finger nucleases (ZFNs) identified in Table 3. Control transfections were conducted with a vector that did not encode a ZFN (“empty vector”).
- ZFNs zinc finger nucleases
- Transfected cells were collected by centrifugation 2 days after transfection. and genomic DNA was isolated using a DNeasy® Tissue kit (Qiagen, Valencia, Calif.), following the manufacturer's protocol. Genomic DNA (200 ng) was used as template for amplification using an AccuPrime® PCR amplification kit (Invitrogen, Carlsbad, Calif.) with primers that yield a 3.8 kilobase pair (kbp) amplification product from a wild-type CTLA4 gene. Amplification products were analyzed by gel electrophoresis.
- Co-expression in cells of either of the first pair of exon 3-targeted nucleases and either of the second pair of exon 5-targeted nucleases results in cleavage events in exon 3 and exon 5 of the IL-2R ⁇ gene. Subsequent rejoining of DNA ends can result in loss of sequences between the cleavage sites, leading to deletion of approximately 1,400 nucleotide pairs of the X chromosome.
Abstract
Disclosed herein are methods and compositions for targeted deletion of double-stranded DNA. The compositions include fusion proteins comprising a cleavage domain (or cleavage half-domain) and an engineered zinc finger domain, and polynucleotides encoding same. Methods for targeted deletion include introduction of such fusion proteins, or polynucleotides encoding same, into a cell such that two targeted cleavage events occur. Subsequent cellular repair mechanisms result in deletion of sequences between the two cleavage sites.
Description
- This application is a divisional of U.S. patent application Ser. No. 15/610,262, filed May 31, 2017, which is a continuation of U.S. patent application Ser. No. 13/784,634, filed Mar. 4, 2013, now U.S. Pat. No. 9,695,442, which is a continuation of U.S. patent application Ser. No. 11/304,981, filed Dec. 15, 2005, now U.S. Pat. No. 8,409,861, which is continuation-in-part of U.S. patent application Ser. No. 10/912,932, filed Aug. 6, 2004, now U.S. Pat. No. 7,888,121, and claims the benefit of U.S. Provisional Application No. 60/649,515, filed Feb. 3, 2005. U.S. application Ser. No. 10/912,932 also claims the benefit of the following U.S. provisional patent applications: 60/493,931, filed Aug. 8, 2003; 60/518,253, filed Nov. 7, 2003; 60/530,541, filed Dec. 18, 2003; 60/542,780, filed Feb. 5, 2004; 60/556,831 filed Mar. 26, 2004; and 60/575,919, filed Jun. 1, 2004. The disclosures of all of the above are hereby incorporated by reference in their entireties for all purposes.
- The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 24, 2020, is named 8325_0036_10_SL.txt and is 12,793 bytes in size.
- Not applicable.
- The present disclosure is in the field of genome engineering and targeted deletion (i.e. “knock-out” technology).
- A major area of interest in genome biology, especially in light of the determination of the complete nucleotide sequences of a number of genomes, is the targeted alteration of genome sequences. One such alteration is deletion, i.e., removal of sequences from a genome. Deletions can be as small as a single nucleotide pair, or can encompass hundreds, thousands or even millions of nucleotide pairs. The ability to reproducibly induce targeted deletions is useful in the identification of gene function (e.g., by gene “knock-out” studies) and can also be useful for inactivating genes (e.g., viral receptors) whose function is required for pathological processes.
- Induction of small deletions by targeted cleavage of chromosomal DNA using zinc finger/nuclease fusion proteins (ZFNs) has been described. See, for example, International Patent Publication No. WO 03/87341 and U.S. Patent Publication No. 2005/0064474. In brief, when a ZFN dimer (or any site-specific nuclease) is expressed within a cell, the process of targeted cleavage, followed by error-prone repair, can lead to generation of deletions, most of which are of fewer than about 20 bases, at or near the site of nuclease cleavage.
- The process of ZFN-mediated mutagenesis as currently implemented using a single ZFN dimer has a number of limitations. First, the sizes of the deletions introduced by this method are generally quite small. Although deletions in excess of 100 bp are occasionally seen, the vast majority of deletions (probably more than 90%) are of fewer than about 20 bp. Therefore the method is unsuitable for generating large deletions at high efficiency. The ability to generate large deletions at high frequency would be required if, for example, it were necessary to eliminate entire regulatory region of a gene.
- A second shortcoming of existing methods for ZFN-mediated mutagenesis is that the heterogeneity of the deletions, coupled with their small sizes, makes it extremely difficult to monitor or quantify the mutagenesis process using conventional approaches such as PCR. By contrast, larger deletions are much more readily detected and quantified in a background of excess unmodified gene sequence using a standard method such as PCR followed by agarose gel electrophoresis.
- Thus, methods for reproducibly obtaining large deletions of chromosomal sequence at high frequency would be useful in a number of areas of genome biology.
- The present disclosure provides compositions and methods for targeted mutagenesis, particularly deletion mutagenesis, of double-stranded DNA sequences. Thus, in one embodiment, a method for deleting sequences in a region of interest in double-stranded DNA is provided, the method comprising expressing first, second, third and fourth fusion proteins in a cell, wherein each of the fusion proteins comprises (i) a zinc finger DNA-binding domain that binds to a target site in the DNA, and (ii) a cleavage half-domain; further wherein (a) the first and second fusion proteins bind to first and second target sites respectively, wherein a first cleavage site lies between the first and second target sites (i.e., the first and second target sites straddle the first cleavage site) and (b) the third and fourth fusion proteins bind to third and fourth target sites respectively, wherein a second cleavage site lies between the third and fourth target sites (i.e., the third and fourth target sites straddle the second cleavage site); such that the first and second fusion proteins cleave the DNA at the first cleavage site, the third and fourth fusion proteins cleave the DNA at the second cleavage site, and DNA ends are rejoined such that sequences between the first and second cleavage sites are deleted.
- Also provided is a method for deleting sequences in a region of interest in double-stranded DNA, the method comprising expressing first and second nucleases in a cell, wherein the first nuclease cleaves a first cleavage site and the second nuclease cleaves a second cleavage site; and DNA ends are rejoined such that sequences between the first and second cleavage sites are deleted.
- In certain embodiments, at least one of the nucleases is a fusion protein comprising (i) a zinc finger DNA-binding domain that binds to a target site in the DNA, wherein the target site is at or adjacent to the first or second cleavage site; and (ii) a cleavage domain.
- Four DNA ends are generated by cleavage at the two cleavage sites. First and second DNA ends are generated by cleavage at the first cleavage site; while third and fourth DNA ends are generated by cleavage at the second cleavage site. In certain embodiments, the first and second cleavage sites are present on the same DNA molecule (e.g., on the same chromosome). In these cases, if the second and third ends, as defined above, are considered to be part of a DNA fragment containing sequences that lie between the first and second cleavage sites (i.e. a fragment that is released by cleavage at the first and second cleavage sites), then rejoining of the first and fourth ends results in deletion of sequences between the first and second cleavage sites. An alternative outcome is inversion of some or all of the sequences located between the first and second cleavage sites.
- If the first and second cleavage sites are located on different chromosomes, chromosomal translocations and/or chromosomal fusions can result. Finally, targeted cleavages and resultant deletion can also occur on extrachromosomal nucleic acids, such as episomes, intracellular vectors, organellar genomes, etc.
- In certain instances, ends generated directly by the cleavage event (e.g., the first and fourth DNA ends) may be rejoined to cause a deletion. In other instances, the ends generated by cleavage may be further processed (e.g., by exonucleolytic resection) and these ends resulting from cleavage can be rejoined. Rejoining can occur by cellular repair mechanisms such as those collectively denoted “non-homologous end-joining.”
- As described above, in certain embodiments, sequences in a region of interest are deleted, wherein the region of interest is in cellular chromatin. In these cases, the first and second cleavage sites can be on the same chromosomes, on different chromosomes, on an extrachromosomal nucleic acid, or the first cleavage sit can be present on a chromosome and the second cleavage site can be present on an extrachromosomal nucleic acid.
- The target sites bound by the fusion proteins are present in pairs wherein, for each pair of target sites, a cleavage site lies therebetween. Thus, the first and second target sites straddle a first cleavage site and the third and fourth target sites straddle a second cleavage site. The target sites can be separated by any number of nucleotide pairs, commensurate with dimerization of the fusion proteins to regenerate a functional cleavage domain. As described elsewhere in this disclosure, maximal cleavage efficiency varies with both the distance between target sites and the length of the linker sequences between the zinc finger portion and the nuclease half-domain portion of the fusion proteins. Accordingly, the first and second target sites can be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide pairs. Similarly, the third and fourth target sites can be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide pairs. When discussing the distance between target sites, this distance is expressed as the number of nucleotide pairs intervening between the near edges of the target sites, and does not include any nucleotide pair that is present in either of the target sites.
- The size of a deletion induced by the disclosed methods and compositions is determined by the distance between the first and second cleavage sites. Accordingly, deletions of any size, in any region of interest, can be obtained. Deletions of 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 nucleotide pairs, or any integral value of nucleotide pairs within this range, can be obtained. In addition deletions of a sequence of any integral value of nucleotide pairs greater than 1,000 nucleotide pairs can be obtained using the methods and compositions disclosed herein.
- The region of interest, in which deletion is induced, can be in a gene. The gene can be a gene involved in a disease or pathological condition. For example, the gene can be a viral receptor. Certain chemokine receptors also function as viral receptors; for example, the chemokine receptor CCR-5 also functions a receptor for human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS). Thus, the present disclosure provides methods for inducing targeted deletions in a CCR-5 gene, optionally a human CCR-5 gene, for treatment of AIDS.
- Also provided are deleted CCR-5 gene sequences and cells comprising deleted CCR-5 genes; optionally, human cells. In certain embodiments, the cells are primary cells obtained from an individual, which may optionally be returned to the same individual or a different individual. In certain embodiments, the primary cells are T-cells or dendritic cells.
- Cells can also include cultured cells, cells in an organism and cells that have been removed from an organism for treatment in cases where the cells and/or their descendants will be returned to the organism after treatment. A region of interest in cellular chromatin can be, for example, a genomic sequence or portion thereof. Compositions include fusion polypeptides comprising an engineered zinc finger binding domain (e.g., a zinc finger binding domain having a novel specificity) and a cleavage domain, and fusion polypeptides comprising an engineered zinc finger binding domain and a cleavage half-domain. Cleavage domains and cleavage half domains can be obtained, for example, from various restriction endonucleases and/or homing endonucleases.
- Cellular chromatin can be present in any type of cell including, but not limited to, prokaryotic and eukaryotic cells, fungal cells, plant cells, animal cells, mammalian cells, primate cells and human cells.
- A protein e.g., a fusion protein, can be expressed in a cell, e.g., by delivering the fusion protein to the cell or by delivering a polynucleotide encoding the fusion protein to a cell, wherein the polynucleotide, if DNA, is transcribed, and an RNA molecule delivered to the cell or a transcript of a DNA molecule delivered to the cell is translated, to generate the protein. Methods for polynucleotide and polypeptide delivery to cells are presented elsewhere in this disclosure.
- In the disclosed methods for targeted deletion, the cleavage half-domains can be derived from the same endonuclease or from different endonucleases. Endonucleases include, but are not limited to, homing endonucleases and restriction endonucleases. Exemplary restriction endonucleases are Type IIS restriction endonucleases; an exemplary Type IIS restriction endonuclease is FokI.
- In certain embodiments, a cleavage domain can comprise two cleavage half-domains that are covalently linked in the same polypeptide. The two cleavage half-domains can be derived from the same endonuclease or from different endonucleases. The cleavage half domain can be derived from, for example, a homing endonuclease or a restriction endonuclease, for example, a Type IIS restriction endonuclease. An exemplary Type IIS restriction endonuclease is FokI.
- In certain embodiments, it is possible to obtain increased cleavage specificity by utilizing fusion proteins in which one or both cleavage half-domains contains an alteration in the amino acid sequence of the dimerization interface.
- In the aforementioned methods for targeted deletion, the target sites for the fusion proteins can comprise any number of nucleotides. Preferably, they are at least nine nucleotides in length, but they can also be larger (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18 and up to 100 nucleotides, including any integral value between 9 and 100 nucleotides); moreover, different target sequences need not necessarily be the same length. The distance between the nearest edges of the target sites can be any integral number of nucleotide pairs between 1 and 50, (e.g., 5 or 6 base pairs) as measured from the near end of one binding site to the closest end of the other binding site.
- In the aforementioned methods for targeted deletion, cellular chromatin can be cleaved at a site located between the target sites of two fusion proteins. In certain embodiments, the target sites are on opposite DNA strands. Moreover, expression of the fusion proteins in the cell can be accomplished either by introduction of the proteins into the cell or by introduction of one or more polynucleotides into the cell, which are optionally transcribed (if the polynucleotide is DNA), and the transcript(s) translated, to produce the fusion proteins. For example, two polynucleotides, each comprising sequences encoding one pair of fusion proteins, can be introduced into a cell. Alternatively, a single polynucleotide comprising sequences encoding both pairs of fusion proteins can be introduced into the cell.
- In any of the methods described herein, a zinc finger binding domain can be engineered, for example designed and/or selected. See, for example, U.S. Pat. Nos. 5,789,538; 6,007,988; 6,013,453; 6,140,466; 6,242,568; 6,410,248; 6,453,242; 6,534,261; 6,733,970; 6,746,838; 6,785,613; 6,790,941; 6,794,136; 6,866,997; and 6,933,113, as well as U.S. Patent Publication No. 2005/0064477. See also International Patent Publication No. WO 02/42459.
- Polynucleotides encoding fusions between a zinc finger binding domain and a cleavage domain or cleavage half-domain can be DNA or RNA, can be linear or circular, and can be single-stranded or double-stranded. They can be delivered to the cell as naked nucleic acid, as a complex with one or more delivery agents (e.g., liposomes, poloxamers) or contained in a viral delivery vehicle, such as, for example, an adenovirus, adeno-associated virus (AAV) or lentivirus. A polynucleotide can encode one or more fusion proteins.
-
FIG. 1 is a photograph of an agarose gel in which amplification products of the human CCR-5 gene were analyzed.Lane 1 shows amplification products of DNA from K562 cells transfected with a plasmid encoding green fluorescent protein.Lane 2 shows amplification products of DNA from K562 cells transfected with a plasmid encoding a pair of ZFNs designed to cleave between +162 and +168 of the human CCR-5 gene.Lane 3 shows amplification products of DNA from K562 cells transfected with a plasmid encoding a pair of ZFNs designed to cleave between +627 and +633 of the human CCR-5 gene.Lane 4 shows amplification products of DNA from K562 cells transfected with both of the aforementioned ZFN pairs. -
FIG. 2 shows nucleotide sequences of amplification products of the CCR-5 gene obtained from K562 cells that had been transfected with two plasmids: one encoding a pair of ZFNs designed to cleave between +162 and +168 of the human CCR-5 gene and the other encoding a pair of ZFNs designed to cleave between +627 and +633 of the human CCR-5 gene (i.e., corresponding tolane 4 ofFIG. 1 ). The topmost line shows a partial nucleotide sequence of the wild-type human CCR-5 gene from +146 to +185 (SEQ ID NO:45) and from +616 to +646 (SEQ ID NO:46), with the ZFN target sites underlined. Coordinates are with respect to the first nucleotide pair of the translation initiation codon. - Each of the remaining lines (SEQ ID NOS:47-62) represents a contiguous nucleotide sequence obtained from an amplification product of DNA obtained from cells that had been transfected as described in the preceding paragraph. In the written representation shown in the Figure, portions of each contiguous sequence have been separated for purposes of alignment with the wild-type sequence. The bottom-most two lines provide descriptions of two additional sequences that were obtained.
-
FIG. 3 shows an autoradiogram of a Southern blot of genomic DNA purified from transfected K562 cells and digested with XhoI and NdeI.Lane 1 shows DNA from cells transfected with two plasmids: one encoding a pair of ZFNs designed to cleave between +162 and +168 of the human CCR-5 gene and the other encoding a pair of ZFNs designed to cleave between +627 and +633 of the human CCR-5 gene.Lane 2 shows DNA from cells transfected with a plasmid encoding GFP. The upper arrow indicates a band derived from amplification of wild-type sequences; the lower arrow identifies a band obtained from amplification of deleted CCR-5 loci. - A schematic diagram of a portion of the human CCR-5 gene, indicating relevant restriction enzyme recognition sites, ZFN target sites and the approximate map position of the fragment used as probe, is shown below the autoradiogram. Numbering is with respect to the first nucleotide pair of the initiation codon.
-
FIG. 4 is a photograph of an agarose gel in which amplification products of the human CCR-5 gene were analyzed.Lane 1 shows size markers.Lane 2 shows amplification products of DNA from human T-cells transfected with a plasmid encoding a pair of ZFNs designed to cleave between +162 and +168 of the human CCR-5 gene and a plasmid encoding a pair of ZFNs designed to cleave between +627 and +633 of the human CCR-5 gene.Lane 3 shows amplification products of DNA from human T-cells transfected with a plasmid encoding green fluorescent protein. The arrow indicates a band representing amplification products of deleted CCR-5 loci. -
FIG. 5 shows nucleotide sequences of amplification products of the CCR-5 gene obtained from primary human T-cells that had been transfected with two plasmids: one encoding a pair of ZFNs designed to cleave between +162 and +168 of the human CCR-5 gene and the other encoding a pair of ZFNs designed to cleave between +627 and +633 of the human CCR-5 gene. The topmost line shows a partial nucleotide sequence of the wild-type human CCR-5 gene from +146 to +185 (SEQ ID NO: 63) and from +616 to +645 (SEQ ID NO: 64). The target sites for the ZFNs are underlined. Coordinates are with respect to the first nucleotide pair of the translation initiation codon. - Each of the remaining lines (SEQ ID NOS:65-84) represents a contiguous nucleotide sequence obtained from an amplification product of DNA obtained from cells that had been transfected as described in the preceding paragraph. In the written representation shown in the Figure, portions of each contiguous sequence have been separated for purposes of alignment with the wild-type sequence.
- Disclosed herein are compositions and methods useful for targeted deletion of sequences in double-stranded DNA (e.g., cellular chromatin). Double-stranded DNA includes that present in chromosomes, episomes, organellar genomes (e.g., mitochondria, chloroplasts), artificial chromosomes and any other type of nucleic acid present in a cell such as, for example, amplified sequences, double minute chromosomes and the genomes of endogenous or infecting bacteria and viruses. Chromosomal sequences can be normal (i.e., wild-type) or mutant; mutant sequences can comprise, for example, insertions, deletions, translocations, rearrangements, and/or point mutations. A chromosomal sequence can also comprise one of a number of different alleles.
- Compositions useful for targeted deletion include pairs of fusion proteins, each fusion protein comprising a cleavage domain (or a cleavage half-domain) and a zinc finger binding domain, polynucleotides encoding these proteins and combinations of polypeptides and polypeptide-encoding polynucleotides. A zinc finger binding domain can comprise one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers), and can be engineered to bind to any sequence. Thus, by identifying a target genomic region, the deletion of which is desired, one can, according to the methods disclosed herein, construct one or more fusion proteins comprising a cleavage domain (or cleavage half-domain) and a zinc finger domain engineered to recognize a target sequence in said genomic region. The presence of such fusion proteins in a cell results in binding of the fusion proteins to their target sites, cleavage at two cleavage sites, and deletion of sequences therebetween.
- Thus, to obtain targeted deletion, cells are treated simultaneously with a pair of ZFN dimers, which stimulates the highly efficient deletion of DNA lying between the sites cleaved by the two dimers. In this context, the term “ZFN dimer” refers to a pair of zinc finger/cleavage half domain fusion proteins, each of which binds to a distinct target site such that DNA is cleaved at a cleavage site which lies between the target sites.
- Advantages of the methods for targeted deletion mutagenesis disclosed herein include:
- 1) The process can be used for highly efficient deletion of large DNA sequences (e.g., several hundred base pairs). This enables disruption of DNA elements (e.g., exons, introns, regulatory sequences) that may not be completely removable via introduction of small (<20 bp) deletions.
- 2) The process is readily monitored using standard molecular biology methods such as PCR followed by agarose gel electrophoresis, as well as Southern blot analysis.
- 3) The induction of large deletions occurs at an efficiency that is substantially enhanced relative to the mutational efficiencies achieved by cleavage with either of the individual ZFN dimers used in the process. Thus use of a pair of ZFN dimers provides a general means for achieving deletion efficiencies which are higher than those achievable using a single ZFN dimer.
- General
- Practice of the methods, as well as preparation and use of the compositions disclosed herein employ, unless otherwise indicated, conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA and related fields as are within the skill of the art. These techniques are fully explained in the literature. See, for example, Sambrook et al. 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; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) Humana Press, Totowa, 1999.
- The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used interchangeably and refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form. For the purposes of the present disclosure, these terms are not to be construed as limiting with respect to the length of a polymer. The terms can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones). In general, an analogue of a particular nucleotide has the same base-pairing specificity; i.e., an analogue of A will base-pair with T.
- The terms “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 (Kd) of 10−6M−1 or lower. “Affinity” refers to the strength of binding: increased binding affinity being correlated with a lower Kd.
- 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). In the case of 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. Non-limiting examples of 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 International Patent Publication Nos. 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. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; and 6,200,759; and International Patent Publication Nos. 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.
- The term “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. The term “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. For example, a polynucleotide comprising the wild-type sequence of a mutant gene is homologous and non-identical to the sequence of the mutant gene. In certain embodiments, 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. For example, an exogenous polynucleotide (i.e., donor polynucleotide) of between 20 and 10,000 nucleotides or nucleotide pairs can be used.
- Techniques for determining 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, whether nucleic acid or amino acid 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 (1981) Advances in Applied Mathematics 2:482-489. 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 (1986) Nucl. Acids Res. 14(6):6745-6763. An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the “BestFit” utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wis.). A preferred method of establishing percent identity in the context of the present disclosure is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of packages 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. Details of these programs can be found online. With respect to sequences described herein, the range of desired degrees of sequence identity is approximately 80% to 100% and any integer value therebetween. Typically the percent identities between sequences are at least 70-75%, preferably 80-82%, more preferably 85-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity.
- Alternatively, 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. As used herein, 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.). 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.
- 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).
- Conditions for hybridization are well-known to those of skill in the art. 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 are well-known to those of skill in the art and 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.
- With respect to 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. For the purposes of this disclosure, “homologous recombination (FIR)” 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. Without wishing to be bound by any particular theory, 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. For the purposes of the present disclosure, the term “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. For example, the sequence 5′-GAATTC-3′ is a target site for the Eco RI restriction endonuclease.
- An “exogenous” molecule is a molecule that is not normally present in a cell, but can be introduced into a cell by one or more genetic, biochemical or other methods. “Normal presence in the cell” is determined with respect to the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic development of muscle is an exogenous molecule with respect to an adult muscle cell. Similarly, a molecule induced by heat shock is an exogenous molecule with respect to a non-heat-shocked cell. An exogenous molecule can comprise, for example, a functioning version of a malfunctioning endogenous molecule or a malfunctioning version of a normally-functioning endogenous molecule.
- An exogenous molecule can be, among other things, a small molecule, such as is generated by a combinatorial chemistry process, or a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules. Nucleic acids include DNA and RNA, can be single- or double-stranded; can be linear, branched or circular; and can be of any length. Nucleic acids include those capable of forming duplexes, as well as triplex-forming nucleic acids. See, for example, U.S. Pat. Nos. 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases and helicases.
- An exogenous molecule can be the same type of molecule as an endogenous molecule, e.g., an exogenous protein or nucleic acid. For example, 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 are known to those of skill in the art and 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.
- By contrast, an “endogenous” molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. For example, 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.
- Expression of a 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.
- A “gene,” for the purposes of the present disclosure, includes a DNA region encoding a gene product (see infra), as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.
- “Gene expression” refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.
- “Modulation” of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression.
- “Eucaryotic” cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells.
- A “region of interest” is any region of cellular chromatin, such as, for example, a gene or a non-coding sequence within or adjacent to a gene, in which it is desirable to bind an exogenous molecule. Binding can be for the purposes of targeted DNA cleavage and/or targeted recombination. A region of interest can be present in a chromosome, an episome, an organellar genome (e.g., mitochondrial, chloroplast), or an infecting viral genome, for example. A region of interest can be within the coding region of a gene, within transcribed non-coding regions such as, for example, leader sequences, trailer sequences or introns, or within non-transcribed regions, either upstream or downstream of the coding region. A region of interest can be as small as a single nucleotide pair or up to 2,000 nucleotide pairs in length, or any integral value of nucleotide pairs.
- The terms “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. By way of illustration, a transcriptional regulatory sequence, such as a promoter, is operatively linked to a coding sequence if the transcriptional regulatory sequence controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. A transcriptional regulatory sequence is generally operatively linked in cis with a coding sequence, but need not be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence that is operatively linked to a coding sequence, even though they are not contiguous.
- With respect to fusion polypeptides, 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. For example, with respect to a fusion polypeptide in which a ZFP DNA-binding domain is fused to a cleavage domain, the ZFP DNA-binding domain and the cleavage domain are in operative linkage if, in the fusion polypeptide, the ZFP DNA-binding domain portion is able to bind its target site and/or its binding site, while the cleavage domain is able to cleave DNA in the vicinity of the target site.
- A “functional fragment” of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, yet retains the same function as the full-length protein, polypeptide or nucleic acid. A functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one ore more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid) are well-known in the art. Similarly, methods for determining protein function are well-known. For example, the 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 International Patent Publication No. WO 98/44350.
- Target Sites
- The disclosed methods and compositions include fusion proteins comprising a cleavage domain (or a cleavage half-domain) and a zinc finger domain, in which the zinc finger domain, by binding to a sequence in cellular chromatin (e.g., a target site or a binding site), directs the activity of the cleavage domain (or cleavage half-domain) to the vicinity of the sequence and, hence, induces cleavage in the vicinity of the target sequence. As set forth elsewhere in this disclosure, a zinc finger domain can be engineered to bind to virtually any desired sequence. Accordingly, after identifying a region of interest containing a sequence at which cleavage or recombination is desired, one or more zinc finger binding domains can be engineered to bind to one or more sequences in the region of interest. Expression of a fusion protein comprising a zinc finger binding domain and a cleavage domain (or of two fusion proteins, each comprising a zinc finger binding domain and a cleavage half-domain), in a cell, effects cleavage in the region of interest.
- Selection of a sequence in cellular chromatin for binding by a zinc finger domain (e.g., a target site) can be accomplished, for example, according to the methods disclosed in co-owned U.S. Pat. No. 6,453,242 (Sep. 17, 2002), which also discloses methods for designing ZFPs to bind to a selected sequence. It will be clear to those skilled in the art that simple visual inspection of a nucleotide sequence can also be used for selection of a target site. Accordingly, any means for target site selection can be used in the claimed methods.
- Target sites are generally composed of a plurality of adjacent target subsites. A target subsite refers to the sequence (usually either a nucleotide triplet, or a nucleotide quadruplet that can overlap by one nucleotide with an adjacent quadruplet) bound by an individual zinc finger. See, for example, International Patent Publication No. 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. However 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. As will be apparent, binding of larger binding domains (e.g., 7-, 8-, 9-finger and more) to longer target sites is also possible.
- It is not necessary for a target site to be a multiple of three nucleotides. For example, in cases in which cross-strand interactions occur (see, e.g., U.S. Pat. No. 6,453,242 and International Patent Publication No. WO 02/077227), one or more of the individual zinc fingers of a multi-finger binding domain can bind to overlapping quadruplet subsites. As a result, 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. For example, the presence of 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 International Patent Publication No. 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. To provide but one example, 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 (e.g., target sites) refers to the number of nucleotides or nucleotide pairs intervening between two sequences, as measured from the edges of the sequences nearest each other.
- In certain embodiments in which cleavage depends on the binding of two zinc finger domain/cleavage half-domain fusion molecules to separate target sites, the two target sites can be on opposite DNA strands. In other embodiments, both target sites are on the same DNA strand.
- Zinc Finger Binding Domains
- A zinc finger binding domain comprises one or more zinc fingers. Miller et al. (1985) EMBO J. 4:1609-1614; Rhodes (1993) Scientific American February: 56-65; U.S. Pat. No. 6,453,242. Typically, a single zinc finger domain is about 30 amino acids in length. Structural studies have demonstrated that each zinc finger domain (motif) contains two beta sheets (held in a beta turn which contains the two invariant cysteine residues) and an alpha helix (containing the two invariant histidine residues), which are held in a particular conformation through coordination of a zinc atom by the two cysteines and the two histidines.
- Zinc fingers include both canonical C2H2 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, C3H zinc fingers (those in which the zinc ion is coordinated by three cysteine residues and one histidine residue) and C4 zinc fingers (those in which the zinc ion is coordinated by four cysteine residues). See also International Patent Publication No. WO 02/057293.
- Zinc finger binding domains can be engineered to bind to a sequence of choice. See, for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416. An engineered zinc finger binding domain can have a novel binding specificity, compared to a naturally-occurring zinc finger protein. 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. See, for example, co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261.
- Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as International Patent Publication Nos. WO 98/37186; WO 98/53057; WO 00/27878; and WO 01/88197 and GB 2,338,237.
- Enhancement of binding specificity for zinc finger binding domains has been described, for example, in co-owned International Patent Publication No. WO 02/077227.
- Since 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) will determine the number of zinc fingers in an engineered zinc finger binding domain. For example, for ZFPs in which the finger motifs do not bind to overlapping subsites, a six-nucleotide target sequence is bound by a two-finger binding domain; a nine-nucleotide target sequence is bound by a three-finger binding domain, etc. As noted herein, 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.
- In a multi-finger zinc 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. See, e.g., co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261. For engineered zinc finger binding domains comprising more than three fingers, 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 International Patent Publication No. WO 01/53480. Accordingly, multi-finger zinc finger binding domains can also be characterized with respect to the presence and location of non-canonical inter-finger linkers. For example, 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. Similarly, 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.
- The presence of 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. For example, 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 International Patent Publication No. WO 01/53480.
- As mentioned previously, a target subsite is a three- or four-nucleotide sequence that is bound by a single zinc finger. For certain purposes, 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. Alternatively, modules can be constructed by assembly of individual zinc fingers. See also International Patent Publication Nos. WO 98/53057 and WO 01/53480.
- Cleavage Domains
- The cleavage domain portion of the fusion proteins disclosed herein can be obtained from any endo- or exonuclease. Exemplary endonucleases from which a cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes which cleave DNA are known (e.g., Si Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains and cleavage half-domains.
- Similarly, a cleavage half-domain (e.g., fusion proteins comprising a zinc finger binding domain and a cleavage half-domain) can be derived from any nuclease or portion thereof, as set forth above, that requires dimerization for cleavage activity. In general, two fusion proteins are required for cleavage if the fusion proteins comprise cleavage half-domains. 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). In addition, the target sites for the two fusion proteins are preferably disposed, with respect to each other, such that binding of the two fusion proteins 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. Thus, in certain embodiments, the near edges of the target sites are separated by 5-8 nucleotides or by 15-18 nucleotides. However 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.
- In general, if two fusion proteins are used, each comprising a cleavage half-domain, the primary contact strand for the zinc finger portion of each fusion protein will be on a different DNA strands and in opposite orientation. That is, for a pair of ZFP/cleavage half-domain fusions, the target sequences are on opposite strands and the two proteins bind in opposite orientation.
- Restriction endonucleases (restriction enzymes) 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) cleave DNA at sites removed from the recognition site and have separable binding and cleavage domains. For example, the Type IIS enzyme FokI 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. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise the cleavage domain (or cleavage half-domain) from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered.
- An exemplary Type IIS restriction enzyme, whose cleavage domain is separable from the binding domain, is FokI. This particular enzyme is active as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95:10,570-10,575. Accordingly, for the purposes of the present disclosure, the portion of the FokI enzyme used in the disclosed fusion proteins is considered a cleavage half-domain. Thus, for targeted double-stranded cleavage and/or targeted replacement of cellular sequences using zinc finger-Fold fusions, two fusion proteins, each comprising a FokI cleavage half-domain, can be used to reconstitute a catalytically active cleavage domain. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two FokI cleavage half-domains can also be used. Parameters for targeted cleavage and targeted sequence alteration using zinc finger-Fold fusions are provided elsewhere in this disclosure.
- Exemplary Type IIS restriction enzymes are listed in Table 1. Additional restriction enzymes also contain separable binding and cleavage domains, and these are contemplated by the present disclosure. See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.
-
TABLE 1 Some Type IIS Restriction Enzymes Aar I BsrB I SspD5 I Ace III BsrD I Sth132 I Aci I BstF5 I Sts I Alo I Btr I TspDT I Bae I Bts I TspGW I Bbr7 I Cdi I Tth111 II Bbv I CjeP I UbaP I Bbv II Drd II Bsa I BbvC I Eci I BsmB I Bcc I Eco31 I Bce83 I Eco57 I BceA I Eco57M I Beef I Esp3 I Bcg I Fau I BciV I Fin I Bfi I Fok I Bin I Gdi II Bmg I Gsu I Bpu10 I Hga I BsaX I Hin4 II Bsb I Hph I BscA I Ksp632 I BscG I Mbo II BseR I Mly I BseY I Mme I Bsi I Mnl I Bsm I Pfl1108 I BsmA I Ple I BsmF I Ppi I Bsp24 I Psr I BspG I RleA I BspM I Sap I BspNC I SfaN I Bsr I Sim I - Methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art. For example, methods for the design and construction of fusion protein comprising zinc finger proteins (and polynucleotides encoding same) are described in co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261. In certain embodiments, polynucleotides encoding such fusion proteins are constructed. These polynucleotides can be inserted into a vector and the vector can be introduced into a cell (see below for additional disclosure regarding vectors and methods for introducing polynucleotides into cells).
- In certain embodiments of the methods described herein, a fusion protein comprises a zinc finger binding domain and a cleavage half-domain from the FokI 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. In additional embodiments, 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.
- In general, the components of the fusion proteins (e.g, ZFP-FokI fusions) are arranged such that the zinc finger domain is nearest the amino terminus of the fusion protein, and the cleavage half-domain is nearest the carboxy-terminus. This mirrors the relative orientation of the cleavage domain in naturally-occurring dimerizing cleavage domains such as those derived from the FokI enzyme, in which the DNA-binding domain is nearest the amino terminus and the cleavage half-domain is nearest the carboxy terminus.
- In the disclosed fusion proteins, the amino acid sequence between the zinc finger binding domain (which is delimited by the N-terminal most of the two conserved cysteine residues and the C-terminal-most of the two conserved histidine residues) and the cleavage domain (or half-domain) is denoted the “ZC linker.” The ZC linker is to be distinguished from the inter-finger linkers discussed above. For instance, in a ZFP-FokI fusion protein (in which the components are arranged: N terminus-zinc finger binding domain-FokI cleavage half domain-C terminus), the ZC linker is located between the second histidine residue of the C-terminal-most zinc finger and the N-terminal-most amino acid residue of the cleavage half-domain (which is generally glutamine (Q) in the sequence QLV). The ZC linker can be any amino acid sequence. To obtain optimal cleavage, the length of the linker and the distance between the target sites (binding sites) are interrelated. See, for example, Smith et al. (2000) Nucleic Acids Res. 28:3361-3369; Bibikova et al. (2001) Mol. Cell. Biol. 21:289-297, noting that their notation for linker length differs from that given here. For example, for ZFP-FokI fusions having a ZC linker length of four amino acids (as defined herein), optimal cleavage occurs when the binding sites for the fusion proteins are located 6 or 16 nucleotides apart (as measured from the near edge of each binding site).
- Methods for Targeted Cleavage
- The disclosed methods and compositions can be used to cleave DNA at a region of interest in cellular chromatin (e.g., at a desired or predetermined site in a genome, for example, in a gene, either mutant or wild-type). For such targeted DNA cleavage, a zinc finger binding domain is engineered to bind a target site at or near the predetermined cleavage site, and a fusion protein comprising the engineered zinc finger binding domain and a cleavage domain is expressed in a cell. Upon binding of the zinc finger portion of the fusion protein to the target site, the DNA is cleaved near the target site by the cleavage domain. The exact site of cleavage can depend on the length of the ZC linker.
- Alternatively, two fusion proteins, each comprising a zinc finger binding domain and a cleavage half-domain, are expressed in a cell, and bind to target sites which are juxtaposed in such a way that a functional cleavage domain is reconstituted and DNA is cleaved in the vicinity of the target sites. In one embodiment, cleavage occurs between the target sites of the two zinc finger binding domains. One or both of the zinc finger binding domains can be engineered.
- For targeted cleavage using a zinc finger binding domain-cleavage domain fusion polypeptide, the binding site can encompass the cleavage site, or the near edge of the binding site can be 1, 2, 3, 4, 5, 6, 10, 25, 50 or more nucleotides (or any integral value between 1 and 50 nucleotides) from the cleavage site. The exact location of the binding site, with respect to the cleavage site, will depend upon the particular cleavage domain, and the length of the ZC linker. For methods in which two fusion polypeptides, each comprising a zinc finger binding domain and a cleavage half-domain, are used, the binding sites generally straddle the cleavage site. Thus the near edge of the first binding site can be 1, 2, 3, 4, 5, 6, 10, 25 or more nucleotides (or any integral value between 1 and 50 nucleotides) on one side of the cleavage site, and the near edge of the second binding site can be 1, 2, 3, 4, 5, 6, 10, 25 or more nucleotides (or any integral value between 1 and 50 nucleotides) on the other side of the cleavage site. Methods for mapping cleavage sites in vitro and in vivo are known to those of skill in the art.
- Thus, the methods described herein can employ an engineered zinc finger binding domain fused to a cleavage domain. In these cases, the binding domain is engineered to bind to a target sequence, at or near which cleavage is desired. The fusion protein, or a polynucleotide encoding same, is introduced into a cell. Once introduced into, or expressed in, the cell, the fusion protein binds to the target sequence and cleaves at or near the target sequence. The exact site of cleavage depends on the nature of the cleavage domain and/or the presence and/or nature of linker sequences between the binding and cleavage domains. In cases where two fusion proteins, each comprising a cleavage half-domain, are used, the distance between the near edges of the binding sites can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25 or more nucleotides (or any integral value between 1 and 50 nucleotides). Optimal levels of cleavage can also depend on both the distance between the binding sites of the two fusion proteins (See, for example, Smith et al. (2000) Nucleic Acids Res. 28:3361-3369; Bibikova et al. (2001) Mol. Cell. Biol. 21:289-297) and the length of the ZC linker in each fusion protein.
- For ZFP-FokI fusion nucleases, the length of the linker between the ZFP and the FokI cleavage half-domain (i.e., the ZC linker) can influence cleavage efficiency. In one experimental system utilizing a ZFP-FokI fusion with a ZC linker of 4 amino acid residues, optimal cleavage was obtained when the near edges of the binding sites for two ZFP-FokI nucleases were separated by 6 base pairs. This particular fusion nuclease comprised the following amino acid sequence between the zinc finger portion and the nuclease half-domain:
-
(SEQ ID NO: 1) HQRTHQNKKQLV
in which the two conserved histidines in the C-terminal portion of the zinc finger and the first three residues in the FokI cleavage half-domain are underlined. Accordingly, the linker sequence in this construct is QNKK. Bibikova et al. (2001) Mol. Cell. Biol. 21:289-297. The present inventors have constructed a number of ZFP-FokI fusion nucleases having a variety of ZC linker lengths and sequences, and analyzed the cleavage efficiencies of these nucleases on a series of substrates having different distances between the ZFP binding sites. See Example 4. - In certain embodiments, the cleavage domain comprises two cleavage half-domains, both of which are part of a single polypeptide comprising a binding domain, a first cleavage half-domain and a second cleavage half-domain. The cleavage half-domains can have the same amino acid sequence or different amino acid sequences, so long as they function to cleave the DNA.
- Cleavage half-domains may also be provided in separate molecules. For example, two fusion polypeptides may be introduced into a cell, wherein each polypeptide comprises a binding domain and a cleavage half-domain. The cleavage half-domains can have the same amino acid sequence or different amino acid sequences, so long as they function to cleave the DNA. Further, the binding domains bind to target sequences which are typically disposed in such a way that, upon binding of the fusion polypeptides, the two cleavage half-domains are presented in a spatial orientation to each other that allows reconstitution of a cleavage domain (e.g., by dimerization of the half-domains), thereby positioning the half-domains relative to each other to form a functional cleavage domain, resulting in cleavage of cellular chromatin in a region of interest. Generally, cleavage by the reconstituted cleavage domain occurs at a site located between the two target sequences. One or both of the proteins can be engineered to bind to its target site.
- The two fusion proteins can bind in the region of interest in the same or opposite polarity, and their binding sites (i.e., target sites) can be separated by any number of nucleotides, e.g., from 0 to 200 nucleotides or any integral value therebetween. In certain embodiments, 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 FokI results from single-strand breaks offset by 4 nucleotides). Thus, cleavage does not necessarily occur at exactly opposite sites on each DNA strand. In addition, the structure of the fusion proteins and the distance between the target sites can influence whether cleavage occurs adjacent a single nucleotide pair, or whether cleavage occurs at several sites. However, for many applications, including targeted recombination (see infra) cleavage within a range of nucleotides is generally sufficient, and cleavage between particular base pairs is not required.
- As noted above, the fusion protein(s) can be introduced as polypeptides and/or polynucleotides. For example, 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. Alternatively, 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.
- To enhance cleavage specificity, additional compositions may also be employed in the methods described herein. For example, single cleavage half-domains can exhibit limited double-stranded cleavage activity. In methods in which two fusion proteins, each containing a three-finger zinc finger domain and a cleavage half-domain, are introduced into the cell, either protein specifies an approximately 9-nucleotide target site. Although the aggregate target sequence of 18 nucleotides is likely to be unique in a mammalian genome, any given 9-nucleotide target site occurs, on average, approximately 23,000 times in the human genome. Thus, non-specific cleavage, due to the site-specific binding of a single half-domain, may occur. Accordingly, the methods described herein contemplate the use of a dominant-negative mutant of a cleavage half-domain such as FokI (or a nucleic acid encoding same) that is expressed in a cell along with the two fusion proteins. The dominant-negative mutant is capable of dimerizing but is unable to cleave, and also blocks the cleavage activity of a half-domain to which it is dimerized. By providing the dominant-negative mutant in molar excess to the fusion proteins, only regions in which both fusion proteins are bound will have a high enough local concentration of functional cleavage half-domains for dimerization and cleavage to occur. At sites where only one of the two fusion proteins are bound, its cleavage half-domain forms a dimer with the dominant negative mutant half-domain, and undesirable, non-specific cleavage does not occur.
- Three catalytic amino acid residues in the FokI cleavage half-domain have been identified: Asp 450, Asp 467 and Lys 469. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575. Thus, one or more mutations at one of these residues can be used to generate a dominant negative mutation. Further, many of the catalytic amino acid residues of other Type IIS endonucleases are known and/or can be determined, for example, by alignment with FokI sequences and/or by generation and testing of mutants for catalytic activity.
- Dimerization Domain Mutations in the Cleavage Half-Domain
- Methods for targeted cleavage which involve the use of fusions between a ZFP and a cleavage half-domain (such as, e.g., a ZFP/FokI fusion) require the use of two such fusion molecules, each generally directed to a distinct target sequence. Target sequences for the two fusion proteins can be chosen so that targeted cleavage is directed to a unique site in a genome, as discussed above. A potential source of reduced cleavage specificity could result from homodimerization of one of the two ZFP/cleavage half-domain fusions. This might occur, for example, due to the presence, in a genome, of inverted repeats of the target sequences for one of the two ZFP/cleavage half-domain fusions, located so as to allow two copies of the same fusion protein to bind with an orientation and spacing that allows formation of a functional dimer.
- One approach for reducing the probability of this type of aberrant cleavage at sequences other than the intended target site involves generating variants of the cleavage half-domain that minimize or prevent homodimerization. Preferably, one or more amino acids in the region of the half-domain involved in its dimerization are altered. In the crystal structure of the FokI protein dimer, the structure of the cleavage half-domains is reported to be similar to the arrangement of the cleavage half-domains during cleavage of DNA by FokI. Wah et al. (1998) Proc. Natl. Acad. Sci. USA 95:10564-10569. This structure indicates that amino acid residues at positions 483 and 487 play a key role in the dimerization of the FokI cleavage half-domains. The structure also indicates that amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 are all close enough to the dimerization interface to influence dimerization. Accordingly, amino acid sequence alterations at one or more of the aforementioned positions will likely alter the dimerization properties of the cleavage half-domain. Such changes can be introduced, for example, by constructing a library containing (or encoding) different amino acid residues at these positions and selecting variants with the desired properties, or by rationally designing individual mutants. In addition to preventing homodimerization, it is also possible that some of these mutations may increase the cleavage efficiency above that obtained with two wild-type cleavage half-domains.
- Accordingly, alteration of a FokI cleavage half-domain at any amino acid residue which affects dimerization can be used to prevent one of a pair of ZFP/FokI fusions from undergoing homodimerization which can lead to cleavage at undesired sequences. Thus, for targeted cleavage using a pair of ZFP FokI fusions, one or both of the fusion proteins can comprise one or more amino acid alterations that inhibit self-dimerization, but allow heterodimerization of the two fusion proteins to occur such that cleavage occurs at the desired target site. In certain embodiments, alterations are present in both fusion proteins, and the alterations have additive effects; i.e., homodimerization of either fusion, leading to aberrant cleavage, is minimized or abolished, while heterodimerization of the two fusion proteins is facilitated compared to that obtained with wild-type cleavage half-domains. See Example 5.
- Methods for Targeted Alteration of Genomic Sequences and Targeted Recombination
- Also described herein are methods of replacing a genomic sequence (e.g., a region of interest in cellular chromatin) with a homologous non-identical sequence (i.e., targeted recombination). Previous attempts to replace particular sequences have involved contacting a cell with a polynucleotide comprising sequences bearing homology to a chromosomal region (i.e., a donor DNA), followed by selection of cells in which the donor DNA molecule had undergone homologous recombination into the genome. The success rate of these methods is low, due to poor efficiency of homologous recombination and a high frequency of non-specific insertion of the donor DNA into regions of the genome other than the target site.
- The present disclosure provides methods of targeted sequence alteration characterized by a greater efficiency of targeted recombination and a lower frequency of non-specific insertion events. The methods involve making and using engineered zinc finger binding domains fused to cleavage domains (or cleavage half-domains) to make one or more targeted double-stranded breaks in cellular DNA. Because double-stranded breaks in cellular DNA stimulate homologous recombination several thousand-fold in the vicinity of the cleavage site, such targeted cleavage allows for the alteration or replacement (via homologous recombination) of sequences at virtually any site in the genome.
- In addition to the fusion molecules described herein, targeted replacement of a selected genomic sequence also requires the introduction of the replacement (or donor) sequence. The donor sequence can be introduced into the cell prior to, concurrently with, or subsequent to, expression of the fusion protein(s). The donor polynucleotide contains sufficient homology to a genomic sequence to support homologous recombination between it and the genomic sequence to which it bears homology. Approximately 25, 50 100 or 200 nucleotides or more of sequence homology between a donor and a genomic sequence (or any integral value between 10 and 200 nucleotides, or more) will support homologous recombination therebetween. Donor sequences can range in length from 10 to 5,000 nucleotides (or any integral value of nucleotides therebetween) or longer. It will be readily apparent that the donor sequence is typically not identical to the genomic sequence that it replaces. For example, the sequence of the donor polynucleotide can contain one or more single base changes, insertions, deletions, inversions or rearrangements with respect to the genomic sequence, so long as sufficient homology is present to support homologous recombination. Alternatively, a donor sequence can contain a non-homologous sequence flanked by two regions of homology. Additionally, donor sequences can comprise a vector molecule containing sequences that are not homologous to the region of interest in cellular chromatin. Generally, the homologous region(s) of a donor sequence will have at least 50% sequence identity to a genomic sequence with which recombination is desired. In certain embodiments, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity is present. Any value between 1% and 100% sequence identity can be present, depending upon the length of the donor polynucleotide.
- A donor molecule can contain several, discontinuous regions of homology to cellular chromatin. For example, for targeted insertion of sequences not normally present in a region of interest, said sequences can be present in a donor nucleic acid molecule and flanked by regions of homology to sequence in the region of interest.
- To simplify assays (e.g., hybridization, PCR, restriction enzyme digestion) for determining successful insertion of the donor sequence, certain sequence differences may be present in the donor sequence as compared to the genomic sequence. Preferably, if located in a coding region, such nucleotide sequence differences will not change the amino acid sequence, or will make silent amino acid changes (i.e., changes which do not affect the structure or function of the protein). The donor polynucleotide can optionally contain changes in sequences corresponding to the zinc finger domain binding sites in the region of interest, to prevent cleavage of donor sequences that have been introduced into cellular chromatin by homologous recombination.
- The donor polynucleotide can be DNA or RNA, single-stranded or double-stranded and can be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3′ terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. A polynucleotide can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. Moreover, donor polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV).
- Without being bound by one theory, it appears that the presence of a double-stranded break in a cellular sequence, coupled with the presence of an exogenous DNA molecule having homology to a region adjacent to or surrounding the break, activates cellular mechanisms which repair the break by transfer of sequence information from the donor molecule into the cellular (e.g., genomic or chromosomal) sequence; i.e., by a processes of homologous recombination. Applicants' methods advantageously combine the powerful targeting capabilities of engineered ZFPs with a cleavage domain (or cleavage half-domain) to specifically target a double-stranded break to the region of the genome at which recombination is desired.
- For alteration of a chromosomal sequence, it is not necessary for the entire sequence of the donor to be copied into the chromosome, as long as enough of the donor sequence is copied to effect the desired sequence alteration.
- The efficiency of insertion of donor sequences by homologous recombination is inversely related to the distance, in the cellular DNA, between the double-stranded break and the site at which recombination is desired. In other words, higher homologous recombination efficiencies are observed when the double-stranded break is closer to the site at which recombination is desired. In cases in which a precise site of recombination is not predetermined (e.g., the desired recombination event can occur over an interval of genomic sequence), the length and sequence of the donor nucleic acid, together with the site(s) of cleavage, are selected to obtain the desired recombination event. In cases in which the desired event is designed to change the sequence of a single nucleotide pair in a genomic sequence, cellular chromatin is cleaved within 10,000 nucleotides on either side of that nucleotide pair. In certain embodiments, cleavage occurs within 500, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, or 2 nucleotides, or any integral value between 2 and 1,000 nucleotides, on either side of the nucleotide pair whose sequence is to be changed.
- As detailed above, the binding sites for two fusion proteins, each comprising a zinc finger binding domain and a cleavage half-domain, can be located 5-8 or 15-18 nucleotides apart, as measured from the edge of each binding site nearest the other binding site, and cleavage occurs between the binding sites. Whether cleavage occurs at a single site or at multiple sites between the binding sites is immaterial, since the cleaved genomic sequences are replaced by the donor sequences. Thus, for efficient alteration of the sequence of a single nucleotide pair by targeted recombination, the midpoint of the region between the binding sites is within 10,000 nucleotides of that nucleotide pair, preferably within 1,000 nucleotides, or 500 nucleotides, or 200 nucleotides, or 100 nucleotides, or 50 nucleotides, or 20 nucleotides, or 10 nucleotides, or 5 nucleotide, or 2 nucleotides, or one nucleotide, or at the nucleotide pair of interest.
- In certain embodiments, a homologous chromosome can serve as the donor polynucleotide. Thus, for example, correction of a mutation in a heterozygote can be achieved by engineering fusion proteins which bind to and cleave the mutant sequence on one chromosome, but do not cleave the wild-type sequence on the homologous chromosome. The double-stranded break on the mutation-bearing chromosome stimulates a homology-based “gene conversion” process in which the wild-type sequence from the homologous chromosome is copied into the cleaved chromosome, thus restoring two copies of the wild-type sequence.
- Methods and compositions are also provided that may enhance levels of targeted recombination including, but not limited to, the use of additional ZFP-functional domain fusions to activate expression of genes involved in homologous recombination, such as, for example, members of the RAD52 epistasis group (e.g., Rad50, Rad51, Rad51B, Rad51C, Rad51D, Rad52, Rad54, Rad54B, Mre11, XRCC2, XRCC3), genes whose products interact with the aforementioned gene products (e.g., BRCA1, BRCA2) and/or genes in the NBS1 complex. Similarly ZFP-functional domain fusions can be used, in combination with the methods and compositions disclosed herein, to repress expression of genes involved in non-homologous end joining (e.g., Ku70/80, XRCC4, poly(ADP ribose) polymerase, DNA ligase 4). See, for example, Yanez et al. (1998) Gene Therapy 5:149-159; Hoeijmakers (2001) Nature 411:366-374; Johnson et al. (2001) Biochem. Soc. Trans. 29:196-201; Tauchi et al. (2002) Oncogene 21:8967-8980. Methods for activation and repression of gene expression using fusions between a zinc finger binding domain and a functional domain are disclosed in co-owned U.S. Pat. No. 6,534,261. Additional repression methods include the use of antisense oligonucleotides and/or small interfering RNA (siRNA or RNAi) targeted to the sequence of the gene to be repressed.
- As an alternative to or, in addition to, activating expression of gene products involved in homologous recombination, fusions of these protein (or functional fragments thereof) with a zinc finger binding domain targeted to the region of interest, can be used to recruit these proteins (recombination proteins) to the region of interest, thereby increasing their local concentration and further stimulating homologous recombination processes. Alternatively, a polypeptide involved in homologous recombination as described above (or a functional fragment thereof) can be part of a triple fusion protein comprising a zinc finger binding domain, a cleavage domain (or cleavage half-domain) and the recombination protein (or functional fragment thereof). Additional proteins involved in gene conversion and recombination-related chromatin remodeling, which can be used in the aforementioned methods and compositions, include histone acetyltransferases (e.g., Esalp, Tip60), histone methyltransferases (e.g., Dotlp), histone kinases and histone phosphatases.
- The p53 protein has been reported to play a central role in repressing homologous recombination (HR). See, for example, Valerie et al., (2003) Oncogene 22:5792-5812; Janz et al. (2002) Oncogene 21:5929-5933. For example, the rate of HR in p53-deficient human tumor lines is 10,000-fold greater than in primary human fibroblasts, and there is a 100-fold increase in HR in tumor cells with a non-functional p53 compared to those with functional p53. Mekeel et al. (1997) Oncogene 14:1847-1857. In addition, overexpression of p53 dominant negative mutants leads to a 20-fold increase in spontaneous recombination. Bertrand et al. (1997) Oncogene 14:1117-1122. Analysis of different p53 mutations has revealed that the roles of p53 in transcriptional transactivation and G1 cell cycle checkpoint control are separable from its involvement in HR. Saintigny et al. (1999) Oncogene 18:3553-3563; Boehden et al. (2003) Oncogene 22:4111-4117. Accordingly, downregulation of p53 activity can serve to increase the efficiency of targeted homologous recombination using the methods and compositions disclosed herein. Any method for downregulation of p53 activity can be used, including but not limited to cotransfection and overexpression of a p53 dominant negative mutant or targeted repression of p53 gene expression according to methods disclosed, e.g., in co-owned U.S. Pat. No. 6,534,261.
- Further increases in efficiency of targeted recombination, in cells comprising a zinc finger/nuclease fusion molecule and a donor DNA molecule, are achieved by blocking the cells in the G2 phase of the cell cycle, when homology-driven repair processes are maximally active. Such arrest can be achieved in a number of ways. For example, cells can be treated with e.g., drugs, compounds and/or small molecules which influence cell-cycle progression so as to arrest cells in G2 phase. 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). Additional increases in recombination efficiency are achieved by the use of histone deacetylase (HDAC) inhibitors (e.g., sodium butyrate, trichostatin A) which alter chromatin structure to make genomic DNA more accessible to the cellular recombination machinery.
- 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.
- Alternatively, in certain cases, targeted cleavage is conducted in the absence of a donor polynucleotide (preferably in S or G2 phase), and recombination occurs between homologous chromosomes.
- Methods to Screen for Cellular Factors that Facilitate Homologous Recombination
- Since homologous recombination is a multi-step process requiring the modification of DNA ends and the recruitment of several cellular factors into a protein complex, the addition of one or more exogenous factors, along with donor DNA and vectors encoding zinc finger-cleavage domain fusions, can be used to facilitate targeted homologous recombination. An exemplary method for identifying such a factor or factors employs analyses of gene expression using microarrays (e.g., Affymetrix Gene Chip® arrays) to compare the mRNA expression patterns of different cells. For example, cells that exhibit a higher capacity to stimulate double strand break-driven homologous recombination in the presence of donor DNA and zinc finger-cleavage domain fusions, either unaided or under conditions known to increase the level of gene correction, can be analyzed for their gene expression patterns compared to cells that lack such capacity. Genes that are upregulated or downregulated in a manner that directly correlates with increased levels of homologous recombination are thereby identified and can be cloned into any one of a number of expression vectors. These expression constructs can be co-transfected along with zinc finger-cleavage domain fusions and donor constructs to yield improved methods for achieving high-efficiency homologous recombination. Alternatively, expression of such genes can be appropriately regulated using engineered zinc finger proteins which modulate expression (either activation or repression) of one or more these genes. See, e.g., co-owned U.S. Pat. No. 6,534,261 for methods for the synthesis of engineered zinc finger proteins for regulation of gene expression.
- As an example, it was observed that the different clones obtained in the experiments described in Example 9 and
FIG. 27 exhibited a wide-range of homologous recombination frequencies, when transfected with donor DNA and plasmids encoding zinc finger-cleavage domain fusions. Gene expression in clones showing a high frequency of targeted recombination can thus be compared to that in clones exhibiting a low frequency, and expression patterns unique to the former clones can be identified. - As an additional example, studies using cell cycle inhibitors (e.g., nocodazole or vinblastine, see e.g., Examples 11, 14 and 15) showed that cells arrested in the G2 phase of the cell cycle carried out homologous recombination at higher rates, indicating that cellular factors responsible for homologous recombination may be preferentially expressed or active in G2. One way to identify these factors is to compare the mRNA expression patterns between the stably transfected HEK 293 cell clones that carry out gene correction at high and low levels (e.g., clone T18 vs. clone T7). Similar comparisons are made between these cell lines in response to compounds that arrest the cells in G2 phase. Candidate genes that are differentially expressed in cells that carry out homologous recombination at a higher rate, either unaided or in response to compounds that arrest the cells in G2, are identified, cloned, and re-introduced into cells to determine whether their expression is sufficient to re-capitulate the improved rates. Alternatively, expression of said candidate genes is activated using engineered zinc finger transcription factors as described, for example, in co-owned U.S. Pat. No. 6,534,261.
- Expression Vectors
- A nucleic acid encoding one or more ZFPs or ZFP fusion proteins can be cloned into a vector for transformation into prokaryotic or eukaryotic cells for replication and/or expression. Vectors can be prokaryotic vectors, e.g., plasmids, or shuttle vectors, insect vectors, or eukaryotic vectors. A nucleic acid encoding a ZFP can also be cloned into an expression vector, for administration to a plant cell, animal cell, preferably a mammalian cell or a human cell, fungal cell, bacterial cell, or protozoal cell.
- To obtain expression of a cloned gene or nucleic acid, sequences encoding a ZFP or ZFP fusion protein are typically subcloned into an expression vector that contains a promoter to direct transcription. Suitable bacterial and eukaryotic promoters are well known in the art and described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989; 3rd ed., 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., supra. Bacterial expression systems for expressing the ZFP are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al. (1983) Gene 22:229-235). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known by those of skill in the art and are also commercially available.
- The promoter used to direct expression of a ZFP-encoding nucleic acid depends on the particular application. For example, a strong constitutive promoter is typically used for expression and purification of ZFP. In contrast, when a ZFP is administered in vivo for gene regulation, either a constitutive or an inducible promoter is used, depending on the particular use of the ZFP. In addition, a preferred promoter for administration of a ZFP can be a weak promoter, such as HSV TK or a promoter having similar activity. The promoter typically can also include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tet-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard (1992) PNAS 89:5547; Oligino et al. (1998) Gene Ther. 5:491-496; Wang et al. (1997) Gene Ther. 4:432-441; Neering et al. (1996) Blood 88:1147-1155; and Rendahl et al. (1998) Nat. Biotechnol. 16:757-761). The MNDU3 promoter can also be used, and is preferentially active in CD34+ hematopoietic stem cells.
- In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in host cells, either prokaryotic or eukaryotic. A typical expression cassette thus contains a promoter operably linked, e.g., to a nucleic acid sequence encoding the ZFP, and signals required, e.g., for efficient polyadenylation of the transcript, transcriptional termination, ribosome binding sites, or translation termination. Additional elements of the cassette may include, e.g., enhancers, and heterologous splicing signals.
- The particular expression vector used to transport the genetic information into the cell is selected with regard to the intended use of the ZFP, e.g., expression in plants, animals, bacteria, fungus, protozoa, etc. (see expression vectors described below). Standard bacterial expression vectors include plasmids such as pBR322-based plasmids, pSKF, pET23D, and commercially available fusion expression systems such as GST and LacZ. An exemplary fusion protein is the maltose binding protein, “MBP.” Such fusion proteins are used for purification of the ZFP. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, for monitoring expression, and for monitoring cellular and subcellular localization, e.g., c-myc or FLAG.
- Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
- Some expression systems have markers for selection of stably transfected cell lines such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. High yield expression systems are also suitable, such as using a baculovirus vector in insect cells, with a ZFP encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.
- The elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of recombinant sequences.
- Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, which are then purified using standard techniques (see, e.g., Colley et al. (1989) J. Biol. Chem. 264:17619-17622; Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison (1977) J. Bact. 132:349-351; Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).
- Any of the well known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, ultrasonic methods (e.g., sonoporation), liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the protein of choice.
- Nucleic Acids Encoding Fusion Proteins and Delivery to Cells
- Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding engineered ZFPs in cells (e.g., mammalian cells) and target tissues. Such methods can also be used to administer nucleic acids encoding ZFPs to cells in vitro. In certain embodiments, nucleic acids encoding ZFPs are administered for in vivo or ex vivo gene therapy uses. Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, (1992) Science 256:808-813; Nabel & Felgner (1993) TIBTECH 11:211-217; Mitani & Caskey (1993) TIBTECH 11:162-166; Dillon (1993) TIBTECH 11:167-175; Miller (1992) Nature 357:455-460; Van Brunt (1988) Biotechnology 6(10):1149-1154; Vigne (1995) Restorative Neurology and Neuroscience 8:35-36; Kremer & Perricaudet (1995) British Medical Bulletin 51(1):31-44; Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bohm (eds) (1995); and Yu et al. (1994) Gene Therapy 1:13-26.
- Methods of non-viral delivery of nucleic acids encoding engineered ZFPs include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids.
- Additional exemplary nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.) and BTX Molecular Delivery Systems (Holliston, Mass.).
- Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, International Patent Publication Nos. WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration).
- The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal (1995) Science 270:404-410; Blaese et al. (1995) Cancer Gene Ther. 2:291-297; Behr et al. (1994) Bioconjugate Chem. 5:382-389; Remy et al. (1994) Bioconjugate Chem. 5:647-654; Gao et al. (1995) Gene Therapy 2:710-722; Ahmad et al. (1992) Cancer Res. 52:4817-4820; U.S. Pat. Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028; and 4,946,787).
- The use of RNA or DNA viral based systems for the delivery of nucleic acids encoding engineered ZFPs take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo). Conventional viral based systems for the delivery of ZFPs include, but are not limited to, retroviral, lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
- The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system depends on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al. (1992) J. Virol. 66:2731-2739; Johann et al. (1992) J. Virol. 66:1635-1640; Sommerfelt et al. (1990) Virol. 176:58-59; Wilson et al. (1989) J. Virol. 63:2374-2378; Miller et al. (1991) J. Virol. 65:2220-2224; International Patent Publication No. WO 94/26877).
- In applications in which transient expression of a ZFP fusion protein is preferred, 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. (1987) Virology 160:38-47; U.S. Pat. No. 4,797,368; International Patent Publication No. WO 93/24641; Kotin (1994) Human Gene Therapy 5:793-801; Muzyczka (1994) J. Clin. Invest. 94:1351. Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260; Tratschin et al. (1984) Mol. Cell. Biol. 4:2072-2081; Hermonat & Muzyczka (1984) PNAS 81:6466-6470; and Samulski et al. (1989) J. Virol. 63:03822-3828.
- Adeno-associated virus vectors include
AAV serotypes AAV 2/1 andAAV 2/5. Both single-stranded and double-stranded (e.g., self-complementary) AAV vectors can be used. - 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. (1995) Blood 85:3048-305; Kohn et al. (1995) Nat. Med. 1:1017-102; Malech et al. (1997) PNAS 94(22):12133-12138). PA317/pLASN was the first therapeutic vector used in a gene therapy trial. (Blaese et al. (1995) Science 270:475-480). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors. (Ellem et al. (1997) Immunol Immunother 44(1):10-20; Dranoff et al. (1997) Hum. Gene Ther. 1:111-2.
- Recombinant adeno-associated virus vectors (rAAV) 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. (1998) Lancet 351(9117):1702-3, Kearns et al. (1996) Gene Ther. 9:748-55). - 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. 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. (1998) Hum. Gene Ther. 7:1083-9). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et al. (1996) Infection 24(1):5-10; Sterman et al. (1998) Hum. Gene Ther. 9(7):1083-1089; Welsh et al. (1995) Hum. Gene Ther. 2:205-18; Alvarez et al. (1997) Hum. Gene Ther. 5:597-613; Topf et al. (1998) Gene Ther. 5:507-513; Sterman et al. (1998) Hum. Gene Ther. 7:1083-1089.
- 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 w2 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. For example, 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. 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.
- In many gene therapy applications, it is desirable that the gene therapy vector be delivered with a high degree of specificity to a particular tissue type. Accordingly, 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. (1995) Proc. Natl. Acad. Sci. USA 92:9747-9751, reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor. 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. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor. Although the above description applies primarily to viral vectors, the same principles can be applied to nonviral vectors. Such vectors can be engineered to contain specific uptake sequences which favor uptake by specific target cells.
- 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. Alternatively, 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 (e.g., via re-infusion of the transfected cells into the host organism) is well known to those of skill in the art. In a preferred embodiment, cells are isolated from the subject organism, transfected with a ZFP nucleic acid (gene or cDNA), and re-infused back into the subject organism (e.g., patient). 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).
- In one embodiment, 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-γ and TNF-α are known (see Inaba et al. (1992) J. Exp. Med. 176:1693-1702).
- 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 Tad (differentiated antigen presenting cells) (see Inaba et al. (1992) J. Exp. Med. 176:1693-1702).
- Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containing therapeutic ZFP nucleic acids can also be administered directly to an organism for transduction of cells in vivo. Alternatively, 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.
- Methods for introduction of DNA into hematopoietic stem cells are disclosed, for example, in U.S. Pat. No. 5,928,638.
- Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions available, as described below (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).
- DNA constructs may be introduced into the genome of a desired plant host by a variety of conventional techniques. For reviews of such techniques see, for example, Weissbach & Weissbach Methods for Plant Molecular Biology (1988, Academic Press, N.Y.) Section VIII, pp. 421-463; and Grierson & Corey, Plant Molecular Biology (1988, 2d Ed.), Blackie, London, Ch. 7-9. For example, 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). Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et al. (1984) Science 233:496-498, and Fraley et al. (1983) Proc. Nat'l. Acad. Sci. USA 80:4803. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria using binary T DNA vector (Bevan (1984) Nuc. Acid Res. 12:8711-8721) or the co-cultivation procedure (Horsch et al. (1985) Science 227:1229-1231). Generally, 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 Hernalsteen 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). Additional methods for plant cell transformation include microinjection, silicon carbide mediated DNA uptake (Kaeppler et al. (1990) Plant Cell Reporter 9:415-418), and microprojectile bombardment (see Klein et al. (1988) Proc. Nat. Acad. Sci. USA 85:4305-4309; and Gordon-Kamm et al. (1990) Plant Cell 2:603-618).
- The disclosed methods and compositions can be used to insert exogenous sequences into a predetermined location in a plant cell genome. This is useful inasmuch as expression of an introduced transgene into a plant genome depends critically on its integration site. Accordingly, genes encoding, e.g., nutrients, antibiotics or therapeutic molecules can be inserted, by targeted recombination, into regions of a plant genome favorable to their expression.
- Transformed plant cells which are produced by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans, et al., “Protoplasts Isolation and Culture” in Handbook of Plant Cell Culture, pp. 124-176, Macmillian Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, pollens, embryos or parts thereof. Such regeneration techniques are described generally in Klee et al. (1987) Ann. Rev. of Plant Phys. 38:467-486.
- Nucleic acids introduced into a plant cell can be used to confer desired traits on essentially any plant. A wide variety of plants and plant cell systems may be engineered for the desired physiological and agronomic characteristics described herein using the nucleic acid constructs of the present disclosure and the various transformation methods mentioned above. In preferred embodiments, 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). Thus, the disclosed methods and compositions have use over a broad range of plants, including, but not limited to, species from the genera Asparagus, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucurbita, Daucus, Glycine, Hordeum, Lactuca, Lycopersicon, Malus, Manihot, Nicotiana, Oryza, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Solanum, Sorghum, Triticum, Vitis, Vigna, and Zea.
- One of skill in the art will recognize that after 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 β-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.
- 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, 51 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. Additional techniques, such as in situ hybridization, enzyme staining, and immunostaining, also may be used to detect the presence or expression of the recombinant construct in specific plant organs and tissues. The methods for doing all these assays are well known to those skilled in the art.
- Effects of gene manipulation using the methods disclosed herein can be observed by, for example, northern blots of the RNA (e.g., mRNA) isolated from the tissues of interest. Typically, if the amount of mRNA has increased, it can be assumed that the corresponding endogenous gene is being expressed at a greater rate than before. Other methods of measuring gene and/or CYP74B activity can be used. Different types of enzymatic assays can be used, depending on the substrate used and the method of detecting the increase or decrease of a reaction product or by-product. In addition, the levels of and/or CYP74B protein expressed can be measured immunochemically, i.e., ELISA, RIA, EIA and other antibody based assays well known to those of skill in the art, such as by electrophoretic detection assays (either with staining or western blotting). The transgene may be selectively expressed in some tissues of the plant or at some developmental stages, or the transgene may be expressed in substantially all plant tissues, substantially along its entire life cycle. However, any combinatorial expression mode is also applicable.
- The present disclosure also encompasses seeds of the transgenic plants described above wherein the seed has the transgene or gene construct. The present disclosure further encompasses the progeny, clones, cell lines or cells of the transgenic plants described above wherein said progeny, clone, cell line or cell has the transgene or gene construct.
- Delivery Vehicles
- An important factor in the administration of polypeptide compounds, such as ZFP fusion proteins, is ensuring that the polypeptide has the ability to traverse the plasma membrane of a cell, or the membrane of an intra-cellular compartment such as the nucleus. Cellular membranes are composed of lipid-protein bilayers that are freely permeable to small, nonionic lipophilic compounds and are inherently impermeable to polar compounds, macromolecules, and therapeutic or diagnostic agents. However, proteins and other compounds such as liposomes have been described, which have the ability to translocate polypeptides such as ZFPs across a cell membrane.
- For example, “membrane translocation polypeptides” have amphiphilic or hydrophobic amino acid subsequences that have the ability to act as membrane-translocating carriers. In one embodiment, homeodomain proteins have the ability to translocate across cell membranes. The shortest internalizable peptide of a homeodomain protein, Antennapedia, was found to be the third helix of the protein, from amino acid position 43 to 58 (see, e.g., Prochiantz (1996) Current Opinion in Neurobiology 6:629-634). Another subsequence, the h (hydrophobic) domain of signal peptides, was found to have similar cell membrane translocation characteristics (see, e.g., Lin et al. (1995) J. Biol. Chem. 270(1):4255-14258).
- Examples of peptide sequences which can be linked to a protein, for facilitating uptake of the protein into cells, include, but are not limited to: an 11 amino acid peptide of the tat protein of HIV; a 20 residue peptide sequence which corresponds to amino acids 84-103 of the p16 protein (see Fahraeus et al. (1996) Current Biology 6:84); the third helix of the 60-amino acid long homeodomain of Antennapedia (Derossi et al. (1994) J. Biol. Chem. 269:10444); the h region of a signal peptide such as the Kaposi fibroblast growth factor (K-FGF) h region (Lin et al., supra); or the VP22 translocation domain from HSV (Elliot & O'Hare (1997) Cell 88:223-233). Other suitable chemical moieties that provide enhanced cellular uptake may also be chemically linked to ZFPs. Membrane translocation domains (i.e., internalization domains) can also be selected from libraries of randomized peptide sequences. See, for example, Yeh et al. (2003) Molecular Therapy 7(5):S461, Abstract #1191.
- Toxin molecules also have the ability to transport polypeptides across cell membranes. Often, such molecules (called “binary toxins”) are composed of at least two parts: a translocation/binding domain or polypeptide and a separate toxin domain or polypeptide. Typically, the translocation domain or polypeptide binds to a cellular receptor, and then the toxin is transported into the cell. Several bacterial toxins, including Clostridium perfringens iota toxin, diphtheria toxin (DT), Pseudomonas exotoxin A (PE), pertussis toxin (PT), Bacillus anthracis toxin, and pertussis adenylate cyclase (CYA), have been used to deliver peptides to the cell cytosol as internal or amino-terminal fusions (Arora et al. (1993) J. Biol. Chem. 268:3334-3341; Perelle et al. (1993) Infect. Immun. 61:5147-5156; Stenmark et al. (1991) J Cell Biol. 113:1025-1032; Donnelly et al. (1993) PNAS 90:3530-3534; Carbonetti et al. (1995) Abstr. Annu. Meet. Am. Soc. Microbiol. 95:295; Sebo et al. (1995) Infect. Immun. 63:3851-3857; Klimpel et al. (1992) PNAS U.S.A. 89:10277-10281; and Novak et al. (1992) J. Biol. Chem. 267:17186-17193).
- Such peptide sequences can be used to translocate ZFPs across a cell membrane. ZFPs can be conveniently fused to or derivatized with such sequences. Typically, the translocation sequence is provided as part of a fusion protein. Optionally, a linker can be used to link the ZFP and the translocation sequence. Any suitable linker can be used, e.g., a peptide linker.
- The ZFP can also be introduced into an animal cell, preferably a mammalian cell, via a liposomes and liposome derivatives such as immunoliposomes. The term “liposome” refers to vesicles comprised of one or more concentrically ordered lipid bilayers, which encapsulate an aqueous phase. The aqueous phase typically contains the compound to be delivered to the cell, i.e., a ZFP.
- The liposome fuses with the plasma membrane, thereby releasing the drug into the cytosol. Alternatively, the liposome is phagocytosed or taken up by the cell in a transport vesicle. Once in the endosome or phagosome, the liposome either degrades or fuses with the membrane of the transport vesicle and releases its contents.
- In current methods of drug delivery via liposomes, the liposome ultimately becomes permeable and releases the encapsulated compound (in this case, a ZFP) at the target tissue or cell. For systemic or tissue specific delivery, this can be accomplished, for example, in a passive manner wherein the liposome bilayer degrades over time through the action of various agents in the body. Alternatively, active drug release involves using an agent to induce a permeability change in the liposome vesicle. Liposome membranes can be constructed so that they become destabilized when the environment becomes acidic near the liposome membrane (see, e.g., Wang et al. (1987) PNAS 84(22):7851-5; Biochemistry 28:908 (1989)). When liposomes are endocytosed by a target cell, for example, they become destabilized and release their contents. This destabilization is termed fusogenesis. Dioleoylphosphatidylethanolamine (DOPE) is the basis of many “fusogenic” systems.
- Such liposomes typically comprise a ZFP and a lipid component, e.g., a neutral and/or cationic lipid, optionally including a receptor-recognition molecule such as an antibody that binds to a predetermined cell surface receptor or ligand (e.g., an antigen). A variety of methods are available for preparing liposomes as described in, e.g., Szoka et al. (1980) Ann. Rev. Biophys. Bioeng. 9:467; U.S. Pat. Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028; 4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028; 4,946,787; International Patent Publication No. WO 91/17424, Deamer & Bangham, Biochim. Biophys. Acta (1976) 443:629-634; Fraley et al. (1979) PNAS 76:3348-3352; Hope et al. (1985) Biochim. Biophys. Acta 812:55-65; Mayer et al. (1986) Biochim. Biophys. Acta 858:161-168; Williams et al. (1988) PNAS 85:242-246; Liposomes (Ostro (ed.), 1983, Chapter 1); Hope et al. (1986) Chem. Phys. Lip. 40:89; Gregoriadis, Liposome Technology (1984) and Lasic, Liposomes: from Physics to Applications (1993)). Suitable methods include, for example, sonication, extrusion, high pressure/homogenization, microfluidization, detergent dialysis, calcium-induced fusion of small liposome vesicles and ether-fusion methods, all of which are known to those of skill in the art.
- In certain embodiments, it is desirable to target liposomes using targeting moieties that are specific to a particular cell type, tissue, and the like. Targeting of liposomes using a variety of targeting moieties (e.g., ligands, receptors, and monoclonal antibodies) has been described. See, e.g., U.S. Pat. Nos. 4,957,773 and 4,603,044.
- Examples of targeting moieties include monoclonal antibodies specific to antigens associated with neoplasms, such as prostate cancer specific antigen and MAGE. Tumors can also be diagnosed by detecting gene products resulting from the activation or over-expression of oncogenes, such as ras or c-erbB2. In addition, many tumors express antigens normally expressed by fetal tissue, such as the alphafetoprotein (AFP) and carcinoembryonic antigen (CEA). Sites of viral infection can be diagnosed using various viral antigens such as hepatitis B core and surface antigens (HBVc, HBVs) hepatitis C antigens, Epstein-Barr virus antigens, human immunodeficiency type-1 virus (HIV1) and papilloma virus antigens. Inflammation can be detected using molecules specifically recognized by surface molecules which are expressed at sites of inflammation such as integrins (e.g., VCAM-1), selectin receptors (e.g., ELAM-1) and the like.
- Standard methods for coupling targeting agents to liposomes can be used. These methods generally involve incorporation into liposomes of lipid components, e.g., phosphatidylethanolamine, which can be activated for attachment of targeting agents, or derivatized lipophilic compounds, such as lipid derivatized bleomycin. Antibody targeted liposomes can be constructed using, for instance, liposomes which incorporate protein A (see Renneisen et al. (1990) J. Biol. Chem. 265:16337-16342 and Leonetti et al. (1990) PNAS 87:2448-2451.
- Dosages
- For therapeutic applications, the dose administered to a patient, or to a cell which will be introduced into a patient, in the context of the present disclosure, should be sufficient to effect a beneficial therapeutic response in the patient over time. In addition, particular dosage regimens can be useful for determining phenotypic changes in an experimental setting, e.g., in functional genomics studies, and in cell or animal models. The dose will be determined by the efficacy and Kd of the particular ZFP employed, the nuclear volume of the target cell, and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound or vector in a particular patient.
- The maximum therapeutically effective dosage of ZFP for approximately 99% binding to target sites is calculated to be in the range of less than about 1.5×105 to 1.5×106 copies of the specific ZFP molecule per cell. The number of ZFPs per cell for this level of binding is calculated as follows, using the volume of a HeLa cell nucleus (approximately 1000 μm3 or 10−12 L; Cell Biology, (Altman & Katz, eds. (1976)). As the HeLa nucleus is relatively large, this dosage number is recalculated as needed using the volume of the target cell nucleus. This calculation also does not take into account competition for ZFP binding by other sites. This calculation also assumes that essentially all of the ZFP is localized to the nucleus. A value of 100×Kd is used to calculate approximately 99% binding of to the target site, and a value of 10×Kd is used to calculate approximately 90% binding of to the target site. For this example, Kd=25 nM
-
- ZFP+target site↔complex
- i.e., DNA+protein↔DNA:protein complex
-
-
-
- When 50% of ZFP is bound, Kd=[protein]
- So when [protein]=25 nM and the nucleus volume is 10−12 L
- [protein]=(25×10−9 moles/L) (10−12 L/nucleus) (6×1023 molecules/mole)
- =15,000 molecules/nucleus for 50% binding
- When 99% target is bound; 100×Kd=[protein]
- 100×Kd=[protein]=2.5 μM
- (2.5×10−6 moles/L) (10−12 L/nucleus) (6×1023 molecules/mole)
- =about 1,500,000 molecules per nucleus for 99% binding of target site.
-
- The appropriate dose of an expression vector encoding a ZFP can also be calculated by taking into account the average rate of ZFP expression from the promoter and the average rate of ZFP degradation in the cell. In certain embodiments, a weak promoter such as a wild-type or mutant HSV TK promoter is used, as described above. The dose of ZFP in micrograms is calculated by taking into account the molecular weight of the particular ZFP being employed.
- In determining the effective amount of the ZFP to be administered in the treatment or prophylaxis of disease, the physician evaluates circulating plasma levels of the ZFP or nucleic acid encoding the ZFP, potential ZFP toxicities, progression of the disease, and the production of anti-ZFP antibodies. Administration can be accomplished via single or divided doses.
- Pharmaceutical Compositions and Administration
- ZFPs and expression vectors encoding ZFPs can be administered directly to the patient for targeted cleavage and/or recombination, and for therapeutic or prophylactic applications, for example, cancer, ischemia, diabetic retinopathy, macular degeneration, rheumatoid arthritis, psoriasis, HIV infection, sickle cell anemia, Alzheimer's disease, muscular dystrophy, neurodegenerative diseases, vascular disease, cystic fibrosis, stroke, and the like. Examples of microorganisms that can be inhibited by ZFP gene therapy include pathogenic bacteria, e.g., chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and conococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme disease bacteria; infectious fungus, e.g., Aspergillus, Candida species; protozoa such as sporozoa (e.g., Plasmodia), rhizopods (e.g., Entamoeba) and flagellates (Trypanosoma, Leishmania, Trichomonas, Giardia, etc.); viral diseases, e.g., hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HSV-6, HSV-II, CMV, and EBV), HIV, Ebola, adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, poliovirus, rabies virus, and arboviral encephalitis virus, etc.
- 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, 17th ed. 1985)).
- The ZFPs, alone or in combination with other suitable components, 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, such as, for example, by intravenous, intramuscular, intradermal, and subcutaneous routes, 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.
- Applications
- The disclosed methods and compositions for targeted cleavage can be used to induce mutations in a genomic sequence, e.g., large deletion mutations. Generation of targeted deletions, as disclosed herein, can be used to create gene knock-outs (e.g., for functional genomics or target validation) and for purposes of cell engineering or protein overexpression.
- Targeted cleavage at a site in chromosomal DNA requires a pair of zinc finger/nuclease half-domain fusion proteins (ZFNs) so that dimerization of the cleavage half-domains occurs. Accordingly, for targeted deletion of long sequences, two pairs of ZFNs are used, to cleave at two sites and delete sequences between the two cleavage sites.
- Targeted deletion of infecting or integrated viral genomes can be used to treat viral infections in a host. Additionally, targeted deletion of genes encoding receptors for viruses can be used to block expression of such receptors, thereby preventing viral infection and/or viral spread in a host organism. Targeted deletion of genes encoding viral receptors (e.g., the CCR5 and CXCR4 receptors for HIV) can be used to render the receptors unable to bind to virus, thereby preventing new infection and blocking the spread of existing infections. Non-limiting examples of viruses or viral receptors that may be targeted include herpes simplex virus (HSV), such as HSV-1 and HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV), HHV6 and HHV7. The hepatitis family of viruses includes hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitis virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV). Other viruses or their receptors may be targeted, including, but not limited to, Picornaviridae (e.g., polioviruses, etc.); Caliciviridae; Togaviridae (e.g., rubella virus, dengue virus, etc.); Flaviviridae; Coronaviridae; Reoviridae; Birnaviridae; Rhabodoviridae (e.g., rabies virus, etc.); Filoviridae; Paramyxoviridae (e.g., mumps virus, measles virus, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g., influenza virus types A, B and C, etc.); Bunyaviridae; Arenaviridae; Retroviradae; lentiviruses (e.g., HTLV-I; HTLV-II; HIV-1 (also known as HTLV-III, LAV, ARV, hTLR, etc.) HIV-II); simian immunodeficiency virus (SIV), human papillomavirus (HPV), influenza virus and the tick-borne encephalitis viruses. See, e.g. Virology, 3rd Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe, eds. 1991), for a description of these and other viruses. Receptors for HIV, for example, include CCR-5 and CXCR-4.
- In similar fashion, the genome of an infecting bacterium can be mutagenized by targeted deletion, to block or ameliorate bacterial infections.
- Certain genetic diseases result from expression of a mutant gene product. In such cases, inactivation of the mutant gene product by targeted deletion of its gene may ameliorate or cure the disease. Exemplary genetic diseases include, but are not limited to, achondroplasia, achromatopsia, acid maltase deficiency, adenosine deaminase deficiency (OMIM No. 102700), 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 6th codon of beta-globin (HbC), hemophilia, Huntington's disease, Hurler Syndrome, hypophosphatasia, Klinefleter syndrome, Krabbes Disease, Langer-Giedion Syndrome, leukocyte adhesion deficiency (LAD, OMIM No. 116920), 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, X-linked lymphoproliferative syndrome (XLP, OMIM No. 308240), 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, α-thalassemia, β-thalassemia) and hemophilias.
- In certain cases, alteration of a genomic sequence in a pluripotent cell (e.g., a hematopoietic stem cell) is desired. Methods for mobilization, enrichment and culture of hematopoietic stem cells are known in the art. See for example, U.S. Pat. Nos. 5,061,620; 5,681,559; 6,335,195; 6,645,489; and 6,667,064. Treated stem cells can be returned to a patient for treatment of various diseases including, but not limited to, AIDS, SCID and sickle-cell anemia.
- As another example, overexpression of an oncogene may be reversed either by mutating the gene or by inactivating its control sequences by deletion. Any pathology dependent upon expression of a particular genomic sequence can be corrected or alleviated by targeted deletion of part or all of the sequence.
- Targeted deletion can also be used to alter non-coding sequences (e.g., regulatory sequences such as promoters, enhancers, initiators, terminators, splice sites) to alter the levels of expression of a gene product. Such methods can be used, for example, for therapeutic purposes, functional genomics and/or target validation studies.
- The compositions and methods described herein also allow for novel approaches and systems to address immune reactions of a host to allogeneic grafts. In particular, a major problem faced when allogeneic stem cells (or any type of allogeneic cell) are grafted into a host recipient is the high risk of rejection by the host's immune system, primarily mediated through recognition of the Major Histocompatibility Complex (MHC) on the surface of the engrafted cells. The MHC comprises the HLA class I protein(s) that function as heterodimers that are comprised of a common β subunit and variable a subunits. It has been demonstrated that tissue grafts derived from stem cells that are devoid of HLA escape the host's immune response. See, e.g., Coffman et al. (1993) J Immunol 151:425-35; Markmann et al. (1992) Transplantation 54:1085-9; Koller et al. (1990) Science 248:1227-30. Using the compositions and methods described herein, genes encoding HLA proteins involved in graft rejection can be mutagenized or altered by deletion, in either their coding or regulatory sequences, so that their expression is blocked or they express a non-functional product. For example, by inactivating the gene encoding the common β subunit gene (β2 microglobulin) using the methods and compositions described herein, HLA class I can be removed from the cells to rapidly and reliably generate HLA class I null stem cells from any donor, thereby reducing the need for closely matched donor/recipient MHC haplotypes during stem cell grafting.
- Thus, inactivation of any gene (e.g., the β2 microglobulin gene, the CTLA4 gene) can be achieved, for example, by cleavage at two sites followed by joining so as to delete the sequence between the two cleavage sites.
- Targeted modification of chromatin structure, as disclosed in co-owned International Patent Publication No. WO 01/83793, can be used to facilitate the binding of fusion proteins to cellular chromatin.
- The following examples show that targeted cleavage at two sites in chromosomal DNA can generate large deletions of genomic sequences, including deletion of sequences between the two cleavage sites. Targeted cleavage is accomplished, in certain embodiments, using fusion proteins (ZFNs) comprising a zinc finger DNA-binding domain and a nuclease half-domain.
- A number of zinc finger proteins were designed to bind to sites in the human CCR5 gene (GenBank® Accession Number AY221093). The proteins were designed in pairs such that, for each pair, target sites occurred on opposite DNA strands and the near edges of the target sites were separated by 5 nucleotide pairs.
- Table 2 shows the nucleotide sequences of the target sites for these zinc finger domains, and the locations of the target sites within the human CCR-5 gene. The amino acid sequences of the recognition regions of their zinc finger portions are also shown.
- Polynucleotides encoding fusions of the zinc finger domains shown in Table 2 to the FokI cleavage half-domain were constructed, in which sequences encoding the zinc finger domain are upstream of sequences encoding the cleavage half-domain, such that, in the encoded proteins, the zinc finger domain is nearest the N-terminus, and the cleavage half-domain is nearest the C-terminus, of the fusion protein.
-
TABLE 2 Zinc Finger Designs for the CCR-5 Gene2 Target Name sequence1 Location3 F1 F2 F3 F4 r162b2 GATGAGGATGAC 151 to DRSNLSR TSANLSR RSDNLAR TSANLSR (SEQ ID NO: 2) 162 (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 3) NO: 4) NO: 5) NO: 4) r162p11 GATGAGGATGAC 151 to DRSNLSR VSSNLTS RSDNLAR TSANLSR (SEQ ID NO: 2) 162 (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 3) NO: 6) NO: 5) NO: 4) 168c4 AAACTGCAAAAG 168 to RSDHLSE QNANRIT RSDVLSE QRNHRTT (SEQ ID NO: 7) 179 (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 8) NO: 9) NO: 10) NO: 11) 168i13 AAACTGCAAAAG 168 to RSDNLSV QKINLQV RSDVLSE QRNHRTT (SEQ ID NO: 7) 179 (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 12) NO: 13) NO: 10) NO: 11) r627s1 GACAAGCAGCGG 616 to RSAHLSE RSANLSE RSANLSV DRANLSR (SEQ ID NO: 14) 627 (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 15) NO: 16) NO: 17) NO: 18) 633a10 CATCTGcTACTCG 633 to RSDSLSK DNSNRIK RSAVLSE TNSNRIT (SEQ ID NO: 19) 645 (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 20) NO: 21) NO: 22) NO: 23) 633b5 CATCTGctACTCGG 633 to RSDHLSE ARSTRTN RSAVLSE TNSNRIT (SEQ ID NO: 24) 646 (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 8) NO: 25) NO: 22) NO: 23) Notes: 1Nucleotides in uppercase represent those present in subsites bound by individual zinc fingers; those in lowercase represent nucleotides not present in a subsite 2The zinc finger amino acid sequencesshown above (in one-letter code) represent residues -1 through +6, with respect to the start of the alpha-helical portion of each zinc finger. Finger F1 is closest to the amino terminus of the protein. 3Numbers in this column refer to nucleotide pairs downstream from the first residue of the translation initiation codon of the human CCR-5 gene - K562 erythroleukemia cells were cultured in RPMI medium with 10% bovine serum. At a density of approximately 1×106 cells/ml, cells were transfected with two DNA constructs, each encoding a pair of zinc finger nucleases (ZFNs), with the ZFN coding sequences separated by a 2A peptide sequence. The first plasmid (denoted 004) encoded the r162b2 and 168c4 ZFNS (see Table 2 above) which were designed to cleave between +162 and +168 (with respect to the translation start) of the human CCR-5 gene; the second plasmid (denoted 043) encoded the r627s1 and 633b5 ZFNs (Table 2) which were designed to cleave between +627 and +633. Control transfections used only one of these two plasmids, or used a plasmid encoding green fluorescent protein (GFP).
- Cells were concentrated 20-fold and transfection by nucleofection, using the AMAXA method (solution V and program T-16 for 2 million cells per 5 ug of each plasmid) following the manufacturer's protocol for K562 cells. Transfection efficiency was close to 90%, as estimated by expression of Green Fluorescent Protein as a control. Forty-eight hours after transfection, cells were harvested and genomic DNA was isolated using a Dneasy® Tissue kit (Qiagen, Valencia, Calif.), following the manufacturer's protocol. The genomic DNA (200-500 ng) was used as template for amplification using an AccuPrime® PCR amplification kit (Invitrogen, Carlsbad, Calif.) with the following primers:
-
CCR5longF: (SEQ ID NO: 26) GATGGTGCTTTCATGAATTCC and CCR5longR: (SEQ ID NO: 27) GTGTCACAAGCCCACAGATA.
Amplification products were analyzed by electrophoresis on 2% agarose e-gels (Invitrogen). - Results are shown in
FIG. 1 . In addition to a band corresponding to the amplification product obtained from chromosomes carrying a wild-type CCR-5 gene (present in all lanes), a lower molecular weight amplification product is obtained from cells transfected with plasmids encoding the two ZFN pairs (lane 4). The size of this low molecular-weight band is consistent with removal of approximately 465 nucleotide pairs from the CCR-5 sequences, which corresponds to the distance between the two targeted cleavage sites. - To confirm that targeted deletions of the endogenous CCR-5 gene had been obtained, amplification products were cloned into the Topo-4® vector (Invitrogen, Carlsbad, Calif.) following the manufacturer's protocol. Two classes of insert size were obtained. Plasmids containing inserts of the smaller size class were analyzed to determine the nucleotide sequence of their inserts. The results are shown in
FIG. 2 , in which a representative number of sequences in the region around and between the two targeted cleavage sites are shown. It can be seen, from the sequences obtained, that sequence alterations induced by cleavage at two cleavage sites can include deletion of some or all of the sequence between the two cleavage sites, and can also include deletion of additional sequences on one or both sides. - Analysis of genomic DNA by blotting was also conducted to provide an estimate of the frequency of deletion events resulting from targeted cleavage at two sites. Genomic DNA was isolated from K562 cells that had been transfected with plasmids encoding the two nuclease pairs described earlier in this example, or from control K562 cells that had been transfected with a plasmid encoding green fluorescent protein. Ten micrograms of DNA was digested with XhoI and NdeI. The digestion products were fractionated on an agarose gel and transferred to a nylon membrane. The membrane was incubated with a labeled probe comprising sequences corresponding to nucleotides −246 through +9, with respect to the first base pair of the translation initiation codon of the human CCR-5 gene.
- The probe used in this experiment identifies a 2.8 kbp XhoI-NdeI fragment in DNA from cells transfected with the GFP-encoding plasmid, corresponding to wild-type CCR-5 sequences (
FIG. 3 , lane 2). In cells expressing the two ZFN pairs, however, a band at approximately 2.3 kbp, corresponding to deleted molecules, is also present (FIG. 3 , lane 1). Quantitation of this lower molecular weight band indicated a deletion frequency of approximately 10%. - Human T-cells were obtained by leukopheresis from a healthy donor, the T cells were depleted in CD8 cells, then activated for two days with PHA+IL2. Transfection was performed by electroporation, using a Maxcyte electroporation device. Cells were transfected with two DNA constructs, each encoding a pair of zinc finger nucleases (ZFNs), with the ZFN coding sequences separated by a 2A peptide sequence. The first plasmid (denoted 149) encoded the r162p11 and 168i13 ZFNS (see Table 2 above) which were designed to cleave between +162 and +168 (with respect to the translation start) of the human CCR-5 gene; the second plasmid (denoted 141) encoded the r627s1 and 633a10 ZFNs (Table 2) which were designed to cleave between +627 and +633. Control transfections used a plasmid encoding green fluorescent protein (GFP).
- 5 million cells+20 ug of DNA (10 ug of each ZFN pair-encoding plasmid) were used per transfection. Transfected cells were collected by
centrifugation 2 days after transfection. and genomic DNA was isolated using a DNeasy® Tissue kit (Qiagen, Valencia, Calif.), following the manufacturer's protocol. The genomic DNA (200-500 ng) was used as template for amplification using an AccuPrime® PCR amplification kit (Invitrogen, Carlsbad, Calif.) with the following primers: -
CCR5longF: (SEQ ID NO: 26) GATGGTGCTTTCATGAATTCC and CCR5longR: (SEQ ID NO: 27) GTGTCACAAGCCCACAGATA
Amplification products were analyzed by electrophoresis on 2% agarose e-gels (Invitrogen). - Results are shown in
FIG. 4 . In addition to a band corresponding to the amplification product obtained from chromosomes carrying a wild-type CCR-5 gene (lane FIG. 4 ), a lower molecular weight amplification product is obtained from cells transfected with plasmids encoding the two ZFN pairs (lane 2 ofFIG. 4 ). The size of this low molecular-weight band is consistent with removal of approximately 465 nucleotide pairs from the CCR-5 sequences, which corresponds to the distance between the two targeted cleavage sites. - To confirm that targeted deletions of the endogenous CCR-5 gene had been obtained, the lower molecular weight band was excised from the gel, DNA was eluted from the band and cloned into the Topo-4® vector (Invitrogen, Carlsbad, Calif.) following the manufacturer's protocol. Resulting plasmids were analyzed to determine the nucleotide sequence of their inserts. The results are shown in
FIG. 5 , in which a representative number of sequences in the region around and between the two targeted cleavage sites are shown. It can be seen, from the sequences obtained, that the amplification products present in the lower band contained deletions of approximately 430 nucleotide pairs, whose endpoints lay at or near the targeted cleavage sites. - A number of zinc finger proteins were designed to bind to sites in the human CTLA4 gene (GenBank® Accession Number NM_005214). The proteins were designed in pairs such that, for each pair, target sites occurred on opposite DNA strands. For one pair, the near edges of the target sites were separated by 5 nucleotide pairs and, for the other pair, the near edges of the target sites were separated by 6 nucleotide pairs.
- Table 3 shows the nucleotide sequences of the target sites for these zinc finger domains, and the locations of the target sites within the human CTLA4 gene. The amino acid sequences of the recognition regions of their zinc finger portions are also shown.
- Polynucleotides encoding fusions of the zinc finger domains shown in Table 3 to the FokI cleavage half-domain were constructed, in which sequences encoding the zinc finger domain are upstream of sequences encoding the cleavage half-domain, such that, in the encoded proteins, the zinc finger domain is nearest the N-terminus, and the cleavage half-domain is nearest the C-terminus, of the fusion protein.
-
TABLE 3 Zinc Finger Designs for the CTLA4 Gene2 Target Name sequence1 Location3 F1 F2 F3 F4 r158a ATGGCTTTATGG 147 to RSDHLSQ TSSARTN QSSDLSR RSDALTQ (SEQ ID NO: 28) 158 (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 29) NO: 30) NO: 31) NO: 32) 164a GCCTTGGATTTC 164 TO TNLPLNN TSSNLSR RSDSLSA DRSDLSR (SEQ ID NO: 33) 175 (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 34) NO: 35) NO: 36) NO: 37) r2902b ACCCGGaCCTCAG 2890 to RSDHLSE TSSTRKT RSDHLSE TSSDRTK (SEQ ID NO: 38) 2902 (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 8) NO: 39) NO: 8) NO: 40) 2909b GCTTCGgCAGGCT 2909 to QSSDLSR RSDNLRE RSDDLSK QSSDLRR (SEQ ID NO: 41) 2921 (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 31) NO: 42) NO: 43) NO: 44) Notes: 1Nucleotides in uppercase represent those present in subsites bound by individual zinc fingers; those in lowercase represent nucleotides not present in a subsite 2The zinc finger amino acid sequences shown above (in one-letter code) represent residues -1 through +6, with respect to the start of the alpha-helical portion of each zinc finger. Finger F1 is closest to the amino terminus of the protein. 3Numbers in this column refer to nucleotide pairs downstream from the first residue of the translation initiation codon of the human CTLA4 gene - K562 cells were cultured and transfected as described in Example 2. Cells were transfected with four DNA constructs, each encoding one of the zinc finger nucleases (ZFNs) identified in Table 3. Control transfections were conducted with a vector that did not encode a ZFN (“empty vector”).
- Transfected cells were collected by
centrifugation 2 days after transfection. and genomic DNA was isolated using a DNeasy® Tissue kit (Qiagen, Valencia, Calif.), following the manufacturer's protocol. Genomic DNA (200 ng) was used as template for amplification using an AccuPrime® PCR amplification kit (Invitrogen, Carlsbad, Calif.) with primers that yield a 3.8 kilobase pair (kbp) amplification product from a wild-type CTLA4 gene. Amplification products were analyzed by gel electrophoresis. - The results indicated that, in addition to a band corresponding to the 3.8 kbp amplification product obtained from chromosomes carrying a wild-type CTLA4 gene, a lower molecular weight amplification product of approximately 1 kbp was obtained from cells that were transfected with plasmids encoding the four ZFNs. The size of this low molecular-weight band is consistent with removal of approximately 2.8 kilobase pairs from the CTLA4 locus, which corresponds to the distance between the two targeted cleavage sites.
- Nucleotide sequence analysis of amplification products, similar to that described in Example 3, confirmed that the endpoints of the deletions lay at or near the targeted cleavage sites in the endogenous CTLA4 gene.
- Similar results were obtained using a second pair of ZFNs designed to cleave between +3104 and +3111, in combination with the r158a and 164a nucleases described in Table 3.
- Similar results were also obtained in primary human T-cells (obtained from AllCells Berkeley, Calif.) using both combinations of ZFN pairs.
- Two pairs of zinc finger/nuclease half-domain fusion proteins, designed to cleave in the third exon of the IL-2Rγ (“common gamma chain”) gene have been disclosed in parent application, U.S. Patent Publication No. 2005/0064474, the disclosure of which is incorporated by reference (See Example 2 of that application). Two pairs of zinc finger/nuclease half-domain fusion proteins designed to cleave in the fifth exon of the IL-2Rγ gene are also disclosed in that application (See Examples 5 and 14 of U.S. Patent Publication No. 2005/0064474, incorporated by reference).
- Co-expression in cells of either of the first pair of exon 3-targeted nucleases and either of the second pair of exon 5-targeted nucleases (e.g., by transfection of cells with plasmids encoding the nucleases), using methods similar to those described in the previous examples, results in cleavage events in
exon 3 and exon 5 of the IL-2Rγ gene. Subsequent rejoining of DNA ends can result in loss of sequences between the cleavage sites, leading to deletion of approximately 1,400 nucleotide pairs of the X chromosome. - All patents, patent applications and publications mentioned herein are hereby incorporated by reference, in their entireties, for all purposes.
- Although disclosure has been provided in some detail by way of illustration and example for the purposes of clarity of understanding, it will be apparent to those skilled in the art that various changes and modifications can be practiced without departing from the spirit or scope of the disclosure. Accordingly, the foregoing descriptions and examples should not be construed as limiting.
Claims (9)
1. A method for deleting a region of interest in an endogenous gene in a plant cell, the method comprising:
cleaving the genome at first and second cleavage sites surrounding the region of interest using first and second pairs of artificial zinc finger nucleases that bind to first, second, third and fourth target sites in the endogenous genome, each zinc finger nuclease comprising:
a fusion protein comprising an engineered zinc finger protein DNA-binding domain that binds to the target site in the endogenous genome;
and a FokI nuclease domain,
wherein each target site comprises at least 3 target subsites and the first cleavage site lies between the first and second target sites and the second cleavage site lies between the third and fourth target sites;
such that region of interest is deleted from the genome and further wherein at least two of the target sites are maintained in the endogenous genome following deletion of the region of interest.
2. The method of claim 1 , wherein the first and second target sites are separated by 2 to 50 nucleotides.
3. The method of claim 1 , wherein the region of interest comprises at least 100 base pairs.
4. The method of claim 1 , wherein the region of interest comprises at least 400 base pairs.
5. The method of claim 1 , wherein the region of interest comprises at least 1000 base pairs.
6. The method of claim 1 , wherein the plant cell is from a monocotyledonous and dicotyledonous plant.
7. The method of claim 1 , wherein the plant cell is a crop plant cell selected from the group consisting of a grain crop, a fruit crop, a forage crop, a vegetable crop, an oil crop, a flowering plant crop and an experimental crop.
8. The method of claim 1 , wherein the plant cell is a seed cell.
9. A plant generated from the plant cell produced by the method of claim 1 .
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US16/859,049 US20200291424A1 (en) | 2003-08-08 | 2020-04-27 | Targeted deletion of cellular dna sequences |
Applications Claiming Priority (12)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US49393103P | 2003-08-08 | 2003-08-08 | |
US51825303P | 2003-11-07 | 2003-11-07 | |
US53054103P | 2003-12-18 | 2003-12-18 | |
US54278004P | 2004-02-05 | 2004-02-05 | |
US55683104P | 2004-03-26 | 2004-03-26 | |
US57591904P | 2004-06-01 | 2004-06-01 | |
US10/912,932 US7888121B2 (en) | 2003-08-08 | 2004-08-06 | Methods and compositions for targeted cleavage and recombination |
US64951505P | 2005-02-03 | 2005-02-03 | |
US11/304,981 US8409861B2 (en) | 2003-08-08 | 2005-12-15 | Targeted deletion of cellular DNA sequences |
US13/784,634 US9695442B2 (en) | 2003-08-08 | 2013-03-04 | Targeted deletion of cellular DNA sequences |
US15/610,262 US10669557B2 (en) | 2003-08-08 | 2017-05-31 | Targeted deletion of cellular DNA sequences |
US16/859,049 US20200291424A1 (en) | 2003-08-08 | 2020-04-27 | Targeted deletion of cellular dna sequences |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/610,262 Division US10669557B2 (en) | 2003-08-08 | 2017-05-31 | Targeted deletion of cellular DNA sequences |
Publications (1)
Publication Number | Publication Date |
---|---|
US20200291424A1 true US20200291424A1 (en) | 2020-09-17 |
Family
ID=36913227
Family Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/304,981 Active 2026-08-27 US8409861B2 (en) | 2003-08-08 | 2005-12-15 | Targeted deletion of cellular DNA sequences |
US13/784,634 Active US9695442B2 (en) | 2003-08-08 | 2013-03-04 | Targeted deletion of cellular DNA sequences |
US15/610,262 Active 2024-09-27 US10669557B2 (en) | 2003-08-08 | 2017-05-31 | Targeted deletion of cellular DNA sequences |
US16/859,049 Pending US20200291424A1 (en) | 2003-08-08 | 2020-04-27 | Targeted deletion of cellular dna sequences |
Family Applications Before (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/304,981 Active 2026-08-27 US8409861B2 (en) | 2003-08-08 | 2005-12-15 | Targeted deletion of cellular DNA sequences |
US13/784,634 Active US9695442B2 (en) | 2003-08-08 | 2013-03-04 | Targeted deletion of cellular DNA sequences |
US15/610,262 Active 2024-09-27 US10669557B2 (en) | 2003-08-08 | 2017-05-31 | Targeted deletion of cellular DNA sequences |
Country Status (1)
Country | Link |
---|---|
US (4) | US8409861B2 (en) |
Families Citing this family (328)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11311574B2 (en) | 2003-08-08 | 2022-04-26 | Sangamo Therapeutics, Inc. | Methods and compositions for targeted cleavage and recombination |
US8409861B2 (en) * | 2003-08-08 | 2013-04-02 | Sangamo Biosciences, Inc. | Targeted deletion of cellular DNA sequences |
US20120196370A1 (en) | 2010-12-03 | 2012-08-02 | Fyodor Urnov | Methods and compositions for targeted genomic deletion |
US7888121B2 (en) | 2003-08-08 | 2011-02-15 | Sangamo Biosciences, Inc. | Methods and compositions for targeted cleavage and recombination |
US20080131962A1 (en) | 2006-05-25 | 2008-06-05 | Sangamo Biosciences, Inc. | Engineered cleavage half-domains |
KR101419729B1 (en) * | 2005-07-26 | 2014-07-17 | 상가모 바이오사이언스 인코포레이티드 | Targeted integration and expression of exogenous nucleic acid sequences |
JP2009537140A (en) * | 2006-05-19 | 2009-10-29 | サンガモ バイオサイエンシーズ, インコーポレイテッド | Methods and compositions for inactivation of dihydrofolate reductase |
WO2007139982A2 (en) * | 2006-05-25 | 2007-12-06 | Sangamo Biosciences, Inc. | Methods and compositions for gene inactivation |
CA2667414C (en) * | 2006-11-13 | 2015-12-29 | Sangamo Biosciences, Inc. | Methods and compositions for modification of the human glucocorticoid receptor locus |
PT2415873E (en) | 2006-12-14 | 2015-03-31 | Sangamo Biosciences Inc | Optimized non-canonical zinc finger proteins |
CA2684378C (en) | 2007-04-26 | 2016-11-29 | Sangamo Biosciences, Inc. | Targeted integration into the ppp1r12c locus |
AU2008262487B2 (en) * | 2007-05-23 | 2013-10-31 | Sangamo Therapeutics, Inc. | Methods and compositions for increased transgene expression |
CN101883850B (en) * | 2007-07-12 | 2014-11-12 | 桑格摩生物科学股份有限公司 | Methods and compositions for inactivating alpha-1,6 fucosyltransferase (FUT 8) gene expression |
US20110014616A1 (en) * | 2009-06-30 | 2011-01-20 | Sangamo Biosciences, Inc. | Rapid screening of biologically active nucleases and isolation of nuclease-modified cells |
US8563314B2 (en) | 2007-09-27 | 2013-10-22 | Sangamo Biosciences, Inc. | Methods and compositions for modulating PD1 |
EP2188384B1 (en) * | 2007-09-27 | 2015-07-15 | Sangamo BioSciences, Inc. | Rapid in vivo identification of biologically active nucleases |
US11235026B2 (en) | 2007-09-27 | 2022-02-01 | Sangamo Therapeutics, Inc. | Methods and compositions for modulating PD1 |
JP2011500082A (en) | 2007-10-25 | 2011-01-06 | サンガモ バイオサイエンシーズ, インコーポレイテッド | Methods and compositions for targeted integration |
CA2720903C (en) | 2008-04-14 | 2019-01-15 | Sangamo Biosciences, Inc. | Linear donor constructs for targeted integration |
JP2011521643A (en) | 2008-05-28 | 2011-07-28 | サンガモ バイオサイエンシーズ, インコーポレイテッド | Composition for linking a DNA binding domain and a cleavage domain |
JP5763530B2 (en) | 2008-06-10 | 2015-08-12 | サンガモ バイオサイエンシーズ, インコーポレイテッド | Methods and compositions for the generation of Bax- and Bak-deficient cell lines |
KR20160015400A (en) | 2008-08-22 | 2016-02-12 | 상가모 바이오사이언스 인코포레이티드 | Methods and compositions for targeted single-stranded cleavage and targeted integration |
CA2741119C (en) * | 2008-10-29 | 2019-02-12 | Sangamo Biosciences, Inc. | Methods and compositions for inactivating glutamine synthetase gene expression |
US20110016539A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genome editing of neurotransmission-related genes in animals |
US20110016543A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genomic editing of genes involved in inflammation |
US20110023158A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Bovine genome editing with zinc finger nucleases |
US20110023141A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved with parkinson's disease |
US20110023150A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genome editing of genes associated with schizophrenia in animals |
US20110030072A1 (en) * | 2008-12-04 | 2011-02-03 | Sigma-Aldrich Co. | Genome editing of immunodeficiency genes in animals |
US20110023139A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in cardiovascular disease |
US20110023149A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in tumor suppression in animals |
US20110023140A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Rabbit genome editing with zinc finger nucleases |
US20110023154A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Silkworm genome editing with zinc finger nucleases |
US20110016542A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Canine genome editing with zinc finger nucleases |
CA2745031C (en) | 2008-12-04 | 2018-08-14 | Sangamo Biosciences, Inc. | Genome editing in rats using zinc-finger nucleases |
US20110023156A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Feline genome editing with zinc finger nucleases |
US20110023157A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Equine genome editing with zinc finger nucleases |
US20110023151A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genome editing of abc transporters |
US20110023143A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of neurodevelopmental genes in animals |
US20110023146A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in secretase-associated disorders |
US20110023147A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of prion disorder-related genes in animals |
US20110023144A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in amyotrophyic lateral sclerosis disease |
US20110023152A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genome editing of cognition related genes in animals |
US20110016540A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genome editing of genes associated with trinucleotide repeat expansion disorders in animals |
US20110023153A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in alzheimer's disease |
US20110016541A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genome editing of sensory-related genes in animals |
US20110023148A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genome editing of addiction-related genes in animals |
US20110023159A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Ovine genome editing with zinc finger nucleases |
US20110016546A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Porcine genome editing with zinc finger nucleases |
US20110023145A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genomic editing of genes involved in autism spectrum disorders |
US8329986B2 (en) * | 2008-12-17 | 2012-12-11 | Dow Agrosciences, Llc | Targeted integration into the Zp15 locus |
CA2749965C (en) | 2009-02-04 | 2018-09-11 | Sangamo Biosciences, Inc. | Methods and compositions for treating neuropathies |
EP2408921B1 (en) * | 2009-03-20 | 2017-04-19 | Sangamo BioSciences, Inc. | Modification of cxcr4 using engineered zinc finger proteins |
EP2419511B1 (en) | 2009-04-09 | 2018-01-17 | Sangamo Therapeutics, Inc. | Targeted integration into stem cells |
US8772008B2 (en) * | 2009-05-18 | 2014-07-08 | Sangamo Biosciences, Inc. | Methods and compositions for increasing nuclease activity |
US20120171771A1 (en) | 2009-07-08 | 2012-07-05 | Cellular Dynamics International, Inc. | Modified ips cells having a mutant form of a human immunodeficiency virus (hiv) cellular entry gene |
EP2461819A4 (en) | 2009-07-28 | 2013-07-31 | Sangamo Biosciences Inc | Methods and compositions for treating trinucleotide repeat disorders |
BR112012003257A2 (en) | 2009-08-11 | 2016-11-22 | Sangamo Biosciences Inc | homozygous organisms for directed modification |
NZ619018A (en) | 2009-10-22 | 2014-06-27 | Dow Agrosciences Llc | Engineered zinc finger proteins targeting plant genes involved in fatty acid biosynthesis |
US8956828B2 (en) | 2009-11-10 | 2015-02-17 | Sangamo Biosciences, Inc. | Targeted disruption of T cell receptor genes using engineered zinc finger protein nucleases |
GEP20176628B (en) * | 2010-01-22 | 2017-02-27 | Sangamo Biosciences Inc | Targeted genomic alteration |
JP6137596B2 (en) | 2010-02-08 | 2017-05-31 | サンガモ セラピューティクス, インコーポレイテッド | Engineered truncated half-domain |
US9255259B2 (en) * | 2010-02-09 | 2016-02-09 | Sangamo Biosciences, Inc. | Targeted genomic modification with partially single-stranded donor molecules |
CN102939377B (en) | 2010-04-26 | 2016-06-08 | 桑格摩生物科学股份有限公司 | Use Zinc finger nuclease to carry out genome editor to Rosa site |
LT2566972T (en) | 2010-05-03 | 2020-03-10 | Sangamo Therapeutics, Inc. | Compositions for linking zinc finger modules |
CN103025344B (en) | 2010-05-17 | 2016-06-29 | 桑格摩生物科学股份有限公司 | Novel DNA-associated proteins and application thereof |
US8945868B2 (en) | 2010-07-21 | 2015-02-03 | Sangamo Biosciences, Inc. | Methods and compositions for modification of a HLA locus |
KR20180121665A (en) | 2010-07-23 | 2018-11-07 | 시그마-알드리치 컴퍼니., 엘엘씨 | Genome editing using targeting endonucleases and single-stranded nucleic acids |
EP3511420B1 (en) | 2010-09-27 | 2021-04-07 | Sangamo Therapeutics, Inc. | Compositions for inhibiting viral entry into cells |
AU2011316575B2 (en) | 2010-10-12 | 2015-10-29 | Sangamo Therapeutics, Inc. | Methods and compositions for treating hemophilia B |
CA2821547A1 (en) | 2010-12-29 | 2012-07-05 | Sigma-Aldrich Co. Llc | Cells having disrupted expression of proteins involved in adme and toxicology processes |
US9267123B2 (en) * | 2011-01-05 | 2016-02-23 | Sangamo Biosciences, Inc. | Methods and compositions for gene correction |
CA2841541C (en) | 2011-07-25 | 2019-11-12 | Sangamo Biosciences, Inc. | Methods and compositions for alteration of a cystic fibrosis transmembrane conductance regulator (cftr) gene |
CN103917644A (en) | 2011-09-21 | 2014-07-09 | 桑格摩生物科学股份有限公司 | Methods and compositions for regulation of transgene expression |
AU2012328682B2 (en) | 2011-10-27 | 2017-09-21 | Sangamo Therapeutics, Inc. | Methods and compositions for modification of the HPRT locus |
EP2780460B1 (en) | 2011-11-16 | 2018-07-11 | Sangamo Therapeutics, Inc. | Modified dna-binding proteins and uses thereof |
US20130216579A1 (en) * | 2012-01-23 | 2013-08-22 | The Trustees Of Columbia University In The City Of New York | Methods and compostitions for gene editing of a pathogen |
WO2013130824A1 (en) | 2012-02-29 | 2013-09-06 | Sangamo Biosciences, Inc. | Methods and compositions for treating huntington's disease |
US9745548B2 (en) | 2012-03-15 | 2017-08-29 | Flodesign Sonics, Inc. | Acoustic perfusion devices |
US10704021B2 (en) | 2012-03-15 | 2020-07-07 | Flodesign Sonics, Inc. | Acoustic perfusion devices |
US9458450B2 (en) | 2012-03-15 | 2016-10-04 | Flodesign Sonics, Inc. | Acoustophoretic separation technology using multi-dimensional standing waves |
US10322949B2 (en) | 2012-03-15 | 2019-06-18 | Flodesign Sonics, Inc. | Transducer and reflector configurations for an acoustophoretic device |
US9950282B2 (en) | 2012-03-15 | 2018-04-24 | Flodesign Sonics, Inc. | Electronic configuration and control for acoustic standing wave generation |
US10967298B2 (en) | 2012-03-15 | 2021-04-06 | Flodesign Sonics, Inc. | Driver and control for variable impedence load |
US9752113B2 (en) | 2012-03-15 | 2017-09-05 | Flodesign Sonics, Inc. | Acoustic perfusion devices |
US10689609B2 (en) | 2012-03-15 | 2020-06-23 | Flodesign Sonics, Inc. | Acoustic bioreactor processes |
US10737953B2 (en) | 2012-04-20 | 2020-08-11 | Flodesign Sonics, Inc. | Acoustophoretic method for use in bioreactors |
CN104364380B (en) | 2012-04-25 | 2018-10-09 | 瑞泽恩制药公司 | The targeting of the big targeting vector of nuclease-mediated use |
UA115875C2 (en) | 2012-05-02 | 2018-01-10 | ДАУ АГРОСАЙЄНСІЗ ЕлЕлСі | Targeted modification of malate dehydrogenase |
AU2013259647B2 (en) | 2012-05-07 | 2018-11-08 | Corteva Agriscience Llc | Methods and compositions for nuclease-mediated targeted integration of transgenes |
WO2013169398A2 (en) | 2012-05-09 | 2013-11-14 | Georgia Tech Research Corporation | Systems and methods for improving nuclease specificity and activity |
JP6329537B2 (en) | 2012-07-11 | 2018-05-23 | サンガモ セラピューティクス, インコーポレイテッド | Methods and compositions for delivery of biological agents |
US10648001B2 (en) | 2012-07-11 | 2020-05-12 | Sangamo Therapeutics, Inc. | Method of treating mucopolysaccharidosis type I or II |
DK2872625T3 (en) | 2012-07-11 | 2017-02-06 | Sangamo Biosciences Inc | METHODS AND COMPOSITIONS FOR TREATING LYSOSOMAL STORAGE DISEASES |
SI2890780T1 (en) | 2012-08-29 | 2020-11-30 | Sangamo Therapeutics, Inc. | Methods and compositions for treatment of a genetic condition |
AU2013312538B2 (en) | 2012-09-07 | 2019-01-24 | Corteva Agriscience Llc | FAD3 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks |
UA119135C2 (en) | 2012-09-07 | 2019-05-10 | ДАУ АГРОСАЙЄНСІЗ ЕлЕлСі | Engineered transgene integration platform (etip) for gene targeting and trait stacking |
UA118090C2 (en) | 2012-09-07 | 2018-11-26 | ДАУ АГРОСАЙЄНСІЗ ЕлЕлСі | Fad2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks |
EP2906684B8 (en) | 2012-10-10 | 2020-09-02 | Sangamo Therapeutics, Inc. | T cell modifying compounds and uses thereof |
JP2016500254A (en) | 2012-12-05 | 2016-01-12 | サンガモ バイオサイエンシーズ, インコーポレイテッド | Methods and compositions for the regulation of metabolic diseases |
WO2014093768A1 (en) | 2012-12-13 | 2014-06-19 | Ainley W Michael | Precision gene targeting to a particular locus in maize |
AU2014207618A1 (en) | 2013-01-16 | 2015-08-06 | Emory University | Cas9-nucleic acid complexes and uses related thereto |
SG10201706741VA (en) | 2013-02-20 | 2017-10-30 | Regeneron Pharma | Genetic modification of rats |
US10227610B2 (en) | 2013-02-25 | 2019-03-12 | Sangamo Therapeutics, Inc. | Methods and compositions for enhancing nuclease-mediated gene disruption |
WO2014152832A1 (en) | 2013-03-14 | 2014-09-25 | Immusoft Corporation | Methods for in vitro memory b cell differentiation and transduction with vsv-g pseudotyped viral vectors |
CN105208866B (en) | 2013-03-21 | 2018-11-23 | 桑格摩生物治疗股份有限公司 | Use engineering zinc finger protein nuclease targeted disruption T cell receptor gene |
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 |
PT3456831T (en) | 2013-04-16 | 2021-09-10 | Regeneron Pharma | Targeted modification of rat genome |
US10604771B2 (en) | 2013-05-10 | 2020-03-31 | Sangamo Therapeutics, Inc. | Delivery methods and compositions for nuclease-mediated genome engineering |
JP2016524464A (en) * | 2013-05-13 | 2016-08-18 | セレクティスCellectis | Method for manipulating highly active T cells for immunotherapy |
CA2910489A1 (en) | 2013-05-15 | 2014-11-20 | Sangamo Biosciences, Inc. | Methods and compositions for treatment of a genetic condition |
WO2015017866A1 (en) | 2013-08-02 | 2015-02-05 | Enevolv, Inc. | Processes and host cells for genome, pathway, and biomolecular engineering |
AU2014312295C1 (en) | 2013-08-28 | 2020-08-13 | Sangamo Therapeutics, Inc. | Compositions for linking DNA-binding domains and cleavage domains |
CN105682452B (en) | 2013-09-04 | 2018-10-16 | 美国陶氏益农公司 | The quick targeting analysis being inserted into for determining donor in crop |
US9745569B2 (en) | 2013-09-13 | 2017-08-29 | Flodesign Sonics, Inc. | System for generating high concentration factors for low cell density suspensions |
WO2015057976A1 (en) | 2013-10-17 | 2015-04-23 | Sangamo Biosciences, Inc. | Delivery methods and compositions for nuclease-mediated genome engineering in hematopoietic stem cells |
US9957526B2 (en) | 2013-10-17 | 2018-05-01 | Sangamo Therapeutics, Inc. | Delivery methods and compositions for nuclease-mediated genome engineering |
CA2926822C (en) | 2013-11-04 | 2022-12-13 | Dow Agrosciences Llc | Optimal soybean loci |
US9909131B2 (en) | 2013-11-04 | 2018-03-06 | Dow Agrosciences Llc | Optimal soybean loci |
JP6633532B2 (en) | 2013-11-04 | 2020-01-22 | ダウ アグロサイエンシィズ エルエルシー | Optimal corn loci |
US10093940B2 (en) | 2013-11-04 | 2018-10-09 | Dow Agrosciences Llc | Optimal maize loci |
NZ719477A (en) | 2013-11-11 | 2022-05-27 | Sangamo Therapeutics Inc | Methods and compositions for treating huntington’s disease |
RS62559B1 (en) | 2013-11-13 | 2021-12-31 | Childrens Medical Center | Nuclease-mediated regulation of gene expression |
WO2015089077A2 (en) | 2013-12-09 | 2015-06-18 | Sangamo Biosciences, Inc. | Methods and compositions for genome engineering |
KR102170502B1 (en) | 2013-12-11 | 2020-10-28 | 리제너론 파마슈티칼스 인코포레이티드 | Methods and compositions for the targeted modification of a genome |
HUE041331T2 (en) | 2013-12-11 | 2019-05-28 | Regeneron Pharma | Methods and compositions for the targeted modification of a genome |
US20160333063A1 (en) | 2013-12-13 | 2016-11-17 | The General Hospital Corporation | Soluble high molecular weight (hmw) tau species and applications thereof |
CA2935960C (en) | 2014-01-08 | 2023-01-10 | Bart Lipkens | Acoustophoresis device with dual acoustophoretic chamber |
US10774338B2 (en) | 2014-01-16 | 2020-09-15 | The Regents Of The University Of California | Generation of heritable chimeric plant traits |
LT3102673T (en) | 2014-02-03 | 2020-08-25 | Sangamo Therapeutics, Inc. | Methods and compositions for treatment of a beta thalessemia |
CN106232814B (en) | 2014-02-13 | 2021-05-11 | 宝生物工程(美国)有限公司 | Methods of depleting target molecules from an initial collection of nucleic acids, and compositions and kits for practicing same |
US10370680B2 (en) | 2014-02-24 | 2019-08-06 | Sangamo Therapeutics, Inc. | Method of treating factor IX deficiency using nuclease-mediated targeted integration |
AU2015231353B2 (en) | 2014-03-18 | 2020-11-05 | Sangamo Therapeutics, Inc. | Methods and compositions for regulation of zinc finger protein expression |
US20150301028A1 (en) | 2014-04-22 | 2015-10-22 | Q-State Biosciences, Inc. | Analysis of compounds for pain and sensory disorders |
US9522936B2 (en) | 2014-04-24 | 2016-12-20 | Sangamo Biosciences, Inc. | Engineered transcription activator like effector (TALE) proteins |
KR102380324B1 (en) | 2014-05-08 | 2022-03-30 | 상가모 테라퓨틱스, 인코포레이티드 | Methods and compositions for treating huntington's disease |
WO2015175642A2 (en) | 2014-05-13 | 2015-11-19 | Sangamo Biosciences, Inc. | Methods and compositions for prevention or treatment of a disease |
JP2017518075A (en) | 2014-05-30 | 2017-07-06 | ザ ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティー | Compositions and methods for treating latent viral infections |
WO2015188056A1 (en) | 2014-06-05 | 2015-12-10 | Sangamo Biosciences, Inc. | Methods and compositions for nuclease design |
SG10201913804WA (en) | 2014-06-06 | 2020-03-30 | Regeneron Pharma | Methods and compositions for modifying a targeted locus |
BR112016030145A8 (en) | 2014-06-23 | 2018-12-11 | Regeneron Pharma | methods for assembling at least two nucleic acids and two or more nucleic acids |
EP3161128B1 (en) | 2014-06-26 | 2018-09-26 | Regeneron Pharmaceuticals, Inc. | Methods and compositions for targeted genetic modifications and methods of use |
US9744483B2 (en) | 2014-07-02 | 2017-08-29 | Flodesign Sonics, Inc. | Large scale acoustic separation device |
EP3166964A1 (en) | 2014-07-08 | 2017-05-17 | Vib Vzw | Means and methods to increase plant yield |
EA039787B1 (en) | 2014-07-14 | 2022-03-14 | Вашингтон Стейт Юниверсити | Livestock animal with ablation of germline cells and method of producing same |
AU2015289644A1 (en) | 2014-07-15 | 2017-02-02 | Juno Therapeutics, Inc. | Engineered cells for adoptive cell therapy |
US9757420B2 (en) | 2014-07-25 | 2017-09-12 | Sangamo Therapeutics, Inc. | Gene editing for HIV gene therapy |
WO2016014794A1 (en) | 2014-07-25 | 2016-01-28 | Sangamo Biosciences, Inc. | Methods and compositions for modulating nuclease-mediated genome engineering in hematopoietic stem cells |
US9616090B2 (en) | 2014-07-30 | 2017-04-11 | Sangamo Biosciences, Inc. | Gene correction of SCID-related genes in hematopoietic stem and progenitor cells |
WO2016021972A1 (en) | 2014-08-06 | 2016-02-11 | College Of Medicine Pochon Cha University Industry-Academic Cooperation Foundation | Immune-compatible cells created by nuclease-mediated editing of genes encoding hla |
HUE055583T2 (en) | 2014-09-16 | 2021-12-28 | Sangamo Therapeutics Inc | Methods and compositions for nuclease-mediated genome engineering and correction in hematopoietic stem cells |
RU2706965C2 (en) | 2014-10-15 | 2019-11-21 | Регенерон Фармасьютикалз, Инк. | Methods and compositions for producing or maintaining pluripotent cells |
US20160120158A1 (en) * | 2014-11-03 | 2016-05-05 | The Johns Hopkins University | Compositions and methods for the study and treatment of acute kidney injury |
US11352666B2 (en) | 2014-11-14 | 2022-06-07 | Institute For Basic Science | Method for detecting off-target sites of programmable nucleases in a genome |
US10889834B2 (en) | 2014-12-15 | 2021-01-12 | Sangamo Therapeutics, Inc. | Methods and compositions for enhancing targeted transgene integration |
JP6840077B2 (en) | 2014-12-19 | 2021-03-10 | リジェネロン・ファーマシューティカルズ・インコーポレイテッドRegeneron Pharmaceuticals, Inc. | Methods and compositions for targeted gene modification through single-step multi-targeting |
US20180002379A1 (en) | 2015-01-21 | 2018-01-04 | Sangamo Therapeutics, Inc. | Methods and compositions for identification of highly specific nucleases |
US10048275B2 (en) | 2015-03-13 | 2018-08-14 | Q-State Biosciences, Inc. | Cardiotoxicity screening methods |
US20160348073A1 (en) * | 2015-03-27 | 2016-12-01 | President And Fellows Of Harvard College | Modified t cells and methods of making and using the same |
US20180094243A1 (en) | 2015-04-03 | 2018-04-05 | Dana-Farber Cancer Institute, Inc. | Composition and methods of genome editing of b-cells |
KR20170135966A (en) * | 2015-04-13 | 2017-12-08 | 맥스시티 인코포레이티드 | Methods and compositions for transforming genomic DNA |
WO2016170484A1 (en) | 2015-04-21 | 2016-10-27 | Novartis Ag | Rna-guided gene editing system and uses thereof |
US11021699B2 (en) | 2015-04-29 | 2021-06-01 | FioDesign Sonics, Inc. | Separation using angled acoustic waves |
US11708572B2 (en) | 2015-04-29 | 2023-07-25 | Flodesign Sonics, Inc. | Acoustic cell separation techniques and processes |
US11377651B2 (en) | 2016-10-19 | 2022-07-05 | Flodesign Sonics, Inc. | Cell therapy processes utilizing acoustophoresis |
US10179918B2 (en) | 2015-05-07 | 2019-01-15 | Sangamo Therapeutics, Inc. | Methods and compositions for increasing transgene activity |
JP2018515139A (en) | 2015-05-08 | 2018-06-14 | プレジデント アンド フェローズ オブ ハーバード カレッジ | Universal donor stem cells and related methods |
BR112017024115A2 (en) | 2015-05-12 | 2018-08-07 | Sangamo Therapeutics Inc | nuclease-mediated gene expression regulation |
WO2016187543A1 (en) | 2015-05-21 | 2016-11-24 | Q-State Biosciences, Inc. | Optogenetics microscope |
US10117911B2 (en) | 2015-05-29 | 2018-11-06 | Agenovir Corporation | Compositions and methods to treat herpes simplex virus infections |
EP3324999A1 (en) | 2015-05-29 | 2018-05-30 | Agenovir Corporation | Compositions and methods for cell targeted hpv treatment |
US9957501B2 (en) | 2015-06-18 | 2018-05-01 | Sangamo Therapeutics, Inc. | Nuclease-mediated regulation of gene expression |
AU2016291778B2 (en) | 2015-07-13 | 2021-05-06 | Sangamo Therapeutics, Inc. | Delivery methods and compositions for nuclease-mediated genome engineering |
MA42895A (en) | 2015-07-15 | 2018-05-23 | Juno Therapeutics Inc | MODIFIED CELLS FOR ADOPTIVE CELL THERAPY |
US11459540B2 (en) | 2015-07-28 | 2022-10-04 | Flodesign Sonics, Inc. | Expanded bed affinity selection |
US11474085B2 (en) | 2015-07-28 | 2022-10-18 | Flodesign Sonics, Inc. | Expanded bed affinity selection |
US10827730B2 (en) | 2015-08-06 | 2020-11-10 | The Curators Of The University Of Missouri | Pathogen-resistant animals having modified CD163 genes |
JP6853257B2 (en) | 2015-09-23 | 2021-03-31 | サンガモ セラピューティクス, インコーポレイテッド | HTT repressor and its use |
CN108473976B (en) | 2015-10-28 | 2022-07-19 | 桑格摩生物治疗股份有限公司 | Liver-specific constructs, factor VIII expression cassettes, and methods of use thereof |
PT3368673T (en) | 2015-10-29 | 2020-08-27 | Amyris Inc | Compositions and methods for production of myrcene |
WO2017091512A1 (en) | 2015-11-23 | 2017-06-01 | Sangamo Biosciences, Inc. | Methods and compositions for engineering immunity |
WO2017106537A2 (en) | 2015-12-18 | 2017-06-22 | Sangamo Biosciences, Inc. | Targeted disruption of the mhc cell receptor |
CN108778297B (en) | 2015-12-18 | 2024-02-09 | 桑格摩生物治疗股份有限公司 | Targeted disruption of T cell receptors |
US20170202931A1 (en) | 2016-01-15 | 2017-07-20 | Sangamo Therapeutics, Inc. | Methods and compositions for the treatment of neurologic disease |
CN109069568B (en) | 2016-02-02 | 2023-07-07 | 桑格摩生物治疗股份有限公司 | Compositions for linking DNA binding domains and cleavage domains |
US11085035B2 (en) | 2016-05-03 | 2021-08-10 | Flodesign Sonics, Inc. | Therapeutic cell washing, concentration, and separation utilizing acoustophoresis |
US11214789B2 (en) | 2016-05-03 | 2022-01-04 | Flodesign Sonics, Inc. | Concentration and washing of particles with acoustics |
ES2938210T3 (en) | 2016-07-13 | 2023-04-05 | Vertex Pharma | Methods, compositions and kits to increase the efficiency of genome editing |
KR20230088522A (en) | 2016-07-21 | 2023-06-19 | 맥스시티 인코포레이티드 | Methods and compositions for modifying genomic dna |
ES2965134T3 (en) | 2016-07-29 | 2024-04-11 | Regeneron Pharma | Mice containing mutations resulting in expression of C-truncated fibrillin-1 |
US11193136B2 (en) | 2016-08-09 | 2021-12-07 | Vib Vzw | Cellulose synthase inhibitors and mutant plants |
EP3496530B1 (en) | 2016-08-11 | 2022-03-30 | The Jackson Laboratory | Methods and compositions relating to improved human red blood cell survival in genetically modified immunodeficient non-human animals |
US11774442B2 (en) | 2016-08-15 | 2023-10-03 | Enevolv, Inc. | Molecule sensor systems |
LT3504229T (en) | 2016-08-24 | 2021-12-10 | Sangamo Therapeutics, Inc. | Regulation of gene expression using engineered nucleases |
WO2018039448A1 (en) | 2016-08-24 | 2018-03-01 | Sangamo Therapeutics, Inc. | Engineered target specific nucleases |
JP7256739B2 (en) | 2016-09-07 | 2023-04-12 | サンガモ セラピューティクス, インコーポレイテッド | Modulation of liver genes |
DK3523326T3 (en) | 2016-10-04 | 2020-08-03 | Prec Biosciences Inc | COSTIMULATING DOMAINS FOR USE IN GENETICALLY MODIFIED CELLS |
CN110050061A (en) | 2016-10-05 | 2019-07-23 | 富士胶片细胞动力公司 | The mature pedigree of generation is induced multi-potent stem cell from what is destroyed with MeCP2 |
JP2019533676A (en) | 2016-10-13 | 2019-11-21 | ジュノー セラピューティクス インコーポレイテッド | Immunotherapy methods and compositions comprising tryptophan metabolic pathway modulators |
EP3529347A1 (en) | 2016-10-19 | 2019-08-28 | Flodesign Sonics, Inc. | Affinity cell extraction by acoustics |
KR20190060829A (en) | 2016-10-20 | 2019-06-03 | 상가모 테라퓨틱스, 인코포레이티드 | Methods and compositions for the treatment of Fabry disease |
WO2018081775A1 (en) | 2016-10-31 | 2018-05-03 | Sangamo Therapeutics, Inc. | Gene correction of scid-related genes in hematopoietic stem and progenitor cells |
KR20190085529A (en) | 2016-12-01 | 2019-07-18 | 상가모 테라퓨틱스, 인코포레이티드 | Tau modulators and methods and compositions for their delivery |
CN110494565A (en) | 2016-12-02 | 2019-11-22 | 朱诺治疗学股份有限公司 | It is engineered B cell and compositions related and method |
WO2018106782A1 (en) | 2016-12-08 | 2018-06-14 | Case Western Reserve University | Methods and compositions for enhancing functional myelin production |
BR112019014841A2 (en) | 2017-01-23 | 2020-04-28 | Regeneron Pharma | guide rna, use of guide rna, antisense rna, sirna or shrna, use of antisense rna, sirna or shrna, isolated nucleic acid, vector, composition, cell, and, methods to modify an hsd17b13 gene in a cell, to decrease the expression of an hsd17b13 gene in a cell, to modify a cell and to treat an individual who does not carry the hsd17b13 variant |
TW201839136A (en) | 2017-02-06 | 2018-11-01 | 瑞士商諾華公司 | Compositions and methods for the treatment of hemoglobinopathies |
WO2018152325A1 (en) | 2017-02-15 | 2018-08-23 | Bluebird Bio, Inc. | Donor repair templates multiplex genome editing |
EP3599842A4 (en) | 2017-03-21 | 2020-12-30 | The Jackson Laboratory | A GENETICALLY MODIFIED MOUSE EXPRESSING HUMAN APOE4 AND MOUSE Trem2.p.R47H AND METHODS OF USE THEREOF |
JP7297676B2 (en) | 2017-04-28 | 2023-06-26 | アクイタス セラピューティクス インコーポレイテッド | Novel carbonyl lipids and lipid nanoparticle formulations for nucleic acid delivery |
RU2019139045A (en) | 2017-05-03 | 2021-06-03 | Сангамо Терапьютикс, Инк. | METHODS AND COMPOSITIONS FOR MODIFICATION OF THE TRANSMEMBRANE CONDUCTIVITY REGULATOR GENE IN CYSTIC FIBROSIS (CFTR) |
CA3062698A1 (en) | 2017-05-08 | 2018-11-15 | Precision Biosciences, Inc. | Nucleic acid molecules encoding an engineered antigen receptor and an inhibitory nucleic acid molecule and methods of use thereof |
US11778994B2 (en) | 2017-05-12 | 2023-10-10 | The Jackson Laboratory | NSG mice lacking MHC class I and class II |
AU2018282072A1 (en) | 2017-06-05 | 2020-01-16 | Regeneron Pharmaceuticals, Inc. | B4GALT1 variants and uses thereof |
US11512287B2 (en) | 2017-06-16 | 2022-11-29 | Sangamo Therapeutics, Inc. | Targeted disruption of T cell and/or HLA receptors |
JP2020529834A (en) | 2017-06-30 | 2020-10-15 | プレシジョン バイオサイエンシズ,インク. | Genetically modified T cells containing modified introns of the T cell receptor alpha gene |
JP7402521B2 (en) | 2017-07-11 | 2023-12-21 | コンパス セラピューティクス リミテッド ライアビリティ カンパニー | Agonistic antibodies that bind to human CD137 and uses thereof |
BR112019027673A2 (en) | 2017-07-31 | 2020-09-15 | Regeneron Pharmaceuticals, Inc. | non-human animal, and, methods to test the recombination induced by crispr / cas and to optimize the ability of crispr / cas |
SG11201912235PA (en) | 2017-07-31 | 2020-01-30 | Regeneron Pharma | Cas-transgenic mouse embryonic stem cells and mice and uses thereof |
EP3585160A2 (en) | 2017-07-31 | 2020-01-01 | Regeneron Pharmaceuticals, Inc. | Crispr reporter non-human animals and uses thereof |
CN111511199A (en) | 2017-08-29 | 2020-08-07 | 科沃施种子欧洲股份两合公司 | Improved blue paste and other separation system |
ES2962277T3 (en) | 2017-09-29 | 2024-03-18 | Regeneron Pharma | Rodents comprising a humanized Ttr locus and methods of use |
EP4269560A3 (en) | 2017-10-03 | 2024-01-17 | Precision Biosciences, Inc. | Modified epidermal growth factor receptor peptides for use in genetically-modified cells |
WO2019089753A2 (en) | 2017-10-31 | 2019-05-09 | Compass Therapeutics Llc | Cd137 antibodies and pd-1 antagonists and uses thereof |
EP3704238B1 (en) | 2017-11-01 | 2024-01-03 | Precision Biosciences, Inc. | Engineered nucleases that target human and canine factor viii genes as a treatment for hemophilia a |
CN111565730A (en) | 2017-11-09 | 2020-08-21 | 桑格摩生物治疗股份有限公司 | Genetic modification of cytokine-induced SH2-containing protein (CISH) gene |
EP3713961A2 (en) | 2017-11-20 | 2020-09-30 | Compass Therapeutics LLC | Cd137 antibodies and tumor antigen-targeting antibodies and uses thereof |
SG11202003907WA (en) | 2017-12-14 | 2020-05-28 | Flodesign Sonics Inc | Acoustic transducer drive and controller |
WO2019143677A1 (en) | 2018-01-17 | 2019-07-25 | Vertex Pharmaceuticals Incorporated | Quinoxalinone compounds, compositions, methods, and kits for increasing genome editing efficiency |
AU2019209293B2 (en) | 2018-01-17 | 2023-07-27 | Vertex Pharmaceuticals Incorporated | DNA-PK inhibitors |
EP3740479A1 (en) | 2018-01-17 | 2020-11-25 | Vertex Pharmaceuticals Incorporated | Dna-pk inhibitors |
WO2019157324A1 (en) | 2018-02-08 | 2019-08-15 | Sangamo Therapeutics, Inc. | Engineered target specific nucleases |
WO2019161133A1 (en) | 2018-02-15 | 2019-08-22 | Memorial Sloan Kettering Cancer Center | Foxp3 targeting agent compositions and methods of use for adoptive cell therapy |
CN111936617A (en) | 2018-03-16 | 2020-11-13 | 益缪索夫特公司 | B cells genetically engineered to secrete follistatin and methods of using the same to treat follistatin-related diseases, conditions, disorders, and enhance muscle growth and strength |
CA3089331A1 (en) | 2018-03-19 | 2019-09-26 | Regeneron Pharmaceuticals, Inc. | Transcription modulation in animals using crispr/cas systems |
KR20210020873A (en) | 2018-04-05 | 2021-02-24 | 주노 쎄러퓨티크스 인코퍼레이티드 | Τ cells expressing recombinant receptors, related polynucleotides and methods |
US20210017249A1 (en) | 2018-04-05 | 2021-01-21 | Juno Therapeutics, Inc. | Methods of producing cells expressing a recombinant receptor and related compositions |
US11421007B2 (en) | 2018-04-18 | 2022-08-23 | 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 |
GB201809273D0 (en) | 2018-06-06 | 2018-07-25 | Vib Vzw | Novel mutant plant cinnamoyl-coa reductase proteins |
US11834686B2 (en) | 2018-08-23 | 2023-12-05 | Sangamo Therapeutics, Inc. | Engineered target specific base editors |
WO2020047282A1 (en) | 2018-08-29 | 2020-03-05 | University Of Copenhagen | Lysosomal enzymes modified by cell based glycoengineering |
JP2022500052A (en) | 2018-09-18 | 2022-01-04 | サンガモ セラピューティクス, インコーポレイテッド | Programmed cell death 1 (PD1) specific nuclease |
MX2021005594A (en) | 2018-11-13 | 2021-10-22 | Compass Therapeutics Llc | Multispecific binding constructs against checkpoint molecules and uses thereof. |
WO2020112870A1 (en) | 2018-11-28 | 2020-06-04 | Forty Seven, Inc. | Genetically modified hspcs resistant to ablation regime |
KR20200071198A (en) | 2018-12-10 | 2020-06-19 | 네오이뮨텍, 인코퍼레이티드 | Development of new adoptive T cell immunotherapy by modification of Nrf2 expression |
GB201820109D0 (en) | 2018-12-11 | 2019-01-23 | Vib Vzw | Plants with a lignin trait and udp-glycosyltransferase mutation |
MX2021007400A (en) | 2018-12-20 | 2021-07-15 | Regeneron Pharma | Nuclease-mediated repeat expansion. |
EP3898661A1 (en) | 2018-12-21 | 2021-10-27 | Precision BioSciences, Inc. | Genetic modification of the hydroxyacid oxidase 1 gene for treatment of primary hyperoxaluria |
EP3908568A1 (en) | 2019-01-11 | 2021-11-17 | Acuitas Therapeutics, Inc. | Lipids for lipid nanoparticle delivery of active agents |
US11857641B2 (en) | 2019-02-06 | 2024-01-02 | Sangamo Therapeutics, Inc. | Method for the treatment of mucopolysaccharidosis type I |
KR20200116044A (en) | 2019-03-26 | 2020-10-08 | 주식회사 툴젠 | HemophiliaB disease model rat |
EP3946384A1 (en) | 2019-04-02 | 2022-02-09 | Sangamo Therapeutics, Inc. | Methods for the treatment of beta-thalassemia |
AU2020256225A1 (en) | 2019-04-03 | 2021-09-02 | Regeneron Pharmaceuticals, Inc. | Methods and compositions for insertion of antibody coding sequences into a safe harbor locus |
EP4332115A2 (en) | 2019-04-03 | 2024-03-06 | Precision Biosciences, Inc. | Genetically-modified immune cells comprising a microrna-adapted shrna (shrnamir) |
KR102487901B1 (en) | 2019-04-04 | 2023-01-12 | 리제너론 파마슈티칼스 인코포레이티드 | Methods for seamless introduction of targeted modifications into targeting vectors |
WO2020206139A1 (en) | 2019-04-04 | 2020-10-08 | Regeneron Pharmaceuticals, Inc. | Non-human animals comprising a humanized coagulation factor 12 locus |
CA3136265A1 (en) | 2019-04-05 | 2020-10-08 | Precision Biosciences, Inc. | Methods of preparing populations of genetically-modified immune cells |
AU2020265749A1 (en) | 2019-05-01 | 2022-01-06 | Juno Therapeutics, Inc. | Cells expressing a chimeric receptor from a modified CD247 locus, related polynucleotides and methods |
SG11202111360YA (en) | 2019-05-01 | 2021-11-29 | Juno Therapeutics Inc | Cells expressing a recombinant receptor from a modified tgfbr2 locus, related polynucleotides and methods |
JP2022534867A (en) | 2019-06-04 | 2022-08-04 | リジェネロン・ファーマシューティカルズ・インコーポレイテッド | Non-human animals containing humanized TTR loci with beta slip mutations and methods of use |
SG11202111256XA (en) | 2019-06-07 | 2021-11-29 | Regeneron Pharma | Non-human animals comprising a humanized albumin locus |
KR20220024053A (en) | 2019-06-14 | 2022-03-03 | 리제너론 파마슈티칼스 인코포레이티드 | model of tauopathy |
EP4004216A1 (en) | 2019-07-25 | 2022-06-01 | Precision BioSciences, Inc. | Compositions and methods for sequential stacking of nucleic acid sequences into a genomic locus |
WO2021035054A1 (en) | 2019-08-20 | 2021-02-25 | Precision Biosciences, Inc. | Lymphodepletion dosing regimens for cellular immunotherapies |
WO2021035170A1 (en) | 2019-08-21 | 2021-02-25 | Precision Biosciences, Inc. | Compositions and methods for tcr reprogramming using fusion proteins |
EP3812472B1 (en) | 2019-10-21 | 2022-11-23 | Albert-Ludwigs-Universität Freiburg | A truly unbiased in vitro assay to profile off-target activity of one or more target-specific programmable nucleases in cells (abnoba-seq) |
US20220411479A1 (en) | 2019-10-30 | 2022-12-29 | Precision Biosciences, Inc. | Cd20 chimeric antigen receptors and methods of use for immunotherapy |
EP4051322A1 (en) | 2019-11-01 | 2022-09-07 | Sangamo Therapeutics, Inc. | Compositions and methods for genome engineering |
JP2022553573A (en) | 2019-11-08 | 2022-12-23 | リジェネロン・ファーマシューティカルズ・インコーポレイテッド | CRISPR and AAV strategies for X-linked juvenile retinal isolation therapy |
WO2021108363A1 (en) | 2019-11-25 | 2021-06-03 | Regeneron Pharmaceuticals, Inc. | Crispr/cas-mediated upregulation of humanized ttr allele |
CA3160096A1 (en) | 2019-12-06 | 2021-06-10 | Bruce J. Mccreedy Jr. | Methods for cancer immunotherapy |
WO2021158915A1 (en) | 2020-02-06 | 2021-08-12 | Precision Biosciences, Inc. | Recombinant adeno-associated virus compositions and methods for producing and using the same |
JP2023515671A (en) | 2020-03-04 | 2023-04-13 | リジェネロン・ファーマシューティカルズ・インコーポレイテッド | Methods and compositions for sensitizing tumor cells to immunotherapy |
CN115697044A (en) | 2020-03-31 | 2023-02-03 | 艾洛生物系统有限公司 | Regulation of endogenous mogroside pathway genes in watermelon and other cucurbitaceae |
JP2023525513A (en) | 2020-05-06 | 2023-06-16 | セレクティス ソシエテ アノニム | Methods for targeted insertion of exogenous sequences in the cellular genome |
WO2021224416A1 (en) | 2020-05-06 | 2021-11-11 | Cellectis S.A. | Methods to genetically modify cells for delivery of therapeutic proteins |
US20230183664A1 (en) | 2020-05-11 | 2023-06-15 | Precision Biosciences, Inc. | Self-limiting viral vectors encoding nucleases |
EP4150057A2 (en) | 2020-05-13 | 2023-03-22 | Juno Therapeutics, Inc. | Process for producing donor-batched cells expressing a recombinant receptor |
GB202007578D0 (en) | 2020-05-21 | 2020-07-08 | Univ Oxford Innovation Ltd | Hdr enhancers |
WO2021234389A1 (en) | 2020-05-21 | 2021-11-25 | Oxford Genetics Limited | Hdr enhancers |
WO2021234388A1 (en) | 2020-05-21 | 2021-11-25 | Oxford Genetics Limited | Hdr enhancers |
GB202007577D0 (en) | 2020-05-21 | 2020-07-08 | Oxford Genetics Ltd | Hdr enhancers |
KR20230042283A (en) | 2020-06-26 | 2023-03-28 | 주노 테라퓨틱스 게엠베하 | Engineered T cells conditionally expressing recombinant receptors, related polynucleotides and methods |
EP4192875A1 (en) | 2020-08-10 | 2023-06-14 | Precision BioSciences, Inc. | Antibodies and fragments specific for b-cell maturation antigen and uses thereof |
WO2022046760A2 (en) | 2020-08-25 | 2022-03-03 | Kite Pharma, Inc. | T cells with improved functionality |
US20230365995A1 (en) | 2020-10-07 | 2023-11-16 | Precision Biosciences, Inc. | Lipid nanoparticle compositions |
MX2023004761A (en) | 2020-10-23 | 2023-07-17 | Elo Life Systems Inc | Methods for producing vanilla plants with improved flavor and agronomic production. |
KR20230090367A (en) | 2020-11-04 | 2023-06-21 | 주노 쎄러퓨티크스 인코퍼레이티드 | Cells Expressing Chimeric Receptors from Modified Invariant CD3 Immunoglobulin Superfamily Chain Loci and Related Polynucleotides and Methods |
US20240000051A1 (en) | 2020-11-16 | 2024-01-04 | Pig Improvement Company Uk Limited | Influenza a-resistant animals having edited anp32 genes |
EP4284823A1 (en) | 2021-01-28 | 2023-12-06 | Precision BioSciences, Inc. | Modulation of tgf beta signaling in genetically-modified eukaryotic cells |
EP4313122A1 (en) | 2021-03-23 | 2024-02-07 | Iovance Biotherapeutics, Inc. | Cish gene editing of tumor infiltrating lymphocytes and uses of same in immunotherapy |
WO2022226316A1 (en) | 2021-04-22 | 2022-10-27 | Precision Biosciences, Inc. | Compositions and methods for generating male sterile plants |
WO2022235911A1 (en) | 2021-05-05 | 2022-11-10 | FUJIFILM Cellular Dynamics, Inc. | Methods and compositions for ipsc-derived microglia |
EP4337769A1 (en) | 2021-05-10 | 2024-03-20 | SQZ Biotechnologies Company | Methods for delivering genome editing molecules to the nucleus or cytosol of a cell and uses thereof |
EP4347796A1 (en) | 2021-05-26 | 2024-04-10 | Fujifilm Cellular Dynamics, Inc. | Methods to prevent rapid silencing of genes in pluripotent stem cells |
WO2022251644A1 (en) | 2021-05-28 | 2022-12-01 | Lyell Immunopharma, Inc. | Nr4a3-deficient immune cells and uses thereof |
WO2022256437A1 (en) | 2021-06-02 | 2022-12-08 | Lyell Immunopharma, Inc. | Nr4a3-deficient immune cells and uses thereof |
CN117716026A (en) | 2021-07-09 | 2024-03-15 | 株式会社图尔金 | Mesenchymal stem cells with oxidative stress resistance, preparation method and application thereof |
WO2023008933A1 (en) | 2021-07-29 | 2023-02-02 | 주식회사 툴젠 | Hemocompatible mesenchymal stem cells, preparation method therefor and use thereof |
US20230183644A1 (en) | 2021-09-10 | 2023-06-15 | FUJIFILM Cellular Dynamics, Inc. | Compositions of induced pluripotent stem cell-derived cells and methods of use thereof |
WO2023064872A1 (en) | 2021-10-14 | 2023-04-20 | Precision Biosciences, Inc. | Combinations of anti-bcma car t cells and gamma secretase inhibitors |
WO2023064924A1 (en) | 2021-10-14 | 2023-04-20 | Codiak Biosciences, Inc. | Modified producer cells for extracellular vesicle production |
CA3235185A1 (en) | 2021-10-19 | 2023-04-27 | Cassandra GORSUCH | Gene editing methods for treating alpha-1 antitrypsin (aat) deficiency |
WO2023070043A1 (en) | 2021-10-20 | 2023-04-27 | Yale University | Compositions and methods for targeted editing and evolution of repetitive genetic elements |
AU2022379633A1 (en) | 2021-10-27 | 2024-04-11 | Regeneron Pharmaceuticals, Inc. | Compositions and methods for expressing factor ix for hemophilia b therapy |
CA3235390A1 (en) | 2021-10-29 | 2023-05-04 | Deepika Rajesh | Dopaminergic neurons comprising mutations and methods of use thereof |
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 |
WO2023081923A1 (en) | 2021-11-08 | 2023-05-11 | Frequency Therapeutics, Inc. | Platelet-derived growth factor receptor (pdgfr) alpha inhibitors and uses thereof |
WO2023091910A1 (en) | 2021-11-16 | 2023-05-25 | Precision Biosciences, Inc. | Methods for cancer immunotherapy |
WO2023108047A1 (en) | 2021-12-08 | 2023-06-15 | Regeneron Pharmaceuticals, Inc. | Mutant myocilin disease model and uses thereof |
WO2023105244A1 (en) | 2021-12-10 | 2023-06-15 | Pig Improvement Company Uk Limited | Editing tmprss2/4 for disease resistance in livestock |
WO2023129974A1 (en) | 2021-12-29 | 2023-07-06 | Bristol-Myers Squibb Company | Generation of landing pad cell lines |
WO2023131616A1 (en) | 2022-01-05 | 2023-07-13 | Vib Vzw | Means and methods to increase abiotic stress tolerance in plants |
WO2023131637A1 (en) | 2022-01-06 | 2023-07-13 | Vib Vzw | Improved silage grasses |
WO2023144199A1 (en) | 2022-01-26 | 2023-08-03 | Vib Vzw | Plants having reduced levels of bitter taste metabolites |
US20230338477A1 (en) | 2022-02-02 | 2023-10-26 | Regeneron Pharmaceuticals, Inc. | Anti-tfr:gaa and anti-cd63:gaa insertion for treatment of pompe disease |
WO2023150798A1 (en) | 2022-02-07 | 2023-08-10 | Regeneron Pharmaceuticals, Inc. | Compositions and methods for defining optimal treatment timeframes in lysosomal disease |
WO2023220603A1 (en) | 2022-05-09 | 2023-11-16 | Regeneron Pharmaceuticals, Inc. | Vectors and methods for in vivo antibody production |
WO2023225665A1 (en) | 2022-05-19 | 2023-11-23 | Lyell Immunopharma, Inc. | Polynucleotides targeting nr4a3 and uses thereof |
US20240003871A1 (en) | 2022-06-29 | 2024-01-04 | FUJIFILM Cellular Dynamics, Inc. | Ipsc-derived astrocytes and methods of use thereof |
WO2024013514A2 (en) | 2022-07-15 | 2024-01-18 | Pig Improvement Company Uk Limited | Gene edited livestock animals having coronavirus resistance |
WO2024026474A1 (en) | 2022-07-29 | 2024-02-01 | Regeneron Pharmaceuticals, Inc. | Compositions and methods for transferrin receptor (tfr)-mediated delivery to the brain and muscle |
WO2024031053A1 (en) | 2022-08-05 | 2024-02-08 | Regeneron Pharmaceuticals, Inc. | Aggregation-resistant variants of tdp-43 |
WO2024064958A1 (en) | 2022-09-23 | 2024-03-28 | Lyell Immunopharma, Inc. | Methods for culturing nr4a-deficient cells |
WO2024064952A1 (en) | 2022-09-23 | 2024-03-28 | Lyell Immunopharma, Inc. | Methods for culturing nr4a-deficient cells overexpressing c-jun |
WO2024073606A1 (en) | 2022-09-28 | 2024-04-04 | Regeneron Pharmaceuticals, Inc. | Antibody resistant modified receptors to enhance cell-based therapies |
WO2024077174A1 (en) | 2022-10-05 | 2024-04-11 | Lyell Immunopharma, Inc. | Methods for culturing nr4a-deficient cells |
Family Cites Families (72)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4942227A (en) * | 1982-01-11 | 1990-07-17 | California Institute Of Technology | Bifunctional molecules having a DNA intercalator or DNA groove binder linked to ethylene diamine tetraacetic acid, their preparation and use to cleave DNA |
US4665184A (en) * | 1983-10-12 | 1987-05-12 | California Institute Of Technology | Bifunctional molecules having a DNA intercalator or DNA groove binder linked to ethylene diamine tetraacetic acid |
FR2598932B1 (en) * | 1986-05-23 | 1988-09-02 | Salomon Sa | DISSYMMETRIC PROFILE SKIING |
US5422251A (en) | 1986-11-26 | 1995-06-06 | Princeton University | Triple-stranded nucleic acids |
US5789155A (en) * | 1987-10-30 | 1998-08-04 | California Institute Of Technology | Process for identifying nucleic acids and triple helices formed thereby |
CA2001815C (en) | 1988-10-31 | 2002-09-03 | Wen-Hwa Lee | Products and methods for controlling the suppression of the neoplastic phenotype |
US5176996A (en) | 1988-12-20 | 1993-01-05 | Baylor College Of Medicine | Method for making synthetic oligonucleotides which bind specifically to target sites on duplex DNA molecules, by forming a colinear triplex, the synthetic oligonucleotides and methods of use |
FR2646438B1 (en) | 1989-03-20 | 2007-11-02 | Pasteur Institut | A METHOD FOR SPECIFIC REPLACEMENT OF A COPY OF A GENE PRESENT IN THE RECEIVER GENOME BY INTEGRATION OF A GENE DIFFERENT FROM THAT OR INTEGRATION |
US5574205A (en) | 1989-07-25 | 1996-11-12 | Cell Genesys | Homologous recombination for universal donor cells and chimeric mammalian hosts |
DE69034263D1 (en) | 1989-07-25 | 2009-04-02 | Cell Genesys Inc | Homologous recombination for universal donor cells and mammalian chimeric cells |
DK0747485T3 (en) | 1989-11-06 | 1999-08-16 | Cell Genesys Inc | Preparation of proteins using homologous recombination |
US5955341A (en) * | 1991-04-10 | 1999-09-21 | The Scripps Research Institute | Heterodimeric receptor libraries using phagemids |
US5641670A (en) | 1991-11-05 | 1997-06-24 | Transkaryotic Therapies, Inc. | Protein production and protein delivery |
US5436150A (en) | 1992-04-03 | 1995-07-25 | The Johns Hopkins University | Functional domains in flavobacterium okeanokoities (foki) restriction endonuclease |
US5356802A (en) * | 1992-04-03 | 1994-10-18 | The Johns Hopkins University | Functional domains in flavobacterium okeanokoites (FokI) restriction endonuclease |
US5487994A (en) * | 1992-04-03 | 1996-01-30 | The Johns Hopkins University | Insertion and deletion mutants of FokI restriction endonuclease |
US5916794A (en) * | 1992-04-03 | 1999-06-29 | Johns Hopkins University | Methods for inactivating target DNA and for detecting conformational change in a nucleic acid |
US5792640A (en) * | 1992-04-03 | 1998-08-11 | The Johns Hopkins University | General method to clone hybrid restriction endonucleases using lig gene |
ES2227511T3 (en) | 1992-04-15 | 2005-04-01 | Sembiosys Genetics Inc. | PROTEINS WITH OIL BODY AS VECTORS OF GREAT VALUE PEPTIDES IN VEGETABLES. |
US5792632A (en) * | 1992-05-05 | 1998-08-11 | Institut Pasteur | Nucleotide sequence encoding the enzyme I-SceI and the uses thereof |
EP0652889A4 (en) | 1992-06-15 | 1997-05-07 | Gene Pharming Europ Bv | Production of recombinant polypeptides by bovine species and transgenic methods. |
US5496720A (en) * | 1993-02-10 | 1996-03-05 | Susko-Parrish; Joan L. | Parthenogenic oocyte activation |
US6331658B1 (en) * | 1993-04-20 | 2001-12-18 | Integris Baptist Medical Center, Inc. | Genetically engineered mammals for use as organ donors |
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 |
AU698152B2 (en) * | 1994-08-20 | 1998-10-22 | Gendaq Limited | Improvements in or relating to binding proteins for recognition of DNA |
US6326166B1 (en) * | 1995-12-29 | 2001-12-04 | Massachusetts Institute Of Technology | Chimeric DNA-binding proteins |
US5789538A (en) * | 1995-02-03 | 1998-08-04 | Massachusetts Institute Of Technology | Zinc finger proteins with high affinity new DNA binding specificities |
WO1996040882A1 (en) | 1995-06-07 | 1996-12-19 | The Ohio State University | Artificial restriction endonuclease |
GB9517780D0 (en) * | 1995-08-31 | 1995-11-01 | Roslin Inst Edinburgh | Biological manipulation |
US6265196B1 (en) * | 1996-05-07 | 2001-07-24 | Johns Hopkins University | Methods for inactivating target DNA and for detecting conformational change in a nucleic acid |
US5928914A (en) | 1996-06-14 | 1999-07-27 | Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University | Methods and compositions for transforming cells |
US5945577A (en) * | 1997-01-10 | 1999-08-31 | University Of Massachusetts As Represented By Its Amherst Campus | Cloning using donor nuclei from proliferating somatic cells |
GB9710807D0 (en) | 1997-05-23 | 1997-07-23 | Medical Res Council | Nucleic acid binding proteins |
GB9710809D0 (en) | 1997-05-23 | 1997-07-23 | Medical Res Council | Nucleic acid binding proteins |
ATE312935T1 (en) | 1997-06-03 | 2005-12-15 | REGULATORY SEQUENCES FOR IN VIVO EXPRESSION OF A HETEROLOGUE DNA SEQUENCE IN ENDOTHELIAL CELLS AND THEIR USES. | |
ATE354654T1 (en) | 1997-07-23 | 2007-03-15 | Roche Diagnostics Gmbh | IDENTIFICATION OF HUMAN CELL LINES FOR THE PRODUCTION OF HUMAN PROTEINS THROUGH ENDOGENE GENE ACTIVATION |
DE69841723D1 (en) | 1997-09-26 | 2010-07-29 | Abt Holding Co | EXPRESSION OF ENDOGENIC GENES THROUGH HOMOLOGOUS RECOMBINATION OF A VECTOR CONSTRUCTURE WITH CELLULAR DNA |
AU757930B2 (en) | 1997-12-01 | 2003-03-13 | Roche Diagnostics Gmbh | Optimization of cells for endogenous gene activation |
FR2772787B1 (en) | 1997-12-24 | 2001-12-07 | Rhone Poulenc Agrochimie | H3C4 PROMOTER OF BUT ASSOCIATED WITH THE FIRST INTRON OF RICE ACTINE, CHIMERIC GENE INCLUDING IT AND TRANSFORMED PLANT |
AU2053999A (en) | 1997-12-15 | 1999-07-05 | Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. | Regulatory sequences involved in pancreas-specific gene expression |
JP4309051B2 (en) * | 1998-03-02 | 2009-08-05 | マサチューセッツ インスティテュート オブ テクノロジー | Polyzinc finger protein with improved linker |
CA2339420A1 (en) | 1998-08-12 | 2000-02-24 | David Zarling | Domain specific gene evolution |
US6140081A (en) * | 1998-10-16 | 2000-10-31 | The Scripps Research Institute | Zinc finger binding domains for GNN |
AU2023700A (en) | 1998-11-10 | 2000-05-29 | Maxygen, Inc. | Modified ribulose 1,5-bisphosphate carboxylase/oxygenase |
US6599692B1 (en) | 1999-09-14 | 2003-07-29 | Sangamo Bioscience, Inc. | Functional genomics using zinc finger proteins |
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 |
EP1147209A2 (en) * | 1999-02-03 | 2001-10-24 | The Children's Medical Center Corporation | Gene repair involving the induction of double-stranded dna cleavage at a chromosomal target site |
WO2000046385A1 (en) | 1999-02-03 | 2000-08-10 | The Children's Medical Center Corporation | Gene repair involving in vivo excision of targeting dna |
WO2000063365A1 (en) | 1999-04-21 | 2000-10-26 | Pangene Corporation | Locked nucleic acid hybrids and methods of use |
CA2373690A1 (en) | 1999-07-14 | 2001-01-25 | Clontech Laboratories, Inc. | Recombinase-based methods for producing expression vectors and compositions for use in practicing the same |
IL150069A0 (en) | 1999-12-06 | 2002-12-01 | Sangamo Biosciences Inc | Methods of using randomized libraries of zinc finger proteins for the identification of gene function |
AU1647501A (en) | 1999-12-10 | 2001-06-18 | Cytos Biotechnology Ag | Replicon based activation of endogenous genes |
AU2001236961A1 (en) * | 2000-02-11 | 2001-08-20 | The Salk Institute For Biological Studies | Method of regulating transcription in a cell by altering remodeling of cromatin |
WO2001066717A2 (en) | 2000-03-03 | 2001-09-13 | The University Of Utah | Gene targeting method |
US6492117B1 (en) | 2000-07-12 | 2002-12-10 | Gendaq Limited | Zinc finger polypeptides capable of binding DNA quadruplexes |
ES2394877T3 (en) | 2000-08-14 | 2013-02-06 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Improved homologous recombination mediated by lambda recombination proteins |
US7091026B2 (en) * | 2001-02-16 | 2006-08-15 | University Of Iowa Research Foundation | Artificial endonuclease |
US7067617B2 (en) * | 2001-02-21 | 2006-06-27 | The Scripps Research Institute | Zinc finger binding domains for nucleotide sequence ANN |
US20040224385A1 (en) * | 2001-08-20 | 2004-11-11 | Barbas Carlos F | Zinc finger binding domains for cnn |
EP2266396A3 (en) | 2001-09-24 | 2011-06-15 | Sangamo BioSciences, Inc. | Modulation of stem cells using zinc finger proteins |
US20060242726A1 (en) | 2001-12-13 | 2006-10-26 | Purdue Research Foundation | Methods and vectors for making knockout animals |
AU2003251286B2 (en) | 2002-01-23 | 2007-08-16 | The University Of Utah Research Foundation | Targeted chromosomal mutagenesis using zinc finger nucleases |
WO2003080809A2 (en) | 2002-03-21 | 2003-10-02 | Sangamo Biosciences, Inc. | Methods and compositions for using zinc finger endonucleases to enhance homologous recombination |
US9447434B2 (en) | 2002-09-05 | 2016-09-20 | California Institute Of Technology | Use of chimeric nucleases to stimulate gene targeting |
EP2559759A1 (en) | 2003-01-28 | 2013-02-20 | Cellectis | Custom-made meganuclease and use thereof |
US8409861B2 (en) * | 2003-08-08 | 2013-04-02 | Sangamo Biosciences, Inc. | Targeted deletion of cellular DNA sequences |
US20120196370A1 (en) * | 2010-12-03 | 2012-08-02 | Fyodor Urnov | Methods and compositions for targeted genomic deletion |
US20080131962A1 (en) * | 2006-05-25 | 2008-06-05 | Sangamo Biosciences, Inc. | Engineered cleavage half-domains |
DK3693384T3 (en) * | 2014-03-11 | 2024-04-15 | Cellectis | Method for generating T cells with compatibility for allogeneic transplantation |
US10975406B2 (en) * | 2014-07-18 | 2021-04-13 | Massachusetts Institute Of Technology | Directed endonucleases for repeatable nucleic acid cleavage |
-
2005
- 2005-12-15 US US11/304,981 patent/US8409861B2/en active Active
-
2013
- 2013-03-04 US US13/784,634 patent/US9695442B2/en active Active
-
2017
- 2017-05-31 US US15/610,262 patent/US10669557B2/en active Active
-
2020
- 2020-04-27 US US16/859,049 patent/US20200291424A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
US9695442B2 (en) | 2017-07-04 |
US20060188987A1 (en) | 2006-08-24 |
US8409861B2 (en) | 2013-04-02 |
US20170298385A1 (en) | 2017-10-19 |
US10669557B2 (en) | 2020-06-02 |
US20140065667A1 (en) | 2014-03-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20200291424A1 (en) | Targeted deletion of cellular dna sequences | |
US10675302B2 (en) | Methods and compositions for targeted cleavage and recombination | |
US7972854B2 (en) | Methods and compositions for targeted cleavage and recombination | |
US9260726B2 (en) | Targeted integration and expression on exogenous nucleic acid sequences | |
EP1651660B1 (en) | Methods and compositions for targeted cleavage and recombination | |
US11311574B2 (en) | Methods and compositions for targeted cleavage and recombination | |
AU2007201649B2 (en) | Methods and Compositions for Targeted Cleavage and Recombination |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |