WO2019161805A1 - Hr knockout non-human animal - Google Patents
Hr knockout non-human animal Download PDFInfo
- Publication number
- WO2019161805A1 WO2019161805A1 PCT/CN2019/076192 CN2019076192W WO2019161805A1 WO 2019161805 A1 WO2019161805 A1 WO 2019161805A1 CN 2019076192 W CN2019076192 W CN 2019076192W WO 2019161805 A1 WO2019161805 A1 WO 2019161805A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- animal
- exon
- intron
- gene
- cells
- Prior art date
Links
- 101150106864 HR gene Proteins 0.000 claims abstract description 130
- 238000000034 method Methods 0.000 claims abstract description 115
- 241001465754 Metazoa Species 0.000 claims description 242
- 210000004027 cell Anatomy 0.000 claims description 139
- 108090000623 proteins and genes Proteins 0.000 claims description 134
- 239000002773 nucleotide Substances 0.000 claims description 133
- 125000003729 nucleotide group Chemical group 0.000 claims description 133
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 118
- 206010028980 Neoplasm Diseases 0.000 claims description 60
- 238000012217 deletion Methods 0.000 claims description 60
- 230000037430 deletion Effects 0.000 claims description 60
- 108700024394 Exon Proteins 0.000 claims description 44
- 239000003795 chemical substances by application Substances 0.000 claims description 39
- 210000000265 leukocyte Anatomy 0.000 claims description 32
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 31
- 108091092195 Intron Proteins 0.000 claims description 27
- 210000001671 embryonic stem cell Anatomy 0.000 claims description 27
- 101710163270 Nuclease Proteins 0.000 claims description 25
- 238000011813 knockout mouse model Methods 0.000 claims description 24
- 210000004881 tumor cell Anatomy 0.000 claims description 23
- 108091033409 CRISPR Proteins 0.000 claims description 20
- 238000011144 upstream manufacturing Methods 0.000 claims description 18
- 102000004169 proteins and genes Human genes 0.000 claims description 17
- 108020005004 Guide RNA Proteins 0.000 claims description 16
- 108010073062 Transcription Activator-Like Effectors Proteins 0.000 claims description 16
- 210000003719 b-lymphocyte Anatomy 0.000 claims description 16
- 210000002459 blastocyst Anatomy 0.000 claims description 16
- 238000010362 genome editing Methods 0.000 claims description 16
- 230000004568 DNA-binding Effects 0.000 claims description 15
- 101710185494 Zinc finger protein Proteins 0.000 claims description 15
- 102100023597 Zinc finger protein 816 Human genes 0.000 claims description 15
- 201000011510 cancer Diseases 0.000 claims description 15
- 230000000694 effects Effects 0.000 claims description 15
- 210000004209 hair Anatomy 0.000 claims description 15
- 210000003958 hematopoietic stem cell Anatomy 0.000 claims description 14
- 210000000822 natural killer cell Anatomy 0.000 claims description 14
- 241000283984 Rodentia Species 0.000 claims description 13
- 102000015736 beta 2-Microglobulin Human genes 0.000 claims description 13
- 108010081355 beta 2-Microglobulin Proteins 0.000 claims description 13
- 102100040678 Programmed cell death protein 1 Human genes 0.000 claims description 12
- 101710089372 Programmed cell death protein 1 Proteins 0.000 claims description 12
- 210000000987 immune system Anatomy 0.000 claims description 12
- 238000011580 nude mouse model Methods 0.000 claims description 12
- 102000003839 Human Proteins Human genes 0.000 claims description 11
- 210000004976 peripheral blood cell Anatomy 0.000 claims description 11
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 claims description 10
- 108020001507 fusion proteins Proteins 0.000 claims description 10
- 102000037865 fusion proteins Human genes 0.000 claims description 10
- 101000851370 Homo sapiens Tumor necrosis factor receptor superfamily member 9 Proteins 0.000 claims description 9
- 102100036856 Tumor necrosis factor receptor superfamily member 9 Human genes 0.000 claims description 9
- 208000002154 non-small cell lung carcinoma Diseases 0.000 claims description 9
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 claims description 8
- 108010074708 B7-H1 Antigen Proteins 0.000 claims description 8
- 101001068052 Homo sapiens Lysine-specific demethylase hairless Proteins 0.000 claims description 8
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 claims description 8
- 206010041067 Small cell lung cancer Diseases 0.000 claims description 8
- 210000002308 embryonic cell Anatomy 0.000 claims description 8
- 230000008175 fetal development Effects 0.000 claims description 8
- 208000000587 small cell lung carcinoma Diseases 0.000 claims description 8
- 101150076800 B2M gene Proteins 0.000 claims description 7
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 claims description 7
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 claims description 7
- 108010038453 Interleukin-2 Receptors Proteins 0.000 claims description 7
- 102000010789 Interleukin-2 Receptors Human genes 0.000 claims description 7
- 210000005260 human cell Anatomy 0.000 claims description 7
- 230000002401 inhibitory effect Effects 0.000 claims description 7
- 210000004698 lymphocyte Anatomy 0.000 claims description 7
- 210000001616 monocyte Anatomy 0.000 claims description 7
- 230000001131 transforming effect Effects 0.000 claims description 7
- 108090000144 Human Proteins Proteins 0.000 claims description 6
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 6
- 206010060862 Prostate cancer Diseases 0.000 claims description 6
- 201000001441 melanoma Diseases 0.000 claims description 6
- 210000000440 neutrophil Anatomy 0.000 claims description 6
- 230000002829 reductive effect Effects 0.000 claims description 6
- 208000026310 Breast neoplasm Diseases 0.000 claims description 5
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 claims description 5
- 108090000172 Interleukin-15 Proteins 0.000 claims description 5
- 102000003812 Interleukin-15 Human genes 0.000 claims description 5
- 102100034466 Lysine-specific demethylase hairless Human genes 0.000 claims description 5
- 229930012538 Paclitaxel Natural products 0.000 claims description 5
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 5
- 229960004562 carboplatin Drugs 0.000 claims description 5
- 190000008236 carboplatin Chemical compound 0.000 claims description 5
- 229960004316 cisplatin Drugs 0.000 claims description 5
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 claims description 5
- 229960002949 fluorouracil Drugs 0.000 claims description 5
- 230000014509 gene expression Effects 0.000 claims description 5
- 208000024908 graft versus host disease Diseases 0.000 claims description 5
- 229960001592 paclitaxel Drugs 0.000 claims description 5
- 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 claims description 5
- 206010005003 Bladder cancer Diseases 0.000 claims description 4
- 206010006187 Breast cancer Diseases 0.000 claims description 4
- GAGWJHPBXLXJQN-UORFTKCHSA-N Capecitabine Chemical compound C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](C)O1 GAGWJHPBXLXJQN-UORFTKCHSA-N 0.000 claims description 4
- GAGWJHPBXLXJQN-UHFFFAOYSA-N Capecitabine Natural products C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1C1C(O)C(O)C(C)O1 GAGWJHPBXLXJQN-UHFFFAOYSA-N 0.000 claims description 4
- 102100028123 Macrophage colony-stimulating factor 1 Human genes 0.000 claims description 4
- ZDZOTLJHXYCWBA-VCVYQWHSSA-N N-debenzoyl-N-(tert-butoxycarbonyl)-10-deacetyltaxol Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=4C=CC=CC=4)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 ZDZOTLJHXYCWBA-VCVYQWHSSA-N 0.000 claims description 4
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 4
- 210000001772 blood platelet Anatomy 0.000 claims description 4
- 229960004117 capecitabine Drugs 0.000 claims description 4
- 229960003668 docetaxel Drugs 0.000 claims description 4
- 206010017758 gastric cancer Diseases 0.000 claims description 4
- 208000010749 gastric carcinoma Diseases 0.000 claims description 4
- 229960005277 gemcitabine Drugs 0.000 claims description 4
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 claims description 4
- 230000001678 irradiating effect Effects 0.000 claims description 4
- 201000005296 lung carcinoma Diseases 0.000 claims description 4
- 210000002540 macrophage Anatomy 0.000 claims description 4
- 229960001756 oxaliplatin Drugs 0.000 claims description 4
- DWAFYCQODLXJNR-BNTLRKBRSA-L oxaliplatin Chemical compound O1C(=O)C(=O)O[Pt]11N[C@@H]2CCCC[C@H]2N1 DWAFYCQODLXJNR-BNTLRKBRSA-L 0.000 claims description 4
- 229960005079 pemetrexed Drugs 0.000 claims description 4
- QOFFJEBXNKRSPX-ZDUSSCGKSA-N pemetrexed Chemical compound C1=N[C]2NC(N)=NC(=O)C2=C1CCC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 QOFFJEBXNKRSPX-ZDUSSCGKSA-N 0.000 claims description 4
- 201000000498 stomach carcinoma Diseases 0.000 claims description 4
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 4
- 101100383038 Homo sapiens CD19 gene Proteins 0.000 claims description 3
- 101000746373 Homo sapiens Granulocyte-macrophage colony-stimulating factor Proteins 0.000 claims description 3
- 108090001005 Interleukin-6 Proteins 0.000 claims description 3
- 241000124008 Mammalia Species 0.000 claims description 3
- FOCAHLGSDWHSAH-UHFFFAOYSA-N difluoromethanethione Chemical compound FC(F)=S FOCAHLGSDWHSAH-UHFFFAOYSA-N 0.000 claims description 3
- 210000003714 granulocyte Anatomy 0.000 claims description 3
- 210000003630 histaminocyte Anatomy 0.000 claims description 3
- 210000000066 myeloid cell Anatomy 0.000 claims description 3
- 239000002243 precursor Substances 0.000 claims description 3
- 238000011725 BALB/c mouse Methods 0.000 claims description 2
- 241000282693 Cercopithecidae Species 0.000 claims description 2
- 101001033279 Homo sapiens Interleukin-3 Proteins 0.000 claims description 2
- 102100039064 Interleukin-3 Human genes 0.000 claims description 2
- 201000005202 lung cancer Diseases 0.000 claims description 2
- 208000020816 lung neoplasm Diseases 0.000 claims description 2
- 102000008096 B7-H1 Antigen Human genes 0.000 claims 1
- 108091011896 CSF1 Proteins 0.000 claims 1
- 102000004889 Interleukin-6 Human genes 0.000 claims 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 109
- 241000699670 Mus sp. Species 0.000 description 73
- 230000006801 homologous recombination Effects 0.000 description 70
- 238000002744 homologous recombination Methods 0.000 description 70
- 235000013601 eggs Nutrition 0.000 description 30
- 230000035772 mutation Effects 0.000 description 19
- 125000003275 alpha amino acid group Chemical group 0.000 description 17
- 210000001519 tissue Anatomy 0.000 description 17
- 241000700159 Rattus Species 0.000 description 14
- 238000010171 animal model Methods 0.000 description 14
- 230000004048 modification Effects 0.000 description 12
- 238000012986 modification Methods 0.000 description 12
- 239000013598 vector Substances 0.000 description 12
- 238000012239 gene modification Methods 0.000 description 11
- 230000005017 genetic modification Effects 0.000 description 11
- 235000013617 genetically modified food Nutrition 0.000 description 11
- 108010002350 Interleukin-2 Proteins 0.000 description 10
- 102000000588 Interleukin-2 Human genes 0.000 description 10
- 239000003814 drug Substances 0.000 description 10
- 230000006870 function Effects 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- 101100284354 Mus musculus Hr gene Proteins 0.000 description 9
- 201000010099 disease Diseases 0.000 description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 9
- 210000004602 germ cell Anatomy 0.000 description 9
- 150000007523 nucleic acids Chemical group 0.000 description 9
- 201000009030 Carcinoma Diseases 0.000 description 8
- -1 TIM-3 Proteins 0.000 description 8
- 238000000338 in vitro Methods 0.000 description 8
- 108020004999 messenger RNA Proteins 0.000 description 8
- 230000008685 targeting Effects 0.000 description 8
- 101000868279 Homo sapiens Leukocyte surface antigen CD47 Proteins 0.000 description 7
- 102100032913 Leukocyte surface antigen CD47 Human genes 0.000 description 7
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 238000004113 cell culture Methods 0.000 description 7
- 238000011161 development Methods 0.000 description 7
- 230000018109 developmental process Effects 0.000 description 7
- 238000011819 knockout animal model Methods 0.000 description 7
- 210000004962 mammalian cell Anatomy 0.000 description 7
- 230000006780 non-homologous end joining Effects 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 102100029822 B- and T-lymphocyte attenuator Human genes 0.000 description 6
- 102100027207 CD27 antigen Human genes 0.000 description 6
- 229940045513 CTLA4 antagonist Drugs 0.000 description 6
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 6
- 102000050627 Glucocorticoid-Induced TNFR-Related Human genes 0.000 description 6
- 101000864344 Homo sapiens B- and T-lymphocyte attenuator Proteins 0.000 description 6
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 6
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 6
- 108091034117 Oligonucleotide Proteins 0.000 description 6
- 102100024834 T-cell immunoreceptor with Ig and ITIM domains Human genes 0.000 description 6
- 101710090983 T-cell immunoreceptor with Ig and ITIM domains Proteins 0.000 description 6
- 101710187882 Tumor necrosis factor receptor superfamily member 18 Proteins 0.000 description 6
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 description 6
- 230000002068 genetic effect Effects 0.000 description 6
- 210000001161 mammalian embryo Anatomy 0.000 description 6
- 102000005962 receptors Human genes 0.000 description 6
- 108020003175 receptors Proteins 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000013518 transcription Methods 0.000 description 6
- 230000035897 transcription Effects 0.000 description 6
- 210000004291 uterus Anatomy 0.000 description 6
- 101100193633 Danio rerio rag2 gene Proteins 0.000 description 5
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 5
- 102000017578 LAG3 Human genes 0.000 description 5
- 101150030213 Lag3 gene Proteins 0.000 description 5
- 241000282560 Macaca mulatta Species 0.000 description 5
- 101100193635 Mus musculus Rag2 gene Proteins 0.000 description 5
- 238000011579 SCID mouse model Methods 0.000 description 5
- 108091027544 Subgenomic mRNA Proteins 0.000 description 5
- 241000282898 Sus scrofa Species 0.000 description 5
- 208000012827 T-B+ severe combined immunodeficiency due to gamma chain deficiency Diseases 0.000 description 5
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 5
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 5
- 208000023940 X-Linked Combined Immunodeficiency disease Diseases 0.000 description 5
- 201000007146 X-linked severe combined immunodeficiency Diseases 0.000 description 5
- 125000000539 amino acid group Chemical group 0.000 description 5
- 230000003698 anagen phase Effects 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 210000000349 chromosome Anatomy 0.000 description 5
- 230000001086 cytosolic effect Effects 0.000 description 5
- 230000002950 deficient Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000003780 insertion Methods 0.000 description 5
- 230000037431 insertion Effects 0.000 description 5
- 230000003993 interaction Effects 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000000520 microinjection Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 210000000287 oocyte Anatomy 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 210000003491 skin Anatomy 0.000 description 5
- 229940124597 therapeutic agent Drugs 0.000 description 5
- 238000011740 C57BL/6 mouse Methods 0.000 description 4
- 102000004127 Cytokines Human genes 0.000 description 4
- 108090000695 Cytokines Proteins 0.000 description 4
- 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 4
- 208000009329 Graft vs Host Disease Diseases 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 4
- 108010076504 Protein Sorting Signals Proteins 0.000 description 4
- 108700022368 Whn Proteins 0.000 description 4
- 230000005540 biological transmission Effects 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000003209 gene knockout Methods 0.000 description 4
- 230000007614 genetic variation Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 102000053615 human HR Human genes 0.000 description 4
- 210000002865 immune cell Anatomy 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 210000004940 nucleus Anatomy 0.000 description 4
- 210000003101 oviduct Anatomy 0.000 description 4
- 210000001082 somatic cell Anatomy 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 230000004614 tumor growth Effects 0.000 description 4
- 102100023371 Forkhead box protein N1 Human genes 0.000 description 3
- 101001055227 Homo sapiens Cytokine receptor common subunit gamma Proteins 0.000 description 3
- 101000907576 Homo sapiens Forkhead box protein N1 Proteins 0.000 description 3
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 3
- 241000699729 Muridae Species 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 108091008731 RAR-related orphan receptors α Proteins 0.000 description 3
- 238000010240 RT-PCR analysis Methods 0.000 description 3
- 241000700157 Rattus norvegicus Species 0.000 description 3
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 210000004748 cultured cell Anatomy 0.000 description 3
- 210000002950 fibroblast Anatomy 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000003779 hair growth Effects 0.000 description 3
- 238000011577 humanized mouse model Methods 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 3
- 229960000318 kanamycin Drugs 0.000 description 3
- 229930182823 kanamycin A Natural products 0.000 description 3
- 238000009630 liquid culture Methods 0.000 description 3
- 230000036210 malignancy Effects 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 230000011664 signaling Effects 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 108020005345 3' Untranslated Regions Proteins 0.000 description 2
- 108020003589 5' Untranslated Regions Proteins 0.000 description 2
- 201000004384 Alopecia Diseases 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 108010071942 Colony-Stimulating Factors Proteins 0.000 description 2
- 241000699800 Cricetinae Species 0.000 description 2
- 102100026234 Cytokine receptor common subunit gamma Human genes 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 241000699694 Gerbillinae Species 0.000 description 2
- 102100039619 Granulocyte colony-stimulating factor Human genes 0.000 description 2
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000746367 Homo sapiens Granulocyte colony-stimulating factor Proteins 0.000 description 2
- 101000679851 Homo sapiens Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 2
- 101000934996 Homo sapiens Tyrosine-protein kinase JAK3 Proteins 0.000 description 2
- 101000863873 Homo sapiens Tyrosine-protein phosphatase non-receptor type substrate 1 Proteins 0.000 description 2
- 102000026633 IL6 Human genes 0.000 description 2
- 206010061598 Immunodeficiency Diseases 0.000 description 2
- 208000029462 Immunodeficiency disease Diseases 0.000 description 2
- 108090000978 Interleukin-4 Proteins 0.000 description 2
- 102000004388 Interleukin-4 Human genes 0.000 description 2
- 108010002586 Interleukin-7 Proteins 0.000 description 2
- 102100021592 Interleukin-7 Human genes 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 108010013709 Leukocyte Common Antigens Proteins 0.000 description 2
- 241000398750 Muroidea Species 0.000 description 2
- 208000006265 Renal cell carcinoma Diseases 0.000 description 2
- 241000121210 Sigmodontinae Species 0.000 description 2
- 208000033040 Somatoform disorder pregnancy Diseases 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102100025387 Tyrosine-protein kinase JAK3 Human genes 0.000 description 2
- 102100029948 Tyrosine-protein phosphatase non-receptor type substrate 1 Human genes 0.000 description 2
- 206010052428 Wound Diseases 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 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 2
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 208000009956 adenocarcinoma Diseases 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 208000010203 atrichia with papular lesions Diseases 0.000 description 2
- 210000003651 basophil Anatomy 0.000 description 2
- 230000029918 bioluminescence Effects 0.000 description 2
- 238000005415 bioluminescence Methods 0.000 description 2
- 210000000481 breast Anatomy 0.000 description 2
- 230000003778 catagen phase Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000002648 combination therapy Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000003979 eosinophil Anatomy 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 238000000799 fluorescence microscopy Methods 0.000 description 2
- 230000000762 glandular Effects 0.000 description 2
- 230000003661 hair follicle regeneration Effects 0.000 description 2
- 210000000777 hematopoietic system Anatomy 0.000 description 2
- 102000049902 human IL2RG Human genes 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000007813 immunodeficiency Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 108010074108 interleukin-21 Proteins 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000012453 sprague-dawley rat model Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000003797 telogen phase Effects 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 102000004217 thyroid hormone receptors Human genes 0.000 description 2
- 108090000721 thyroid hormone receptors Proteins 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 102000009310 vitamin D receptors Human genes 0.000 description 2
- 108050000156 vitamin D receptors Proteins 0.000 description 2
- 238000011714 129 mouse Methods 0.000 description 1
- 102100023340 3-ketodihydrosphingosine reductase Human genes 0.000 description 1
- 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 1
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 1
- 241000699725 Acomys Species 0.000 description 1
- 208000008190 Agammaglobulinemia Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 1
- 235000017491 Bambusa tulda Nutrition 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 1
- 241000398949 Calomyscidae Species 0.000 description 1
- 241000700193 Calomyscus Species 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000017897 Carcinoma of esophagus Diseases 0.000 description 1
- 201000000274 Carcinosarcoma Diseases 0.000 description 1
- 241000282994 Cervidae Species 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 241000398985 Cricetidae Species 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- 101710189311 Cytokine receptor common subunit gamma Proteins 0.000 description 1
- 238000011765 DBA/2 mouse Methods 0.000 description 1
- 108010092160 Dactinomycin Proteins 0.000 description 1
- 241001095404 Dipodoidea Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 241001416537 Gliridae Species 0.000 description 1
- 102000003693 Hedgehog Proteins Human genes 0.000 description 1
- 108090000031 Hedgehog Proteins Proteins 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101001050680 Homo sapiens 3-ketodihydrosphingosine reductase Proteins 0.000 description 1
- 101100067102 Homo sapiens FOXN1 gene Proteins 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 1
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 1
- 101000578932 Homo sapiens Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2 Proteins 0.000 description 1
- 101000615488 Homo sapiens Methyl-CpG-binding domain protein 2 Proteins 0.000 description 1
- 206010020983 Hypogammaglobulinaemia Diseases 0.000 description 1
- 102000039990 IL-2 family Human genes 0.000 description 1
- 108091069192 IL-2 family Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 108010066719 Interleukin Receptor Common gamma Subunit Proteins 0.000 description 1
- 102000018682 Interleukin Receptor Common gamma Subunit Human genes 0.000 description 1
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 1
- 102100030703 Interleukin-22 Human genes 0.000 description 1
- 108010038498 Interleukin-7 Receptors Proteins 0.000 description 1
- 102000010782 Interleukin-7 Receptors Human genes 0.000 description 1
- 108010002335 Interleukin-9 Proteins 0.000 description 1
- 102000000585 Interleukin-9 Human genes 0.000 description 1
- 241000581650 Ivesia Species 0.000 description 1
- 108050006228 JmjC domains Proteins 0.000 description 1
- 102000016624 JmjC domains Human genes 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 241001046461 Lophiomys imhausi Species 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 101710095950 Lysine-specific demethylase hairless Proteins 0.000 description 1
- 102100028328 Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2 Human genes 0.000 description 1
- 102100021299 Methyl-CpG-binding domain protein 2 Human genes 0.000 description 1
- 229930192392 Mitomycin Natural products 0.000 description 1
- 241000699669 Mus saxicola Species 0.000 description 1
- 241000282341 Mustela putorius furo Species 0.000 description 1
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 1
- 241000398990 Nesomyidae Species 0.000 description 1
- 108020004485 Nonsense Codon Proteins 0.000 description 1
- 108010077850 Nuclear Localization Signals Proteins 0.000 description 1
- 102000008297 Nuclear Matrix-Associated Proteins Human genes 0.000 description 1
- 108010035916 Nuclear Matrix-Associated Proteins Proteins 0.000 description 1
- 108020005497 Nuclear hormone receptor Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 244000082204 Phyllostachys viridis Species 0.000 description 1
- 235000015334 Phyllostachys viridis Nutrition 0.000 description 1
- 241001338313 Platacanthomyidae Species 0.000 description 1
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 102000016971 Proto-Oncogene Proteins c-kit Human genes 0.000 description 1
- 108010014608 Proto-Oncogene Proteins c-kit Proteins 0.000 description 1
- 108091008680 RAR-related orphan receptors Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 241000398956 Spalacidae Species 0.000 description 1
- 102000043168 TGF-beta family Human genes 0.000 description 1
- 108091085018 TGF-beta family Proteins 0.000 description 1
- 208000024313 Testicular Neoplasms Diseases 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 102000013814 Wnt Human genes 0.000 description 1
- 108050003627 Wnt Proteins 0.000 description 1
- 210000001766 X chromosome Anatomy 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 229940009456 adriamycin Drugs 0.000 description 1
- 231100000360 alopecia Toxicity 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000011425 bamboo Substances 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000027455 binding Effects 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
- 210000004556 brain Anatomy 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 230000005907 cancer growth Effects 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 230000021617 central nervous system development Effects 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 210000003679 cervix uteri Anatomy 0.000 description 1
- 229960004630 chlorambucil Drugs 0.000 description 1
- JCKYGMPEJWAADB-UHFFFAOYSA-N chlorambucil Chemical compound OC(=O)CCCC1=CC=C(N(CCCl)CCCl)C=C1 JCKYGMPEJWAADB-UHFFFAOYSA-N 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 230000009194 climbing Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000000749 co-immunoprecipitation Methods 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 229960000640 dactinomycin Drugs 0.000 description 1
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin 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(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 1
- 229960000975 daunorubicin Drugs 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 1
- 208000018554 digestive system carcinoma Diseases 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 208000037765 diseases and disorders Diseases 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 210000000750 endocrine system Anatomy 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 230000001973 epigenetic effect Effects 0.000 description 1
- YJGVMLPVUAXIQN-UHFFFAOYSA-N epipodophyllotoxin Natural products COC1=C(OC)C(OC)=CC(C2C3=CC=4OCOC=4C=C3C(O)C3C2C(OC3)=O)=C1 YJGVMLPVUAXIQN-UHFFFAOYSA-N 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
- 230000037433 frameshift Effects 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 230000031774 hair cycle Effects 0.000 description 1
- 210000003780 hair follicle Anatomy 0.000 description 1
- 230000023643 hair follicle morphogenesis Effects 0.000 description 1
- 208000024963 hair loss Diseases 0.000 description 1
- 230000003676 hair loss Effects 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 230000003118 histopathologic effect Effects 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 229960001101 ifosfamide Drugs 0.000 description 1
- HOMGKSMUEGBAAB-UHFFFAOYSA-N ifosfamide Chemical compound ClCCNP1(=O)OCCCN1CCCl HOMGKSMUEGBAAB-UHFFFAOYSA-N 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 238000011563 immunodeficient animal model Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 241001515942 marmosets Species 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229960004961 mechlorethamine Drugs 0.000 description 1
- HAWPXGHAZFHHAD-UHFFFAOYSA-N mechlorethamine Chemical compound ClCCN(C)CCCl HAWPXGHAZFHHAD-UHFFFAOYSA-N 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229960001924 melphalan Drugs 0.000 description 1
- SGDBTWWWUNNDEQ-LBPRGKRZSA-N melphalan Chemical compound OC(=O)[C@@H](N)CC1=CC=C(N(CCCl)CCCl)C=C1 SGDBTWWWUNNDEQ-LBPRGKRZSA-N 0.000 description 1
- 210000003071 memory t lymphocyte Anatomy 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- 210000003739 neck Anatomy 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 230000037434 nonsense mutation Effects 0.000 description 1
- 210000000299 nuclear matrix Anatomy 0.000 description 1
- 102000006255 nuclear receptors Human genes 0.000 description 1
- 108020004017 nuclear receptors Proteins 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 210000004789 organ system Anatomy 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000000242 pagocytic effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 229960001237 podophyllotoxin Drugs 0.000 description 1
- YJGVMLPVUAXIQN-XVVDYKMHSA-N podophyllotoxin Chemical compound COC1=C(OC)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@H](O)[C@@H]3[C@@H]2C(OC3)=O)=C1 YJGVMLPVUAXIQN-XVVDYKMHSA-N 0.000 description 1
- YVCVYCSAAZQOJI-UHFFFAOYSA-N podophyllotoxin Natural products COC1=C(O)C(OC)=CC(C2C3=CC=4OCOC=4C=C3C(O)C3C2C(OC3)=O)=C1 YVCVYCSAAZQOJI-UHFFFAOYSA-N 0.000 description 1
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 description 1
- 229960000624 procarbazine Drugs 0.000 description 1
- CPTBDICYNRMXFX-UHFFFAOYSA-N procarbazine Chemical compound CNNCC1=CC=C(C(=O)NC(C)C)C=C1 CPTBDICYNRMXFX-UHFFFAOYSA-N 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 230000004850 protein–protein interaction Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000007420 reactivation Effects 0.000 description 1
- 102000027426 receptor tyrosine kinases Human genes 0.000 description 1
- 108091008598 receptor tyrosine kinases Proteins 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 210000003289 regulatory T cell Anatomy 0.000 description 1
- 201000010174 renal carcinoma Diseases 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 210000004918 root sheath Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000037380 skin damage Effects 0.000 description 1
- 208000017520 skin disease Diseases 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 229960001603 tamoxifen Drugs 0.000 description 1
- 230000002381 testicular Effects 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000004565 tumor cell growth Effects 0.000 description 1
- 210000002229 urogenital system Anatomy 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- JXLYSJRDGCGARV-CFWMRBGOSA-N vinblastine 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-CFWMRBGOSA-N 0.000 description 1
- 229960003048 vinblastine Drugs 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 238000007482 whole exome sequencing Methods 0.000 description 1
- 238000012070 whole genome sequencing analysis Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/12—Animals modified by administration of exogenous cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/15—Humanized animals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0337—Animal models for infectious diseases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
- C12N2015/8527—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic for producing animal models, e.g. for tests or diseases
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
Definitions
- This disclosure relates to genetically modified animals that have a disruption at the endogenous HR gene (e.g., HR knockout) , and methods of use thereof.
- HR gene e.g., HR knockout
- Immunodeficient animals are very important for disease modeling and drug developments. In recent years, immunodeficient mice are routinely used as model organisms for research of the immune system, cell transplantation strategies, and the mechanisms of diseases. They have also been extensively used as hosts for normal and malignant tissue transplants, and are widely used to test the safety and efficacy of therapeutic agents.
- This disclosure is related to genetically modified animals that have a disruption at the endogenous HR gene (e.g., HR knockout) , and methods of making and use thereof.
- HR gene e.g., HR knockout
- the disclosure relates to a genetically-modified, non-human animal whose genome comprise a disruption in the animal’s endogenous HR lysine demethylase and nuclear receptor corepressor (HR) gene.
- the disruption of the endogenous HR gene comprises deletion of one or more exons of the endogenous HR gene.
- the disruption of the endogenous HR gene comprises deletion of one or more exons selected from exon 3, exon 4, exon 5, exon 6, and exon 7 of the endogenous HR gene.
- the disruption of the endogenous HR gene comprises deletion of exon 3, exon 4, exon 5, exon 6, and exon 7 of the endogenous HR gene.
- the disruption of the endogenous HR gene further comprises deletion of one or more exons or part of exons selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, and exon 20 of the endogenous HR gene.
- the disruption of the endogenous HR gene comprises deletion of one or more introns of the endogenous HR gene.
- the disruption of the endogenous HR gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, intron 17, intron 18, and intron 19 of the endogenous HR gene.
- the disruption consists of deletion of at least 10 nucleotides in intron 2; deletion of the entirety of exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, and exon 7; and deletion of at least 10 nucleotides in intron 7.
- the animal is homozygous with respect to the disruption of the endogenous HR gene. In some embodiments, the animal is heterozygous with respect to the disruption of the endogenous HR gene.
- the disruption prevents the expression of functional HR protein.
- the length of the remaining exon sequences at the endogenous HR gene locus is less than 70%of the total length of all exon sequences of the endogenous HR gene. In some embodiments, the length of the remaining sequences at that the endogenous HR gene locus is less than 65%of the full sequence of the endogenous HR gene.
- the disclosure relates to a genetically-modified, non-human animal, wherein the genome of the animal does not have one or more exons of HR gene at the animal’s endogenous HR gene locus.
- the genome of the animal does not have one or more exons or part of exons selected from the group consisting of exon 3, exon 4, exon 5, exon 6, and exon 7.
- the genome of the animal does not have one or more introns or part of introns selected from the group consisting of intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7.
- the disclosure relates to a HR knockout non-human animal, wherein the genome of the animal comprises from 5’ to 3’ at the endogenous HR gene locus, (a) a first DNA sequence; optionally (b) a second DNA sequence comprising an exogenous sequence; (c) a third DNA sequence.
- the first DNA sequence, the optional second DNA sequence, and the third DNA sequence are linked.
- the first DNA sequence comprises an endogenous HR gene sequence that is located upstream of intron 2
- the second DNA sequence can have a length of 0 nucleotides to 100 nucleotides
- the third DNA sequence comprises an endogenous HR gene sequence that is located downstream of intron 7.
- the first DNA sequence comprises a sequence that has a length (5’ to 3’) of from 10 to 1500 nucleotides.
- the length of the sequence refers to the length from the first nucleotide in exon 1 of the HR gene to the last nucleotide of the first DNA sequence.
- the first DNA sequence comprises at least 10 nucleotides from intron 2 of the endogenous HR gene.
- the first DNA sequence comprise exon 1 and exon 2 of the endogenous HR gene.
- the third DNA sequence comprises a sequence that has a length (5’ to 3’) of from 10 to 11000 nucleotides.
- the length of the sequence refers to the length from the first nucleotide in the third DNA sequence to the last nucleotide in the last exon of the endogenous HR gene.
- the third DNA sequence comprises at least 10 nucleotides from intron 7 of the endogenous HR gene.
- the third DNA sequence comprises exons 8-20, and introns 8-19.
- the disclosure relates to a genetically-modified, non-human animal produced by a method comprising knocking out one or more exons of endogenous HR gene by using (1) a first nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a target sequence in intron 2 of the endogenous HR gene, and (2) a second nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a sequence in intron 7 of the endogenous HR gene.
- a first nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a target sequence in intron 2 of the endogenous HR gene
- sgRNA single guide RNA
- the nuclease is CRISPR associated protein 9 (Cas9) .
- the target sequence in intron 2 of the endogenous HR gene is set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, and the target sequence in intron 7 of the endogenous HR gene is set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14.
- the first nuclease comprises a sgRNA that targets SEQ ID NO: 4 and the second nuclease comprises a sgRNA that targets SEQ ID NO: 10.
- the animal does not express a functional HR protein.
- the animal does not express a functional interleukin-2 receptor.
- the animal has one or more of the following characteristics:
- the percentage of T cells is less than 5%, 2%, 1.5%, 1%, 0.7%, or 0.5%of leukocytes in the animal;
- the percentage of B cells is less than 1%, 0.1%or 0.05%of leukocytes in the animal;
- the percentage of NK cells is less than 5%, 2%or 1.5%of leukocytes in the animal;
- the percentage of CD4+ T cells is less than 1%, 0.5%, 0.3%, or 0.1%of T cells;
- the percentage of CD8+ T cells is less than 1%, 0.5%, 0.3%, or 0.1%of T cells
- the percentage of CD3+ CD4+ cells, CD3+ CD8+ cells, CD3-CD19+ cells is less than 5%, 1%or 0.5%of leukocytes in the animal;
- the percentage of T cells, B cells, and NK cells is less than 5%, 4%, 3%, 2%or 1%of leukocytes in the animal.
- the animal after being engrafted with human hematopoietic stem cells to develop a human immune system has one or more of the following characteristics:
- the percentage of human CD45+ cells is about or at least 10%, 20%, 30%, 40%, or 50%of leukocytes of the animal;
- the percentage of human CD19+ cells is about or at least 10%, 20%, 30%, 40%, or 50%of leukocytes in the animal.
- the animal does not have hair.
- the animal has one or more of the following characteristics:
- the animal has no functional T-cells and/or no functional B-cells;
- the animal is a mammal, e.g., a monkey, a rodent, a rat, or a mouse.
- the animal is a C57 mouse, a C57BL mouse, a BALB/c mouse, a NOD/scid mouse, or a NOD/scid nude mouse, or a NOD-Prkdc scid IL-2r ⁇ null mouse.
- the animal is an immune deficient animal. In some embodiments, the animal is not an immune deficient animal.
- the animal further comprises a sequence encoding a human or chimeric protein.
- the human or chimeric protein is programmed cell death protein 1 (PD-1) , PD-L1, IL3, IL6, IL15, CSF1, or CSF2.
- the animal further comprises a disruption in the animal’s endogenous Beta-2-Microglobulin (B2M) gene.
- the disclosure relates to methods of determining effectiveness of an agent or a combination of agents for the treatment of cancer.
- the methods involve engrafting tumor cells to the animal as described herein, thereby forming one or more tumors in the animal; administering the agent or the combination of agents to the animal; and determining the inhibitory effects on the tumors.
- human peripheral blood cells hPBMC
- human hematopoietic stem cells are injected to the animal.
- the tumor cells are from cancer cell lines. In some embodiments, the tumor cells are from a tumor sample obtained from a human patient.
- the inhibitory effects are determined by measuring the tumor volume in the animal.
- the tumor cells are melanoma cells, lung cancer cells, primary lung carcinoma cells, non-small cell lung carcinoma (NSCLC) cells, small cell lung cancer (SCLC) cells, primary gastric carcinoma cells, bladder cancer cells, breast cancer cells, and/or prostate cancer cells.
- NSCLC non-small cell lung carcinoma
- SCLC small cell lung cancer
- the agent is an anti-PD-1 antibody. In some embodiments, the agent is an anti-PD-L1 antibody.
- the combination of agents comprises one or more agents selected from the group consisting of paclitaxel, cisplatin, carboplatin, pemetrexed, 5-FU, gemcitabine, oxaliplatin, docetaxel, and capecitabine.
- the disclosure relates to methods of producing an animal comprising a human hemato-lymphoid system.
- the methods involve engrafting a population of cells comprising human hematopoietic cells or human peripheral blood cells into the animal as described herein.
- the human hemato-lymphoid system comprises human cells selected from the group consisting of hematopoietic stem cells, myeloid precursor cells, myeloid cells, dendritic cells, monocytes, granulocytes, neutrophils, mast cells, lymphocytes, and platelets.
- the methods further comprise irradiating the animal prior to the engrafting.
- the disclosure relates to methods of producing a HR knockout mouse.
- the methods involve:
- the disclosure relates to methods of producing a HR knockout mouse.
- the methods involve
- the gene editing system comprises a first nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a target sequence in intron 2 of the endogenous HR gene, and a second nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a sequence in intron 7 of the endogenous HR gene.
- sgRNA single guide RNA
- the nuclease is CRISPR associated protein 9 (Cas9) .
- the target sequence in intron 2 of the endogenous HR gene is set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, and the target sequence in intron 7 of the endogenous HR gene is set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14.
- the mouse embryonic stem cell or the fertilized egg has a C57 background, a C57BL background, a BALB background (e.g., BALB/c background) , a NOD/scid background, a NOD/scid nude, or a NOD-Prkdc scid IL-2r ⁇ null background.
- a BALB background e.g., BALB/c background
- NOD/scid background e.g., BALB/c background
- NOD/scid nude e.g., NOD/scid nude
- NOD-Prkdc scid IL-2r ⁇ null background e.g., NOD-Prkdc scid IL-2r ⁇ null background.
- the mouse embryonic stem cell or the fertilized egg comprises a sequence encoding a human or chimeric protein.
- the human or chimeric protein is PD-1 or CD137.
- the mouse embryonic stem cell or the fertilized egg has a genome comprising a disruption in the animal’s endogenous B2M gene.
- the disclosure relates to a non-human mammalian cell, comprising a disruption, a deletion, or a genetic modification as described herein.
- the cell includes Cas9 mRNA or an in vitro transcript thereof.
- the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell. In some embodiments, the cell is a germ cell. In some embodiments, the cell is a blastocyst.
- the disclosure relates to methods for establishing a HR knockout animal model.
- the methods include the steps of:
- the cell is a fertilized egg cell
- step (d) identifying the germline transmission in the offspring of the pregnant female in step (c) .
- the establishment of a HR knockout animal involves a gene editing technique that is based on CRISPR/Cas9.
- the non-human mammal is a mouse. In some embodiments, the non-human mammal in step (c) is a female with false pregnancy.
- the disclosure relates to a tumor bearing non-human mammal model, characterized in that the non-human mammal model is obtained through the methods as described herein.
- the disclosure also relates to a cell or cell line, or a primary cell culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal.
- the disclosure further relates to the tissue, organ or a culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal.
- the disclosure relates to a tumor tissue derived from the non-human mammal or an offspring thereof when it bears a tumor, or the tumor bearing non-human mammal.
- the disclosure further relates to the use of the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal, the animal model generated through the method as described herein in the development of a product related to an immunization processes of human cells, the manufacture of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.
- the disclosure also relates to the use of the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal, the animal model generated through the method as described herein in the production and utilization of an animal experimental disease model of an immunization processes involving human cells, the study on a pathogen, or the development of a new diagnostic strategy and/or a therapeutic strategy.
- the disclosure further relates to the use of the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal, the animal model generated through the methods as described herein, in the screening, verifying, evaluating or studying the HR gene function, and the drugs for immune-related diseases and antitumor drugs.
- FIGS. 1A-1B are bar graphs showing activity testing results for sgRNA1-sgRNA14 (NC is a negative control; PC is a positive control) .
- FIG. 2 is a schematic diagram showing pT7-sgRNA G2 plasmid map.
- FIG. 3 shows PCR identification results for samples collected from tails of F0 generation mice (WT is a NOD-Prkdc scid IL-2rg null (B-NDG) mouse) .
- WT is a NOD-Prkdc scid IL-2rg null (B-NDG) mouse
- F0-1, F0-4, F0-18, F0-21, F0-28, F0-35, F0-37, F0-38, F0-41, and F0-45 are positive.
- FIG. 4 shows PCR identification results for samples collected from tails of F1 generation mice (offspring of F0-18 and a B-NDG mouse) .
- M is the Marker;
- WT is B-NDG mouse, + is a positive control, H 2 O is a negative control.
- FIG. 5 shows PCR identification results for samples collected from tails of F1 generation mice (offspring of F0-28 and a B-NDG mouse) .
- M is the Marker;
- WT is B-NDG mouse, + is a positive control, H 2 O is a negative control.
- FIG. 6 is an image of a HR knockout mouse with NOD-Prkdc scid IL-2rg null mutations.
- FIG. 7 is an image of a NOD-Prkdc scid IL-2rg null (B-NDG) mouse.
- FIG. 8 is an image of HR knockout mouse with NOD-Prkdc scid IL-2rg null mutations (B-NDG background) with human tumor cells.
- FIG. 9 is a diagram showing the mouse HR locus.
- This disclosure relates to HR knockout non-human animals, and methods of use thereof.
- Immunodeficient animals are an indispensable research tool for studying the mechanism of diseases, and methods of treating such diseases. They can easily accept exogenous cells or tissues due to their immunodeficiency, and have been widely used in the research.
- the commonly used immunodeficient animals include e.g., NOD-Prkdc scid IL-2r ⁇ nul mice, NOD-Rag 1 -/- -IL2rg -/- (NRG) , Rag 2 -/- -IL2rg -/- (RG) , NOD/SCID (NOD-Prkdc scid ) , and NOD/SCID nude mice.
- NOD-Prkdc scid IL-2r ⁇ nul mice may be the best recipient mice for transplantation.
- These immunodeficient mice are described in detail e.g., in Ito et al. "Current advances in humanized mouse models. " Cellular &molecular immunology 9.3 (2012) : 208, which is incorporated herein by reference in its entirety.
- hair needs to be removed, e.g., before exogenous cells or tissues are implanted to these animals. If hair removal is incomplete or the hair removal causes some skin damage, it may interfere with subsequent observations or studies, e.g., bioluminescence and fluorescence imaging and measuring tumor size.
- the skin of hairless animals e.g., mice
- it can also be used to test cosmetics, and various therapeutic agents to heal wounds.
- Hair is maintained through a cyclic process that includes periodic regeneration of hair follicles in a stem-cell-dependent manner.
- the hair cycle consists of three defined stages: growth (anagen) , followed by regression (catagen) and rest (telogen) .
- Growth of a new hair requires reentry into anagen, a process involving activation of multipotent epithelial stem cells residing in a specialized part of the follicle outer root sheath (ORS) .
- Activating signals emanate from adjacent mesenchymal cells (dermal papilla) , directing epithelial stem cells to migrate and differentiate to regenerate the hair bulb.
- Multiple signaling pathways, including Wnts, Sonic hedgehog (Shh) , and TGF- ⁇ family members have been shown to promote anagen initiation.
- Hr mutant mice Hair follicle morphogenesis and initial hair growth is normal. However, after the follicles regress (catagen) and the hair is shed, around postnatal day (P) 17, telogen stage follicles never reenter anagen, and no new hair is produced, resulting in alopecia.
- the defect in anagen initiation may reflect a loss of the relevant epithelial stem cell population or an inability to generate and/or interpret the necessary signals.
- the Hairless (also known as HR, HR lysine demethylase and nuclear receptor corepressor, or lysine-specific demethylase hairless) gene can encode an approximately 130 kDa nuclear transcription factor.
- the HR protein contains functional domains that include a nuclear localization signal domain, a nuclear matrix targeting motif, a putative zinc-finger, and a Jumonji C (JmjC) domain.
- HR can directly interact with several nuclear transcription factors and chromatin modulators.
- Rodent Hr has been shown to interact with thyroid hormone receptors TR ⁇ and TR ⁇ and with RAR-related orphan receptors (RORs) , especially ROR ⁇ to repress their transactivation activity.
- Human and rodent HRs have also been shown to undergo direct protein-protein interactions with the vitamin D receptor VDR.
- Regions in HR which mediate interactions with nuclear receptors include four motifs of hydrophobic amino acids, two of the form LXXLL (where L is leucine and X is any amino acid) and two ⁇ XX ⁇ motifs (where ⁇ can be leucine, isoleucine or valine) . These four hydrophobic motifs are also referred to as interaction domains (IDs) .
- HR has been shown to interact with ROR ⁇ and TRs via the LXXLL motif pair and the ⁇ XX ⁇ motif pair respectively, whereas all four motifs participate in interactions with VDR as revealed by coimmunoprecipitation and functional studies. Through its interactions with TRs, HR has also been implicated as playing a role in mammalian CNS development. Similarly, HR interactions with ROR ⁇ have been shown to be important in cerebellar development.
- HR may regulate epidermal homeostasis via direct control of a set of target genes that includes KDSR, MAGI2, and CSNK2A.
- APL atrichia with papular lesions
- HR gene (Gene ID: 55806) is located on chromosome 8, and has 19 exons.
- the nucleotide sequence for human HR mRNA is NM_005144.4, and the amino acid sequence for human HR is NP_005135.2.
- HR gene locus has 20 exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, and exon 20 (FIG. 9) .
- the nucleotide sequence for mouse HR cDNA is NM_021877.3 (SEQ ID NO: 26)
- the amino acid sequence for mouse HR is NP_068677.2 (SEQ ID NO: 27) .
- the location for each exon in the mouse HR nucleotide sequence and amino acid sequence is listed below:
- the mouse HR gene (Gene ID: 15460) is located in Chromosome 14 of the mouse genome, which is located from70554056 to 70573548 of NC_000080.6 (GRCm38. p4 (GCF_000001635.24) ) .
- the 5’-UTR is from 70, 554, 056 to 70, 554, 112 and 70, 554, 512 to 70, 555, 107 and 70, 556, 348 to 70, 556, 388, exon 1 is from 70, 554, 056 to 70, 554, 112, the first intron is from 70, 554, 113 to 70, 554, 511, exon 2 is from 70, 554, 512 to 70, 555, 107, the second intron is from 70, 555, 108 to 70, 556, 347, exon 3 is from 70, 556, 348 to 70, 557, 000, the third intron is from 70, 557, 001 to 70, 557, 628, exon 4 is from 70, 557, 629 to 70, 558, 409, the fourth intron is from 70, 558, 410 to 70, 559, 638, exon 5 is from 70, 559, 639 to 70, 559, 789, the fifth intron is from 70, 559, 790
- HR genes, proteins, and locus of the other species are also known in the art.
- the gene ID for HR in Rattus norvegicus is 60563
- the gene ID for HR in Macaca mulatta (Rhesus monkey) is 574164
- the gene ID for HR in Sus scrofa is 397617.
- the relevant information for these genes can be found, e.g., in NCBI database.
- the present disclosure provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal’s endogenous HR gene, wherein the disruption of the endogenous HR gene comprises deletion of one or more exons, or part of the one or more exons, wherein the one or more exons are selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, and exon 20 of the endogenous HR gene.
- the disclosure provides a genetically-modified, non-human animal that does not have one or more exons that are selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, and exon 20 of the endogenous HR gene.
- the animals do not have exons 3-7.
- deletion of an exon refers to the deletion the entire exon.
- deletion of exon 2 means that all sequences in exon 2 are deleted.
- the term “deletion of part of an exon” refers to at least one nucleotide, but not all nucleotides in the exon is deleted. In some embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nucleotides in the exon are deleted.
- the disruption comprises deletion of one or more introns, or part of the one or more introns, wherein the one or more introns are selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, intron 17, intron 18, and intron 19 of the endogenous HR gene.
- the disclosure provides a genetically-modified, non-human animal does not have one or more introns that are selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, intron 17, intron 18, and intron 19 of the endogenous HR gene.
- the animal does not have part of intron 2, intron 3, intron 4, intron 5, intron 6, and/or part of intron 7.
- deletion of an intron refers to the deletion the entire intron.
- deletion of intron 3 means that all sequences in intron 3 are deleted.
- the term “deletion of part of an intron” refers to at least one nucleotide, but not all nucleotides in the intron is deleted. In some embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, or 3000 nucleotides in the intron are deleted.
- the disruption of the endogenous HR gene comprises deletion of one or more exons selected from the group consisting of exon 3, exon 4, exon 5, exon 6, and exon 7 of the endogenous HR gene. In some embodiments, the disruption of the endogenous HR gene further comprises deletion of exon 1, exon 2, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, and/or exon 20 of the endogenous HR gene.
- the entire sequence of mouse exon 3, exon 4, exon 5, exon 6, and exon 7 are deleted.
- a “region” or “portion” of mouse exons or introns of HR gene are deleted.
- the term “region” or “portion” can refer to e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, or 3000 nucleotides.
- the “region” or “portion” can be at least 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, intron 17, intron 18, or intron 19.
- a region, a portion, or the entire sequence of exon 3, exon 4, exon 5, exon 6, and/or exon 7 are deleted. In some embodiments, a region, a portion, or the entire sequence of mouse intron 2, intron 3, intron 4, intron 5, intron 6, and/or intron 7 are deleted.
- the disruption comprises or consists of deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nucleotides in exon 3, exon 4, exon 5, exon 6, and/or exon 7.
- the disruption comprises or consists of deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, or 3000 nucleotides in intron 2, intron 3, intron 4, intron 5, intron 6, and/or intron 7.
- the disruption comprises or consists of deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, or 1000 in intron 2; deletion of the entirety of exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, and exon 7; and/or deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, or 1000 nucleotides in intron 7.
- the length of the remaining exon sequences at the endogenous HR gene locus is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, or 80%of the total length of all exon sequences of the endogenous HR gene.
- the length of the remaining exon sequences at the endogenous HR gene locus is about or at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, or 80%of the total length of all exon sequences of the endogenous HR gene.
- the length of the remaining sequences at that the endogenous HR gene locus is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, or 80%of the full sequence of the endogenous HR gene.
- the length of the remaining sequences at that the endogenous HR gene locus is about or at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, or 80%of the full sequence of the endogenous HR gene.
- the sequence starts from the first nucleotide of exon 1. In some embodiments, the sequence ends at the last nucleotide of the last exon.
- the present disclosure further relates to the genomic DNA sequence of a HR knockout animal (e.g., a rodent, a mouse) .
- the genome of the animal comprises from 5’ to 3’ at the endogenous HR gene locus, (a) a first DNA sequence; optionally, (b) a second DNA sequence comprising an exogenous sequence; (c) a third DNA sequence, wherein the first DNA sequence, the optional second DNA sequence, and the third DNA sequence are linked.
- the second DNA sequence can have a length of 0 nucleotides to 1000 nucleotides (e.g., at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nucleotides) .
- 1000 nucleotides e.g., at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,
- the second DNA sequence has only 0 nucleotides, which means that there is no extra sequence between the first DNA sequence and the third DNA sequence. In some embodiments, random or exogenous sequences are added. In some embodiments, the second DNA sequence has a length of 1 nucleotide to 100 nucleotides (e.g., 1 to 20 nucleotides) .
- the second DNA sequence has about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nucleotides.
- the second DNA sequence has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nucleotides.
- the first DNA sequence comprises an endogenous HR gene sequence that is located upstream of intron 2, and can include all or just part of sequences that is located upstream of intron 2. In some embodiments, the first DNA sequence comprises an endogenous HR gene sequence that is located upstream of exon 2.
- the first DNA sequence comprises a sequence that has a length (5’ to 3’) of from 10 to 1500 nucleotides (e.g., from 10 to 100 nucleotides, from 100 to 500 nucleotides, from 500 to 1000 nucleotides, from 1000 to 1500 nucleotides, from 1000 to 2000 nucleotides, or from 1400 to 1500 nucleotides) starting from the first nucleotide in exon 1 of the HR gene to the last nucleotide of the first DNA sequence.
- 10 to 1500 nucleotides e.g., from 10 to 100 nucleotides, from 100 to 500 nucleotides, from 500 to 1000 nucleotides, from 1000 to 1500 nucleotides, from 1000 to 2000 nucleotides, or from 1400 to 1500 nucleotides
- the first DNA sequence comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 nucleotides from exon 1, exon 2, or the combination of exon 1 and exon 2.
- the first DNA sequence has at most , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 nucleotides from exon 1, exon 2, or the combination of exon 1 and exon 2.
- the third DNA sequence comprises an endogenous HR gene sequence that is located downstream of intron 7, and can include all or just part of sequences that is located downstream of intron 7.
- the third DNA sequence comprises a sequence that has a length (5’ to 3’) of from 1 to 1351 nucleotides (e.g., from 1 to 1000 nucleotides, or from 500 to 1300 nucleotides) starting from the first nucleotide in the third DNA sequence to the last nucleotide in the intron 7 (e.g., intron 7 in mouse) of the endogenous HR gene.
- the third DNA sequence comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, or 1300 nucleotides from intron 7.
- the third DNA sequence has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, or 1350 nucleotides from intron 7.
- the third DNA sequence comprises an endogenous HR gene sequence that is located downstream of the last intron (e.g., intron 19 in mouse) , and can include all or just part of sequences that is located downstream of intron 19.
- the third DNA sequence comprises a sequence that has a length (5’ to 3’) of from 10 to 11000 nucleotides (e.g., from 100 to 11000 nucleotides, from 1000 to 11000 nucleotides, or from 5000 to 11000 nucleotides) starting from the first nucleotide in the third DNA sequence to the last nucleotide in the last exon (e.g., exon 20 in mouse) of the endogenous HR gene.
- the third DNA sequence comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or 11000 nucleotides.
- the third DNA sequence has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or 11000 nucleotides.
- the third DNA sequence comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, or 1300 nucleotides from the last exon (e.g., exon 20 in mouse) .
- the third DNA sequence has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, or 1306 nucleotides from the last exon (e.g., exon 20 in mouse) .
- the last exon e.g., exon 20 in mouse
- the HR gene sequence at the endogens HR locus is set forth in SEQ ID NO: 32 (70554056-70573548 of NC_000080.6) .
- 70555486-70562742 of NC_000080.6 (SEQ ID NO: 33) is deleted.
- 70555492-70562744 of NC_000080.6 (SEQ ID NO: 34) is deleted.
- the genetic modified non-human animal comprises a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical or 100%identical to SEQ ID NO: 28.
- the genetic modified non-human animal comprises a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical or 100%identical to SEQ ID NO: 35.
- the sequence is located at the endogenous HR locus.
- the disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any nucleotide sequence as described herein (e.g., exon sequences, intron sequences, the remaining exon sequences, the deleted sequences) , and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
- the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein.
- the nucleic acid sequence is less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or 11000 nucleotides.
- the amino acid sequence is less than , 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400 or 1500 amino acid residues.
- the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.
- the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.
- the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) .
- the length of a reference sequence aligned for comparison purposes is at least 80%of the length of the reference sequence, and in some embodiments is at least 90%, 95%, or 100%.
- the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
- the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
- the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
- Cells, tissues, and animals are also provided that comprise a disruption of the endogenous HR gene as described herein, as well as cells, tissues, and animals (e.g., mouse) that have any nucleic acid sequence as described herein.
- the term “genetically-modified non-human animal” refers to a non-human animal having a modified sequence (e.g., deletion of endogenous sequence or insertion of exogenous sequence) in at least one chromosome of the animal’s genome.
- at least one or more cells e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%of cells of the genetically-modified non-human animal have the modified sequence in its genome.
- the cell having the modified sequence can be various kinds of cells, e.g., an endogenous cell, a somatic cell, an immune cell, a T cell, a B cell, a germ cell, a blastocyst, or an endogenous tumor cell.
- genetically-modified non-human animals are provided that comprise a disruption or a deletion at the endogenous HR locus. The animals are generally able to pass the modification to progeny, i.e., through germline transmission.
- the genetically-modified non-human animal does not express HR (e.g., intact or functional HR protein) . Because HR is involved in hair growth and hair follicle regeneration, the genetically-modified non-human animal does not have hair. In some embodiments, the hair coverage of the genetically-modified non-human animal is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.
- HR e.g., intact or functional HR protein
- the genetically-modified non-human animal is an immunodeficient animal.
- the animal is a NOD-Prkdc scid IL-2r ⁇ nul , NOD-Rag 1 -/- -IL2rg -/- (NRG) , Rag 2 -/- -IL2rg -/- (RG) , NOD/SCID (NOD-Prkdc scid ) , NOD/SCID nude, or NOD-Prkdc scid IL-2rg null animal (e.g., a rodent, a rat, or a mouse) .
- the genetically-modified non-human animal is not an immunodeficient animal.
- the genetically-modified non-human animal lack functional T cells, B cells, and/or NK cells.
- the animal can have one or more of the following characteristics:
- the percentage of T cells is less than 5%, 4%, 3%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%or 0.1%of leukocytes in the animal;
- the percentage of B cells is less than 5%, 4%, 3%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%or 0.01%of leukocytes in the animal;
- the percentage of NK cells is less than 5%, 4%, 3%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, or 0.5%of leukocytes in the animal;
- CD4+ T cells CD3+ CD4+ cells
- percentage of CD4+ T cells is less than 5%, 4%, 3%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%of T cells;
- the percentage of CD8+ T cells is less than 5%, 4%, 3%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%of T cells;
- the percentage of CD3+ CD4+ cells, CD3+ CD8+ cells, CD3-CD19+ cells is less than 5%, 4%, 3%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%of leukocytes in the animal;
- the percentage of T cells (CD3+ cells) and NK cells (CD3-CD49b+ cells) is less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%of leukocytes in the animal.
- leukocytes or “white blood cells” include neutrophils, eosinophils (acidophilus) , basophils, lymphocytes, and monocytes. All leukocytes have nuclei, which distinguishes them from the anucleated red blood cells (RBCs) and platelets.
- CD45 also known as leukocyte common antigen (LCA) , is a cell surface marker for leukocytes. Among leukocytes, monocytes and neutrophils are phagocytic.
- Lymphocytes is a subtype of leukocytes. Lymphocytes include natural killer (NK) cells (which function in cell-mediated, cytotoxic innate immunity) , T cells, and B cells.
- NK natural killer
- the variations among individual mice are very small.
- the standard deviations of the percentages are less than 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%or 0.01%.
- the genetically-modified non-human animal has a NOD-Prkdc scid IL-2rg null background.
- the genetically-modified animal can also have one or more of the following characteristics:
- the genetically-modified mouse has no functional T-cells and/or no functional B-cells;
- the genetically-modified mouse exhibits reduced macrophage function relative to a NOD/scid mouse, or a NOD/scid nude mouse;
- the genetically-modified mouse exhibits reduced dendritic function relative to a NOD/scid mouse, or a NOD/scid nude mouse;
- the genetically-modified mouse has an enhanced engraftment capacity of exogenous cells relative to a NOD/scid mouse, or a NOD/scid nude mouse.
- the genetically modified non-human animal can also be various other animals, e.g., a rat, rabbit, pig, bovine (e.g., cow, bull, buffalo) , deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey) .
- a rat, rabbit, pig, bovine e.g., cow, bull, buffalo
- deer sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey)
- a non-human animals where suitable genetically modifiable ES cells are not readily available, other methods are employed to make a non-human animal comprising the genetic modification.
- Such methods include, e.g., modifying a non-ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and employing nuclear transfer to transfer the modified genome to a suitable cell, e.g., an oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo.
- a suitable cell e.g., an oocyte
- gestating the modified cell e.g., the modified oocyte
- the animal is a mammal, e.g., of the superfamily Dipodoidea or Muroidea.
- the genetically modified animal is a rodent.
- the rodent can be selected from a mouse, a rat, and a hamster.
- the rodent is selected from the superfamily Muroidea.
- the genetically modified animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters) , Cricetidae (e.g., hamster, New World rats and mice, voles) , Muridae (true mice and rats, gerbils, spiny mice, crested rats) , Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasy rats and mice) , Platacanthomyidae (e.g., spiny dormice) , and Spalacidae (e.g., mole rates, bamboo rats, and zokors) .
- Calomyscidae e.g., mouse-like hamsters
- Cricetidae e.g., hamster, New World rats and mice, voles
- Muridae true mice and rats, gerbils, spiny mice, crested rats
- the genetically modified rodent is selected from a true mouse or rat (family Muridae) , a gerbil, a spiny mouse, and a crested rat.
- the non-human animal is a mouse.
- the animal is a mouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola.
- a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola.
- the mouse is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm) , 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac) , 129S7, 129S8, 129T1, 129T2.
- a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm) , 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac) , 129S7, 129S8, 129T1, 129T2.
- the genetically modified mouse is a mix of the 129 strain and the C57BL/6 strain.
- the mouse is a mix of the 129 strains, or a mix of the BL/6 strains.
- the mouse is a BALB strain, e.g., BALB/c strain.
- the mouse is a mix of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid line (e.g., 50%BALB/c-50%12954/Sv; or 50%C57BL/6-50%129) .
- a hybrid line e.g., 50%BALB/c-50%12954/Sv; or 50%C57BL/6-50%129
- the animal is a rat.
- the rat can be selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti.
- the rat strain is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.
- the animal can have one or more other genetic modifications, and/or other modifications, that are suitable for the particular purpose for which the HR knockout animal is made.
- suitable mice for maintaining a xenograft e.g., a human cancer or tumor
- mice for maintaining a xenograft can have one or more modifications that compromise, inactivate, or destroy the immune system of the non-human animal in whole or in part.
- Compromise, inactivation, or destruction of the immune system of the non-human animal can include, for example, destruction of hematopoietic cells and/or immune cells by chemical means (e.g., administering a toxin) , physical means (e.g., irradiating the animal) , and/or genetic modification (e.g., knocking out one or more genes) .
- chemical means e.g., administering a toxin
- physical means e.g., irradiating the animal
- genetic modification e.g., knocking out one or more genes
- mice include, e.g., NOD mice, SCID mice, NOD/SCID mice, nude mice, NOD/SCID nude mice, NOD-Prkdc scid IL-2r ⁇ null , and Rag1 and/or Rag2 knockout mice. These mice can optionally be irradiated, or otherwise treated to destroy one or more immune cell type.
- a genetically modified mouse is provided that can include a disruption of the endogenous non-human HR locus, and further comprises a modification that compromises, inactivates, or destroys the immune system (or one or more cell types of the immune system) of the non-human animal in whole or in part.
- modification is, e.g., selected from the group consisting of a modification that results in NOD mice, SCID mice, NOD/SCID mice, nude mice, NOD-Prkdc scid IL-2r ⁇ null mice, Rag1 and/or Rag2 knockout mice, and a combination thereof.
- genetically modified cells are also provided that can comprise the modifications (e.g., disruption) described herein (e.g., ES cells, somatic cells)
- the genetically modified non-human animals comprise the modification of the endogenous HR locus in the germline of the animal.
- the genetically modified animal can be homozygous with respect to the disruption of the endogenous HR gene. In some embodiments, the animal can be heterozygous with respect to the disruption of the endogenous HR gene.
- the present disclosure further relates to a non-human mammal generated through the methods as described herein.
- the genome thereof contains human gene (s) .
- the present disclosure also relates to a tumor bearing non-human mammal model, characterized in that the non-human mammal model is obtained through the methods as described herein.
- the non-human mammal is a rodent (e.g., a mouse) .
- the present disclosure further relates to a cell or cell line, or a primary cell culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal; the tissue, organ or a culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal; and the tumor tissue derived from the non-human mammal or an offspring thereof when it bears a tumor, or the tumor bearing non-human mammal.
- the present disclosure also provides non-human mammals produced by any of the methods described herein.
- a non-human mammal is provided; and the genetically modified animal contains a disruption of the HR gene in the genome of the animal.
- the present disclosure also relates to the progeny produced by the non-human mammal provided by the present disclosure mated with the same or other genotypes.
- the present disclosure also provides a cell line or primary cell culture derived from the non-human mammal or a progeny thereof.
- a model based on cell culture can be prepared, for example, by the following methods. Cell cultures can be obtained by way of isolation from a non-human mammal, alternatively cell can be obtained from the cell culture established using the same constructs and the standard cell transfection techniques. The disruption of HR gene can be detected by a variety of methods.
- RNA quantification approaches using reverse transcriptase polymerase chain reaction (RT-PCR) or Southern blotting, and in situ hybridization methods at the protein level (including histochemistry, immunoblot analysis and in vitro binding studies) .
- RT-PCR reverse transcriptase polymerase chain reaction
- Southern blotting methods at the protein level
- protein level including histochemistry, immunoblot analysis and in vitro binding studies.
- Many standard analysis methods can be used to complete quantitative measurements. For example, transcription levels of wildtype HR can be measured using RT-PCR and hybridization methods including RNase protection, Southern blot analysis, RNA dot analysis (RNAdot) analysis. Immunohistochemical staining, flow cytometry, Western blot analysis can also be used to assess the presence of human proteins.
- the disclosure also provides vectors for constructing a HR animal model.
- the vectors comprise sgRNA sequence, wherein the sgRNA sequence target HR gene, and the sgRNA is unique on the target sequence of the HR gene to be altered.
- the sequence meets the sequence arrangement rule of 5’-NNN (20) -NGG3’ or 5’-CCN-N (20) -3’; and in some embodiments, the targeting site of the sgRNA in the mouse HR gene is located on the exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, and exon 20, intron 1, intron 2, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, intron 17, intron 18, intron 19, upstream of exon 1, or downstream of exon 20 of the mouse HR gene.
- the 5’ targeting sequence for the knockout sequence is shown as SEQ ID NOs: 1-7, and the sgRNA sequence recognizes the 5’ targeting site.
- the 3’ targeting sequence for the knockout sequence is shown as SEQ ID NOs: 8-14 and the sgRNA sequence recognizes the 3’ targeting site.
- the disclosure provides sgRNA sequences for constructing a HR knockout animal model.
- the oligonucleotide sgRNA sequences are set forth in SEQ ID NOs: 15-22.
- the disclosure relates to a plasmid construct (e.g., pT7-sgRNA) including the sgRNA sequence, and/or a cell including the construct.
- a plasmid construct e.g., pT7-sgRNA
- the present disclosure further relates to a non-human mammalian cell, having any one of the foregoing targeting vectors, and one or more in vitro transcripts of the sgRNA construct as described herein.
- the cell includes Cas9 mRNA or an in vitro transcript thereof.
- the genes in the cell are heterozygous. In some embodiments, the genes in the cell are homozygous.
- the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell.
- Genetically modified animals can be made by several techniques that are known in the art, including, e.g., nonhomologous end-joining (NHEJ) , homologous recombination (HR) , zinc finger nucleases (ZFNs) , transcription activator-like effector-based nucleases (TALEN) , and the clustered regularly interspaced short palindromic repeats (CRISPR) -Cas system.
- NHEJ nonhomologous end-joining
- HR homologous recombination
- ZFNs zinc finger nucleases
- TALEN transcription activator-like effector-based nucleases
- CRISPR clustered regularly interspaced short palindromic repeats
- homologous recombination is used.
- CRISPR-Cas9 genome editing is used to generate genetically modified animals.
- genome editing techniques are known in the art, and is described, e.g., in Yin et al., "Delivery technologies for genome editing, " Nature Reviews Drug Discovery 16.6 (2017) : 387-399, which is incorporated by reference in its entirety.
- Many other methods are also provided and can be used in genome editing, e.g., micro-injecting a genetically modified nucleus into an enucleated oocyte, and fusing an enucleated oocyte with another genetically modified cell.
- the disclosure provides knocking out in at least one cell of the animal, at an endogenous HR gene locus, one or more exons (e.g., about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 exons) and/or one or more introns (e.g., about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 introns) of the endogenous HR gene.
- the modification occurs in a germ cell, a somatic cell, a blastocyst, or a fibroblast, etc.
- the nucleus of a somatic cell or the fibroblast can also be inserted into an enucleated oocyte.
- cleavages at the upstream and the downstream of the knockout sequence by a nuclease can result in DNA double strands break, and non-homologous end joining (NHEJ) occurs and ligates the break ends, thereby knocking out the sequence of interest.
- NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately.
- imprecise repair occurs, and in some cases, leading to loss of nucleotides or insertion of random nucleotides.
- Zinc finger proteins, TAL-effector domains, or single guide RNA (sgRNA) DNA-binding domains can be designed to target the upstream and the downstream of the knockout sequence.
- SEQ ID NOs: 1-14 are exemplary target sequences for the modification. Among them, SEQ ID NOs: 1-7 are located within intron 2 of mouse endogenous HR gene. SEQ ID NOs: 8-14 are located within intron 7 of mouse endogenous HR gene.
- the nuclease cleaves the genomic DNA, and triggers NHEJ.
- the nuclease is CRISPR associated protein 9 (Cas9) .
- the methods of producing a HR knockout mouse can involve one or more of the following steps: transforming a mouse embryonic stem cell with a gene editing system that targets endogenous HR gene, thereby producing a transformed embryonic stem cell; introducing the transformed embryonic stem cell into a mouse blastocyst; implanting the mouse blastocyst into a pseudopregnant female mouse; and allowing the blastocyst to undergo fetal development to term.
- the transformed embryonic cell is directly implanted into a pseudopregnant female mouse instead, and the embryonic cell undergoes fetal development.
- the gene editing system can involve zinc finger proteins, TAL-effector domains, or single guide RNA (sgRNA) DNA-binding domains.
- sgRNA single guide RNA
- the present disclosure further provides a method for establishing a HR gene knockout animal model, involving the following steps:
- step (d) identifying the germline transmission in the offspring genetically modified humanized non-human mammal of the pregnant female in step (c) .
- the non-human mammal in the foregoing method is a mouse (e.g., a C57BL/6 mouse, a NOD/scid mouse, a NOD/scid nude mouse, or a NOD-Prkdc scid IL-2r ⁇ null mouse) .
- the non-human mammal is a NOD/scid mouse.
- the SCID mutation has been transferred onto a non-obese diabetic (NOD) background. Animals homozygous for the SCID mutation have impaired T and B cell lymphocyte development. The NOD background additionally results in deficient natural killer (NK) cell function.
- NOD non-obese diabetic
- the non-human mammal is a NOD/scid nude mouse.
- the NOD/scid nude mouse additionally has a disruption of FOXN1 gene on chromosome 11 in mice.
- the animal can comprise an additional disruption in the animal’s endogenous Beta-2-Microglobulin (B2M) gene.
- B2M Beta-2-Microglobulin
- the fertilized eggs for the methods described above are NOD/scid fertilized eggs, NOD/scid nude fertilized eggs, or NOD-Prkdc scid IL-2r ⁇ null fertilized eggs.
- Other fertilized eggs that can also be used in the methods as described herein include, but are not limited to, C57BL/6 fertilized eggs, FVB/N fertilized eggs, BALB/c fertilized eggs, DBA/1 fertilized eggs and DBA/2 fertilized eggs.
- Fertilized eggs can come from any non-human animal, e.g., any non-human animal as described herein.
- the fertilized egg cells are derived from rodents.
- the genetic construct can be introduced into a fertilized egg by microinjection of DNA. For example, by way of culturing a fertilized egg after microinjection, a cultured fertilized egg can be transferred to a false pregnant non-human animal, which then gives birth of a non-human mammal, so as to generate the non-human mammal mentioned in the method described above.
- the genetically modified animals e.g., mice
- the genetically modified mice do not require backcrossing, and thus have a more defined background (e.g., less genetic variations among individual subjects) as compared to some other HR knockout or immunodeficient mice.
- a defined background or a pure background is beneficial to obtain consistent experiment results.
- the genetic variation among the mice across the entire genome (e.g., autosomes) among different individuals is less than 3%, 2%, 1%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%or 0.01%.
- the methods to determine the genetic variations are known in the art.
- the genetic variations can be measured or determined by sequencing, whole genome sequencing, exome sequencing, detecting variations at some selected sites (e.g., by using SNP microarrays, by detecting copy number variations) etc.
- the mice have a genome (e.g., autosomes) that is about or at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%identical to NOD-Prkdc scid IL-2r ⁇ null mice (e.g., B-NDG mice) except for certain mutations (e.g., the HR knockout mutation) .
- NOD-Prkdc scid IL-2r ⁇ null mice e.g., B-NDG mice
- certain mutations e.g., the HR knock
- the mice have a genome (e.g., autosomes) that is about or at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%identical to NOD/SCID mice (or NOD-Prkdc scid mice) except for certain mutations (e.g., the HR knockout mutation and/or IL-2r ⁇ mutation) .
- a genome e.g., autosomes
- the mice have a genome (e.g., autosomes) that is about or at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%identical to NOD mice except for certain mutations (e.g., the HR knockout mutation, the SCID mutation, and/or IL-2r ⁇ mutation) .
- a genome e.g., autosomes
- mice are also relatively healthy, and in some cases, they have a relatively long life span (e.g., about or at least 1 year, 1.5 years, 2 years, 2.5 years, or 3 years) .
- Genetically modified animals with a disruption at endogenous HR gene can provide a variety of uses that include, but are not limited to, observing tumor growth, testing various therapeutic agents (e.g., to treat tumors, wounds, and various skin diseases) , and testing cosmetics.
- the animal is an immunodeficient animal, e.g., a NOD-Prkdc scid IL-2r ⁇ nul , NOD-Rag 1 -/- -IL2rg -/- (NRG) , Rag 2 -/- -IL2rg -/- (RG) , NOD/SCID (NOD-Prkdc scid ) , NOD/SCID nude, or NOD-Prkdc scid IL-2rg null animal (e.g., a rodent, a rat, or a mouse) .
- the animal further comprises an additional disruption in the animal’s endogenous Beta-2-Microglobulin (B2M) gene.
- B2M Beta-2-Microglobulin
- the hairless immunodeficient animal can be used to establish a human hemato-lymphoid animal model, develop therapeutics for human diseases and disorders, and assess the efficacy of these therapeutic agents in the animal models.
- the genetically modified animals can be used for establishing a human hemato-lymphoid system.
- the methods involve engrafting a population of cells comprising human hematopoietic cells (CD34+ cells) or human peripheral blood cells into the genetically modified animal described herein.
- the methods further include the step of irradiating the animal prior to the engrafting.
- the human hemato-lymphoid system in the genetically modified animals can include various human cells, e.g., hematopoietic stem cells, myeloid precursor cells, myeloid cells, dendritic cells, monocytes, granulocytes, neutrophils, mast cells, lymphocytes, and platelets.
- the genetically modified animals described herein are also an excellent animal model for establishing the human hemato-lymphoid system.
- the animal after being engrafted with human hematopoietic stem cells or human peripheral blood cells to develop a human immune system has one or more of the following characteristics:
- the percentage of human CD45+ cells is about or at least 10%, 20%, 30%, 40%, or 50%of leukocytes or CD45+ cells of the animal;
- the percentage of human CD3+ cells is about or at least 10%, 20%, 30%, 40%, or 50%of leukocytes or CD45+ cells in the animal;
- the percentage of human CD19+ cells is about or at least 10%, 20%, 30%, 40%, or 50%of leukocytes or CD45+ cells in the animal.
- the genetically modified animals described herein are less likely to develop graft-versus-host disease (GVHD) or xenogeneic graft-versus-host disease (X-GVHD) .
- GVHD graft-versus-host disease
- X-GVHD xenogeneic graft-versus-host disease
- the genetically modified animals described herein can live for about or at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 days.
- the genetically modified animals described herein do not have a NSG or NOG background. In some embodiments, the genetically modified animals described herein are better animal models for establishing the human hemato-lymphoid system (e.g., having a higher percentage of human leukocytes, human T cells, human B cells, or human NK cells) .
- NSG mice and NOD mice A detailed description of the NSG mice and NOD mice can be found, e.g., in Ishikawa et al. "Development of functional human blood and immune systems in NOD/SCID/IL2 receptor ⁇ chainnull mice. " Blood 106.5 (2005) : 1565-1573; Katano et al. "NOD-Rag2null IL-2R ⁇ null mice: an alternative to NOG mice for generation of humanized mice. " Experimental animals 63.3 (2014) : 321-330, both of which are incorporated herein by reference in the entirety.
- the genetically modified animals can be used to determine the effectiveness of an agent or a combination of agents for the treatment of cancer.
- the methods involve engrafting tumor cells to the animal as described herein, administering the agent or the combination of agents to the animal; and determining the inhibitory effects on the tumors.
- the tumor cells are from a tumor sample obtained from a human patient.
- These animal models are also known as patient derived xenografts (PDX) models.
- PDX models are often used to create an environment that resembles the natural growth of cancer, for the study of cancer progression and treatment.
- patient tumor samples grow in physiologically-relevant tumor microenvironments that mimic the oxygen, nutrient, and hormone levels that are found in the patient’s primary tumor site.
- implanted tumor tissue maintains the genetic and epigenetic abnormalities found in the patient and the xenograft tissue can be excised from the patient to include the surrounding human stroma.
- PDX models can often exhibit similar responses to anti-cancer agents as seen in the actual patient who provide the tumor sample.
- the genetically modified animals do not have functional T cells or B cells, the genetically modified animals still have functional phagocytic cells, e.g., neutrophils, eosinophils (acidophilus) , basophils, or monocytes. Macrophages can be derived from monocytes, and can engulf and digest cellular debris, foreign substances, microbes, cancer cells.
- an agent e.g., anti-CD47 antibodies or anti-SIRPa antibodies
- human peripheral blood cells hPBMC
- human hematopoietic stem cells are injected to the animal to develop human hematopoietic system.
- the genetically modified animals described herein can be used to determine the effect of an agent in human hematopoietic system, and the effects of the agent to inhibit tumor cell growth or tumor growth.
- the methods as described herein are also designed to determine the effects of the agent on human immune cells (e.g., human T cells, B cells, or NK cells) , e.g., whether the agent can stimulate T cells or inhibit T cells, whether the agent can upregulate the immune response or downregulate immune response.
- the genetically modified animals can be used for determining the effective dosage of a therapeutic agent for treating a disease in the subject, e.g., cancer, or autoimmune diseases.
- the tested agent or the combination of tested agents is designed for treating various cancers.
- cancer refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
- tumor refers to cancerous cells, e.g., a mass of cancerous cells.
- Cancers that can be treated or diagnosed using the methods described herein include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
- the agents described herein are designed for treating or diagnosing a carcinoma in a subject.
- carcinoma is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas.
- the cancer is renal carcinoma or melanoma.
- Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.
- carcinosarcomas e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues.
- an “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
- the term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.
- the tested agent is designed for the treating melanoma, primary lung carcinoma, non-small cell lung carcinoma (NSCLC) , small cell lung cancer (SCLC) , primary gastric carcinoma, bladder cancer, breast cancer, and/or prostate cancer.
- NSCLC non-small cell lung carcinoma
- SCLC small cell lung cancer
- the injected tumor cells are human tumor cells.
- the injected tumor cells are melanoma cells, primary lung carcinoma cells, non-small cell lung carcinoma (NSCLC) cells, small cell lung cancer (SCLC) cells, primary gastric carcinoma cells, bladder cancer cells, breast cancer cells, and/or prostate cancer cells.
- NSCLC non-small cell lung carcinoma
- SCLC small cell lung cancer
- the inhibitory effects on tumors can also be determined by any methods known in the art.
- the tumor cells can be labeled by a luciferase gene.
- the number of the tumor cells or the size of the tumor in the animal can be determined by an in vivo imaging system (e.g., the intensity of fluorescence) .
- the inhibitory effects on tumors can also be determined by measuring the tumor volume in the animal, and/or determining tumor (volume) inhibition rate (TGI TV ) .
- the tested agent can be one or more agents selected from the group consisting of paclitaxel, cisplatin, carboplatin, pemetrexed, 5-FU, gemcitabine, oxaliplatin, docetaxel, and capecitabine.
- the tested agent can be an antibody, for example, an antibody that binds to CD47, PD-1, CTLA-4, LAG-3, TIM-3, BTLA, PD-L1, 4-1BB, CD27, CD28, CD47, TIGIT, CD27, GITR, or OX40.
- the antibody is a human antibody.
- the present disclosure also relates to the use of the animal model generated through the methods as described herein in the development of a product related to an immunization processes of human cells, the manufacturing of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.
- the disclosure provides the use of the animal model generated through the methods as described herein in the production and utilization of an animal experimental disease model of an immunization processes involving human cells, the study on a pathogen, or the development of a new diagnostic strategy and/or a therapeutic strategy.
- the present disclosure further relates to methods for generating genetically modified animal models described herein with some additional modifications (e.g., human or chimeric genes or additional gene knockout) .
- the animal can comprise a disruption at the endogenous HR gene and a sequence encoding a human or chimeric protein.
- the human or chimeric protein can be programmed cell death protein 1 (PD-1) , TNF Receptor Superfamily Member 9 (4-1BB or CD137) , cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) , LAG-3, T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) , B And T Lymphocyte Associated (BTLA) , Programmed Cell Death 1 Ligand 1 (PD-L1) , CD27, CD28, CD47, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , Glucocorticoid-Induced TNFR-Related Protein (GITR) , or TNF Receptor Superfamily Member 4 (TNFRSF4; or OX40) .
- PD-1BB TNF Receptor Superfamily Member 9
- CTLA-4 cytotoxic T-ly
- the animal can further comprise a disruption (e.g., knockout) at one or more of the following endogenous genes, e.g., IL6, IL15, colony stimulating factor (CSF) , colony stimulating factor 1 (CSF1) , colony stimulating factor 2 (CSF2 or GM-CSF) , colony stimulating factor 3 (CSF3) , signal regulatory protein alpha (SIRPA) , B2M, and KIT proto-oncogene receptor tyrosine kinase (C-KIT) genes.
- the animal can comprise a disruption at the endogenous CD132 gene (interleukin-2 receptor subunit gamma) .
- the animal can comprise a disruption at some other endogenous genes (e.g., Forkhead Box N1 (Foxn1) ) .
- the animal can comprise a disruption at the endogenous B2M gene.
- the methods of HR knockout animal model with additional genetic modifications can include the following steps:
- the genetically modified animal in step (b) of the method, can be mated with a genetically modified non-human animal with human or chimeric PD-1, CTLA-4, LAG-3, TIM-3, BTLA, PD-L1, 4-1BB, CD27, CD28, CD47, TIGIT, GITR, or OX40, or an animal with NOD-Prkdc scid IL-2r ⁇ null mutation and/or B2M mutation.
- the HR knockout can be directly performed on a genetically modified animal having a human or chimeric PD-1, CTLA-4, LAG-3, BTLA, TIM-3, PD-L1, 4-1BB, CD27, CD28, CD47, TIGIT, GITR, or OX40 gene, or an animal with NOD-Prkdc scid IL-2r ⁇ null mutation and/or B2M mutation.
- the mouse further has a disruption of B2M gene (e.g., deletion of one or more exons or part of exons selected from the group consisting of exon 1, exon 2, exon 3, or exon 4 of the endogenous B2M gene) .
- the HR knockout can be directly performed on a CD132 knockout mouse or a Foxn1 knockout mouse.
- a combination therapy that targets two or more of these proteins thereof may be a more effective treatment.
- many related clinical trials are in progress and have shown a good effect.
- the HR knockout animal model, and/or the HR knockout animal model with additional genetic modifications can be used for determining effectiveness of a combination therapy.
- the combination of agents can include one or more agents selected from the group consisting of paclitaxel, cisplatin, carboplatin, pemetrexed, 5-FU, gemcitabine, oxaliplatin, docetaxel, and capecitabine.
- the combination of agents can include one or more agents selected from the group consisting of campothecin, doxorubicin, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, adriamycin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, bleomycin, plicomycin, mitomycin, etoposide, verampil, podophyllotoxin, tamoxifen, taxol, transplatinum, 5-flurouracil, vincristin, vinblastin, and methotrexate.
- campothecin campothecin
- doxorubicin doxorubicin
- cisplatin carboplatin
- procarbazine mechlorethamine
- cyclophosphamide adriamycin
- the combination of agents can include one or more antibodies that bind to CD47, PD-1, CTLA-4, LAG-3, BTLA, TIM-3, PD-L1, 4-1BB, CD27, CD28, CD47, TIGIT, GITR, and/or OX40.
- the methods can also include performing surgery on the subject to remove at least a portion of the cancer, e.g., to remove a portion of or all of a tumor (s) , from the subject.
- Interleukin-2 is a 15.5 kDa type 1 four ⁇ -helical bundle cytokine produced primarily by CD4+ T cells following their activation by antigen.
- IL-2 was the first type 1 cytokine cloned and the first cytokine for which a receptor component was cloned.
- the ligand-specific IL-2 receptor ⁇ chain (IL-2R ⁇ , CD25, Tac antigen) , which is expressed on activated but not non-activated lymphocytes, binds IL-2 with low affinity (Kd ⁇ 10 -8 M) ; the combination of IL-2R ⁇ (CD122) and IL-2R ⁇ (CD132) together form an IL-2R ⁇ / ⁇ c complex mainly on memory T cells and NK cells that binds IL-2 with intermediate affinity (Kd ⁇ 10 -9 M) ; and when all three receptor chains are co-expressed on activated T cells and Treg cells, IL-2 is bound with high affinity (Kd ⁇ 10 -11 M) .
- the three dimensional structure of the quaternary complex supports a model wherein IL-2 initially bind IL-2R ⁇ , then IL-2R ⁇ is recruited, and finally IL-2R ⁇ .
- the intermediate and high affinity receptor forms are functional, transducing IL-2 signals.
- CD132 is also an essential component shared by the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.
- IL-2R ⁇ is encoded by the gene, IL2RG (CD132) , that is mutated in humans with X-linked severe combined immunodeficiency (XSCID) and physically recruits JAK3, which when mutated also causes an XSCID-like T-B+NK-form of SCID.
- XSCID and JAK3-deficient SCID the lack of signaling by IL-7 and IL-15, respectively, explains the lack of T and NK cell development, whereas defective signaling by IL-4 and IL-21 together explain the non-functional B cells and hypogammaglobulinemia.
- CD132 A detailed description of CD132 and its function can be found, e.g., in Liao et al. "IL-2 family cytokines: new insights into the complex roles of IL-2 as a broad regulator of T helper cell differentiation, " Current opinion in immunology 23.5 (2011) : 598-604; Noguchi et al. "Interleukin-2 receptor gamma chain: a functional component of the interleukin-7 receptor, " Science 262.5141 (1993) : 1877-1880; Henthorn et al. "IL-2R ⁇ gene microdeletion demonstrates that canine X-linked severe combined immunodeficiency is a homologue of the human disease, " Genomics 23.1 (1994) : 69-74; and US Patent No. 7145055; each of which is incorporated herein by reference in its entirety.
- CD132 gene In human genomes, CD132 gene (Gene ID: 3561) is located on X chromosome, and has eight exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8.
- the CD132 protein also has an extracellular region, a transmembrane region, and a cytoplasmic region.
- the nucleotide sequence for human CD132 mRNA is NM_000206.2
- amino acid sequence for human CD132 is NP_000197.1.
- CD132 gene locus has eight exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8.
- the CD132 protein also has an extracellular region, a transmembrane region, and a cytoplasmic region, and the signal peptide is located at the extracellular region of CD132.
- the nucleotide sequence for mouse CD132 cDNA is NM_013563.4 (SEQ ID NO: 29)
- amino acid sequence for mouse CD132 is NP_038591.1 (SEQ ID NO: 30) .
- the location for each exon and each region in the mouse CD132 nucleotide sequence and amino acid sequence is listed below:
- the mouse CD132 gene (Gene ID: 16186) is located in Chromosome X of the mouse genome, which is located from 101, 268, 255 to 101, 264, 385 of NC_000086.7 (GRCm38. p4 (GCF_000001635.24) ) .
- the 5’-UTR is from 101, 268, 255 to 101, 268, 170, exon 1 is from 101, 268, 255 to 101, 268, 055, the first intron (intron 1) is from 101, 268, 054 to 101, 267, 865, exon 2 is from 101, 267, 864 to 101, 267, 711, the second intron (intron 2) is from 101, 267, 710 to 101, 267, 496, exon 3 is from 101, 267, 495 to 101, 267, 311, the third intron (intron 3) is from 101, 267, 310 to 101, 267, 121, exon 4 is from 101, 267, 120 to 101, 266, 978, the fourth intron (intron 4) is from 101, 266, 977 to 101, 266, 344, exon 5 is from 101, 266, 343 to 101, 266, 181, the fifth intron (intron 5) is from 101, 266, 180 to 101, 265, 727, exon 6 is from 101,
- CD132 genes, proteins, and locus of the other species are also known in the art.
- the gene ID for CD132 in Rattus norvegicus is 140924
- the gene ID for CD132 in Macaca mulatta (Rhesus monkey) is 641338,
- the gene ID for CD132 in Sus scrofa (pig) is 397156.
- the relevant information for these genes can be found, e.g., intron sequences, exon sequences, amino acid residues of these proteins
- NCBI database e.g., NCBI database.
- the present disclosure provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal’s endogenous CD132 gene, wherein the disruption of the endogenous CD132 gene comprises deletion of one or more exons, or part of the one or more exons, wherein the one or more exons are selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 of the endogenous CD132 gene.
- the disclosure provides a genetically-modified, non-human animal that does not have one or more exons that are selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 of the endogenous CD132 gene.
- At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides in the exon of CD132 are deleted.
- the disruption comprises deletion of one or more introns, or part of the one or more introns, wherein the one or more introns are selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7 of the endogenous CD132 gene.
- the disclosure provides a genetically-modified, non-human animal does not have one or more introns that are selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7 of the endogenous CD132 gene.
- the disruption of the endogenous CD132 gene comprises deletion of exon 2 of the endogenous CD132 gene. In some embodiments, the disruption of the endogenous CD132 gene further comprises deletion of exon 1, or part of exon 1 of the endogenous CD132 gene.
- the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 are deleted.
- the signal peptide region, extracellular region, transmembrane region, and/or cytoplasmic region of CD132 are deleted.
- a “region” or “portion” of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7, signal peptide region, extracellular region, transmembrane region, and/or cytoplasmic region are deleted.
- the “region” or “portion” can be at least 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7, signal peptide region, extracellular region, transmembrane region, or cytoplasmic region of CD132.
- a region, a portion, or the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8 of CD132 are deleted.
- a region, a portion, or the entire sequence of mouse intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and/or intron 7 are deleted.
- the disruption comprises or consists of deletion of more than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 nucleotides in exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8.
- the disruption comprises or consists of deletion of more than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, or 1000 nucleotides in intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and/or intron 7.
- the disruption comprises or consists of deletion of more than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (e.g., about 150 or 160 nucleotides) in exon 1; deletion of the entirety of intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7; and/or deletion of more than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, or 650 nucleotides (e.g., about 200, 250 or 270 nucleotides) in exon 8.
- nucleotides e.g., about 150 or 160 nucleotides in exon 8.
- the length of the remaining exon sequences at the endogenous CD132 gene locus is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, or 50%of the total length of all exon sequences of the endogenous CD132 gene.
- the length of the remaining exon sequences at the endogenous CD132 gene locus is more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, or 50%of the total length of all exon sequences of the endogenous CD132 gene.
- the length of the remaining sequences at that the endogenous CD132 gene locus is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, or 50%of the full sequence of the endogenous CD132 gene.
- the length of the remaining sequences at that the endogenous CD132 gene locus is more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, or 50%of the full sequence of the endogenous CD132 gene.
- the present disclosure further relates to the genomic DNA sequence of a CD132 knockout mouse.
- the genome of the animal comprises from 5’ to 3’ at the endogenous CD132 gene locus, (a) a first DNA sequence; optionally (b) a second DNA sequence comprising an exogenous sequence; (c) a third DNA sequence, wherein the first DNA sequence, the optional second DNA sequence, and the third DNA sequence are linked.
- the second DNA sequence can have a length of 0 nucleotides to 300 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 nucleotides) .
- the second DNA sequence has only 0 nucleotides, which means that there is no extra sequence between the first DNA sequence and the third DNA sequence.
- the second DNA sequence has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 nucleotides.
- the second DNA sequence has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 nucleotides.
- the first DNA sequence comprises an endogenous CD132 gene sequence that is located upstream of intron 1, and can include all or just part of sequences that is located upstream of intron 1. In some embodiments, the first DNA sequence comprises an endogenous CD132 gene sequence that is located upstream of exon 1. In some embodiments, the first DNA sequence comprises a sequence that has a length (5’ to 3’) of from 10 to 200 nucleotides (e.g., from 10 to 100 nucleotides, or from 10 to 20 nucleotides) starting from the first nucleotide in exon 1 of the CD132 gene to the last nucleotide of the first DNA sequence.
- the first DNA sequence comprises at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides from exon 1. In some embodiments, the first DNA sequence has at most 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides from exon 1.
- the third DNA sequence comprises an endogenous CD132 gene sequence that is located downstream of the last intron (e.g., intron 7 in mouse) , and can include all or just part of sequences that is located downstream of intron 7.
- the third DNA sequence comprises a sequence that has a length (5’ to 3’) of from 200 to 600 nucleotides (e.g., from 300 to 400 nucleotides, or from 350 to 400 nucleotides) starting from the first nucleotide in the third DNA sequence to the last nucleotide in the last exon (e.g., exon 8 in mouse) of the endogenous CD132 gene.
- the third DNA sequence comprises at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 nucleotides from the last exon (e.g., exon 8 in mouse) . In some embodiments, the third DNA sequence has at most 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 nucleotides from the last exon (e.g., exon 8 in mouse) .
- the disclosure relates to a genetically-modified, non-human animal whose genome comprise a disruption in the animal’s endogenous CD132 gene, wherein the disruption of the endogenous CD132 gene comprises deletion of exon 2 of the endogenous CD132 gene.
- the disruption of the endogenous CD132 gene further comprises deletion of exon 1 of the endogenous CD132 gene. In some embodiments, the disruption of the endogenous CD132 gene comprises deletion of part of exon 1 of the endogenous CD132 gene.
- the disruption of the endogenous CD132 gene further comprises deletion of one or more exons or part of exons selected from the group consisting of exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 of the endogenous CD132 gene. In some embodiments, the disruption of the endogenous CD132 gene comprises deletion of exons 1-8 of the endogenous CD132 gene.
- the disruption of the endogenous CD132 gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7 of the endogenous CD132 gene.
- the disruption consists of deletion of more than 150 nucleotides in exon 1; deletion of the entirety of intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7; and deletion of more than 250 nucleotides in exon 8.
- the animal is homozygous with respect to the disruption of the endogenous CD132 gene. In some embodiments, the animal is heterozygous with respect to the disruption of the endogenous CD132 gene.
- the disruption prevents the expression of functional CD132 protein.
- the length of the remaining exon sequences at the endogenous CD132 gene locus is less than 30%of the total length of all exon sequences of the endogenous CD132 gene. In some embodiments, the length of the remaining sequences at that the endogenous CD132 gene locus is less than 15%of the full sequence of the endogenous CD132 gene.
- the disclosure relates to a genetically-modified, non-human animal, wherein the genome of the animal does not have exon 2 of CD132 gene at the animal’s endogenous CD132 gene locus.
- the genome of the animal does not have one or more exons or part of exons selected from the group consisting of exon 1, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8. In some embodiments, the genome of the animal does not have one or more introns or part of introns selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7.
- the disclosure also provides a CD132 knockout non-human animal, wherein the genome of the animal comprises from 5’ to 3’ at the endogenous CD132 gene locus, (a) a first DNA sequence; optionally (b) a second DNA sequence comprising an exogenous sequence; (c) a third DNA sequence, wherein the first DNA sequence, the optional second DNA sequence, and the third DNA sequence are linked, wherein the first DNA sequence comprises an endogenous CD132 gene sequence that is located upstream of intron 1, the second DNA sequence can have a length of 0 nucleotides to 300 nucleotides, and the third DNA sequence comprises an endogenous CD132 gene sequence that is located downstream of intron 7.
- the first DNA sequence comprises a sequence that has a length (5’ to 3’) of from 10 to 100 nucleotides (e.g., approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides) , wherein the length of the sequence refers to the length from the first nucleotide in exon 1 of the CD132 gene to the last nucleotide of the first DNA sequence.
- the first DNA sequence comprises at least 10 nucleotides from exon 1 of the endogenous CD132 gene. In some embodiments, the first DNA sequence has at most 100 nucleotides from exon 1 of the endogenous CD132 gene.
- the third DNA sequence comprises a sequence that has a length (5’ to 3’) of from 200 to 600 nucleotides (e.g., approximately 200, 250, 300, 350, 400, 450, 500, 550, 600 nucleotides) , wherein the length of the sequence refers to the length from the first nucleotide in the third DNA sequence to the last nucleotide in exon 8 of the endogenous CD132 gene.
- the third DNA sequence comprises at least 300 nucleotides from exon 8 of the endogenous CD132 gene. In some embodiments, the third DNA sequence has at most 400 nucleotides from exon 8 of the endogenous CD132 gene.
- the genetic modified non-human animal comprises a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical or 100%identical to the following sequence: aggaaatgtatggtggggagggcttgtgggagagctaagtttcgatttcctgtcccatgtaactgctttttctgtccatatgccctact tgagagtgtcccttgcctctttccctgcacaagccctcccatgcccagcctaacacctttccactttctttgaagagagtcttaccct gtagcccagggtggctgggagctcactatgtaggccaggttggctgggagctcactatgtagg
- the disclosure also relates to a genetically-modified, non-human animal produced by a method comprising knocking out one or more exons of endogenous CD132 gene by using (1) a first nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a target sequence in exon 1 of the endogenous CD132 gene or upstream of exon 1 of the endogenous CD132 gene, and (2) a second nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a sequence in exon 8 of the endogenous CD132 gene.
- a first nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a target sequence in exon 1 of the endogenous CD132 gene or
- the nuclease is CRISPR associated protein 9 (Cas9) .
- the target sequence is in exon 1 of the endogenous CD132 gene or upstream of exon 1 of the endogenous CD132 gene. In some embodiments, the target sequence is in exon 8 of the endogenous CD132 gene.
- the animal does not express a functional CD132 protein. In some embodiments, the animal does not express a functional interleukin-2 receptor.
- the animal further comprises a disruption in the animal’s endogenous Beta-2-Microglobulin (B2m) gene and/or a disruption in the animal’s endogenous Forkhead Box N1 (Foxn1) gene.
- B2m Beta-2-Microglobulin
- Foxn1 Endogenous Forkhead Box N1
- the disclosure is also related to methods of producing a CD132 knockout mouse.
- the methods involve
- the disclosure also provides methods of producing a CD132 knockout mouse.
- the methods include the steps of
- the gene editing system comprises a first nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a target sequence in exon 1 of the endogenous CD132 gene or upstream of exon 1 of the endogenous CD132 gene, and a second nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a sequence in exon 8 of the endogenous CD132 gene.
- sgRNA single guide RNA
- the mouse embryonic stem cell has a Nod/scid background, or a NOD/scid nude background.
- the mouse embryonic stem cell has a genome comprising a disruption in the animal’s endogenous Beta-2-Microglobulin (B2m) gene and/or a disruption in the animal’s endogenous Forkhead Box N1 (Foxn1) gene.
- B2m Beta-2-Microglobulin
- Foxn1 Endogenous Forkhead Box N1
- the disclosure relates to a non-human mammalian cell, comprising a disruption, a deletion, or a genetic modification as described herein.
- the cell includes Cas9 mRNA or an in vitro transcript thereof.
- the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell. In some embodiments, the cell is a germ cell. In some embodiments, the cell is a blastocyst.
- the disclosure relates to methods for establishing a CD132 knockout animal model.
- the methods include the steps of:
- the cell is a fertilized egg cell
- step (d) identifying the germline transmission in the offspring of the pregnant female in step (c) .
- the establishment of a CD132 knockout animal involves a gene editing technique that is based on CRISPR/Cas9.
- the non-human mammal is mouse. In some embodiments, the non-human mammal in step (c) is a female with false pregnancy.
- NOD-Prkdc scid IL-2rg null (B-NDG) mice were obtained from Beijing Biocytogen Co., Ltd (Catalog number: B-CM-002) .
- Ambion TM in vitro transcription kit was purchased from Ambion, Inc. The catalog number is AM1354.
- E. coli TOP10 competent cells were purchased from the Tiangen Biotech (Beijing) Co. The catalog number is CB104-02.
- EcoRI, BamHI, and BbsI were purchased from NEB. The catalog numbers are R3101M, R3136M, and R0539L.
- Kanamycin was purchased from Amresco. The catalog number is 0408.
- Cas9 mRNA was obtained from SIGMA.
- the catalog number is CAS9MRNA-1EA.
- UCA kit was obtained from Beijing Biocytogen Co., Ltd. The catalog number is BCG-DX-001.
- pHSG299 plasmids were purchased from Takara. The catalog number is 3299.
- the target sequence determines the targeting specificity of small guide RNA (sgRNA) and the efficiency of Cas9 cleavage at the target site. Therefore, target sequence selection is important for sgRNA vector construction.
- sgRNA small guide RNA
- sgRNAs were designed for the mouse HR gene (NCBI Gene ID: 15460) .
- the target sequences for these sgRNAs are shown below:
- sgRNA1 target sequence (SEQ ID NO: 1) : 5’-acccgacaggctcgagtcactgg-3’
- sgRNA2 target sequence (SEQ ID NO: 2) : 5’-cctggcactgccgtcgggcttgg -3’
- sgRNA3 target sequence (SEQ ID NO: 3) : 5’-ccccagagagacgcaagcgaggg-3’
- sgRNA4 target sequence (SEQ ID NO: 4) : 5’-cgctgctaactgaagcccggagg-3’
- sgRNA5 target sequence (SEQ ID NO: 5) : 5’-ttccctcgcttgcgtctctctgg -3’
- sgRNA6 target sequence (SEQ ID NO: 6) : 5’-ggtgccctggcactgccgtcggg-3’
- sgRNA7 target sequence (SEQ ID NO: 7) : 5’-cccagtgactcgagcctgtcggg-3’
- sgRNA8 target sequence (SEQ ID NO: 8) : 5’-ggtgctagggaccggaacgtagg-3’
- sgRNA9 target sequence (SEQ ID NO: 9) : 5’-aaacaggaggacctacgttccgg-3’
- sgRNA10 target sequence (SEQ ID NO: 10) : 5’-gcaatgtttaagtcgagccaggg-3’
- sgRNA11 target sequence (SEQ ID NO: 11) : 5’-gcatgtatgacggtcagatttgg-3’
- sgRNA12 target sequence (SEQ ID NO: 12) : 5’-tgcacgtgcacgcatgccctcgg-3’
- sgRNA13 target sequence (SEQ ID NO: 13) : 5’-tctacattaacatcgtgaaatgg-3’
- sgRNA14 target sequence (SEQ ID NO: 14) : 5’-attcagtccgatccttctcaagg-3’
- sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5, sgRNA6, and sgRNA7 target the 5’-end target site and sgRNA8, sgRNA9, sgRNA10, sgRNA11, sgRNA12, sgRNA13, and sgRNA14 target the 3’-end target site.
- the target sites for sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5, sgRNA6, and sgRNA7 are located within intron 2 of the mouse endogenous HR gene (Gene ID: 15460) .
- the target sites for sgRNA8, sgRNA9, sgRNA10, sgRNA11, sgRNA12, sgRNA13, and sgRNA14 are located within intron 7 of HR (based on the sequence of NM_021877.3 ⁇ NP_068677.2) .
- the UCA kit was used to detect the activities of sgRNAs (FIG. 1 and Table 4) .
- the results show that the sgRNAs had different activities. Two of them (sgRNA4 and sgRNA10) were selected for further experiments.
- Single strand oligonucleotides were synthesized for sgRNA4 and sgRNA10.
- TAGG was first added to the 5’ end of the upstream sequence of sgRNA4 and sgRNA10 target sequences to obtain a forward oligonucleotide sequence
- AAAC was added to the 5’ end of the complementary strand to obtain a reverse oligonucleotide sequence.
- pT7-sgRNA G2 vector map is shown in FIG. 2.
- the DNA fragment containing T7 promoter and sgRNA scaffold was synthesized, and linked to the backbone vector pHSG299 by restriction enzyme digestion (EcoRI and BamHI) and ligation.
- the plasmid sequences were confirmed by sequencing.
- the DNA fragment containing the T7 promoter and sgRNA scaffold (SEQ ID NO: 23) is shown below:
- the product was ligated into the pT7-sgRNA G2 plasmid (the plasmid was first treated by BbsI restriction enzyme) .
- the ligation reaction was carried out at room temperature for 10 to 30 minutes.
- the ligation product was then transferred to 30 ⁇ L of TOP10 competent cells.
- the cells were then plated on a petri dish with Kanamycin, and then cultured at 37 °C for at least 12 hours and then two clones were selected and added to LB medium with Kanamycin (5 ml) , and then cultured at 37 °C at 250 rpm for at least 12 hours.
- Clones were randomly selected and sequenced to verify their sequences.
- the pT7-HR-4 and pT7-HR-10 vectors with correct sequences were selected for subsequent experiments.
- the pre-mixed Cas9 mRNA, in vitro transcription products of pT7-HR-4 and pT7-HR-10 plasmids were injected into the cytoplasm or nucleus of B-NDG mouse fertilized eggs with a microinjection instrument (using Ambion in vitro transcription kit to carry out the transcription according to the method provided in the product instruction) .
- the embryo microinjection was carried out according to the method described, e.g., in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition) , ” Cold Spring Harbor Laboratory Press, 2003.
- the injected fertilized eggs were then transferred to a culture medium to culture for a short time and then was transplanted into the oviduct of the recipient mouse to produce the genetically modified mice (F0 generation) .
- the mouse population was further expanded by cross-mating and self-mating to establish stable mouse lines.
- genomic DNA was extracted from the tail of the F0 generation mice obtained in Example 5.
- PCR was performed with the primer MSD-F (SEQ ID NO: 24) and MSD-R (SEQ ID NO: 25) .
- MSD-F is located on the left of the sgRNA4 target site.
- MSD-R is located on the right of the sgRNA10 target site.
- the length of PCR amplification products in the wildtype (WT) mice should be about 7859 bp. In the knockout mice, the length of the PCR amplification products should be about 610 bp.
- the sequence for the primers are shown below:
- MSD-F (SEQ ID NO: 24) : 5’-gctcacgtacatccatccctcttgg -3’
- MSD-R (SEQ ID NO: 25) : 5’-tagaattcttgtttttggaacgcaga -3’
- ⁇ indicates knockout, e.g., ⁇ 7258 indicates a deletion of 7258bp, and ⁇ 7252 indicates a deletion of 7252bp.
- F0-28 a sequence of 7257 nucleotides (SEQ ID NO: 33) at the endogens HR locus is deleted.
- the mouse has a sequence that is identical to SEQ ID NO: 28 at the endogens HR locus.
- F0-18 a sequence of 7253 nucleotides (SEQ ID NO: 34) at the endogens HR locus is deleted.
- the mouse has a sequence that is identical to SEQ ID NO: 35 at the endogens HR locus.
- F0 generation mice were then mated with B-NDG mice to obtain F1 generation mice. Both F0-18 and F0-28 were mated with wild-type B-NDG mice, and their offspring were tested. Gene identification showed that a total of six F1 generation mice were positive.
- the PCR results were shown in FIGS. 4 and 5.
- the mice labeled with F1-2, F1-3 and F1-7 were F0-18 offspring.
- the mice labeled with F1-10, F1-11, F1-12 were offspring of F0-28.
- the six mice were further sequenced and verified, and the sequencing results are shown in Table 9. The results indicate that the method can be used to make a Hr knockout mouse.
- FIG. 6 shows B-NDG mice that have wildtype Hr genes.
- mice obtained by the methods as described herein a human immune system was constructed by engraftment with human peripheral blood cells (hPBMC) .
- hPBMC human peripheral blood cells
- hPBMCs human peripheral blood cells
- mice peripheral blood cells engraftment on these mice can create a humanized mouse model with the human immune system. Furthermore, human tumor cells were injected into these mice (FIG. 8) . These mice can be used to screen new drugs, and test drug efficacy.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Environmental Sciences (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The genetically modified non-human animals that have a disruption at the endogenous HR gene (e.g., HR knockout), and methods of use thereof are provided.
Description
CLAIM OF PRIORITY
This application claims the benefit of Chinese Patent Application App. No. 201810163134.3, filed on February 26, 2018, and Chinese Patent Application App. No. 201811543165.8, filed on December 17, 2018. The entire contents of the foregoing are incorporated herein by reference.
This disclosure relates to genetically modified animals that have a disruption at the endogenous HR gene (e.g., HR knockout) , and methods of use thereof.
Immunodeficient animals are very important for disease modeling and drug developments. In recent years, immunodeficient mice are routinely used as model organisms for research of the immune system, cell transplantation strategies, and the mechanisms of diseases. They have also been extensively used as hosts for normal and malignant tissue transplants, and are widely used to test the safety and efficacy of therapeutic agents.
In many experiments that involve immunodeficient animals (e.g., mice) , bioluminescence and fluorescence imaging are often used to observe the transplanted cells (e.g., tumor cells) . However, hair on the animal may interfere with the measurement and observation. There is a need for hairless immunodeficient animal models.
SUMMARY
This disclosure is related to genetically modified animals that have a disruption at the endogenous HR gene (e.g., HR knockout) , and methods of making and use thereof.
In one aspect, the disclosure relates to a genetically-modified, non-human animal whose genome comprise a disruption in the animal’s endogenous HR lysine demethylase and nuclear receptor corepressor (HR) gene. In some embodiments, the disruption of the endogenous HR gene comprises deletion of one or more exons of the endogenous HR gene.
In some embodiments, the disruption of the endogenous HR gene comprises deletion of one or more exons selected from exon 3, exon 4, exon 5, exon 6, and exon 7 of the endogenous HR gene.
In some embodiments, the disruption of the endogenous HR gene comprises deletion of exon 3, exon 4, exon 5, exon 6, and exon 7 of the endogenous HR gene.
In some embodiments, the disruption of the endogenous HR gene further comprises deletion of one or more exons or part of exons selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, and exon 20 of the endogenous HR gene.
In some embodiments, the disruption of the endogenous HR gene comprises deletion of one or more introns of the endogenous HR gene.
In some embodiments, the disruption of the endogenous HR gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, intron 17, intron 18, and intron 19 of the endogenous HR gene.
In some embodiments, the disruption consists of deletion of at least 10 nucleotides in intron 2; deletion of the entirety of exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, and exon 7; and deletion of at least 10 nucleotides in intron 7.
In some embodiments, the animal is homozygous with respect to the disruption of the endogenous HR gene. In some embodiments, the animal is heterozygous with respect to the disruption of the endogenous HR gene.
In some embodiments, the disruption prevents the expression of functional HR protein.
In some embodiments, the length of the remaining exon sequences at the endogenous HR gene locus is less than 70%of the total length of all exon sequences of the endogenous HR gene. In some embodiments, the length of the remaining sequences at that the endogenous HR gene locus is less than 65%of the full sequence of the endogenous HR gene.
In one aspect, the disclosure relates to a genetically-modified, non-human animal, wherein the genome of the animal does not have one or more exons of HR gene at the animal’s endogenous HR gene locus.
In some embodiments, the genome of the animal does not have one or more exons or part of exons selected from the group consisting of exon 3, exon 4, exon 5, exon 6, and exon 7.
In some embodiments, the genome of the animal does not have one or more introns or part of introns selected from the group consisting of intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7.
In one aspect, the disclosure relates to a HR knockout non-human animal, wherein the genome of the animal comprises from 5’ to 3’ at the endogenous HR gene locus, (a) a first DNA sequence; optionally (b) a second DNA sequence comprising an exogenous sequence; (c) a third DNA sequence. In some embodiments, the first DNA sequence, the optional second DNA sequence, and the third DNA sequence are linked.
In some embodiments, the first DNA sequence comprises an endogenous HR gene sequence that is located upstream of intron 2, the second DNA sequence can have a length of 0 nucleotides to 100 nucleotides, and the third DNA sequence comprises an endogenous HR gene sequence that is located downstream of intron 7.
In some embodiments, the first DNA sequence comprises a sequence that has a length (5’ to 3’) of from 10 to 1500 nucleotides.
In some embodiments, the length of the sequence refers to the length from the first nucleotide in exon 1 of the HR gene to the last nucleotide of the first DNA sequence.
In some embodiments, the first DNA sequence comprises at least 10 nucleotides from intron 2 of the endogenous HR gene.
In some embodiments, the first DNA sequence comprise exon 1 and exon 2 of the endogenous HR gene.
In some embodiments, the third DNA sequence comprises a sequence that has a length (5’ to 3’) of from 10 to 11000 nucleotides.
In some embodiments, the length of the sequence refers to the length from the first nucleotide in the third DNA sequence to the last nucleotide in the last exon of the endogenous HR gene.
In some embodiments, the third DNA sequence comprises at least 10 nucleotides from intron 7 of the endogenous HR gene.
In some embodiments, the third DNA sequence comprises exons 8-20, and introns 8-19.
In one aspect, the disclosure relates to a genetically-modified, non-human animal produced by a method comprising knocking out one or more exons of endogenous HR gene by using (1) a first nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a target sequence in intron 2 of the endogenous HR gene, and (2) a second nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a sequence in intron 7 of the endogenous HR gene.
In some embodiments, the nuclease is CRISPR associated protein 9 (Cas9) .
In some embodiments, the target sequence in intron 2 of the endogenous HR gene is set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, and the target sequence in intron 7 of the endogenous HR gene is set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14.
In some embodiments, the first nuclease comprises a sgRNA that targets SEQ ID NO: 4 and the second nuclease comprises a sgRNA that targets SEQ ID NO: 10.
In some embodiments, the animal does not express a functional HR protein.
In some embodiments, the animal does not express a functional interleukin-2 receptor.
In some embodiments, the animal has one or more of the following characteristics:
(a) the percentage of T cells (CD3+ cells) is less than 5%, 2%, 1.5%, 1%, 0.7%, or 0.5%of leukocytes in the animal;
(b) the percentage of B cells (e.g., CD3-CD19+ cells) is less than 1%, 0.1%or 0.05%of leukocytes in the animal;
(c) the percentage of NK cells (e.g., CD3-CD49b+ cells) is less than 5%, 2%or 1.5%of leukocytes in the animal;
(d) the percentage of CD4+ T cells is less than 1%, 0.5%, 0.3%, or 0.1%of T cells;
(e) the percentage of CD8+ T cells is less than 1%, 0.5%, 0.3%, or 0.1%of T cells;
(f) the percentage of CD3+ CD4+ cells, CD3+ CD8+ cells, CD3-CD19+ cells is less than 5%, 1%or 0.5%of leukocytes in the animal;
(g) the percentage of T cells, B cells, and NK cells is less than 5%, 4%, 3%, 2%or 1%of leukocytes in the animal.
In some embodiments, the animal after being engrafted with human hematopoietic stem cells to develop a human immune system has one or more of the following characteristics:
(a) the percentage of human CD45+ cells is about or at least 10%, 20%, 30%, 40%, or 50%of leukocytes of the animal;
(b) the percentage of human CD3+ cells about or at least 10%, 20%, 30%, 40%, or 50%of leukocytes in the animal;
(c) the percentage of human CD19+ cells is about or at least 10%, 20%, 30%, 40%, or 50%of leukocytes in the animal.
In some embodiments, the animal does not have hair.
In some embodiments, the animal has one or more of the following characteristics:
(a) the animal has no functional T-cells and/or no functional B-cells;
(b) the animal exhibits reduced macrophage function relative to a NOD/scid mouse;
(c) the animal exhibits no NK cell activity;
(d) the animal exhibits reduced dendritic function relative to a NOD/scid mouse; and
(e) the animal does not have xenogeneic GVHD.
In some embodiments, the animal is a mammal, e.g., a monkey, a rodent, a rat, or a mouse.
In some embodiments, the animal is a C57 mouse, a C57BL mouse, a BALB/c mouse, a NOD/scid mouse, or a NOD/scid nude mouse, or a NOD-Prkdc
scid IL-2rγ
null mouse.
In some embodiments, the animal is an immune deficient animal. In some embodiments, the animal is not an immune deficient animal.
In some embodiments, the animal further comprises a sequence encoding a human or chimeric protein. In some embodiments, the human or chimeric protein is programmed cell death protein 1 (PD-1) , PD-L1, IL3, IL6, IL15, CSF1, or CSF2. In some embodiments, the animal further comprises a disruption in the animal’s endogenous Beta-2-Microglobulin (B2M) gene.
In one aspect, the disclosure relates to methods of determining effectiveness of an agent or a combination of agents for the treatment of cancer. The methods involve engrafting tumor cells to the animal as described herein, thereby forming one or more tumors in the animal; administering the agent or the combination of agents to the animal; and determining the inhibitory effects on the tumors.
In some embodiments, before engrafting the tumor cells to the animal, human peripheral blood cells (hPBMC) or human hematopoietic stem cells are injected to the animal.
In some embodiments, the tumor cells are from cancer cell lines. In some embodiments, the tumor cells are from a tumor sample obtained from a human patient.
In some embodiments, the inhibitory effects are determined by measuring the tumor volume in the animal.
In some embodiments, the tumor cells are melanoma cells, lung cancer cells, primary lung carcinoma cells, non-small cell lung carcinoma (NSCLC) cells, small cell lung cancer (SCLC) cells, primary gastric carcinoma cells, bladder cancer cells, breast cancer cells, and/or prostate cancer cells.
In some embodiments, the agent is an anti-PD-1 antibody. In some embodiments, the agent is an anti-PD-L1 antibody.
In some embodiments, the combination of agents comprises one or more agents selected from the group consisting of paclitaxel, cisplatin, carboplatin, pemetrexed, 5-FU, gemcitabine, oxaliplatin, docetaxel, and capecitabine.
In one aspect, the disclosure relates to methods of producing an animal comprising a human hemato-lymphoid system. The methods involve engrafting a population of cells comprising human hematopoietic cells or human peripheral blood cells into the animal as described herein.
In some embodiments, the human hemato-lymphoid system comprises human cells selected from the group consisting of hematopoietic stem cells, myeloid precursor cells, myeloid cells, dendritic cells, monocytes, granulocytes, neutrophils, mast cells, lymphocytes, and platelets.
In some embodiments, the methods further comprise irradiating the animal prior to the engrafting.
In one aspect, the disclosure relates to methods of producing a HR knockout mouse. The methods involve:
(a) transforming a mouse embryonic stem cell or a fertilized egg with a gene editing system that targets endogenous HR gene, thereby producing a transformed embryonic stem cell;
(b) introducing the transformed embryonic stem cell or fertilized egg into a mouse blastocyst;
(c) implanting the mouse blastocyst into a pseudopregnant female mouse; and
(d) allowing the blastocyst to undergo fetal development to term,
thereby obtaining the HR knockout mouse.
In one aspect, the disclosure relates to methods of producing a HR knockout mouse. The methods involve
(a) transforming a mouse embryonic stem cell or a fertilized egg with a gene editing system that targets endogenous HR gene, thereby producing a transformed embryonic stem cell;
(b) implanting the transformed embryonic cell or the fertilized egg into a pseudopregnant female mouse; and
(c) allowing the transformed embryonic cell to undergo fetal development to term, thereby obtaining the HR knockout mouse.
In some embodiments, the gene editing system comprises a first nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a target sequence in intron 2 of the endogenous HR gene, and a second nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a sequence in intron 7 of the endogenous HR gene.
In some embodiments, the nuclease is CRISPR associated protein 9 (Cas9) .
In some embodiments, the target sequence in intron 2 of the endogenous HR gene is set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, and the target sequence in intron 7 of the endogenous HR gene is set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14.
In some embodiments, the mouse embryonic stem cell or the fertilized egg has a C57 background, a C57BL background, a BALB background (e.g., BALB/c background) , a NOD/scid background, a NOD/scid nude, or a NOD-Prkdc
scid IL-2rγ
null background.
In some embodiments, the mouse embryonic stem cell or the fertilized egg comprises a sequence encoding a human or chimeric protein.
In some embodiments, the human or chimeric protein is PD-1 or CD137.
In some embodiments, the mouse embryonic stem cell or the fertilized egg has a genome comprising a disruption in the animal’s endogenous B2M gene.
In another aspect, the disclosure relates to a non-human mammalian cell, comprising a disruption, a deletion, or a genetic modification as described herein.
In some embodiments, the cell includes Cas9 mRNA or an in vitro transcript thereof.
In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell. In some embodiments, the cell is a germ cell. In some embodiments, the cell is a blastocyst.
In another aspect, the disclosure relates to methods for establishing a HR knockout animal model. The methods include the steps of:
(a) providing the cell with a disruption in the endogenous HR gene, and
preferably the cell is a fertilized egg cell;
(b) culturing the cell in a liquid culture medium;
(c) transplanting the cultured cell to the fallopian tube or uterus of the recipient female non-human mammal, allowing the cell to develop in the uterus of the female non-human mammal;
(d) identifying the germline transmission in the offspring of the pregnant female in step (c) .
In some embodiments, the establishment of a HR knockout animal involves a gene editing technique that is based on CRISPR/Cas9.
In some embodiments, the non-human mammal is a mouse. In some embodiments, the non-human mammal in step (c) is a female with false pregnancy.
In another aspect, the disclosure relates to a tumor bearing non-human mammal model, characterized in that the non-human mammal model is obtained through the methods as described herein.
The disclosure also relates to a cell or cell line, or a primary cell culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal.
The disclosure further relates to the tissue, organ or a culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal.
In another aspect, the disclosure relates to a tumor tissue derived from the non-human mammal or an offspring thereof when it bears a tumor, or the tumor bearing non-human mammal.
The disclosure further relates to the use of the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal, the animal model generated through the method as described herein in the development of a product related to an immunization processes of human cells, the manufacture of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.
The disclosure also relates to the use of the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal, the animal model generated through the method as described herein in the production and utilization of an animal experimental disease model of an immunization processes involving human cells, the study on a pathogen, or the development of a new diagnostic strategy and/or a therapeutic strategy.
The disclosure further relates to the use of the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal, the animal model generated through the methods as described herein, in the screening, verifying, evaluating or studying the HR gene function, and the drugs for immune-related diseases and antitumor drugs.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
DESCRIPTION OF DRAWINGS
FIGS. 1A-1B are bar graphs showing activity testing results for sgRNA1-sgRNA14 (NC is a negative control; PC is a positive control) .
FIG. 2 is a schematic diagram showing pT7-sgRNA G2 plasmid map.
FIG. 3 shows PCR identification results for samples collected from tails of F0 generation mice (WT is a NOD-Prkdc
scid IL-2rg
null (B-NDG) mouse) . Among them, F0-1, F0-4, F0-18, F0-21, F0-28, F0-35, F0-37, F0-38, F0-41, and F0-45 are positive.
FIG. 4 shows PCR identification results for samples collected from tails of F1 generation mice (offspring of F0-18 and a B-NDG mouse) . M is the Marker; WT is B-NDG mouse, + is a positive control, H
2O is a negative control.
FIG. 5 shows PCR identification results for samples collected from tails of F1 generation mice (offspring of F0-28 and a B-NDG mouse) . M is the Marker; WT is B-NDG mouse, + is a positive control, H
2O is a negative control.
FIG. 6 is an image of a HR knockout mouse with NOD-Prkdc
scid IL-2rg
null mutations.
FIG. 7 is an image of a NOD-Prkdc
scid IL-2rg
null (B-NDG) mouse.
FIG. 8 is an image of HR knockout mouse with NOD-Prkdc
scid IL-2rg
null mutations (B-NDG background) with human tumor cells.
FIG. 9 is a diagram showing the mouse HR locus.
This disclosure relates to HR knockout non-human animals, and methods of use thereof.
Immunodeficient animals are an indispensable research tool for studying the mechanism of diseases, and methods of treating such diseases. They can easily accept exogenous cells or tissues due to their immunodeficiency, and have been widely used in the research. The commonly used immunodeficient animals include e.g., NOD-Prkdc
scid IL-2rγ
nul mice, NOD-Rag 1
-/--IL2rg
-/- (NRG) , Rag 2
-/--IL2rg
-/- (RG) , NOD/SCID (NOD-Prkdc
scid) , and NOD/SCID nude mice. Among them, NOD-Prkdc
scid IL-2rγ
nul mice may be the best recipient mice for transplantation. These immunodeficient mice are described in detail e.g., in Ito et al. "Current advances in humanized mouse models. " Cellular &molecular immunology 9.3 (2012) : 208, which is incorporated herein by reference in its entirety.
Before experiments can be performed on these immunodeficient animals (e.g., mice) or some other transgenic animals, usually hair needs to be removed, e.g., before exogenous cells or tissues are implanted to these animals. If hair removal is incomplete or the hair removal causes some skin damage, it may interfere with subsequent observations or studies, e.g., bioluminescence and fluorescence imaging and measuring tumor size. Furthermore, the skin of hairless animals (e.g., mice) is similar to human skin. Thus, it can also be used to test cosmetics, and various therapeutic agents to heal wounds.
Unless otherwise specified, the practice of the methods described herein can take advantage of the techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology. These techniques are explained in detail in the following literature, for examples: Molecular Cloning A Laboratory Manual, 2nd Ed., ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989) ; DNA Cloning, Volumes I and II (D.N. Glovered., 1985) ; Oligonucleotide Synthesis (M.J. Gaited., 1984) ; Mullisetal U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B.D. Hames&S.J. Higginseds. 1984) ; Transcription And Translation (B.D. Hames&S.J. Higginseds. 1984) ; Culture Of Animal Cell (R.I. Freshney, Alan R. Liss, Inc., 1987) ; Immobilized Cells And Enzymes (IRL Press, 1986) ; B. Perbal, A Practical Guide To Molecular Cloning (1984) , the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds. -in-chief, Academic Press, Inc., New York) , specifically, Vols. 154 and 155 (Wuetal. eds. ) and Vol. 185, “Gene Expression Technology” (D. Goeddel, ed. ) ; Gene Transfer Vectors For Mammalian Cells (J.H. Miller and M.P. Caloseds., 1987, Cold Spring Harbor Laboratory) ; Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987) ; Hand book Of Experimental Immunology, Volumes V (D.M. Weir and C.C. Blackwell, eds., 1986) ; and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986) ; each of which is incorporated herein by reference in its entirety.
HR Lysine demethylase and nuclear receptor corepressor (HR)
Hair is maintained through a cyclic process that includes periodic regeneration of hair follicles in a stem-cell-dependent manner. The hair cycle consists of three defined stages: growth (anagen) , followed by regression (catagen) and rest (telogen) . Growth of a new hair requires reentry into anagen, a process involving activation of multipotent epithelial stem cells residing in a specialized part of the follicle outer root sheath (ORS) . Activating signals emanate from adjacent mesenchymal cells (dermal papilla) , directing epithelial stem cells to migrate and differentiate to regenerate the hair bulb. Multiple signaling pathways, including Wnts, Sonic hedgehog (Shh) , and TGF-β family members have been shown to promote anagen initiation.
Disruption of Hairless (HR) gene function causes a complex skin phenotype that includes a specific defect in hair follicle regeneration in both humans and mice. In Hr mutant mice, hair follicle morphogenesis and initial hair growth is normal. However, after the follicles regress (catagen) and the hair is shed, around postnatal day (P) 17, telogen stage follicles never reenter anagen, and no new hair is produced, resulting in alopecia. Alternatively, the defect in anagen initiation may reflect a loss of the relevant epithelial stem cell population or an inability to generate and/or interpret the necessary signals.
The Hairless (also known as HR, HR lysine demethylase and nuclear receptor corepressor, or lysine-specific demethylase hairless) gene can encode an approximately 130 kDa nuclear transcription factor. The HR protein contains functional domains that include a nuclear localization signal domain, a nuclear matrix targeting motif, a putative zinc-finger, and a Jumonji C (JmjC) domain.
HR can directly interact with several nuclear transcription factors and chromatin modulators. Rodent Hr has been shown to interact with thyroid hormone receptors TRα and TRβ and with RAR-related orphan receptors (RORs) , especially RORα to repress their transactivation activity. Human and rodent HRs have also been shown to undergo direct protein-protein interactions with the vitamin D receptor VDR. Regions in HR which mediate interactions with nuclear receptors include four motifs of hydrophobic amino acids, two of the form LXXLL (where L is leucine and X is any amino acid) and two ΦXXΦΦ motifs (where Φ can be leucine, isoleucine or valine) . These four hydrophobic motifs are also referred to as interaction domains (IDs) . HR has been shown to interact with RORα and TRs via the LXXLL motif pair and the ΦXXΦΦ motif pair respectively, whereas all four motifs participate in interactions with VDR as revealed by coimmunoprecipitation and functional studies. Through its interactions with TRs, HR has also been implicated as playing a role in mammalian CNS development. Similarly, HR interactions with RORα have been shown to be important in cerebellar development.
HR may regulate epidermal homeostasis via direct control of a set of target genes that includes KDSR, MAGI2, and CSNK2A. Moreover, HR mutations D1020N and V1056M from patients with atrichia with papular lesions (APL) markedly diminished the HDM activity compared to wild-type HR. Such findings provide positive evidence that the disruption of the HR JmjC domain is relevant to hair cycling and skin maintenance.
A detailed description of HR and its function can be found, e.g., in Liu et al., "Hairless is a histone H3K9 demethylase. " The FASEB Journal 28.4 (2014) : 1534-1542; Maatough et al., "Human hairless protein roles in skin/hair and emerging connections to brain and other cancers. " Journal of cellular biochemistry 119.1 (2018) : 69-80; Beaudoin, et al. "Hairless triggers reactivation of hair growth by promoting Wnt signaling. " Proceedings of the National Academy of Sciences 102.41 (2005) : 14653-14658; Liu et al. "Molecular basis for hair loss in mice carrying a novel nonsense mutation (Hrrh-R) in the hairless gene (Hr) . " Veterinary pathology 47.1 (2010) : 167-176; each of which is incorporated herein by reference in its entirety.
In human genomes, HR gene (Gene ID: 55806) is located on chromosome 8, and has 19 exons. The nucleotide sequence for human HR mRNA is NM_005144.4, and the amino acid sequence for human HR is NP_005135.2.
In mice, HR gene locus has 20 exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, and exon 20 (FIG. 9) . The nucleotide sequence for mouse HR cDNA is NM_021877.3 (SEQ ID NO: 26) , the amino acid sequence for mouse HR is NP_068677.2 (SEQ ID NO: 27) . The location for each exon in the mouse HR nucleotide sequence and amino acid sequence is listed below:
Table 1
The mouse HR gene (Gene ID: 15460) is located in Chromosome 14 of the mouse genome, which is located from70554056 to 70573548 of NC_000080.6 (GRCm38. p4 (GCF_000001635.24) ) . The 5’-UTR is from 70, 554, 056 to 70, 554, 112 and 70, 554, 512 to 70, 555, 107 and 70, 556, 348 to 70, 556, 388, exon 1 is from 70, 554, 056 to 70, 554, 112, the first intron is from 70, 554, 113 to 70, 554, 511, exon 2 is from 70, 554, 512 to 70, 555, 107, the second intron is from 70, 555, 108 to 70, 556, 347, exon 3 is from 70, 556, 348 to 70, 557, 000, the third intron is from 70, 557, 001 to 70, 557, 628, exon 4 is from 70, 557, 629 to 70, 558, 409, the fourth intron is from 70, 558, 410 to 70, 559, 638, exon 5 is from 70, 559, 639 to 70, 559, 789, the fifth intron is from 70, 559, 790 to 70, 559, 872, exon 6 is from 70, 559, 873 to 70, 560, 063, the sixth intron is from 70, 560, 064 to 70, 561, 811, exon 7 is from 70, 561, 812 to 70, 561, 976, the seventh intron is from 70, 561, 977 to 70, 563, 328, exon 8 is from 70, 563, 329 to 70, 563, 418 , the eighth intron is from 70, 563, 419 to 70, 563, 566, exon 9 is from 70, 563, 567 to 70, 563, 682, the ninth intron is from 70, 563, 683 to 70, 565, 301, exon 10 is from 70, 565, 302 to 70, 565, 383, the tenth intron is from 70, 565, 384 to 70, 566, 028, exon 11 is from 70, 566, 029 to 70, 566, 192, the eleventh intron is from 70, 566, 193 to 70, 566, 309, exon 12 is from 70, 566, 310 to 70, 566, 546, the twelfth intron is from 70, 566, 547 to 70, 566, 779, exon 13 is from 70, 566, 780 to 70, 566, 945, the thirteenth intron is from 70, 566, 946 to 70, 567, 148, exon 14 is from 70, 567, 149 to 70, 567, 218, the fourteenth intron is from 70, 567, 219 to 70, 567, 501, exon 15 is from 70, 567, 502 to 70, 567, 632, the fifteenth intron is from 70, 567, 633 to 70, 567, 775, exon 16 is from 70, 567, 776 to 70, 567, 895, the sixteenth intron is from 70, 567, 896 to 70, 567, 976, exon 17 is from 70, 567, 977 to 70, 568, 092, the seventeenth intron is from 70, 568, 093 to 70, 571, 377, exon 18 is from 70, 571, 378 to 70, 571, 542, the eighteenth intron is from 70, 571, 543 to 70, 571, 907, exon 19 is from 70, 571, 908 to 70, 572, 036, the nineteenth intron is from 70, 572, 037 to 70, 572, 241, exon 20 is from 70, 572, 242 to 70, 573, 548, the 3’-UTR is from 70, 572, 305 to 70, 573, 548, based on transcript NM_021877.3. All relevant information for mouse HR locus can be found in the NCBI website with Gene ID: 15460, which is incorporated by reference herein in its entirety.
Other HR genes, proteins, and locus of the other species are also known in the art. For example, the gene ID for HR in Rattus norvegicus is 60563, the gene ID for HR in Macaca mulatta (Rhesus monkey) is 574164, the gene ID for HR in Sus scrofa (pig) is 397617. The relevant information for these genes (e.g., intron sequences, exon sequences, amino acid residues of these proteins) can be found, e.g., in NCBI database.
The present disclosure provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal’s endogenous HR gene, wherein the disruption of the endogenous HR gene comprises deletion of one or more exons, or part of the one or more exons, wherein the one or more exons are selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, and exon 20 of the endogenous HR gene. Thus, the disclosure provides a genetically-modified, non-human animal that does not have one or more exons that are selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, and exon 20 of the endogenous HR gene. In some embodiments, the animals do not have exons 3-7.
As used herein, the term “deletion of an exon” refers to the deletion the entire exon. For example, deletion of exon 2 means that all sequences in exon 2 are deleted.
As used herein, the term “deletion of part of an exon” refers to at least one nucleotide, but not all nucleotides in the exon is deleted. In some embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nucleotides in the exon are deleted.
In some embodiments, the disruption comprises deletion of one or more introns, or part of the one or more introns, wherein the one or more introns are selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, intron 17, intron 18, and intron 19 of the endogenous HR gene. Thus, the disclosure provides a genetically-modified, non-human animal does not have one or more introns that are selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, intron 17, intron 18, and intron 19 of the endogenous HR gene. In some embodiments, the animal does not have part of intron 2, intron 3, intron 4, intron 5, intron 6, and/or part of intron 7.
As used herein, the term “deletion of an intron” refers to the deletion the entire intron. For example, deletion of intron 3 means that all sequences in intron 3 are deleted.
As used herein, the term “deletion of part of an intron” refers to at least one nucleotide, but not all nucleotides in the intron is deleted. In some embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, or 3000 nucleotides in the intron are deleted.
In some embodiments, the disruption of the endogenous HR gene comprises deletion of one or more exons selected from the group consisting of exon 3, exon 4, exon 5, exon 6, and exon 7 of the endogenous HR gene. In some embodiments, the disruption of the endogenous HR gene further comprises deletion of exon 1, exon 2, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, and/or exon 20 of the endogenous HR gene.
In some embodiments, the entire sequence of mouse exon 3, exon 4, exon 5, exon 6, and exon 7 are deleted.
In some embodiments, a “region” or “portion” of mouse exons or introns of HR gene are deleted. The term “region” or “portion” can refer to e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, or 3000 nucleotides.
In some embodiments, the “region” or “portion” can be at least 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, intron 17, intron 18, or intron 19. In some embodiments, a region, a portion, or the entire sequence of exon 3, exon 4, exon 5, exon 6, and/or exon 7 are deleted. In some embodiments, a region, a portion, or the entire sequence of mouse intron 2, intron 3, intron 4, intron 5, intron 6, and/or intron 7 are deleted.
In some embodiments, the disruption comprises or consists of deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nucleotides in exon 3, exon 4, exon 5, exon 6, and/or exon 7. In some embodiments, the disruption comprises or consists of deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, or 3000 nucleotides in intron 2, intron 3, intron 4, intron 5, intron 6, and/or intron 7.
In some embodiments, the disruption comprises or consists of deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, or 1000 in intron 2; deletion of the entirety of exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, and exon 7; and/or deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, or 1000 nucleotides in intron 7.
In some embodiments, the length of the remaining exon sequences at the endogenous HR gene locus is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, or 80%of the total length of all exon sequences of the endogenous HR gene. In some embodiments, the length of the remaining exon sequences at the endogenous HR gene locus is about or at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, or 80%of the total length of all exon sequences of the endogenous HR gene.
In some embodiments, the length of the remaining sequences at that the endogenous HR gene locus is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, or 80%of the full sequence of the endogenous HR gene. In some embodiments, the length of the remaining sequences at that the endogenous HR gene locus is about or at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, or 80%of the full sequence of the endogenous HR gene.
In some embodiments, the sequence starts from the first nucleotide of exon 1. In some embodiments, the sequence ends at the last nucleotide of the last exon.
The present disclosure further relates to the genomic DNA sequence of a HR knockout animal (e.g., a rodent, a mouse) . In some embodiments, the genome of the animal comprises from 5’ to 3’ at the endogenous HR gene locus, (a) a first DNA sequence; optionally, (b) a second DNA sequence comprising an exogenous sequence; (c) a third DNA sequence, wherein the first DNA sequence, the optional second DNA sequence, and the third DNA sequence are linked.
The second DNA sequence can have a length of 0 nucleotides to 1000 nucleotides (e.g., at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nucleotides) . In some embodiments, the second DNA sequence has only 0 nucleotides, which means that there is no extra sequence between the first DNA sequence and the third DNA sequence. In some embodiments, random or exogenous sequences are added. In some embodiments, the second DNA sequence has a length of 1 nucleotide to 100 nucleotides (e.g., 1 to 20 nucleotides) .
In some embodiments, the second DNA sequence has about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nucleotides. In some embodiments, the second DNA sequence has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nucleotides.
In some embodiments, the first DNA sequence comprises an endogenous HR gene sequence that is located upstream of intron 2, and can include all or just part of sequences that is located upstream of intron 2. In some embodiments, the first DNA sequence comprises an endogenous HR gene sequence that is located upstream of exon 2.
In some embodiments, the first DNA sequence comprises a sequence that has a length (5’ to 3’) of from 10 to 1500 nucleotides (e.g., from 10 to 100 nucleotides, from 100 to 500 nucleotides, from 500 to 1000 nucleotides, from 1000 to 1500 nucleotides, from 1000 to 2000 nucleotides, or from 1400 to 1500 nucleotides) starting from the first nucleotide in exon 1 of the HR gene to the last nucleotide of the first DNA sequence. In some embodiments, the first DNA sequence comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 nucleotides from exon 1, exon 2, or the combination of exon 1 and exon 2. In some embodiments, the first DNA sequence has at most , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 nucleotides from exon 1, exon 2, or the combination of exon 1 and exon 2.
In some embodiments, the third DNA sequence comprises an endogenous HR gene sequence that is located downstream of intron 7, and can include all or just part of sequences that is located downstream of intron 7. In some embodiments, the third DNA sequence comprises a sequence that has a length (5’ to 3’) of from 1 to 1351 nucleotides (e.g., from 1 to 1000 nucleotides, or from 500 to 1300 nucleotides) starting from the first nucleotide in the third DNA sequence to the last nucleotide in the intron 7 (e.g., intron 7 in mouse) of the endogenous HR gene. In some embodiments, the third DNA sequence comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, or 1300 nucleotides from intron 7. In some embodiments, the third DNA sequence has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, or 1350 nucleotides from intron 7.
In some embodiments, the third DNA sequence comprises an endogenous HR gene sequence that is located downstream of the last intron (e.g., intron 19 in mouse) , and can include all or just part of sequences that is located downstream of intron 19. In some embodiments, the third DNA sequence comprises a sequence that has a length (5’ to 3’) of from 10 to 11000 nucleotides (e.g., from 100 to 11000 nucleotides, from 1000 to 11000 nucleotides, or from 5000 to 11000 nucleotides) starting from the first nucleotide in the third DNA sequence to the last nucleotide in the last exon (e.g., exon 20 in mouse) of the endogenous HR gene. In some embodiments, the third DNA sequence comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or 11000 nucleotides. In some embodiments, the third DNA sequence has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or 11000 nucleotides.
In some embodiments, the third DNA sequence comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, or 1300 nucleotides from the last exon (e.g., exon 20 in mouse) . In some embodiments, the third DNA sequence has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, or 1306 nucleotides from the last exon (e.g., exon 20 in mouse) .
In some embodiments, the HR gene sequence at the endogens HR locus is set forth in SEQ ID NO: 32 (70554056-70573548 of NC_000080.6) . In some embodiments, 70555486-70562742 of NC_000080.6 (SEQ ID NO: 33) is deleted. In some embodiments, 70555492-70562744 of NC_000080.6 (SEQ ID NO: 34) is deleted. In some embodiments, the genetic modified non-human animal comprises a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical or 100%identical to SEQ ID NO: 28. In some embodiments, the genetic modified non-human animal comprises a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical or 100%identical to SEQ ID NO: 35.
In some embodiments, the sequence is located at the endogenous HR locus.
The disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any nucleotide sequence as described herein (e.g., exon sequences, intron sequences, the remaining exon sequences, the deleted sequences) , and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any amino acid sequence as described herein (e.g., amino acid sequences encoded by exons) . In some embodiments, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein. In some embodiments, the nucleic acid sequence is less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or 11000 nucleotides. In some embodiments, the amino acid sequence is less than , 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400 or 1500 amino acid residues.
In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.
In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.
To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) . The length of a reference sequence aligned for comparison purposes is at least 80%of the length of the reference sequence, and in some embodiments is at least 90%, 95%, or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
Cells, tissues, and animals (e.g., mouse) are also provided that comprise a disruption of the endogenous HR gene as described herein, as well as cells, tissues, and animals (e.g., mouse) that have any nucleic acid sequence as described herein.
Genetically modified animals
As used herein, the term “genetically-modified non-human animal” refers to a non-human animal having a modified sequence (e.g., deletion of endogenous sequence or insertion of exogenous sequence) in at least one chromosome of the animal’s genome. In some embodiments, at least one or more cells, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%of cells of the genetically-modified non-human animal have the modified sequence in its genome. The cell having the modified sequence can be various kinds of cells, e.g., an endogenous cell, a somatic cell, an immune cell, a T cell, a B cell, a germ cell, a blastocyst, or an endogenous tumor cell. In some embodiments, genetically-modified non-human animals are provided that comprise a disruption or a deletion at the endogenous HR locus. The animals are generally able to pass the modification to progeny, i.e., through germline transmission.
In some embodiments, the genetically-modified non-human animal does not express HR (e.g., intact or functional HR protein) . Because HR is involved in hair growth and hair follicle regeneration, the genetically-modified non-human animal does not have hair. In some embodiments, the hair coverage of the genetically-modified non-human animal is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.
In some embodiments, the genetically-modified non-human animal is an immunodeficient animal. In some embodiments, the animal is a NOD-Prkdc
scid IL-2rγ
nul, NOD-Rag 1
-/--IL2rg
-/- (NRG) , Rag 2
-/--IL2rg
-/- (RG) , NOD/SCID (NOD-Prkdc
scid) , NOD/SCID nude, or NOD-Prkdc
scid IL-2rg
null animal (e.g., a rodent, a rat, or a mouse) . In some embodiments, the genetically-modified non-human animal is not an immunodeficient animal.
In some embodiments, the genetically-modified non-human animal lack functional T cells, B cells, and/or NK cells.
In some embodiments, the animal can have one or more of the following characteristics:
(a) the percentage of T cells (CD3+ cells) is less than 5%, 4%, 3%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%or 0.1%of leukocytes in the animal;
(b) the percentage of B cells (e.g., CD3-CD19+ cells) is less than 5%, 4%, 3%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%or 0.01%of leukocytes in the animal;
(c) the percentage of NK cells (e.g., CD3-CD49b+ cells) is less than 5%, 4%, 3%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, or 0.5%of leukocytes in the animal;
(d) the percentage of CD4+ T cells (CD3+ CD4+ cells) is less than 5%, 4%, 3%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%of T cells;
(e) the percentage of CD8+ T cells (CD3+ CD8+ cells) is less than 5%, 4%, 3%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%of T cells;
(f) the percentage of CD3+ CD4+ cells, CD3+ CD8+ cells, CD3-CD19+ cells is less than 5%, 4%, 3%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%of leukocytes in the animal;
(g) the percentage of T cells (CD3+ cells) and NK cells (CD3-CD49b+ cells) is less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%of leukocytes in the animal.
As used herein, the term “leukocytes” or “white blood cells” include neutrophils, eosinophils (acidophilus) , basophils, lymphocytes, and monocytes. All leukocytes have nuclei, which distinguishes them from the anucleated red blood cells (RBCs) and platelets. CD45, also known as leukocyte common antigen (LCA) , is a cell surface marker for leukocytes. Among leukocytes, monocytes and neutrophils are phagocytic.
Lymphocytes is a subtype of leukocytes. Lymphocytes include natural killer (NK) cells (which function in cell-mediated, cytotoxic innate immunity) , T cells, and B cells.
In some embodiments, the variations among individual mice are very small. In some embodiments, the standard deviations of the percentages are less than 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%or 0.01%.
In some embodiments, the genetically-modified non-human animal has a NOD-Prkdc
scid IL-2rg
null background. The genetically-modified animal can also have one or more of the following characteristics:
(a) the genetically-modified mouse has no functional T-cells and/or no functional B-cells;
(b) the genetically-modified mouse exhibits reduced macrophage function relative to a NOD/scid mouse, or a NOD/scid nude mouse;
(c) the genetically-modified mouse exhibits no NK cell activity;
(d) the genetically-modified mouse exhibits reduced dendritic function relative to a NOD/scid mouse, or a NOD/scid nude mouse; and
(e) the genetically-modified mouse has an enhanced engraftment capacity of exogenous cells relative to a NOD/scid mouse, or a NOD/scid nude mouse.
The genetically modified non-human animal can also be various other animals, e.g., a rat, rabbit, pig, bovine (e.g., cow, bull, buffalo) , deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey) . For the non-human animals where suitable genetically modifiable ES cells are not readily available, other methods are employed to make a non-human animal comprising the genetic modification. Such methods include, e.g., modifying a non-ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and employing nuclear transfer to transfer the modified genome to a suitable cell, e.g., an oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo. These methods are known in the art, and are described, e.g., in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition) , ” Cold Spring Harbor Laboratory Press, 2003, which is incorporated by reference herein in its entirety.
In one aspect, the animal is a mammal, e.g., of the superfamily Dipodoidea or Muroidea. In some embodiments, the genetically modified animal is a rodent. The rodent can be selected from a mouse, a rat, and a hamster. In some embodiment, the rodent is selected from the superfamily Muroidea. In some embodiments, the genetically modified animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters) , Cricetidae (e.g., hamster, New World rats and mice, voles) , Muridae (true mice and rats, gerbils, spiny mice, crested rats) , Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasy rats and mice) , Platacanthomyidae (e.g., spiny dormice) , and Spalacidae (e.g., mole rates, bamboo rats, and zokors) . In some embodiments, the genetically modified rodent is selected from a true mouse or rat (family Muridae) , a gerbil, a spiny mouse, and a crested rat. In one embodiment, the non-human animal is a mouse.
In some embodiments, the animal is a mouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In some embodiments, the mouse is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm) , 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac) , 129S7, 129S8, 129T1, 129T2. These mice are described, e.g., in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999) ; Auerbach et al., Establishment and Chimera Analysis of 129/SvEv-and C57BL/6-Derived Mouse Embryonic Stem Cell Lines (2000) , both of which are incorporated herein by reference in the entirety. In some embodiments, the genetically modified mouse is a mix of the 129 strain and the C57BL/6 strain. In some embodiments, the mouse is a mix of the 129 strains, or a mix of the BL/6 strains. In some embodiment, the mouse is a BALB strain, e.g., BALB/c strain. In some embodiments, the mouse is a mix of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid line (e.g., 50%BALB/c-50%12954/Sv; or 50%C57BL/6-50%129) .
In some embodiments, the animal is a rat. The rat can be selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti. In some embodiments, the rat strain is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.
The animal can have one or more other genetic modifications, and/or other modifications, that are suitable for the particular purpose for which the HR knockout animal is made. For example, suitable mice for maintaining a xenograft (e.g., a human cancer or tumor) , can have one or more modifications that compromise, inactivate, or destroy the immune system of the non-human animal in whole or in part. Compromise, inactivation, or destruction of the immune system of the non-human animal can include, for example, destruction of hematopoietic cells and/or immune cells by chemical means (e.g., administering a toxin) , physical means (e.g., irradiating the animal) , and/or genetic modification (e.g., knocking out one or more genes) .
Non-limiting examples of such mice include, e.g., NOD mice, SCID mice, NOD/SCID mice, nude mice, NOD/SCID nude mice, NOD-Prkdc
scid IL-2rγ
null, and Rag1 and/or Rag2 knockout mice. These mice can optionally be irradiated, or otherwise treated to destroy one or more immune cell type. Thus, in various embodiments, a genetically modified mouse is provided that can include a disruption of the endogenous non-human HR locus, and further comprises a modification that compromises, inactivates, or destroys the immune system (or one or more cell types of the immune system) of the non-human animal in whole or in part. In some embodiments, modification is, e.g., selected from the group consisting of a modification that results in NOD mice, SCID mice, NOD/SCID mice, nude mice, NOD-Prkdc
scid IL-2rγ
null mice, Rag1 and/or Rag2 knockout mice, and a combination thereof. These genetically modified animals are described, e.g., in US20150106961; and PCT/CN2018/079365, which are incorporated herein by reference in the entirety.
Although genetically modified cells are also provided that can comprise the modifications (e.g., disruption) described herein (e.g., ES cells, somatic cells) , in many embodiments, the genetically modified non-human animals comprise the modification of the endogenous HR locus in the germline of the animal.
Furthermore, the genetically modified animal can be homozygous with respect to the disruption of the endogenous HR gene. In some embodiments, the animal can be heterozygous with respect to the disruption of the endogenous HR gene.
The present disclosure further relates to a non-human mammal generated through the methods as described herein. In some embodiments, the genome thereof contains human gene (s) .
In addition, the present disclosure also relates to a tumor bearing non-human mammal model, characterized in that the non-human mammal model is obtained through the methods as described herein. In some embodiments, the non-human mammal is a rodent (e.g., a mouse) .
The present disclosure further relates to a cell or cell line, or a primary cell culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal; the tissue, organ or a culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal; and the tumor tissue derived from the non-human mammal or an offspring thereof when it bears a tumor, or the tumor bearing non-human mammal.
The present disclosure also provides non-human mammals produced by any of the methods described herein. In some embodiments, a non-human mammal is provided; and the genetically modified animal contains a disruption of the HR gene in the genome of the animal.
Genetic, molecular and behavioral analyses for the non-human mammals described above can be performed. The present disclosure also relates to the progeny produced by the non-human mammal provided by the present disclosure mated with the same or other genotypes.
The present disclosure also provides a cell line or primary cell culture derived from the non-human mammal or a progeny thereof. A model based on cell culture can be prepared, for example, by the following methods. Cell cultures can be obtained by way of isolation from a non-human mammal, alternatively cell can be obtained from the cell culture established using the same constructs and the standard cell transfection techniques. The disruption of HR gene can be detected by a variety of methods.
There are also many analytical methods that can be used to detect DNA expression, including methods at the level of RNA (including the mRNA quantification approaches using reverse transcriptase polymerase chain reaction (RT-PCR) or Southern blotting, and in situ hybridization) and methods at the protein level (including histochemistry, immunoblot analysis and in vitro binding studies) . Many standard analysis methods can be used to complete quantitative measurements. For example, transcription levels of wildtype HR can be measured using RT-PCR and hybridization methods including RNase protection, Southern blot analysis, RNA dot analysis (RNAdot) analysis. Immunohistochemical staining, flow cytometry, Western blot analysis can also be used to assess the presence of human proteins.
Vectors
The disclosure also provides vectors for constructing a HR animal model. In some embodiments, the vectors comprise sgRNA sequence, wherein the sgRNA sequence target HR gene, and the sgRNA is unique on the target sequence of the HR gene to be altered. In some embodiments, the sequence meets the sequence arrangement rule of 5’-NNN (20) -NGG3’ or 5’-CCN-N (20) -3’; and in some embodiments, the targeting site of the sgRNA in the mouse HR gene is located on the exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, and exon 20, intron 1, intron 2, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, intron 17, intron 18, intron 19, upstream of exon 1, or downstream of exon 20 of the mouse HR gene.
In some embodiments, the 5’ targeting sequence for the knockout sequence is shown as SEQ ID NOs: 1-7, and the sgRNA sequence recognizes the 5’ targeting site. In some embodiments, the 3’ targeting sequence for the knockout sequence is shown as SEQ ID NOs: 8-14 and the sgRNA sequence recognizes the 3’ targeting site.
Thus, the disclosure provides sgRNA sequences for constructing a HR knockout animal model. In some embodiments, the oligonucleotide sgRNA sequences are set forth in SEQ ID NOs: 15-22.
In some embodiments, the disclosure relates to a plasmid construct (e.g., pT7-sgRNA) including the sgRNA sequence, and/or a cell including the construct.
In addition, the present disclosure further relates to a non-human mammalian cell, having any one of the foregoing targeting vectors, and one or more in vitro transcripts of the sgRNA construct as described herein. In some embodiments, the cell includes Cas9 mRNA or an in vitro transcript thereof.
In some embodiments, the genes in the cell are heterozygous. In some embodiments, the genes in the cell are homozygous.
In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell.
Methods of making genetically modified animals
Genetically modified animals can be made by several techniques that are known in the art, including, e.g., nonhomologous end-joining (NHEJ) , homologous recombination (HR) , zinc finger nucleases (ZFNs) , transcription activator-like effector-based nucleases (TALEN) , and the clustered regularly interspaced short palindromic repeats (CRISPR) -Cas system. In some embodiments, homologous recombination is used. In some embodiments, CRISPR-Cas9 genome editing is used to generate genetically modified animals. Many of these genome editing techniques are known in the art, and is described, e.g., in Yin et al., "Delivery technologies for genome editing, " Nature Reviews Drug Discovery 16.6 (2017) : 387-399, which is incorporated by reference in its entirety. Many other methods are also provided and can be used in genome editing, e.g., micro-injecting a genetically modified nucleus into an enucleated oocyte, and fusing an enucleated oocyte with another genetically modified cell.
Thus, in some embodiments, the disclosure provides knocking out in at least one cell of the animal, at an endogenous HR gene locus, one or more exons (e.g., about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 exons) and/or one or more introns (e.g., about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 introns) of the endogenous HR gene. In some embodiments, the modification occurs in a germ cell, a somatic cell, a blastocyst, or a fibroblast, etc. The nucleus of a somatic cell or the fibroblast can also be inserted into an enucleated oocyte.
In some embodiments, cleavages at the upstream and the downstream of the knockout sequence by a nuclease (e.g., by zinc finger nucleases, TALEN or CRISPR) can result in DNA double strands break, and non-homologous end joining (NHEJ) occurs and ligates the break ends, thereby knocking out the sequence of interest. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. When the break ends located at the upstream and the downstream of the target sequence are ligated, imprecise repair occurs, and in some cases, leading to loss of nucleotides or insertion of random nucleotides.
Zinc finger proteins, TAL-effector domains, or single guide RNA (sgRNA) DNA-binding domains can be designed to target the upstream and the downstream of the knockout sequence. SEQ ID NOs: 1-14 are exemplary target sequences for the modification. Among them, SEQ ID NOs: 1-7 are located within intron 2 of mouse endogenous HR gene. SEQ ID NOs: 8-14 are located within intron 7 of mouse endogenous HR gene. After the zinc finger proteins, TAL-effector domains, or single guide RNA (sgRNA) DNA-binding domains bind to the target sequences, the nuclease cleaves the genomic DNA, and triggers NHEJ. In some embodiments, the nuclease is CRISPR associated protein 9 (Cas9) .
Thus, the methods of producing a HR knockout mouse can involve one or more of the following steps: transforming a mouse embryonic stem cell with a gene editing system that targets endogenous HR gene, thereby producing a transformed embryonic stem cell; introducing the transformed embryonic stem cell into a mouse blastocyst; implanting the mouse blastocyst into a pseudopregnant female mouse; and allowing the blastocyst to undergo fetal development to term.
In some embodiments, the transformed embryonic cell is directly implanted into a pseudopregnant female mouse instead, and the embryonic cell undergoes fetal development.
In some embodiments, the gene editing system can involve zinc finger proteins, TAL-effector domains, or single guide RNA (sgRNA) DNA-binding domains.
The present disclosure further provides a method for establishing a HR gene knockout animal model, involving the following steps:
(a) providing the cell (e.g. a fertilized egg cell) with the genetic modification based on the methods described herein;
(b) culturing the cell in a liquid culture medium;
(c) transplanting the cultured cell to the fallopian tube or uterus of the recipient female non-human mammal, allowing the cell to develop in the uterus of the female non-human mammal;
(d) identifying the germline transmission in the offspring genetically modified humanized non-human mammal of the pregnant female in step (c) .
In some embodiments, the non-human mammal in the foregoing method is a mouse (e.g., a C57BL/6 mouse, a NOD/scid mouse, a NOD/scid nude mouse, or a NOD-Prkdc
scid IL-2rγ
null mouse) . In some embodiments, the non-human mammal is a NOD/scid mouse. In the NOD/scid mouse, the SCID mutation has been transferred onto a non-obese diabetic (NOD) background. Animals homozygous for the SCID mutation have impaired T and B cell lymphocyte development. The NOD background additionally results in deficient natural killer (NK) cell function. In some embodiments, the non-human mammal is a NOD/scid nude mouse. The NOD/scid nude mouse additionally has a disruption of FOXN1 gene on chromosome 11 in mice. In some embodiments, the animal can comprise an additional disruption in the animal’s endogenous Beta-2-Microglobulin (B2M) gene.
In some embodiments, the fertilized eggs for the methods described above are NOD/scid fertilized eggs, NOD/scid nude fertilized eggs, or NOD-Prkdc
scid IL-2rγ
null fertilized eggs. Other fertilized eggs that can also be used in the methods as described herein include, but are not limited to, C57BL/6 fertilized eggs, FVB/N fertilized eggs, BALB/c fertilized eggs, DBA/1 fertilized eggs and DBA/2 fertilized eggs.
Fertilized eggs can come from any non-human animal, e.g., any non-human animal as described herein. In some embodiments, the fertilized egg cells are derived from rodents. The genetic construct can be introduced into a fertilized egg by microinjection of DNA. For example, by way of culturing a fertilized egg after microinjection, a cultured fertilized egg can be transferred to a false pregnant non-human animal, which then gives birth of a non-human mammal, so as to generate the non-human mammal mentioned in the method described above.
The genetically modified animals (e.g., mice) generated by the methods described herein can have several advantages. For example, the genetically modified mice do not require backcrossing, and thus have a more defined background (e.g., less genetic variations among individual subjects) as compared to some other HR knockout or immunodeficient mice. A defined background or a pure background is beneficial to obtain consistent experiment results. In some embodiments, the genetic variation among the mice across the entire genome (e.g., autosomes) among different individuals is less than 3%, 2%, 1%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%or 0.01%. The methods to determine the genetic variations are known in the art. For example, the genetic variations can be measured or determined by sequencing, whole genome sequencing, exome sequencing, detecting variations at some selected sites (e.g., by using SNP microarrays, by detecting copy number variations) etc. In some embodiments, the mice have a genome (e.g., autosomes) that is about or at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%identical to NOD-Prkdc
scid IL-2rγ
null mice (e.g., B-NDG mice) except for certain mutations (e.g., the HR knockout mutation) . In some embodiments, the mice have a genome (e.g., autosomes) that is about or at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%identical to NOD/SCID mice (or NOD-Prkdc
scid mice) except for certain mutations (e.g., the HR knockout mutation and/or IL-2rγ mutation) . In some embodiments, the mice have a genome (e.g., autosomes) that is about or at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%identical to NOD mice except for certain mutations (e.g., the HR knockout mutation, the SCID mutation, and/or IL-2rγ mutation) .
Furthermore, despite the immunodeficiency, these mice are also relatively healthy, and in some cases, they have a relatively long life span (e.g., about or at least 1 year, 1.5 years, 2 years, 2.5 years, or 3 years) .
Methods of using genetically modified animals
Genetically modified animals with a disruption at endogenous HR gene can provide a variety of uses that include, but are not limited to, observing tumor growth, testing various therapeutic agents (e.g., to treat tumors, wounds, and various skin diseases) , and testing cosmetics.
In some embodiments, the animal is an immunodeficient animal, e.g., a NOD-Prkdc
scid IL-2rγ
nul, NOD-Rag 1
-/--IL2rg
-/- (NRG) , Rag 2
-/--IL2rg
-/- (RG) , NOD/SCID (NOD-Prkdc
scid) , NOD/SCID nude, or NOD-Prkdc
scid IL-2rg
null animal (e.g., a rodent, a rat, or a mouse) . In some embodiments, the animal further comprises an additional disruption in the animal’s endogenous Beta-2-Microglobulin (B2M) gene.
In some embodiments, the hairless immunodeficient animal can be used to establish a human hemato-lymphoid animal model, develop therapeutics for human diseases and disorders, and assess the efficacy of these therapeutic agents in the animal models.
In some embodiments, the genetically modified animals can be used for establishing a human hemato-lymphoid system. The methods involve engrafting a population of cells comprising human hematopoietic cells (CD34+ cells) or human peripheral blood cells into the genetically modified animal described herein. In some embodiments, the methods further include the step of irradiating the animal prior to the engrafting. The human hemato-lymphoid system in the genetically modified animals can include various human cells, e.g., hematopoietic stem cells, myeloid precursor cells, myeloid cells, dendritic cells, monocytes, granulocytes, neutrophils, mast cells, lymphocytes, and platelets.
The genetically modified animals described herein are also an excellent animal model for establishing the human hemato-lymphoid system. In some embodiments, the animal after being engrafted with human hematopoietic stem cells or human peripheral blood cells to develop a human immune system has one or more of the following characteristics:
(a) the percentage of human CD45+ cells is about or at least 10%, 20%, 30%, 40%, or 50%of leukocytes or CD45+ cells of the animal;
(b) the percentage of human CD3+ cells is about or at least 10%, 20%, 30%, 40%, or 50%of leukocytes or CD45+ cells in the animal; and
(c) the percentage of human CD19+ cells is about or at least 10%, 20%, 30%, 40%, or 50%of leukocytes or CD45+ cells in the animal.
In some embodiments, the genetically modified animals described herein are less likely to develop graft-versus-host disease (GVHD) or xenogeneic graft-versus-host disease (X-GVHD) .
In some embodiments, after being engrafted with human hematopoietic stem cells or human peripheral blood cells, the genetically modified animals described herein can live for about or at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 days.
In some embodiments, the genetically modified animals described herein do not have a NSG or NOG background. In some embodiments, the genetically modified animals described herein are better animal models for establishing the human hemato-lymphoid system (e.g., having a higher percentage of human leukocytes, human T cells, human B cells, or human NK cells) . A detailed description of the NSG mice and NOD mice can be found, e.g., in Ishikawa et al. "Development of functional human blood and immune systems in NOD/SCID/IL2 receptor γ chainnull mice. " Blood 106.5 (2005) : 1565-1573; Katano et al. "NOD-Rag2null IL-2Rγnull mice: an alternative to NOG mice for generation of humanized mice. " Experimental animals 63.3 (2014) : 321-330, both of which are incorporated herein by reference in the entirety.
In some embodiments, the genetically modified animals can be used to determine the effectiveness of an agent or a combination of agents for the treatment of cancer. The methods involve engrafting tumor cells to the animal as described herein, administering the agent or the combination of agents to the animal; and determining the inhibitory effects on the tumors.
In some embodiments, the tumor cells are from a tumor sample obtained from a human patient. These animal models are also known as patient derived xenografts (PDX) models. PDX models are often used to create an environment that resembles the natural growth of cancer, for the study of cancer progression and treatment. Within PDX models, patient tumor samples grow in physiologically-relevant tumor microenvironments that mimic the oxygen, nutrient, and hormone levels that are found in the patient’s primary tumor site. Furthermore, implanted tumor tissue maintains the genetic and epigenetic abnormalities found in the patient and the xenograft tissue can be excised from the patient to include the surrounding human stroma. As a result, PDX models can often exhibit similar responses to anti-cancer agents as seen in the actual patient who provide the tumor sample.
While the genetically modified animals do not have functional T cells or B cells, the genetically modified animals still have functional phagocytic cells, e.g., neutrophils, eosinophils (acidophilus) , basophils, or monocytes. Macrophages can be derived from monocytes, and can engulf and digest cellular debris, foreign substances, microbes, cancer cells. Thus, the genetically modified animals described herein can be used to determine the effect of an agent (e.g., anti-CD47 antibodies or anti-SIRPa antibodies) on phagocytosis, and the effects of the agent to inhibit the growth of tumor cells.
In some embodiments, human peripheral blood cells (hPBMC) or human hematopoietic stem cells are injected to the animal to develop human hematopoietic system. The genetically modified animals described herein can be used to determine the effect of an agent in human hematopoietic system, and the effects of the agent to inhibit tumor cell growth or tumor growth. Thus, in some embodiments, the methods as described herein are also designed to determine the effects of the agent on human immune cells (e.g., human T cells, B cells, or NK cells) , e.g., whether the agent can stimulate T cells or inhibit T cells, whether the agent can upregulate the immune response or downregulate immune response. In some embodiments, the genetically modified animals can be used for determining the effective dosage of a therapeutic agent for treating a disease in the subject, e.g., cancer, or autoimmune diseases.
In some embodiments, the tested agent or the combination of tested agents is designed for treating various cancers. As used herein, the term “cancer” refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term “tumor” as used herein refers to cancerous cells, e.g., a mass of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In some embodiments, the agents described herein are designed for treating or diagnosing a carcinoma in a subject. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the cancer is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.
In some embodiments, the tested agent is designed for the treating melanoma, primary lung carcinoma, non-small cell lung carcinoma (NSCLC) , small cell lung cancer (SCLC) , primary gastric carcinoma, bladder cancer, breast cancer, and/or prostate cancer.
In some embodiments, the injected tumor cells are human tumor cells. In some embodiments, the injected tumor cells are melanoma cells, primary lung carcinoma cells, non-small cell lung carcinoma (NSCLC) cells, small cell lung cancer (SCLC) cells, primary gastric carcinoma cells, bladder cancer cells, breast cancer cells, and/or prostate cancer cells.
The inhibitory effects on tumors can also be determined by any methods known in the art. In some embodiments, the tumor cells can be labeled by a luciferase gene. Thus, the number of the tumor cells or the size of the tumor in the animal can be determined by an in vivo imaging system (e.g., the intensity of fluorescence) . In some embodiments, the inhibitory effects on tumors can also be determined by measuring the tumor volume in the animal, and/or determining tumor (volume) inhibition rate (TGI
TV) . The tumor growth inhibition rate can be calculated using the formula TGI
TV (%) = (1 –TVt/TVc) x 100, wherein TVt and TVc are the mean tumor volume (or weight) of treated and control groups.
In some embodiments, the tested agent can be one or more agents selected from the group consisting of paclitaxel, cisplatin, carboplatin, pemetrexed, 5-FU, gemcitabine, oxaliplatin, docetaxel, and capecitabine.
In some embodiments, the tested agent can be an antibody, for example, an antibody that binds to CD47, PD-1, CTLA-4, LAG-3, TIM-3, BTLA, PD-L1, 4-1BB, CD27, CD28, CD47, TIGIT, CD27, GITR, or OX40. In some embodiments, the antibody is a human antibody.
The present disclosure also relates to the use of the animal model generated through the methods as described herein in the development of a product related to an immunization processes of human cells, the manufacturing of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.
In some embodiments, the disclosure provides the use of the animal model generated through the methods as described herein in the production and utilization of an animal experimental disease model of an immunization processes involving human cells, the study on a pathogen, or the development of a new diagnostic strategy and/or a therapeutic strategy.
HR knockout animal model with additional genetic modifications
The present disclosure further relates to methods for generating genetically modified animal models described herein with some additional modifications (e.g., human or chimeric genes or additional gene knockout) .
In some embodiments, the animal can comprise a disruption at the endogenous HR gene and a sequence encoding a human or chimeric protein. In some embodiments, the human or chimeric protein can be programmed cell death protein 1 (PD-1) , TNF Receptor Superfamily Member 9 (4-1BB or CD137) , cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) , LAG-3, T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) , B And T Lymphocyte Associated (BTLA) , Programmed Cell Death 1 Ligand 1 (PD-L1) , CD27, CD28, CD47, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , Glucocorticoid-Induced TNFR-Related Protein (GITR) , or TNF Receptor Superfamily Member 4 (TNFRSF4; or OX40) .
In some embodiments, the animal can further comprise a disruption (e.g., knockout) at one or more of the following endogenous genes, e.g., IL6, IL15, colony stimulating factor (CSF) , colony stimulating factor 1 (CSF1) , colony stimulating factor 2 (CSF2 or GM-CSF) , colony stimulating factor 3 (CSF3) , signal regulatory protein alpha (SIRPA) , B2M, and KIT proto-oncogene receptor tyrosine kinase (C-KIT) genes. In some embodiments, the animal can comprise a disruption at the endogenous CD132 gene (interleukin-2 receptor subunit gamma) . In some embodiments, the animal can comprise a disruption at some other endogenous genes (e.g., Forkhead Box N1 (Foxn1) ) . In some embodiments, the animal can comprise a disruption at the endogenous B2M gene.
The methods of HR knockout animal model with additional genetic modifications (e.g., humanized genes or additional gene knockout) can include the following steps:
(a) using the methods as described herein to obtain a HR knockout animal;
(b) mating the HR knockout animal with another genetically modified non-human animal with the desired genetic modifications, and then screening the progeny to obtain a HR animal with the desired genetic modifications.
In some embodiments, in step (b) of the method, the genetically modified animal can be mated with a genetically modified non-human animal with human or chimeric PD-1, CTLA-4, LAG-3, TIM-3, BTLA, PD-L1, 4-1BB, CD27, CD28, CD47, TIGIT, GITR, or OX40, or an animal with NOD-Prkdc
scid IL-2rγ
null mutation and/or B2M mutation.
Some of these genetically modified non-human animals are described, e.g., in PCT/CN2017/090320, PCT/CN2017/099577, PCT/CN2017/099575, PCT/CN2017/099576, PCT/CN2017/099574, PCT/CN2017/106024, PCT/CN2018/079365; PCT/CN2019/072406; each of which is incorporated herein by reference in its entirety.
In some embodiments, the HR knockout can be directly performed on a genetically modified animal having a human or chimeric PD-1, CTLA-4, LAG-3, BTLA, TIM-3, PD-L1, 4-1BB, CD27, CD28, CD47, TIGIT, GITR, or OX40 gene, or an animal with NOD-Prkdc
scid IL-2rγ
null mutation and/or B2M mutation. In some embodiments, the mouse further has a disruption of B2M gene (e.g., deletion of one or more exons or part of exons selected from the group consisting of exon 1, exon 2, exon 3, or exon 4 of the endogenous B2M gene) .
In some embodiments, the HR knockout can be directly performed on a CD132 knockout mouse or a Foxn1 knockout mouse.
As these proteins may involve different mechanisms, a combination therapy that targets two or more of these proteins thereof may be a more effective treatment. In fact, many related clinical trials are in progress and have shown a good effect. The HR knockout animal model, and/or the HR knockout animal model with additional genetic modifications can be used for determining effectiveness of a combination therapy.
In some embodiments, the combination of agents can include one or more agents selected from the group consisting of paclitaxel, cisplatin, carboplatin, pemetrexed, 5-FU, gemcitabine, oxaliplatin, docetaxel, and capecitabine.
In some embodiments, the combination of agents can include one or more agents selected from the group consisting of campothecin, doxorubicin, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, adriamycin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, bleomycin, plicomycin, mitomycin, etoposide, verampil, podophyllotoxin, tamoxifen, taxol, transplatinum, 5-flurouracil, vincristin, vinblastin, and methotrexate.
In some embodiments, the combination of agents can include one or more antibodies that bind to CD47, PD-1, CTLA-4, LAG-3, BTLA, TIM-3, PD-L1, 4-1BB, CD27, CD28, CD47, TIGIT, GITR, and/or OX40.
Alternatively or in addition, the methods can also include performing surgery on the subject to remove at least a portion of the cancer, e.g., to remove a portion of or all of a tumor (s) , from the subject.
CD132 knockout non-human animal
Interleukin-2 (IL-2) is a 15.5 kDa type 1 four α-helical bundle cytokine produced primarily by CD4+ T cells following their activation by antigen. IL-2 was the first type 1 cytokine cloned and the first cytokine for which a receptor component was cloned. Three different IL-2 receptor chains exist that together generate low, intermediate, and high affinity IL-2 receptors. The ligand-specific IL-2 receptor α chain (IL-2Rα, CD25, Tac antigen) , which is expressed on activated but not non-activated lymphocytes, binds IL-2 with low affinity (Kd ~ 10
-8 M) ; the combination of IL-2Rβ (CD122) and IL-2Rγ (CD132) together form an IL-2Rβ/γc complex mainly on memory T cells and NK cells that binds IL-2 with intermediate affinity (Kd ~ 10
-9 M) ; and when all three receptor chains are co-expressed on activated T cells and Treg cells, IL-2 is bound with high affinity (Kd ~ 10
-11 M) .
For the high affinity receptor, the three dimensional structure of the quaternary complex supports a model wherein IL-2 initially bind IL-2Rα, then IL-2Rβ is recruited, and finally IL-2Rγ. The intermediate and high affinity receptor forms are functional, transducing IL-2 signals.
CD132 is also an essential component shared by the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.
IL-2Rγ is encoded by the gene, IL2RG (CD132) , that is mutated in humans with X-linked severe combined immunodeficiency (XSCID) and physically recruits JAK3, which when mutated also causes an XSCID-like T-B+NK-form of SCID. In XSCID and JAK3-deficient SCID, the lack of signaling by IL-7 and IL-15, respectively, explains the lack of T and NK cell development, whereas defective signaling by IL-4 and IL-21 together explain the non-functional B cells and hypogammaglobulinemia.
A detailed description of CD132 and its function can be found, e.g., in Liao et al. "IL-2 family cytokines: new insights into the complex roles of IL-2 as a broad regulator of T helper cell differentiation, " Current opinion in immunology 23.5 (2011) : 598-604; Noguchi et al. "Interleukin-2 receptor gamma chain: a functional component of the interleukin-7 receptor, " Science 262.5141 (1993) : 1877-1880; Henthorn et al. "IL-2Rγ gene microdeletion demonstrates that canine X-linked severe combined immunodeficiency is a homologue of the human disease, " Genomics 23.1 (1994) : 69-74; and US Patent No. 7145055; each of which is incorporated herein by reference in its entirety.
In human genomes, CD132 gene (Gene ID: 3561) is located on X chromosome, and has eight exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8. The CD132 protein also has an extracellular region, a transmembrane region, and a cytoplasmic region. The nucleotide sequence for human CD132 mRNA is NM_000206.2, and the amino acid sequence for human CD132 is NP_000197.1.
Similarly, in mice, CD132 gene locus has eight exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8. The CD132 protein also has an extracellular region, a transmembrane region, and a cytoplasmic region, and the signal peptide is located at the extracellular region of CD132. The nucleotide sequence for mouse CD132 cDNA is NM_013563.4 (SEQ ID NO: 29) , the amino acid sequence for mouse CD132 is NP_038591.1 (SEQ ID NO: 30) . The location for each exon and each region in the mouse CD132 nucleotide sequence and amino acid sequence is listed below:
Table 2
The mouse CD132 gene (Gene ID: 16186) is located in Chromosome X of the mouse genome, which is located from 101, 268, 255 to 101, 264, 385 of NC_000086.7 (GRCm38. p4 (GCF_000001635.24) ) . The 5’-UTR is from 101, 268, 255 to 101, 268, 170, exon 1 is from 101, 268, 255 to 101, 268, 055, the first intron (intron 1) is from 101, 268, 054 to 101, 267, 865, exon 2 is from 101, 267, 864 to 101, 267, 711, the second intron (intron 2) is from 101, 267, 710 to 101, 267, 496, exon 3 is from 101, 267, 495 to 101, 267, 311, the third intron (intron 3) is from 101, 267, 310 to 101, 267, 121, exon 4 is from 101, 267, 120 to 101, 266, 978, the fourth intron (intron 4) is from 101, 266, 977 to 101, 266, 344, exon 5 is from 101, 266, 343 to 101, 266, 181, the fifth intron (intron 5) is from 101, 266, 180 to 101, 265, 727, exon 6 is from 101, 265, 726 to 101, 265, 630, the sixth intron (intron 6) is from 101, 265, 629 to 101, 265, 443, exon 7 is from 101, 265, 442 to 101, 265, 376, the seventh intron (intron 7) is from 101, 265, 375 to 101, 265, 038, exon 8 is from 101, 265, 037 to 101, 264, 378, and the 3’-UTR is from 101, 264, 851 to 101, 264, 378, based on transcript NM_013563.4. All relevant information for mouse CD132 locus can be found in the NCBI website with Gene ID: 16186, which is incorporated by reference herein in its entirety.
CD132 genes, proteins, and locus of the other species are also known in the art. For example, the gene ID for CD132 in Rattus norvegicus is 140924, the gene ID for CD132 in Macaca mulatta (Rhesus monkey) is 641338, the gene ID for CD132 in Sus scrofa (pig) is 397156. The relevant information for these genes (e.g., intron sequences, exon sequences, amino acid residues of these proteins) can be found, e.g., in NCBI database.
The present disclosure provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal’s endogenous CD132 gene, wherein the disruption of the endogenous CD132 gene comprises deletion of one or more exons, or part of the one or more exons, wherein the one or more exons are selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 of the endogenous CD132 gene. Thus, the disclosure provides a genetically-modified, non-human animal that does not have one or more exons that are selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 of the endogenous CD132 gene. In some embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides in the exon of CD132 are deleted.
In some embodiments, the disruption comprises deletion of one or more introns, or part of the one or more introns, wherein the one or more introns are selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7 of the endogenous CD132 gene. Thus, the disclosure provides a genetically-modified, non-human animal does not have one or more introns that are selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7 of the endogenous CD132 gene.
In some embodiments, the disruption of the endogenous CD132 gene comprises deletion of exon 2 of the endogenous CD132 gene. In some embodiments, the disruption of the endogenous CD132 gene further comprises deletion of exon 1, or part of exon 1 of the endogenous CD132 gene.
In some embodiments, the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 are deleted. In some embodiments, the signal peptide region, extracellular region, transmembrane region, and/or cytoplasmic region of CD132 are deleted.
In some embodiments, a “region” or “portion” of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7, signal peptide region, extracellular region, transmembrane region, and/or cytoplasmic region are deleted.
In some embodiments, the “region” or “portion” can be at least 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7, signal peptide region, extracellular region, transmembrane region, or cytoplasmic region of CD132. In some embodiments, a region, a portion, or the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8 of CD132 are deleted. In some embodiments, a region, a portion, or the entire sequence of mouse intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and/or intron 7 are deleted.
In some embodiments, the disruption comprises or consists of deletion of more than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 nucleotides in exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, and/or exon 8. In some embodiments, the disruption comprises or consists of deletion of more than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, or 1000 nucleotides in intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and/or intron 7.
In some embodiments, the disruption comprises or consists of deletion of more than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (e.g., about 150 or 160 nucleotides) in exon 1; deletion of the entirety of intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7; and/or deletion of more than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, or 650 nucleotides (e.g., about 200, 250 or 270 nucleotides) in exon 8.
In some embodiments, the length of the remaining exon sequences at the endogenous CD132 gene locus is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, or 50%of the total length of all exon sequences of the endogenous CD132 gene. In some embodiments, the length of the remaining exon sequences at the endogenous CD132 gene locus is more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, or 50%of the total length of all exon sequences of the endogenous CD132 gene.
In some embodiments, the length of the remaining sequences at that the endogenous CD132 gene locus is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, or 50%of the full sequence of the endogenous CD132 gene. In some embodiments, the length of the remaining sequences at that the endogenous CD132 gene locus is more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, or 50%of the full sequence of the endogenous CD132 gene.
The present disclosure further relates to the genomic DNA sequence of a CD132 knockout mouse. In some embodiments, the genome of the animal comprises from 5’ to 3’ at the endogenous CD132 gene locus, (a) a first DNA sequence; optionally (b) a second DNA sequence comprising an exogenous sequence; (c) a third DNA sequence, wherein the first DNA sequence, the optional second DNA sequence, and the third DNA sequence are linked.
The second DNA sequence can have a length of 0 nucleotides to 300 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 nucleotides) . In some embodiments, the second DNA sequence has only 0 nucleotides, which means that there is no extra sequence between the first DNA sequence and the third DNA sequence. In some embodiments, the second DNA sequence has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 nucleotides. In some embodiments, the second DNA sequence has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 nucleotides.
In some embodiments, the first DNA sequence comprises an endogenous CD132 gene sequence that is located upstream of intron 1, and can include all or just part of sequences that is located upstream of intron 1. In some embodiments, the first DNA sequence comprises an endogenous CD132 gene sequence that is located upstream of exon 1. In some embodiments, the first DNA sequence comprises a sequence that has a length (5’ to 3’) of from 10 to 200 nucleotides (e.g., from 10 to 100 nucleotides, or from 10 to 20 nucleotides) starting from the first nucleotide in exon 1 of the CD132 gene to the last nucleotide of the first DNA sequence. In some embodiments, the first DNA sequence comprises at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides from exon 1. In some embodiments, the first DNA sequence has at most 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides from exon 1.
In some embodiments, the third DNA sequence comprises an endogenous CD132 gene sequence that is located downstream of the last intron (e.g., intron 7 in mouse) , and can include all or just part of sequences that is located downstream of intron 7. In some embodiments, the third DNA sequence comprises a sequence that has a length (5’ to 3’) of from 200 to 600 nucleotides (e.g., from 300 to 400 nucleotides, or from 350 to 400 nucleotides) starting from the first nucleotide in the third DNA sequence to the last nucleotide in the last exon (e.g., exon 8 in mouse) of the endogenous CD132 gene. In some embodiments, the third DNA sequence comprises at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 nucleotides from the last exon (e.g., exon 8 in mouse) . In some embodiments, the third DNA sequence has at most 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 nucleotides from the last exon (e.g., exon 8 in mouse) .
Thus, in one aspect, the disclosure relates to a genetically-modified, non-human animal whose genome comprise a disruption in the animal’s endogenous CD132 gene, wherein the disruption of the endogenous CD132 gene comprises deletion of exon 2 of the endogenous CD132 gene.
In some embodiments, the disruption of the endogenous CD132 gene further comprises deletion of exon 1 of the endogenous CD132 gene. In some embodiments, the disruption of the endogenous CD132 gene comprises deletion of part of exon 1 of the endogenous CD132 gene.
In some embodiments, the disruption of the endogenous CD132 gene further comprises deletion of one or more exons or part of exons selected from the group consisting of exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 of the endogenous CD132 gene. In some embodiments, the disruption of the endogenous CD132 gene comprises deletion of exons 1-8 of the endogenous CD132 gene.
In some embodiments, the disruption of the endogenous CD132 gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7 of the endogenous CD132 gene.
In some embodiments, the disruption consists of deletion of more than 150 nucleotides in exon 1; deletion of the entirety of intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7; and deletion of more than 250 nucleotides in exon 8.
In some embodiments, the animal is homozygous with respect to the disruption of the endogenous CD132 gene. In some embodiments, the animal is heterozygous with respect to the disruption of the endogenous CD132 gene.
In some embodiments, the disruption prevents the expression of functional CD132 protein.
In some embodiments, the length of the remaining exon sequences at the endogenous CD132 gene locus is less than 30%of the total length of all exon sequences of the endogenous CD132 gene. In some embodiments, the length of the remaining sequences at that the endogenous CD132 gene locus is less than 15%of the full sequence of the endogenous CD132 gene.
In another aspect, the disclosure relates to a genetically-modified, non-human animal, wherein the genome of the animal does not have exon 2 of CD132 gene at the animal’s endogenous CD132 gene locus.
In some embodiments, the genome of the animal does not have one or more exons or part of exons selected from the group consisting of exon 1, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8. In some embodiments, the genome of the animal does not have one or more introns or part of introns selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7.
In one aspect, the disclosure also provides a CD132 knockout non-human animal, wherein the genome of the animal comprises from 5’ to 3’ at the endogenous CD132 gene locus, (a) a first DNA sequence; optionally (b) a second DNA sequence comprising an exogenous sequence; (c) a third DNA sequence, wherein the first DNA sequence, the optional second DNA sequence, and the third DNA sequence are linked, wherein the first DNA sequence comprises an endogenous CD132 gene sequence that is located upstream of intron 1, the second DNA sequence can have a length of 0 nucleotides to 300 nucleotides, and the third DNA sequence comprises an endogenous CD132 gene sequence that is located downstream of intron 7.
In some embodiments, the first DNA sequence comprises a sequence that has a length (5’ to 3’) of from 10 to 100 nucleotides (e.g., approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides) , wherein the length of the sequence refers to the length from the first nucleotide in exon 1 of the CD132 gene to the last nucleotide of the first DNA sequence.
In some embodiments, the first DNA sequence comprises at least 10 nucleotides from exon 1 of the endogenous CD132 gene. In some embodiments, the first DNA sequence has at most 100 nucleotides from exon 1 of the endogenous CD132 gene.
In some embodiments, the third DNA sequence comprises a sequence that has a length (5’ to 3’) of from 200 to 600 nucleotides (e.g., approximately 200, 250, 300, 350, 400, 450, 500, 550, 600 nucleotides) , wherein the length of the sequence refers to the length from the first nucleotide in the third DNA sequence to the last nucleotide in exon 8 of the endogenous CD132 gene.
In some embodiments, the third DNA sequence comprises at least 300 nucleotides from exon 8 of the endogenous CD132 gene. In some embodiments, the third DNA sequence has at most 400 nucleotides from exon 8 of the endogenous CD132 gene.
In some embodiments, the genetic modified non-human animal comprises a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical or 100%identical to the following sequence: aggaaatgtatggtggggagggcttgtgggagagctaagtttcgatttcctgtcccatgtaactgcttttctgttccatatgccctact tgagagtgtcccttgccctctttccctgcacaagccctcccatgcccagcctaacacctttccactttctttgaagagagtcttaccct gtagcccagggtggctgggagctcactatgtaggccaggttggcctccaactcacaggctatcctcccacctctgcctcataaga gttggggttactggcatgcaccaccacacccagcatggtccttctcttttataggattctccctccctttttctacctatgattcaactgt ttccaaatcaacaagaaataaagtttttaaccaatgatca (SEQ ID NO: 31) . In some embodiments, the sequence is located at the endogenous CD132 locus.
In one aspect, the disclosure also relates to a genetically-modified, non-human animal produced by a method comprising knocking out one or more exons of endogenous CD132 gene by using (1) a first nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a target sequence in exon 1 of the endogenous CD132 gene or upstream of exon 1 of the endogenous CD132 gene, and (2) a second nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a sequence in exon 8 of the endogenous CD132 gene.
In some embodiments, the nuclease is CRISPR associated protein 9 (Cas9) . In some embodiments, the target sequence is in exon 1 of the endogenous CD132 gene or upstream of exon 1 of the endogenous CD132 gene. In some embodiments, the target sequence is in exon 8 of the endogenous CD132 gene.
In some embodiments, the animal does not express a functional CD132 protein. In some embodiments, the animal does not express a functional interleukin-2 receptor.
In some embodiments, the animal further comprises a disruption in the animal’s endogenous Beta-2-Microglobulin (B2m) gene and/or a disruption in the animal’s endogenous Forkhead Box N1 (Foxn1) gene.
In one aspect, the disclosure is also related to methods of producing a CD132 knockout mouse. The methods involve
(a) transforming a mouse embryonic stem cell with a gene editing system that targets endogenous CD132 gene, thereby producing a transformed embryonic stem cell;
(b) introducing the transformed embryonic stem cell into a mouse blastocyst;
(c) implanting the mouse blastocyst into a pseudopregnant female mouse; and
(d) allowing the blastocyst to undergo fetal development to term, thereby obtaining the CD132 knockout mouse.
In another aspect, the disclosure also provides methods of producing a CD132 knockout mouse. The methods include the steps of
(a) transforming a mouse embryonic stem cell with a gene editing system that targets endogenous CD132 gene, thereby producing a transformed embryonic stem cell;
(b) implanting the transformed embryonic cell into a pseudopregnant female mouse; and
(c) allowing the transformed embryonic cell to undergo fetal development to term, thereby obtaining the CD132 knockout mouse.
In some embodiments, the gene editing system comprises a first nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a target sequence in exon 1 of the endogenous CD132 gene or upstream of exon 1 of the endogenous CD132 gene, and a second nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a sequence in exon 8 of the endogenous CD132 gene.
In some embodiments, the mouse embryonic stem cell has a Nod/scid background, or a NOD/scid nude background.
In some embodiments, the mouse embryonic stem cell has a genome comprising a disruption in the animal’s endogenous Beta-2-Microglobulin (B2m) gene and/or a disruption in the animal’s endogenous Forkhead Box N1 (Foxn1) gene.
In another aspect, the disclosure relates to a non-human mammalian cell, comprising a disruption, a deletion, or a genetic modification as described herein.
In some embodiments, the cell includes Cas9 mRNA or an in vitro transcript thereof.
In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell. In some embodiments, the cell is a germ cell. In some embodiments, the cell is a blastocyst.
In another aspect, the disclosure relates to methods for establishing a CD132 knockout animal model. The methods include the steps of:
(a) providing the cell with a disruption in the endogenous CD132 gene, and
preferably the cell is a fertilized egg cell;
(b) culturing the cell in a liquid culture medium;
(c) transplanting the cultured cell to the fallopian tube or uterus of the recipient female non-human mammal, allowing the cell to develop in the uterus of the female non-human mammal;
(d) identifying the germline transmission in the offspring of the pregnant female in step (c) .
In some embodiments, the establishment of a CD132 knockout animal involves a gene editing technique that is based on CRISPR/Cas9.
In some embodiments, the non-human mammal is mouse. In some embodiments, the non-human mammal in step (c) is a female with false pregnancy.
EXAMPLES
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Materials and Methods
The following materials were used in the following examples.
NOD-Prkdc
scid IL-2rg
null (B-NDG) mice were obtained from Beijing Biocytogen Co., Ltd (Catalog number: B-CM-002) .
Ambion
TM in vitro transcription kit was purchased from Ambion, Inc. The catalog number is AM1354.
E. coli TOP10 competent cells were purchased from the Tiangen Biotech (Beijing) Co. The catalog number is CB104-02.
EcoRI, BamHI, and BbsI were purchased from NEB. The catalog numbers are R3101M, R3136M, and R0539L.
Kanamycin was purchased from Amresco. The catalog number is 0408.
Cas9 mRNA was obtained from SIGMA. The catalog number is CAS9MRNA-1EA.
UCA kit was obtained from Beijing Biocytogen Co., Ltd. The catalog number is BCG-DX-001.
pHSG299 plasmids were purchased from Takara. The catalog number is 3299.
EXAMPLE 1: sgRNAs for HR
The target sequence determines the targeting specificity of small guide RNA (sgRNA) and the efficiency of Cas9 cleavage at the target site. Therefore, target sequence selection is important for sgRNA vector construction.
Several sgRNAs were designed for the mouse HR gene (NCBI Gene ID: 15460) . The target sequences for these sgRNAs are shown below:
sgRNA1 target sequence (SEQ ID NO: 1) : 5’-acccgacaggctcgagtcactgg-3’
sgRNA2 target sequence (SEQ ID NO: 2) : 5’-cctggcactgccgtcgggcttgg -3’
sgRNA3 target sequence (SEQ ID NO: 3) : 5’-ccccagagagacgcaagcgaggg-3’
sgRNA4 target sequence (SEQ ID NO: 4) : 5’-cgctgctaactgaagcccggagg-3’
sgRNA5 target sequence (SEQ ID NO: 5) : 5’-ttccctcgcttgcgtctctctgg -3’
sgRNA6 target sequence (SEQ ID NO: 6) : 5’-ggtgccctggcactgccgtcggg-3’
sgRNA7 target sequence (SEQ ID NO: 7) : 5’-cccagtgactcgagcctgtcggg-3’
sgRNA8 target sequence (SEQ ID NO: 8) : 5’-ggtgctagggaccggaacgtagg-3’
sgRNA9 target sequence (SEQ ID NO: 9) : 5’-aaacaggaggacctacgttccgg-3’
sgRNA10 target sequence (SEQ ID NO: 10) : 5’-gcaatgtttaagtcgagccaggg-3’
sgRNA11 target sequence (SEQ ID NO: 11) : 5’-gcatgtatgacggtcagatttgg-3’
sgRNA12 target sequence (SEQ ID NO: 12) : 5’-tgcacgtgcacgcatgccctcgg-3’
sgRNA13 target sequence (SEQ ID NO: 13) : 5’-tctacattaacatcgtgaaatgg-3’
sgRNA14 target sequence (SEQ ID NO: 14) : 5’-attcagtccgatccttctcaagg-3’
sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5, sgRNA6, and sgRNA7 target the 5’-end target site and sgRNA8, sgRNA9, sgRNA10, sgRNA11, sgRNA12, sgRNA13, and sgRNA14 target the 3’-end target site. The target sites for sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5, sgRNA6, and sgRNA7 are located within intron 2 of the mouse endogenous HR gene (Gene ID: 15460) . The target sites for sgRNA8, sgRNA9, sgRNA10, sgRNA11, sgRNA12, sgRNA13, and sgRNA14 are located within intron 7 of HR (based on the sequence of NM_021877.3→NP_068677.2) .
EXAMPLE 2. sgRNA selection
The UCA kit was used to detect the activities of sgRNAs (FIG. 1 and Table 4) . The results show that the sgRNAs had different activities. Two of them (sgRNA4 and sgRNA10) were selected for further experiments.
Single strand oligonucleotides were synthesized for sgRNA4 and sgRNA10. TAGG was first added to the 5’ end of the upstream sequence of sgRNA4 and sgRNA10 target sequences to obtain a forward oligonucleotide sequence, and AAAC was added to the 5’ end of the complementary strand to obtain a reverse oligonucleotide sequence.
Table 3. Oligonucleotide sequences for sgRNA4 and sgRNA10
Table 4. sgRNA activities
EXAMPLE 3. Construction of pT7-sgRNA G2 vector
pT7-sgRNA G2 vector map is shown in FIG. 2. The DNA fragment containing T7 promoter and sgRNA scaffold was synthesized, and linked to the backbone vector pHSG299 by restriction enzyme digestion (EcoRI and BamHI) and ligation. The plasmid sequences were confirmed by sequencing.
The DNA fragment containing the T7 promoter and sgRNA scaffold (SEQ ID NO: 23) is shown below:
EXAMPLE 4. Construction of pT7-HR-4 and pT7-HR-10 vectors
After annealing the oligonucleotides obtained in Example 2, the product was ligated into the pT7-sgRNA G2 plasmid (the plasmid was first treated by BbsI restriction enzyme) .
Table 5. The ligation reaction conditions (10μL)
Double stranded fragment | 1μL (0.5μM) |
pT7-sgRNA G2 vector | 1μL (10 ng) |
T4 DNA Ligase | 1μL (5U) |
10×T4 DNA Ligase buffer | 1μL |
50%PEG4000 | 1μL |
H 2O | Add to 10μL |
The ligation reaction was carried out at room temperature for 10 to 30 minutes. The ligation product was then transferred to 30 μL of TOP10 competent cells. The cells were then plated on a petri dish with Kanamycin, and then cultured at 37 ℃ for at least 12 hours and then two clones were selected and added to LB medium with Kanamycin (5 ml) , and then cultured at 37 ℃ at 250 rpm for at least 12 hours.
Clones were randomly selected and sequenced to verify their sequences. The pT7-HR-4 and pT7-HR-10 vectors with correct sequences were selected for subsequent experiments.
EXAMPLE 5. Microinjection and embryo transfer
The pre-mixed Cas9 mRNA, in vitro transcription products of pT7-HR-4 and pT7-HR-10 plasmids were injected into the cytoplasm or nucleus of B-NDG mouse fertilized eggs with a microinjection instrument (using Ambion in vitro transcription kit to carry out the transcription according to the method provided in the product instruction) . The embryo microinjection was carried out according to the method described, e.g., in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition) , ” Cold Spring Harbor Laboratory Press, 2003. The injected fertilized eggs were then transferred to a culture medium to culture for a short time and then was transplanted into the oviduct of the recipient mouse to produce the genetically modified mice (F0 generation) . The mouse population was further expanded by cross-mating and self-mating to establish stable mouse lines.
EXAMPLE 6. Genotype verification
1. Genotype identification for F0 generation mice
In order to confirm the genotype of the F0-generation Hr gene knockout heterozygous mouse, genomic DNA was extracted from the tail of the F0 generation mice obtained in Example 5. PCR was performed with the primer MSD-F (SEQ ID NO: 24) and MSD-R (SEQ ID NO: 25) . MSD-F is located on the left of the sgRNA4 target site. MSD-R is located on the right of the sgRNA10 target site. The length of PCR amplification products in the wildtype (WT) mice should be about 7859 bp. In the knockout mice, the length of the PCR amplification products should be about 610 bp. The sequence for the primers are shown below:
MSD-F (SEQ ID NO: 24) : 5’-gctcacgtacatccatccctcttgg -3’
MSD-R (SEQ ID NO: 25) : 5’-tagaattcttgtttttggaacgcaga -3’
The PCR reaction conditions are shown in the tables below. The results are shown in FIG. 3.
Table 6. The PCR reaction system (20 μL)
2× PCR buffer | 10 μL |
dNTP (2 mM) | 4 μL |
Upstream primer (10μM) | 0.6μL |
Downstream primer (10μM) | 0.6μL |
Mouse tail genomic DNA | 100ng |
KOD-FX (1U/μL) | 0.4μL |
H 2O | Add to 20μL |
Table 7. The PCR reaction conditions
Sequencing was performed on the PCR amplification products of the mice that were tested positive. The sequencing results for F0 generation mice (Table 8) confirmed that several Hr knockout mice were obtained by the method described herein, and they had different mutations.
Table 8. Genotype verification for F0 generation mice
Label | Sex | Results |
F0-1 | M | △7258 |
F0-4 | M | △7252 |
F0-18 | M | △7253 |
F0-21 | M | in15△7248 |
F0-28 | M | △7257 |
F0-35 | F | △7265 |
F0-37 | F | in1△7254 |
F0-38 | F | in4△7263 |
F0-41 | F | △7252 |
△ indicates knockout, e.g., △7258 indicates a deletion of 7258bp, and △7252 indicates a deletion of 7252bp. In indicates random insertion, e.g., in15 indicates a random insertion of 15bp of nucleotides and in1 a random insertion of 1bp of nucleotide. In F0-28, a sequence of 7257 nucleotides (SEQ ID NO: 33) at the endogens HR locus is deleted. The mouse has a sequence that is identical to SEQ ID NO: 28 at the endogens HR locus. In F0-18, a sequence of 7253 nucleotides (SEQ ID NO: 34) at the endogens HR locus is deleted. The mouse has a sequence that is identical to SEQ ID NO: 35 at the endogens HR locus.
2. Genotype identification for F1 generation mice
F0 generation mice were then mated with B-NDG mice to obtain F1 generation mice. Both F0-18 and F0-28 were mated with wild-type B-NDG mice, and their offspring were tested. Gene identification showed that a total of six F1 generation mice were positive. The PCR results were shown in FIGS. 4 and 5. The mice labeled with F1-2, F1-3 and F1-7 were F0-18 offspring. The mice labeled with F1-10, F1-11, F1-12 were offspring of F0-28. The six mice were further sequenced and verified, and the sequencing results are shown in Table 9. The results indicate that the method can be used to make a Hr knockout mouse.
Table 9. Genotype identification for F1 generation mice
These F1 generation mice were mated with each other. Homozygous F2 generation mice having a hairless phenotype were identified. The results are shown in FIG. 6. In contrast, FIG. 7 shows B-NDG mice that have wildtype Hr genes.
EXAMPLE 8. Construction of Human Immune System in Immunodeficient Mice
In the mice obtained by the methods as described herein, a human immune system was constructed by engraftment with human peripheral blood cells (hPBMC) .
Three immunodeficient HR knockout mice were selected and 5 x 10
6 human peripheral blood cells (hPBMCs) were injected into the tail vein of each mouse. Blood was taken 24 days later for flow cytometry analysis. The flow cytometry results showed that cells expressing human leukocyte surface molecular markers (human CD45) were detected in all three mice.
The results show that human peripheral blood cells engraftment on these mice can create a humanized mouse model with the human immune system. Furthermore, human tumor cells were injected into these mice (FIG. 8) . These mice can be used to screen new drugs, and test drug efficacy.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
Claims (58)
- A genetically-modified, non-human animal whose genome comprise a disruption in the animal’s endogenous HR lysine demethylase and nuclear receptor corepressor (HR) gene, wherein the disruption of the endogenous HR gene comprises deletion of one or more exons of the endogenous HR gene.
- The animal of claim 1, wherein the disruption of the endogenous HR gene comprises deletion of one or more exons selected from exon 3, exon 4, exon 5, exon 6, and exon 7 of the endogenous HR gene.
- The animal of claim 1, wherein the disruption of the endogenous HR gene comprises deletion of exon 3, exon 4, exon 5, exon 6, and exon 7 of the endogenous HR gene.
- The animal of any one of claims 1-3, wherein the disruption of the endogenous HR gene further comprises deletion of one or more exons or part of exons selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, and exon 20 of the endogenous HR gene.
- The animal of any one of claims 1-4, wherein the disruption of the endogenous HR gene comprises deletion of one or more introns of the endogenous HR gene.
- The animal of any one of claims 1-5, wherein the disruption of the endogenous HR gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, intron 17, intron 18, and intron 19 of the endogenous HR gene.
- The animal of any one of claims 1-6, wherein the disruption consists ofdeletion of at least 10 nucleotides in intron 2,deletion of the entirety of exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, and exon 7; anddeletion of at least 10 nucleotides in intron 7.
- The animal of any one of claims 1-7, wherein the animal is homozygous with respect to the disruption of the endogenous HR gene.
- The animal of any one of claims 1-7, wherein the animal is heterozygous with respect to the disruption of the endogenous HR gene.
- The animal of any one of claims 1-9, wherein the disruption prevents the expression of functional HR protein.
- The animal of any one of claims 1-10, wherein the length of the remaining exon sequences at the endogenous HR gene locus is less than 70%of the total length of all exon sequences of the endogenous HR gene.
- The animal of any one of claims 1-10, wherein the length of the remaining sequences at that the endogenous HR gene locus is less than 65%of the full sequence of the endogenous HR gene.
- A genetically-modified, non-human animal, wherein the genome of the animal does not have one or more exons of HR gene at the animal’s endogenous HR gene locus.
- The animal of claim 13, wherein the genome of the animal does not have one or more exons or part of exons selected from the group consisting of exon 3, exon 4, exon 5, exon 6, and exon 7.
- The animal of claim 13, wherein the genome of the animal does not have one or more introns or part of introns selected from the group consisting of intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7.
- A HR knockout non-human animal, wherein the genome of the animal comprises from 5’ to 3’ at the endogenous HR gene locus, (a) a first DNA sequence; optionally (b) a second DNA sequence comprising an exogenous sequence; (c) a third DNA sequence, wherein the first DNA sequence, the optional second DNA sequence, and the third DNA sequence are linked,wherein the first DNA sequence comprises an endogenous HR gene sequence that is located upstream of intron 2,the second DNA sequence can have a length of 0 nucleotides to 100 nucleotides, and the third DNA sequence comprises an endogenous HR gene sequence that is located downstream of intron 7.
- The animal of claim 16, wherein the first DNA sequence comprises a sequence that has a length (5’ to 3’) of from 10 to 1500 nucleotides, wherein the length of the sequence refers to the length from the first nucleotide in exon 1 of the HR gene to the last nucleotide of the first DNA sequence.
- The animal of claim 16 or 17, wherein the first DNA sequence comprises at least 10 nucleotides from intron 2 of the endogenous HR gene.
- The animal of any one of claims 16-18, wherein the first DNA sequence comprise exon 1 and exon 2 of the endogenous HR gene.
- The animal of any one of claims 16-19, wherein the third DNA sequence comprises a sequence that has a length (5’ to 3’) of from 10 to 11000 nucleotides, wherein the length of the sequence refers to the length from the first nucleotide in the third DNA sequence to the last nucleotide in the last exon of the endogenous HR gene.
- The animal of any one of claims 16-20, wherein the third DNA sequence comprises at least 10 nucleotides from intron 7 of the endogenous HR gene.
- The animal of any one of claims 16-19, wherein the third DNA sequence comprises exons 8-20, and introns 8-19.
- A genetically-modified, non-human animal produced by a method comprising knocking out one or more exons of endogenous HR gene by using (1) a first nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a target sequence in intron 2 of the endogenous HR gene, and (2) a second nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a sequence in intron 7 of the endogenous HR gene.
- The animal of claim 23, wherein the nuclease is CRISPR associated protein 9 (Cas9) .
- The animal of claim 23, wherein the target sequence in intron 2 of the endogenous HR gene is set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, and the target sequence in intron 7 of the endogenous HR gene is set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14.
- The animal of claim 23, wherein the first nuclease comprises a sgRNA that targets SEQ ID NO: 4 and the second nuclease comprises a sgRNA that targets SEQ ID NO: 10.
- The animal of any one of claims 1-26, wherein the animal does not express a functional HR protein.
- The animal of any one of claims 1-27, wherein the animal does not express a functional interleukin-2 receptor.
- The animal of any one of claims 1-28, wherein the animal has one or more of the following characteristics:(a) the percentage of T cells (CD3+ cells) is less than 5%, 2%, 1.5%, 1%, 0.7%, or 0.5%of leukocytes in the animal;(b) the percentage of B cells (e.g., CD3-CD19+ cells) is less than 1%, 0.1%or 0.05%of leukocytes in the animal;(c) the percentage of NK cells (e.g., CD3-CD49b+ cells) is less than 5%, 2%or 1.5%of leukocytes in the animal;(d) the percentage of CD4+ T cells is less than 1%, 0.5%, 0.3%, or 0.1%of T cells;(e) the percentage of CD8+ T cells is less than 1%, 0.5%, 0.3%, or 0.1%of T cells;(f) the percentage of CD3+ CD4+ cells, CD3+ CD8+ cells, CD3-CD19+ cells is less than 5%, 1%or 0.5%of leukocytes in the animal;(g) the percentage of T cells, B cells, and NK cells is less than 5%, 4%, 3%, 2%or 1%of leukocytes in the animal.
- The animal of any one of claims 1-28, wherein the animal after being engrafted with human hematopoietic stem cells to develop a human immune system has one or more of the following characteristics:(a) the percentage of human CD45+ cells is about or at least 10%, 20%, 30%, 40%, or 50%of leukocytes of the animal;(b) the percentage of human CD3+ cells about or at least 10%, 20%, 30%, 40%, or 50%of leukocytes in the animal;(c) the percentage of human CD19+ cells is about or at least 10%, 20%, 30%, 40%, or 50%of leukocytes in the animal.
- The animal of any one of claims 1-28, wherein the animal does not have hair.
- The animal of any one of claims 1-28, wherein the animal has one or more of the following characteristics:(a) the animal has no functional T-cells and/or no functional B-cells;(b) the animal exhibits reduced macrophage function relative to a NOD/scid mouse;(c) the animal exhibits no NK cell activity;(d) the animal exhibits reduced dendritic function relative to a NOD/scid mouse; and(e) the animal does not have xenogeneic GVHD.
- The animal of any one of claims 1-32, wherein the animal is a mammal, e.g., a monkey, a rodent, a rat, or a mouse.
- The animal of any one of claims 1-32, wherein the animal is a C57 mouse, a C57BL mouse, a BALB/c mouse, a NOD/scid mouse, or a NOD/scid nude mouse, or a NOD-Prkdc scid IL-2rγ null mouse.
- The animal of any one of claims 1-34, wherein the animal further comprises a sequence encoding a human or chimeric protein.
- The animal of claim 35, wherein the human or chimeric protein is programmed cell death protein 1 (PD-1) , PD-L1, IL3, IL6, IL15, CSF1, or CSF2.
- The animal of any one of claims 1-36, wherein the animal further comprises a disruption in the animal’s endogenous Beta-2-Microglobulin (B2M) gene.
- A method of determining effectiveness of an agent or a combination of agents for the treatment of cancer, comprising:engrafting tumor cells to the animal of any one of claims 1-37, thereby forming one or more tumors in the animal;administering the agent or the combination of agents to the animal; anddetermining the inhibitory effects on the tumors.
- The method of claim 38, wherein before engrafting the tumor cells to the animal, human peripheral blood cells (hPBMC) or human hematopoietic stem cells are injected to the animal.
- The method of claim 38, wherein the tumor cells are from cancer cell lines.
- The method of claim 38, wherein the tumor cells are from a tumor sample obtained from a human patient.
- The method of claim 38, wherein the inhibitory effects are determined by measuring the tumor volume in the animal.
- The method of claim 38, wherein the tumor cells are melanoma cells, lung cancer cells, primary lung carcinoma cells, non-small cell lung carcinoma (NSCLC) cells, small cell lung cancer (SCLC) cells, primary gastric carcinoma cells, bladder cancer cells, breast cancer cells, and/or prostate cancer cells.
- The method of claim 38, wherein the agent is an anti-PD-1 antibody.
- The method of claim 38, wherein the agent is an anti-PD-L1 antibody.
- The method of claim 38, wherein the combination of agents comprises one or more agents selected from the group consisting of paclitaxel, cisplatin, carboplatin, pemetrexed, 5-FU, gemcitabine, oxaliplatin, docetaxel, and capecitabine.
- A method of producing an animal comprising a human hemato-lymphoid system, the method comprising:engrafting a population of cells comprising human hematopoietic cells or human peripheral blood cells into the animal of any one of claims 1-37.
- The method of claim 47, wherein the human hemato-lymphoid system comprises human cells selected from the group consisting of hematopoietic stem cells, myeloid precursor cells, myeloid cells, dendritic cells, monocytes, granulocytes, neutrophils, mast cells, lymphocytes, and platelets.
- The method of claim 47, further comprising: irradiating the animal prior to the engrafting.
- A method of producing a HR knockout mouse, the method comprising the steps of:(a) transforming a mouse embryonic stem cell or a fertilized egg with a gene editing system that targets endogenous HR gene, thereby producing a transformed embryonic stem cell;(b) introducing the transformed embryonic stem cell or the fertilized egg into a mouse blastocyst;(c) implanting the mouse blastocyst into a pseudopregnant female mouse; and(d) allowing the blastocyst to undergo fetal development to term, thereby obtaining the HR knockout mouse.
- A method of producing a HR knockout mouse, the method comprising the steps of:(a) transforming a mouse embryonic stem cell or a fertilized egg with a gene editing system that targets endogenous HR gene, thereby producing a transformed embryonic stem cell;(b) implanting the transformed embryonic cell or the fertilized egg into a pseudopregnant female mouse; and(c) allowing the transformed embryonic cell to undergo fetal development to term, thereby obtaining the HR knockout mouse.
- The method of claim 50 or claim 51, wherein the gene editing system comprises a first nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a target sequence in intron 2 of the endogenous HR gene, and a second nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a sequence in intron 7 of the endogenous HR gene.
- The method of claim 52, wherein the nuclease is CRISPR associated protein 9 (Cas9) .
- The method of claim 52, wherein the target sequence in intron 2 of the endogenous HR gene is set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, and the target sequence in intron 7 of the endogenous HR gene is set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14.
- The method of claim 52, wherein the mouse embryonic stem cell or the fertilized egg has a C57 background, a C57BL background, a BALB/c background, a NOD/scid background, a NOD/scid nude, or a NOD-Prkdc scid IL-2rγ null background.
- The method of claim 52, wherein the mouse embryonic stem cell or the fertilized egg comprises a sequence encoding a human or chimeric protein.
- The method of claim 56, wherein the human or chimeric protein is PD-1 or CD137.
- The method of claim 52, wherein the mouse embryonic stem cell or the fertilized egg has a genome comprising a disruption in the animal’s endogenous B2M gene.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810163134 | 2018-02-26 | ||
CN201810163134.3 | 2018-02-26 | ||
CN201811543165.8A CN110195057B (en) | 2018-02-26 | 2018-12-17 | Preparation method and application of genetically modified non-human animal or progeny thereof with Hr gene |
CN201811543165.8 | 2018-12-17 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2019161805A1 true WO2019161805A1 (en) | 2019-08-29 |
Family
ID=67686693
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2019/076192 WO2019161805A1 (en) | 2018-02-26 | 2019-02-26 | Hr knockout non-human animal |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2019161805A1 (en) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999038965A1 (en) * | 1998-01-29 | 1999-08-05 | The Trustees Of Columbia University In The City Of New York | Human hairless gene, protein and uses thereof |
US20160273000A1 (en) * | 2010-02-11 | 2016-09-22 | Recombinetics, Inc. | Methods and materials for producing transgenic artiodactyls |
CN106957856A (en) * | 2016-01-12 | 2017-07-18 | 中国科学院广州生物医药与健康研究院 | The reconstructed eggs and its construction method and the construction method of swine model of hairless swine model |
-
2019
- 2019-02-26 WO PCT/CN2019/076192 patent/WO2019161805A1/en active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999038965A1 (en) * | 1998-01-29 | 1999-08-05 | The Trustees Of Columbia University In The City Of New York | Human hairless gene, protein and uses thereof |
US20160273000A1 (en) * | 2010-02-11 | 2016-09-22 | Recombinetics, Inc. | Methods and materials for producing transgenic artiodactyls |
CN106957856A (en) * | 2016-01-12 | 2017-07-18 | 中国科学院广州生物医药与健康研究院 | The reconstructed eggs and its construction method and the construction method of swine model of hairless swine model |
Non-Patent Citations (3)
Title |
---|
CHEN YING-YING ET AL.: "Model construction of Hr-knockout mouse and its biological characteristics primary research", CHINESE MASTER'S THESES FULL-TEXT DATABASE, 15 November 2017 (2017-11-15), pages 1 - 77, ISSN: 1674-0246 * |
GERARD M. J. BEAUDOIN III ET AL.: "Hairless triggers reactivation of hair growth by promoting Wnt signaling", PNAS, vol. 102, no. 41, 11 October 2005 (2005-10-11), pages 14563 - 14568, XP055633771, ISSN: 0027-8424 * |
ZHU KUI-CHENG ET AL.: "Construction of a Hr mutant knockout mouse model and phenotypic analysis", CHINESE JOURNAL OF COMPAR ATIVE MEDICINE, vol. 26, no. 8, 31 August 2016 (2016-08-31), pages 75 - 78, ISSN: 1671-7856 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11317611B2 (en) | Genetically modified non-human animal with human or chimeric PD-L1 | |
US10945418B2 (en) | Genetically modified non-human animal with human or chimeric PD-L1 | |
WO2018041121A1 (en) | Genetically modified non-human animal with human or chimeric ctla-4 | |
US11071290B2 (en) | Genetically modified non-human animal with human or chimeric CTLA-4 | |
US11505806B2 (en) | Genetically modified non-human animal with human or chimeric OX40 | |
WO2018041120A1 (en) | Genetically modified non-human animal with human or chimeric tigit | |
WO2018041119A1 (en) | Genetically modified non-human animal with human or chimeric ox40 | |
US10820580B2 (en) | Immunodeficient non-human animal | |
US11234421B2 (en) | Genetically modified non-human animal with human or chimeric IL15 | |
WO2018086583A1 (en) | Genetically modified non-human animal with human or chimeric lag-3 | |
US11464876B2 (en) | Genetically modified mouse comprising a chimeric TIGIT | |
WO2020125639A1 (en) | Genetically modified non-human animal with human or chimeric genes | |
WO2019141251A1 (en) | Immunodeficient non-human animal | |
US20190335728A1 (en) | Genetically modified non-human animal with human or chimeric cd28 | |
WO2019179439A1 (en) | Foxn1 knockout non-human animal | |
US20230227531A1 (en) | Genetically modified non-human animal expressing a b2m/fcrn fusion protein | |
WO2021083366A1 (en) | Genetically modified non-human animals with human or chimeric thpo | |
WO2021139799A1 (en) | Genetically modified non-human animal with human or chimeric mhc protein complex | |
WO2021136537A1 (en) | GENETICALLY MODIFIED IMMUNODEFICIENT NON-HUMAN ANIMAL WITH HUMAN OR CHIMERIC SIRPα/CD47 | |
WO2019161805A1 (en) | Hr knockout non-human animal | |
WO2021043289A1 (en) | Genetically modified non-human animals with kit mutations | |
WO2018233608A1 (en) | Genetically modified non-human animal with human or chimeric cd28 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 19757904 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 19757904 Country of ref document: EP Kind code of ref document: A1 |