CN113322275A - 一种巨噬细胞的CRISPR/Cas9表观编辑系统、方法、编辑后的巨噬细胞和应用 - Google Patents
一种巨噬细胞的CRISPR/Cas9表观编辑系统、方法、编辑后的巨噬细胞和应用 Download PDFInfo
- Publication number
- CN113322275A CN113322275A CN202110312960.1A CN202110312960A CN113322275A CN 113322275 A CN113322275 A CN 113322275A CN 202110312960 A CN202110312960 A CN 202110312960A CN 113322275 A CN113322275 A CN 113322275A
- Authority
- CN
- China
- Prior art keywords
- sgrna
- gene
- ezh2
- pcp
- macrophage
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000002540 macrophage Anatomy 0.000 title claims abstract description 124
- 108091033409 CRISPR Proteins 0.000 title claims abstract description 68
- 238000010354 CRISPR gene editing Methods 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 17
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 70
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 42
- 239000002773 nucleotide Substances 0.000 claims abstract description 39
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 39
- 108091027544 Subgenomic mRNA Proteins 0.000 claims abstract description 36
- 101150090105 Ezh2 gene Proteins 0.000 claims abstract description 19
- 101150015232 pcp gene Proteins 0.000 claims abstract description 19
- 108091023037 Aptamer Proteins 0.000 claims abstract description 16
- 230000004614 tumor growth Effects 0.000 claims abstract description 11
- 230000004927 fusion Effects 0.000 claims abstract description 10
- 230000000259 anti-tumor effect Effects 0.000 claims abstract description 8
- 239000013604 expression vector Substances 0.000 claims description 44
- 101001046870 Homo sapiens Hypoxia-inducible factor 1-alpha Proteins 0.000 claims description 30
- 239000013598 vector Substances 0.000 claims description 25
- 230000008685 targeting Effects 0.000 claims description 19
- 239000003814 drug Substances 0.000 claims description 12
- 230000002401 inhibitory effect Effects 0.000 claims description 11
- 108700039691 Genetic Promoter Regions Proteins 0.000 claims description 10
- 210000002865 immune cell Anatomy 0.000 claims description 10
- 230000010287 polarization Effects 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 8
- 230000001737 promoting effect Effects 0.000 claims description 8
- 206010062016 Immunosuppression Diseases 0.000 claims description 6
- 230000002708 enhancing effect Effects 0.000 claims description 6
- 230000001506 immunosuppresive effect Effects 0.000 claims description 6
- 206010057249 Phagocytosis Diseases 0.000 claims description 5
- 230000001973 epigenetic effect Effects 0.000 claims description 5
- 230000008782 phagocytosis Effects 0.000 claims description 5
- 230000005747 tumor angiogenesis Effects 0.000 claims description 5
- 230000005909 tumor killing Effects 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 102100022875 Hypoxia-inducible factor 1-alpha Human genes 0.000 claims 2
- 210000001744 T-lymphocyte Anatomy 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 8
- 230000028993 immune response Effects 0.000 abstract description 5
- 102100032742 Histone-lysine N-methyltransferase SETD2 Human genes 0.000 description 28
- 210000004981 tumor-associated macrophage Anatomy 0.000 description 26
- 230000014509 gene expression Effects 0.000 description 18
- 230000006870 function Effects 0.000 description 13
- 238000002347 injection Methods 0.000 description 10
- 239000007924 injection Substances 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 230000008859 change Effects 0.000 description 9
- 230000030279 gene silencing Effects 0.000 description 9
- 238000011529 RT qPCR Methods 0.000 description 8
- 238000013459 approach Methods 0.000 description 8
- 238000010586 diagram Methods 0.000 description 8
- 101000882127 Homo sapiens Histone-lysine N-methyltransferase EZH2 Proteins 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 230000002147 killing effect Effects 0.000 description 7
- 230000000242 pagocytic effect Effects 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 102100038970 Histone-lysine N-methyltransferase EZH2 Human genes 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 206010027476 Metastases Diseases 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 210000001185 bone marrow Anatomy 0.000 description 5
- 230000009401 metastasis Effects 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- 101000868279 Homo sapiens Leukocyte surface antigen CD47 Proteins 0.000 description 4
- 102100032913 Leukocyte surface antigen CD47 Human genes 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 230000033115 angiogenesis Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000002560 therapeutic procedure Methods 0.000 description 4
- 206010021143 Hypoxia Diseases 0.000 description 3
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 239000012636 effector Substances 0.000 description 3
- 238000009169 immunotherapy Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 201000001441 melanoma Diseases 0.000 description 3
- 210000001616 monocyte Anatomy 0.000 description 3
- 238000010172 mouse model Methods 0.000 description 3
- 210000000822 natural killer cell Anatomy 0.000 description 3
- 210000005259 peripheral blood Anatomy 0.000 description 3
- 239000011886 peripheral blood Substances 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 102100021723 Arginase-1 Human genes 0.000 description 2
- 108010012236 Chemokines Proteins 0.000 description 2
- 102000019034 Chemokines Human genes 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102220605874 Cytosolic arginine sensor for mTORC1 subunit 2_D10A_mutation Human genes 0.000 description 2
- 101001124991 Homo sapiens Nitric oxide synthase, inducible Proteins 0.000 description 2
- 108010063738 Interleukins Proteins 0.000 description 2
- 102000015696 Interleukins Human genes 0.000 description 2
- 102100029438 Nitric oxide synthase, inducible Human genes 0.000 description 2
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 101150063416 add gene Proteins 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 210000004979 bone marrow derived macrophage Anatomy 0.000 description 2
- 230000009400 cancer invasion Effects 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 230000010460 detection of virus Effects 0.000 description 2
- 239000001177 diphosphate Substances 0.000 description 2
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical class [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 2
- 235000011180 diphosphates Nutrition 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000001146 hypoxic effect Effects 0.000 description 2
- 230000006303 immediate early viral mRNA transcription Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 230000011987 methylation Effects 0.000 description 2
- 238000007069 methylation reaction Methods 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000005748 tumor development Effects 0.000 description 2
- 102000004452 Arginase Human genes 0.000 description 1
- 101710129000 Arginase-1 Proteins 0.000 description 1
- 108700024123 Arginases Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 102100028728 Bone morphogenetic protein 1 Human genes 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 102100036848 C-C motif chemokine 20 Human genes 0.000 description 1
- 108091011896 CSF1 Proteins 0.000 description 1
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 1
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 1
- 229940045513 CTLA4 antagonist Drugs 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102000004091 Caspase-8 Human genes 0.000 description 1
- 108090000538 Caspase-8 Proteins 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 102100021758 E3 ubiquitin-protein transferase MAEA Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000007569 Giant Cell Tumors Diseases 0.000 description 1
- 208000032612 Glial tumor Diseases 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101000752037 Homo sapiens Arginase-1 Proteins 0.000 description 1
- 101000713099 Homo sapiens C-C motif chemokine 20 Proteins 0.000 description 1
- 101000797762 Homo sapiens C-C motif chemokine 5 Proteins 0.000 description 1
- 101000616009 Homo sapiens E3 ubiquitin-protein transferase MAEA Proteins 0.000 description 1
- 101000971513 Homo sapiens Natural killer cells antigen CD94 Proteins 0.000 description 1
- 101001095308 Homo sapiens Periostin Proteins 0.000 description 1
- 101000800287 Homo sapiens Tubulointerstitial nephritis antigen-like Proteins 0.000 description 1
- 101000863873 Homo sapiens Tyrosine-protein phosphatase non-receptor type substrate 1 Proteins 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 108010017736 Leukocyte Immunoglobulin-like Receptor B1 Proteins 0.000 description 1
- 102000004569 Leukocyte Immunoglobulin-like Receptor B1 Human genes 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 102100028123 Macrophage colony-stimulating factor 1 Human genes 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 108010031099 Mannose Receptor Proteins 0.000 description 1
- 102100021462 Natural killer cells antigen CD94 Human genes 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 108010077850 Nuclear Localization Signals Proteins 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 102100037765 Periostin Human genes 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 1
- 102000009609 Pyrophosphatases Human genes 0.000 description 1
- 108010009413 Pyrophosphatases Proteins 0.000 description 1
- 101150036449 SIRPA gene Proteins 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 108010046722 Thrombospondin 1 Proteins 0.000 description 1
- 102100036034 Thrombospondin-1 Human genes 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 102100040247 Tumor necrosis factor Human genes 0.000 description 1
- 206010064390 Tumour invasion Diseases 0.000 description 1
- 102100029948 Tyrosine-protein phosphatase non-receptor type substrate 1 Human genes 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000006536 aerobic glycolysis Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000005809 anti-tumor immunity Effects 0.000 description 1
- 230000006023 anti-tumor response Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 230000005975 antitumor immune response Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 210000003050 axon Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004791 biological behavior Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000003995 blood forming stem cell Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000002449 bone cell Anatomy 0.000 description 1
- 210000002805 bone matrix Anatomy 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 210000003690 classically activated macrophage Anatomy 0.000 description 1
- ACSIXWWBWUQEHA-UHFFFAOYSA-N clodronic acid Chemical compound OP(O)(=O)C(Cl)(Cl)P(O)(O)=O ACSIXWWBWUQEHA-UHFFFAOYSA-N 0.000 description 1
- 229960002286 clodronic acid Drugs 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000000779 depleting effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 210000003162 effector t lymphocyte Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 238000005206 flow analysis Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000000777 hematopoietic system Anatomy 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000002390 hyperplastic effect Effects 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 238000011502 immune monitoring Methods 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000010185 immunofluorescence analysis Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 231100001252 long-term toxicity Toxicity 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000000581 natural killer T-cell Anatomy 0.000 description 1
- 210000002501 natural regulatory T cell Anatomy 0.000 description 1
- 210000002997 osteoclast Anatomy 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000013259 porous coordination polymer Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 230000014493 regulation of gene expression Effects 0.000 description 1
- 210000003289 regulatory T cell Anatomy 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000001743 silencing effect Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 229940037128 systemic glucocorticoids Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 210000001325 yolk sac Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
-
- 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
- C12N2510/00—Genetically modified cells
Abstract
本发明涉及一种巨噬细胞的CRISPR/Cas9表观编辑系统、方法、编辑后的巨噬细胞和应用,属于抗肿瘤技术领域。本发明所述巨噬细胞的CRISPR/Cas9表观编辑系统包括以下组分:PCP‑EZH2、sgRNA‑X‑PP7和dCas9;所述PCP‑EZH2为PCP基因和EZH2基因的融合基因;所述sgRNA‑X‑PP7为用于插入sgRNA的含PP7适配子茎环的基因,核苷酸序列如SEQ ID NO.1所示。本发明所述编辑系统能够从多方面增强巨噬细胞的抗肿瘤效应,促进T细胞等免疫应答,抑制肿瘤生长,显著改善肿瘤的治疗效果。
Description
技术领域
本发明涉及抗肿瘤技术领域,具体涉及一种巨噬细胞的CRISPR/Cas9 表观编辑系统、方法、编辑后的巨噬细胞和应用。
背景技术
肿瘤是危害人类健康的重要危险因素之一。其发病率仅次于心脑血管疾病,位居第二位。然而传统的放疗、化疗及靶向药物等治疗方式都很难做到完全抑制肿瘤的作用。近年来,肿瘤免疫治疗受到越来越广泛的关注。免疫治疗是与以往的传统治疗完全不同的一类全新肿瘤治疗模式,其主要机制是改变肿瘤微环境,从而使机体自身的免疫功能重新发挥作用,达到杀伤肿瘤细胞的目的。正常机体的免疫系统具有免疫监视功能,即能够识别、杀伤并及时清除体内包括肿瘤细胞在内的异常增生细胞。随着肿瘤的进展,多种免疫细胞可以参与抑制抗肿瘤免疫,从而促进肿瘤的生长、侵袭和转移。
肿瘤相关巨噬细胞(Tumor-Associated Macrophages,TAMs)是肿瘤免疫微环境中所占比例最多的免疫细胞,参与了肿瘤的生成、增殖、转移和耐药等多种生物学行为。长久以来研究认为巨噬细胞的发育来源是成年之后的造血系统,即骨髓中的HSC和外周血中的单核前体细胞。随着遗传修饰小鼠的应用,研究发现了定植于组织中的成熟巨噬细胞,随着个体的发育,巨噬细胞分别来源于三种不同的途径(来自卵黄囊和胎肝中的红细胞祖细胞(Erythro-myeloid progenitor,EMP),以及来自骨髓中的巨噬细胞/树突细胞祖细胞(macrophage/dendritic cell progenitor cells,MDP)。在肿瘤发展过程中,TAMs可能来源于胚胎或单核细胞来源的组织定植巨噬细胞,这些巨噬细胞可能在癌发生过程中发生表型和活化状态的变化(组织驻留TAMs),或来自经历明显分化的单核细胞,最终成为促进肿瘤生长的巨噬细胞。
机体组织中的巨噬细胞具有表型可变和功能多样的重要特征,在不同的环境刺激下,巨噬细胞可极化成具有不同分子表型和功能的两个亚型:M1 型(经典活化型)和M2型(替代活化型)。M1巨噬细胞由IFNγ及其他促炎刺激因素诱导,其表型特点为白介素(interleukin,IL)12,IL-23,IL-10 等。M1型的主要功能是有效呈递抗原,特异性表达一氧化氮合成酶2 (NOS2)、肿瘤坏死因子α(TNFα)、IL-6等标记分子。除此之外,M1型巨噬细胞可激活Th1反应,并且通常介导抗肿瘤反应。相反,M2型巨噬细胞由IL-10,糖皮质激素,IL-4和IL-13等因素诱导,其特异性表达精氨酸酶1(ARG1)、血管内皮生长因子(VEGF)、甘露糖受体(CD206)和TGF β等标记分子,这些表型参与受损组织的重塑和修复,寄生虫抗性,免疫调节和促肿瘤增殖。对于TAMs而言,肿瘤微环境中影响其极化的主要因素有三种:1)免疫相关信号是TAMs极化的关键决定因素;这些包括通过免疫细胞以及肿瘤和基质细胞本身在肿瘤部位释放的细胞因子,趋化因子和其他调节分子。值得注意的是,干细胞也可以是这些信号的来源(例如,通过神经胶质瘤干细胞释放POSTN)。2)代谢信号如乳酸(有氧糖酵解的副产物)也可以调控TAM影响肿瘤的发生发展。3)在自发(对于免疫原性肿瘤)或治疗诱发的肿瘤细胞死亡时释放的死亡信号可以激活TAMs,可以介导其促炎功能。
研究表明,表达经典巨噬细胞标记物的TAMs在不同环境下具有不同的功能,包括增加血管生成,引起肿瘤免疫抑制,增强肿瘤侵袭迁移等。其中, TAMs的主要致病活性是抑制肿瘤免疫应答。TAMs表达一系列抑制抗肿瘤免疫反应的效应分子;这包括细胞表面受体,细胞因子,趋化因子和酶。TAMs 表达PD-1和CTLA-4的配体,其在激活时抑制T细胞,NKT细胞和NK细胞的细胞毒性功能;TAMs表达死亡受体FAS和TRAIL的配体,其在靶细胞中触发caspase8依赖性细胞死亡(细胞凋亡);TAMs表达与T细胞上的 ILT2和NK细胞上的CD94结合的配体从而抑制NK细胞和T细胞的功能;TAMs分泌细胞因子IL-10和TGFβ,从而抑制T细胞效应功能和诱导调节功能;TAMs还分泌趋化因子CCL5,CCL20,CCL22,从而募集nTreg细胞;TAMs分泌精氨酸酶I,其通过消耗精氨酸使T细胞中的TCR失活。综上,巨噬细胞可以作为建立肿瘤干预策略的新方向。
现有研究干预TAMs的方法有以下三个类型:第一种方法是对TAMs的消耗。其中,靶向CSF1-CSF1R轴是一种很有前景的策略,尤其是在高表达CSF1的肿瘤中,例如滑膜巨细胞瘤,这种靶向方式将作为一种有效的治疗思路。然而,由于长时间耗尽体内所有的巨噬细胞存在毒性作用,限制了药物剂量的增加,使该方法存在显著的局限性。另一种方法是应用二磷酸盐选择性地消耗TAMs。由于二磷酸盐是稳定的,并且它的结构与骨基质的焦磷酸酶相同,因此可以被破骨细胞快速代谢并抑制其再吸收。研究表明,将氯膦酸盐包裹在例如脂质体(clodrolip)中,由于它们的吞噬活性会优先被巨噬细胞摄取。会显著减少肺癌的骨转移和乳腺癌的肺转移的实验模型中的巨噬细胞肿瘤浸润,从而限制了肿瘤转移。第二种方法是抑制TAMs的募集。最常用的方式是对CCL2-CCR2轴的抑制,然而CCL2的抑制剂会引起单核细胞反馈性增加,并且会出现组织中巨噬细胞增殖,从而增加巨噬细胞的浸润。第三种方法是巨噬细胞的重构。该方式可以重新平衡肿瘤微环境的免疫浸润,并且可以克服巨噬细胞清除剂引起的长期毒性以及CCL2-CCR2抑制剂引起的反馈性巨噬细胞增多。重构巨噬细胞的方式众多,其中最具代表性的是抗CD47抗体的应用。CD47是一种调节细胞迁移,轴突延伸,细胞因子的产生和T细胞活化的蛋白。CD47与血小板反应蛋白1和信号调节蛋白- α(SIRPα;也称为SHPS1)相互作用,这种相互作用的结果是“不要吃我”信号,以防止自体细胞在稳态条件下的吞噬作用。肿瘤细胞中的CD47过表达,CD47的抗体可有效增强巨噬细胞的吞噬作用,进一步促进抗肿瘤免疫效应。然而,该类方法存在一个共同问题是,巨噬细胞在减少肿瘤的同时,也会消灭血液循环中的血细胞,引起贫血甚至是自身免疫系统疾病。因此,寻找一种新的靶向TAMs的方法是目前研究的重要方向。
肿瘤相关的巨噬细胞是肿瘤免疫微环境的重要组成部分,恢复巨噬细胞的抗肿瘤作用有望成为肿瘤治疗的重要方向。
发明内容
本发明的目的在于提供一种巨噬细胞的CRISPR/Cas9表观编辑系统、方法、编辑后的巨噬细胞和应用。本发明所述编辑系统能够从多方面增强巨噬细胞的抗肿瘤效应,促进T细胞等免疫应答,抑制肿瘤生长,显著改善肿瘤的治疗效果。
本发明提供了一种巨噬细胞的CRISPR/Cas9表观编辑系统,所述系统包括以下组分:PCP-EZH2、sgRNA-X-PP7和dCas9;所述PCP-EZH2为PCP 基因和EZH2基因的融合基因,所述PCP基因的核苷酸序列如SEQ ID NO.3 所示;所述EZH2基因的核苷酸序列如SEQ ID NO.4所示;所述sgRNA-X-PP7 为用于插入sgRNA的含PP7适配子茎环的基因,核苷酸序列如SEQID NO.1 所示;所述dCas9的核苷酸序列如SEQ ID NO.2所示。
本发明还提供了一种靶向巨噬细胞特定基因的CRISPR/Cas9表观编辑系统,所述系统包括以下组分:PCP-EZH2、sgRNA-PP7和dCas9;所述 PCP-EZH2为PCP基因和EZH2基因的融合基因,所述PCP基因的核苷酸序列如SEQ ID NO.3所示;所述EZH2基因的核苷酸序列如SEQ ID NO.4 所示;所述sgRNA-PP7为插入能靶向巨噬细胞特定基因的sgRNA的含PP7 适配子茎环的基因,还未插入靶向巨噬细胞特定基因的sgRNA的含PP7适配子茎环的基因的核苷酸序列如SEQ ID NO.1所示;所述dCas9的核苷酸序列如SEQ ID NO.2所示。
本发明还提供了一种靶向巨噬细胞HIF1α启动子区的CRISPR/Cas9表观编辑系统,所述系统包括以下组分:PCP-EZH2、sgRNA-HIF1α-PP7和 dCas9;所述PCP-EZH2为PCP基因和EZH2基因的融合基因,所述PCP基因的核苷酸序列如SEQ ID NO.3所示;所述EZH2基因的核苷酸序列如SEQ ID NO.4所示;所述sgRNA-HIF1α-PP7为插入了靶向巨噬细胞HIF1α的sgRNA的含PP7适配子茎环的基因,核苷酸序列如SEQ ID NO.5所示;所述dCas9的核苷酸序列如SEQ ID NO.2所示。
本发明还提供了基于上述技术方案所述系统的巨噬细胞的 CRISPR/Cas9表观编辑载体系统,所述载体系统包括:sgRNA-X-PP7表达载体或sgRNA-PP7表达载体或sgRNA-HIF1α-PP7表达载体和PCP-EZH2表达载体、dCas9表达载体。
本发明还提供了基于上述技术方案所述载体系统的巨噬细胞的编辑方法,包括以下步骤:将sgRNA-X-PP7表达载体或sgRNA-PP7表达载体或 sgRNA-HIF1α-PP7表达载体和PCP-EZH2表达载体、dCas9表达载体转染到巨噬细胞中。
本发明还提供了利用上述技术方案所述编辑方法制备得到的巨噬细胞。
本发明还提供了上述技术方案所述系统或上述技术方案所述载体系统在制备促进巨噬细胞向M1极化和/或增强巨噬细胞吞噬能力和/或恢复巨噬细胞杀伤肿瘤能力的药物中的应用。
本发明还提供了上述技术方案所述系统或上述技术方案所述载体系统或上述技术方案所述巨噬细胞在制备抗肿瘤药物中的应用。
本发明还提供了上述技术方案所述系统或上述技术方案所述载体系统或上述技术方案所述巨噬细胞在制备抑制肿瘤生长和/或减少肿瘤血管生成的药物中的应用。
本发明还提供了上述技术方案所述系统或上述技术方案所述载体系统或上述技术方案所述巨噬细胞在制备解除肿瘤免疫抑制和/或促进免疫细胞对肿瘤的杀伤作用的药物中的应用。
本发明提供了一种巨噬细胞的CRISPR/Cas9表观编辑系统。本发明应用 CRISPR/Cas9表观遗传编辑技术,针对多个基因对巨噬细胞进行改造,将巨噬细胞作为基因递送的载体,使肿瘤微环境发生功能性的多元改变:1)抑制T细胞PD1表达,增强T细胞对肿瘤的杀伤作用;2)促进巨噬细胞向 M1型极化,增强其对肿瘤的杀伤作用;3)抑制肿瘤血管的生成;从而从多个方面增强巨噬细胞的抗肿瘤效应,促进T细胞免疫应答,抑制肿瘤生长,显著改善肿瘤的治疗效果。
本发明所述系统还具有以下有益效果:
(1)巨噬细胞来源广泛:巨噬细胞是肿瘤微环境中含量最多的免疫细胞,可来源于外周血或者骨髓;
(2)稳定性:表观遗传学水平稳定调控基因表达;
(3)无MHC限制性:巨噬细胞作为一类固有免疫细胞,无MHC限制性;
(4)发挥多元免疫效应:改造后的巨噬细胞可促进内源性巨噬细胞吞噬功能,抑制血管生成,解除肿瘤微环境的抑制效应,全方位抑制肿瘤进展。
附图说明
图1为本发明提供的CRISPR/Cas9-EZH2系统各载体结构示意图;
图2为本发明提供的CRISPR/Cas9-EZH2系统组成示意图;
图3为本发明提供的sgRNA序列示意图;
图4为本发明提供的验证CRISPR/Cas9-EZH2系统成功构建,其中,A 为WesternBlot检测dCas9和PCP-EZH2的表达;B为qRT-PCR检测sgRNA 的表达;C为感染表达CRISPR/dCas9-EZH2系统的病毒后,HIF1αmRNA 水平的表达变化;D为Western Blot检测感染表达CRISPR/dCas9-EZH2系统的病毒后,HIF1α蛋白水平的表达变化;
图5为本发明提供的CRISPR/Cas9-EZH2系统持续性验证,其中,A为实验流程图,B为qRT-PCR对CRISPR/Cas9-EZH2系统持续时间的检测;
图6为本发明提供的小鼠模型、肿瘤变化及小鼠生存情况图,A为构建 Luc+B16-F10荷瘤小鼠模型,于荷瘤后第12天注射巨噬细胞;B于荷瘤后第12天注射MΦdCas9+EZH2+sg-Hif1α通过体内生物成像检测小鼠肿瘤大小;C和 D用游标卡尺测量小鼠肿瘤的长径和短径,并用V=(长径x短径2)/2的公式计算肿瘤体积;E为注射MΦdCas9+EZH2+sg-Hif1α后统计小鼠的生存情况。
具体实施方式
本发明提供了一种巨噬细胞的CRISPR/Cas9表观编辑系统,所述系统包括以下组分:PCP-EZH2、sgRNA-X-PP7和dCas9;所述PCP-EZH2为PCP 基因和EZH2基因的融合基因;所述PCP基因的核苷酸序列如SEQ ID NO.3 所示: ATGTCTCTCGAGATGCCCAAAAAGAAAAGAAAAGTGGGTAGTATGGGT TCCAAAACCATCGTTCTTTCGGTCGGCGAGGCTACTCGCACTCTGACT GAGATCCAGTCCACCGCAGACCGTCAGATCTTCGAAGAGAAGGTCGG GCCTCTGGTGGGTCGGCTGCGCCTCACGGCTTCGCTCCGTCAAAACGG AGCCAAGACCGCGTATCGCGTCAACCTAAAACTGGATCAGGCGGACGT CGTTGATTCCGGACTTCCGAAAGTGCGCTACACTCAGGTATGGTCGCA CGACGTGACAATCGTTGCGAATAGCACCGAGGCCTCGCGCAAATCGTT GTACGATTTGACCAAGTCCCTCGTCGCGACCTCGCAGGTCGAAGATCT TGTCGTCAACCTTGTGCCGCTGGGCCGTGGTGGCGGAGGGACTAGTGG AGGT,所述EZH2基因的核苷酸序列如SEQ ID NO.4所示(EZH2CDS NM_001203247.1): ATGGGCCAGACTGGGAAGAAATCTGAGAAGGGACCAGTTTGTTGGCG GAAGCGTGTAAAATCAGAGTACATGCGACTGAGACAGCTCAAGAGGT TCAGACGAGCTGATGAAGTAAAGAGTATGTTTAGTTCCAATCGTCAGA AAATTTTGGAAAGAACGGAAATCTTAAACCAAGAATGGAAACAGCGA AGGATACAGCCTGTGCACATCCTGACTTCTGTGAGCTCATTGCGCGGG ACTAGGGAGTGTTCGGTGACCAGTGACTTGGATTTTCCAACACAAGTC ATCCCATTAAAGACTCTGAATGCAGTTGCTTCAGTACCCATAATGTATTC TTGGTCTCCCCTACAGCAGAATTTTATGGTGGAAGATGAAACTGTTTTA CATAACATTCCTTATATGGGAGATGAAGTTTTAGATCAGGATGGTACTTT CATTGAAGAACTAATAAAAAATTATGATGGGAAAGTACACGGGGATAG AGAATGTGGGTTTATAAATGATGAAATTTTTGTGGAGTTGGTGAATGCC CTTGGTCAATATAATGATGATGACGATGATGATGATGGAGACGATCCTG AAGAAAGAGAAGAAAAGCAGAAAGATCTGGAGGATCACCGAGATGAT AAAGAAAGCCGCCCACCTCGGAAATTTCCTTCTGATAAAATTTTTGAA GCCATTTCCTCAATGTTTCCAGATAAGGGCACAGCAGAAGAACTAAAG GAAAAATATAAAGAACTCACCGAACAGCAGCTCCCAGGCGCACTTCCT CCTGAATGTACCCCCAACATAGATGGACCAAATGCTAAATCTGTTCAGA GAGAGCAAAGCTTACACTCCTTTCATACGCTTTTCTGTAGGCGATGTTT TAAATATGACTGCTTCCTACATCCTTTTCATGCAACACCCAACACTTATA AGCGGAAGAACACAGAAACAGCTCTAGACAACAAACCTTGTGGACCA CAGTGTTACCAGCATTTGGAGGGAGCAAAGGAGTTTGCTGCTGCTCTC ACCGCTGAGCGGATAAAGACCCCACCAAAACGTCCAGGAGGCCGCAG AAGAGGACGGCTTCCCAATAACAGTAGCAGGCCCAGCACCCCCACCAT TAATGTGCTGGAATCAAAGGATACAGACAGTGATAGGGAAGCAGGGAC TGAAACGGGGGGAGAGAACAATGATAAAGAAGAAGAAGAGAAGAAA GATGAAACTTCGAGCTCCTCTGAAGCAAATTCTCGGTGTCAAACACCA ATAAAGATGAAGCCAAATATTGAACCTCCTGAGAATGTGGAGTGGAGT GGTGCTGAAGCCTCAATGTTTAGAGTCCTCATTGGCACTTACTATGACA ATTTCTGTGCCATTGCTAGGTTAATTGGGACCAAAACATGTAGACAGGT GTATGAGTTTAGAGTCAAAGAATCTAGCATCATAGCTCCAGCTCCCGCT GAGGATGTGGATACTCCTCCAAGGAAAAAGAAGAGGAAACACCGGTT GTGGGCTGCACACTGCAGAAAGATACAGCTGAAAAAGGACGGCTCCT CTAACCATGTTTACAACTATCAACCCTGTGATCATCCACGGCAGCCTTG TGACAGTTCGTGCCCTTGTGTGATAGCACAAAATTTTTGTGAAAAGTTT TGTCAATGTAGTTCAGAGTGTCAAAACCGCTTTCCGGGATGCCGCTGC AAAGCACAGTGCAACACCAAGCAGTGCCCGTGCTACCTGGCTGTCCG AGAGTGTGACCCTGACCTCTGTCTTACTTGTGGAGCCGCTGACCATTG GGACAGTAAAAATGTGTCCTGCAAGAACTGCAGTATTCAGCGGGGCTC CAAAAAGCATCTATTGCTGGCACCATCTGACGTGGCAGGCTGGGGGAT TTTTATCAAAGATCCTGTGCAGAAAAATGAATTCATCTCAGAATACTGT GGAGAGATTATTTCTCAAGATGAAGCTGACAGAAGAGGGAAAGTGTAT GATAAATACATGTGCAGCTTTCTGTTCAACTTGAACAATGATTTTGTGG TGGATGCAACCCGCAAGGGTAACAAAATTCGTTTTGCAAATCATTCGGT AAATCCAAACTGCTATGCAAAAGTTATGATGGTTAACGGTGATCACAG GATAGGTATTTTTGCCAAGAGAGCCATCCAGACTGGCGAAGAGCTGTT TTTTGATTACAGATACAGCCAGGCTGATGCCCTGAAGTATGTCGGCATC GAAAGAGAAATGGAAATCCCTTGA;所述sgRNA-X-PP7为用于插入 sgRNA的含PP7适配子茎环的基因,核苷酸序列如SEQ ID NO.1所示: GAAGACTTCACCGGAGACGGGATACCGTCTCTGTTTTAGAGCTATAAG GAGTTTATATGGAAACCCTTATAGCAAGTTAAAATAAGGCTAGTCCGTT ATCAACTTGGCCTAAGGAGTTTATATGGAAACCCTTAGGCCAAGTGGC ACCGAGTCGGTGCTTTTTTTGTTTAAGTCTTC;所述dCas9的核苷酸序列如SEQ ID NO.2所示 GAAAAGGCCGGCGGCCACGAAAAAGGCCGAGCGCCAGGCAAAAAAG AAAAAGGACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACTC TGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGA AATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAAC CTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCAC CCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAAC CGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTG GACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGA GGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACG AGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGA AACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGG CCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCG ACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTG GTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAG CGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCA GACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAAT GGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAAC TTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAG CAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCG GCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACG CCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGG CCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGG ACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGT ACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACA TTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCA TCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAAC AGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCAT CCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCA GGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAA GATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGG AAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCA CCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAG AGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAG AAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCC CGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTT CAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACT TCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAG ATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTAT CAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGG AAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCG AGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATG AAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCG GAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCC TGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCC AGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGG CCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTG GTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACAT CGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGA AGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGA GCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGC TGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATA TGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATG TGGACCACATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACA ACAAGGTGCTGACCAGAAGCGACAAGGCCCGGGGCAAGAGCGACAA CGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGC AGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGA CCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTC ATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGC ACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAA GCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTC CGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAA CTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGC CCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGG AAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGC GGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGAT AAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCA AGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCA AAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAA AGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACC GTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCC AAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGA AAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGG GCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACT CCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGT GAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCC CGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACT ACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGA TCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGC ACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGT TTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACAC CACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACG CCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGAC。
本发明还提供了一种靶向巨噬细胞特定基因的CRISPR/Cas9表观编辑系统,所述系统包括以下组分:PCP-EZH2、sgRNA-PP7和dCas9;所述 PCP-EZH2为PCP基因和EZH2基因的融合基因,所述PCP基因的核苷酸序列如SEQ ID NO.3所示;所述EZH2基因的核苷酸序列如SEQ ID NO.4 所示;所述sgRNA-PP7为插入能靶向巨噬细胞特定基因的sgRNA的含PP7 适配子茎环的基因,还未插入靶向巨噬细胞特定基因的sgRNA的含PP7适配子茎环的基因的核苷酸序列如SEQ ID NO.1所示;所述dCas9的核苷酸序列如SEQ ID NO.2所示。sgRNA-PP7中插入了能靶向巨噬细胞特定基因的 sgRNA后,能够特异靶向巨噬细胞效应基因的启动子区,增强巨噬细胞的抗肿瘤效应,促进T细胞等免疫应答,抑制肿瘤生长,显著改善肿瘤的治疗效果。
本发明还提供了一种靶向巨噬细胞HIF1α启动子区的CRISPR/Cas9表观编辑系统,所述系统包括以下组分:PCP-EZH2、sgRNA-HIF1α-PP7和 dCas9;所述PCP-EZH2为PCP基因和EZH2基因的融合基因,所述PCP基因的核苷酸序列如SEQ ID NO.3所示;所述EZH2基因的核苷酸序列如SEQ ID NO.4所示;所述sgRNA-HIF1α-PP7为插入了靶向巨噬细胞HIF1α的sgRNA的含PP7适配子茎环的基因,核苷酸序列如SEQ ID NO.5所示 CACCGACGTGGGCTGGGGTGGGGCCGTTTTAGAGCTATAAGGAGTTTA TATGGAAACCCTTATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT TGGCCTAAGGAGTTTATATGGAAACCCTTAGGCCAAGTGGCACCGAGT CGGTGCTTTTTTTGTTT;所述dCas9的核苷酸序列如SEQ ID NO.2所示。在本发明中,所述sgRNA-HIF1α-PP7为sgRNA-PP7中插入了能靶向巨噬细胞HIF1α启动子区的sgRNA序列(Mus-Hif1a-sgRNA,如表1所示),本发明所述系统为靶向HIF1α启动子区的CRISPR/dCas9-EZH2系统,本发明构建的靶向HIF1α启动子区的CRISPR/dCas9-EZH2系统可将组蛋白H3K27甲基转移酶EZH2带至靶点,实现HIF1α启动子区组蛋白甲基化修饰,如图2 所示。本发明所述系统对巨噬细胞进行改造后,可促进内源性巨噬细胞吞噬功能,抑制血管生成,解除肿瘤微环境的抑制效应,全方位抑制肿瘤进展。
本发明还提供了基于上述技术方案所述系统的巨噬细胞的 CRISPR/Cas9表观编辑载体系统,所述载体系统包括:sgRNA-X-PP7表达载体或sgRNA-PP7表达载体或sgRNA-HIF1α-PP7表达载体和PCP-EZH2表达载体、dCas9表达载体。如图1所示,图1上图表示的是插入sgRNA的载体结构示意图,具体为插入了HIF1αsgRNA的载体结构示意图,中间的图为插入了dCas9的载体结构示意图,最下边的图为插入了PCP-EZH2的载体示意图。
本发明还提供了基于上述技术方案所述载体系统的巨噬细胞的编辑方法,包括以下步骤:将sgRNA-X-PP7表达载体或sgRNA-PP7表达载体或 sgRNA-HIF1α-PP7表达载体和PCP-EZH2表达载体、dCas9表达载体转染到巨噬细胞中。在本发明中,所述PCP-EZH2表达载体的骨架载体优选包括 pWPI载体(Addgene#12254)。在本发明中,所述dCas9表达载体的骨架载体优选包括pWPI载体(Addgene#12254)。本发明对PCP-EZH2表达载体和 dCas9表达载体的制备方法没有特殊的限定,采用常规表达载体制备方法进行制备即可。在本发明中,所述sgRNA-X-PP7表达载体或sgRNA-PP7表达载体或sgRNA-HIF1α-PP7表达载体的骨架载体优选包括lenti sgRNA(MS2)_Zeo backbone。在本发明中,所述sgRNA-X-PP7表达载体的制备方法优选包括:设计酶切位点(表2中标记下划线的序列:GAAGACTT和AAGTCTTC)将sgRNA-X-PP7的核苷酸序列替换lenti sgRNA(MS2)_Zeo backbone质粒中的MS2适配子,得到sgRNA-X-PP7表达载体。在本发明中, sgRNA-PP7表达载体的制备方法优选包括以下步骤:将上述制备得到的 sgRNA-X-PP7表达载体中插入能靶向巨噬细胞特定基因的sgRNA,得到 sgRNA-PP7表达载体;当所述sgRNA的核苷选序列为Mus-Hif1a-sgRNA(如表1所示)时,得到的就是sgRNA-HIF1α-PP7表达载体。在本发明中,sgRNA 优选插入表2所示加粗酶切位点序列和之间。本发明对转染前的巨噬细胞的来源没有特殊限定,巨噬细胞是肿瘤微环境中含量最多的免疫细胞,可来源于外周血或者骨髓。
本发明还提供了利用上述技术方案所述编辑方法制备得到的巨噬细胞。本发明制备得到的巨噬细胞吞噬功能得到提升,能够抑制血管生成,解除肿瘤微环境的抑制效应,全方位抑制肿瘤进展。
本发明还提供了上述技术方案所述系统或上述技术方案所述载体系统在制备促进巨噬细胞向M1极化和/或增强巨噬细胞吞噬能力和/或恢复巨噬细胞杀伤肿瘤能力的药物中的应用。
本发明还提供了上述技术方案所述系统或上述技术方案所述载体系统或上述技术方案所述巨噬细胞在制备抗肿瘤药物中的应用。
本发明还提供了上述技术方案所述系统或上述技术方案所述载体系统或上述技术方案所述巨噬细胞在制备抑制肿瘤生长和/或减少肿瘤血管生成的药物中的应用。
本发明还提供了上述技术方案所述系统或上述技术方案所述载体系统或上述技术方案所述巨噬细胞在制备解除肿瘤免疫抑制和/或促进免疫细胞对肿瘤的杀伤作用的药物中的应用。
下面结合具体实施例对本发明所述的一种巨噬细胞的CRISPR/Cas9表观编辑系统、方法、编辑后的巨噬细胞和应用做进一步详细的介绍,本发明的技术方案包括但不限于以下实施例。
实施例1
构建基于CRISPR/Cas9表观编辑系统的巨噬细胞
首先设计对照基因以及能够结合在HIF1α启动子区域的sgRNA(表1)。用sgRNA-X-PP7序列(表2)替换lenti sgRNA(MS2)_Zeo backbone (Addgene##61427)质粒中的MS2适配子,将设计好的sgRNA(如表1所示,包括Mus-Hif1a-sgRNA和对照Mus-luciferase-sgRNA)分别连接到重组后的lenti sgRNA(PP7)_Zeo backbone质粒中,通过测序确认构建成功(图3),图3为本发明提供的sgRNA序列示意图,说明sgRNA成功插入至lenti sgRNA(PP7)_Zeobackbone载体的sgRNA-X-PP7中。
表1 sgRNA序列
表2 sgRNA-X-PP7序列
其次,从lenti dCAS-VP64_Blast(Addgene#61425)质粒中扩增含有D10A 和H840A的dCas9并将其插入到pWPI载体中。表达PCP-EZH2序列的pWPI 质粒由南京金斯瑞公司合成。
构建得到靶向HIF1α启动子区的CRISPR/dCas9-EZH2载体系统:(1) sgRNAs靶向的目的基因座以及2×PP7适配子茎环;(2)含有D10A和H840A 的核酸酶无效的Cas9蛋白(dCas9)以及其后的核定位信号;(3)与PP7外壳蛋白(PCP)融合的EZH2。具体结构如图2所示,图2为CRISPR/Cas9-EZH2 系统组成示意图。PCP结合sgRNA-PP7的茎环结构,一个茎环可以结合两个PCP蛋白,即一个sgRNA-PP7结构可以结合四个PCP-EZH2。
同时转染以上质粒进入小鼠骨髓来源巨噬细胞,并筛选出稳定表达的细胞株。通过qRT-PCR检测sgRNA的表达,用Western Blot检测dcas9和 PCP-EZH2的表达;利用ChIP分析HIF1α启动子甲基化,通过qRT-PCR和 WesternBlot检测HIF1α在转染CRISPR/dCas9-EZH2前后的表达差异,从而确定CRISPR/dCas9-EZH2系统是否成功构建以及其在慢病毒共感染的细胞中是否稳定表达。图4为验证CRISPR/Cas9-EZH2系统成功构建,其中, A为WesternBlot检测dCas9和PCP-EZH2的表达,结果表明相较于对照组,感染表达CRISPR/Cas9-EZH2系统的病毒组,其dCas9及PCP-EZH2蛋白表达水平明显升高;B为qRT-PCR检测sgRNA的表达,结果发现,感染表达 lenti sgRNA(PP7)_Zeo backbone病毒组的sgRNA表达水平显著升高;C qRT-PCR表明感染表达CRISPR/dCas9-EZH2系统的病毒后,HIF1α在mRNA水平的表达显著下降;D为Western Blot检测感染表达CRISPR/ dCas9-EZH2系统的病毒后,HIF1α蛋白水平的表达明显降低。综上,本发明成功构建了靶向沉默HIF1α的CRISPR/Cas9-EZH2的表观编辑系统。
实施例2
构建好的靶向沉默HIF1α的表观编辑系统感染至原代巨噬细胞中,发现该系统对巨噬细胞HIF1α有至少3天的沉默作用。结果如图5所示,图5 为CRISPR/Cas9-EZH2系统持续性验证;其中,A为实验流程图,分别在感染后第0h,6h,12h,24h,36h,48h,72h检测HIF1α的表达。B为qRT-PCR 对CRISPR/Cas9-EZH2系统持续时间的检测。
实施例3
检测HIF1α表观沉默修饰对肿瘤相关巨噬细胞免疫调控功能的改变
将表观沉默系统感染至CoCl2诱导的原代巨噬细胞缺氧模型中,分别诱导缺氧原代巨噬细胞的极化,流式细胞术及qRT-PCR结果表明HIF1α表观沉默修饰可以诱导缺氧原代巨噬细胞向M1极化。将HIF1α表观沉默修饰的巨噬细胞(HERMs)注射入B16-F10黑色素瘤荷瘤小鼠肿瘤内,在荷瘤第 32天通过流式和免疫荧光检测巨噬细胞极化方向的变化,结果表明,HERMs 可以促进TAMs向M1方向极化,并抑制其向M2方向极化。同时,通过感染HIF1α表观沉默系统的RAW264.7细胞及由化疗药物诱导凋亡的B16细胞共培养检测表观沉默系统对巨噬细胞吞噬功能的影响,结果表明HIF1α表观沉默系统可以显著促进巨噬细胞的吞噬能力。综上所述,HIF1α表观沉默系统可以促进TAMs向M1极化,增强其吞噬能力,恢复巨噬细胞杀伤肿瘤的能力。
实施例4
利用B16-F10黑色素瘤荷瘤模型在体内验证HERMs的治疗效果。
首先,将GFP转基因小鼠骨髓来源的巨噬细胞过继性注射至肿瘤内,检测巨噬细胞在肿瘤内的分布情况。流式及免疫荧光结果表明GFP小鼠骨髓来源的巨噬细胞可以在肿瘤组织中持续存在至少12天。本发明研究表明在荷瘤后第12天HIF1α的表达发生变化,因此本发明选择于第12天过继性注射HERMs,通过活体成像和大体形态观察HERMs注射后肿瘤大小的变化,并对小鼠进行生存分析,结果表示过继性注射HERMs可以显著抑制肿瘤生长。随后本发明通过免疫荧光及流式分析了HERMs对肿瘤免疫的改变,结果表明过继性注射HERMs显著增强效应T细胞的数量,抑制了Treg 的数量以及与肿瘤免疫抑制相关的PD1的表达。同时,过继性HERMs治疗可以显著减少肿瘤血管生成。综上,过继性HERMs治疗解除肿瘤免疫抑制,促进免疫细胞对肿瘤的杀伤作用,从而产生杀伤肿瘤的作用。
将表观编辑的巨噬细胞输注入B16-F10黑色素瘤小鼠体内,观察肿瘤变化及小鼠生存情况,结果如图6所示,其中,A为构建Luc+B16-F10荷瘤小鼠模型,于荷瘤后第12天注射巨噬细胞;B于荷瘤后第12天注射 MΦdCas9+EZH2+sg-Hif1α通过体内生物成像检测小鼠肿瘤大小;C和D用游标卡尺测量小鼠肿瘤的长径和短径,并用V=(长径x短径2)/2的公式计算肿瘤体积,并进行统计,以上结果说明过继性注射HERMs显著抑制肿瘤生长,E 为注射MΦdCas9+EZH2+sg-Hif1α后统计小鼠的生存情况,结果说明过继性注射 HERMs显著延长生存率。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国人民解放军空军军医大学
<120> 一种巨噬细胞的CRISPR/Cas9表观编辑系统、方法、编辑后的巨噬细胞和应用
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 177
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gaagacttca ccggagacgg gataccgtct ctgttttaga gctataagga gtttatatgg 60
aaacccttat agcaagttaa aataaggcta gtccgttatc aacttggcct aaggagttta 120
tatggaaacc cttaggccaa gtggcaccga gtcggtgctt tttttgttta agtcttc 177
<210> 2
<211> 4153
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gaaaaggccg gcggccacga aaaaggccga gcgccaggca aaaaagaaaa aggacaagaa 60
gtacagcatc ggcctggcca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga 120
gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa 180
gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct 240
gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga 300
gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc 360
cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga 420
cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga 480
cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt 540
ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct 600
gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag 660
cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa 720
tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggcaacc tgattgccct 780
gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact 840
gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga 900
ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga 960
catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag 1020
atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc 1080
tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga 1140
cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga 1200
cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac 1260
cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg 1320
gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct 1380
gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg 1440
gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa 1500
gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa 1560
cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct 1620
gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca 1680
gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct 1740
gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga 1800
agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa 1860
ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac 1920
actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga 1980
cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg 2040
gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa 2100
gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt 2160
taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat 2220
tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt 2280
ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc 2340
cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat 2400
cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac 2460
ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt 2520
ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccaca tcgtgcctca 2580
gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaaggcccg 2640
gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg 2700
gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga 2760
gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac 2820
ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga 2880
cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc 2940
cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc 3000
ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct 3060
ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa 3120
gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa 3180
ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga 3240
gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg 3300
gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg 3360
cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa 3420
ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct 3480
ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct 3540
ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc 3600
caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt 3660
cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa 3720
cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa 3780
gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca 3840
ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga 3900
cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga 3960
gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt 4020
caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga 4080
cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca 4140
gctgggaggc gac 4153
<210> 3
<211> 435
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgtctctcg agatgcccaa aaagaaaaga aaagtgggta gtatgggttc caaaaccatc 60
gttctttcgg tcggcgaggc tactcgcact ctgactgaga tccagtccac cgcagaccgt 120
cagatcttcg aagagaaggt cgggcctctg gtgggtcggc tgcgcctcac ggcttcgctc 180
cgtcaaaacg gagccaagac cgcgtatcgc gtcaacctaa aactggatca ggcggacgtc 240
gttgattccg gacttccgaa agtgcgctac actcaggtat ggtcgcacga cgtgacaatc 300
gttgcgaata gcaccgaggc ctcgcgcaaa tcgttgtacg atttgaccaa gtccctcgtc 360
gcgacctcgc aggtcgaaga tcttgtcgtc aaccttgtgc cgctgggccg tggtggcgga 420
gggactagtg gaggt 435
<210> 4
<211> 2241
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgggccaga ctgggaagaa atctgagaag ggaccagttt gttggcggaa gcgtgtaaaa 60
tcagagtaca tgcgactgag acagctcaag aggttcagac gagctgatga agtaaagagt 120
atgtttagtt ccaatcgtca gaaaattttg gaaagaacgg aaatcttaaa ccaagaatgg 180
aaacagcgaa ggatacagcc tgtgcacatc ctgacttctg tgagctcatt gcgcgggact 240
agggagtgtt cggtgaccag tgacttggat tttccaacac aagtcatccc attaaagact 300
ctgaatgcag ttgcttcagt acccataatg tattcttggt ctcccctaca gcagaatttt 360
atggtggaag atgaaactgt tttacataac attccttata tgggagatga agttttagat 420
caggatggta ctttcattga agaactaata aaaaattatg atgggaaagt acacggggat 480
agagaatgtg ggtttataaa tgatgaaatt tttgtggagt tggtgaatgc ccttggtcaa 540
tataatgatg atgacgatga tgatgatgga gacgatcctg aagaaagaga agaaaagcag 600
aaagatctgg aggatcaccg agatgataaa gaaagccgcc cacctcggaa atttccttct 660
gataaaattt ttgaagccat ttcctcaatg tttccagata agggcacagc agaagaacta 720
aaggaaaaat ataaagaact caccgaacag cagctcccag gcgcacttcc tcctgaatgt 780
acccccaaca tagatggacc aaatgctaaa tctgttcaga gagagcaaag cttacactcc 840
tttcatacgc ttttctgtag gcgatgtttt aaatatgact gcttcctaca tccttttcat 900
gcaacaccca acacttataa gcggaagaac acagaaacag ctctagacaa caaaccttgt 960
ggaccacagt gttaccagca tttggaggga gcaaaggagt ttgctgctgc tctcaccgct 1020
gagcggataa agaccccacc aaaacgtcca ggaggccgca gaagaggacg gcttcccaat 1080
aacagtagca ggcccagcac ccccaccatt aatgtgctgg aatcaaagga tacagacagt 1140
gatagggaag cagggactga aacgggggga gagaacaatg ataaagaaga agaagagaag 1200
aaagatgaaa cttcgagctc ctctgaagca aattctcggt gtcaaacacc aataaagatg 1260
aagccaaata ttgaacctcc tgagaatgtg gagtggagtg gtgctgaagc ctcaatgttt 1320
agagtcctca ttggcactta ctatgacaat ttctgtgcca ttgctaggtt aattgggacc 1380
aaaacatgta gacaggtgta tgagtttaga gtcaaagaat ctagcatcat agctccagct 1440
cccgctgagg atgtggatac tcctccaagg aaaaagaaga ggaaacaccg gttgtgggct 1500
gcacactgca gaaagataca gctgaaaaag gacggctcct ctaaccatgt ttacaactat 1560
caaccctgtg atcatccacg gcagccttgt gacagttcgt gcccttgtgt gatagcacaa 1620
aatttttgtg aaaagttttg tcaatgtagt tcagagtgtc aaaaccgctt tccgggatgc 1680
cgctgcaaag cacagtgcaa caccaagcag tgcccgtgct acctggctgt ccgagagtgt 1740
gaccctgacc tctgtcttac ttgtggagcc gctgaccatt gggacagtaa aaatgtgtcc 1800
tgcaagaact gcagtattca gcggggctcc aaaaagcatc tattgctggc accatctgac 1860
gtggcaggct gggggatttt tatcaaagat cctgtgcaga aaaatgaatt catctcagaa 1920
tactgtggag agattatttc tcaagatgaa gctgacagaa gagggaaagt gtatgataaa 1980
tacatgtgca gctttctgtt caacttgaac aatgattttg tggtggatgc aacccgcaag 2040
ggtaacaaaa ttcgttttgc aaatcattcg gtaaatccaa actgctatgc aaaagttatg 2100
atggttaacg gtgatcacag gataggtatt tttgccaaga gagccatcca gactggcgaa 2160
gagctgtttt ttgattacag atacagccag gctgatgccc tgaagtatgt cggcatcgaa 2220
agagaaatgg aaatcccttg a 2241
<210> 5
<211> 162
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
caccgacgtg ggctggggtg gggccgtttt agagctataa ggagtttata tggaaaccct 60
tatagcaagt taaaataagg ctagtccgtt atcaacttgg cctaaggagt ttatatggaa 120
acccttaggc caagtggcac cgagtcggtg ctttttttgt tt 162
<210> 6
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
caccgagttc gcgacattca ttctt 25
<210> 7
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aaacaagaat gaatgtcgcg aactc 25
<210> 8
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
caccgacgtg ggctggggtg gggcc 25
<210> 9
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
aaacggcccc accccagccc acgtc 25
Claims (10)
1.一种巨噬细胞的CRISPR/Cas9表观编辑系统,其特征在于,所述系统包括以下组分:PCP-EZH2、sgRNA-X-PP7和dCas9;所述PCP-EZH2为PCP基因和EZH2基因的融合基因,所述PCP基因的核苷酸序列如SEQ ID NO.3所示;所述EZH2基因的核苷酸序列如SEQ ID NO.4所示;所述sgRNA-X-PP7为用于插入sgRNA的含PP7适配子茎环的基因,核苷酸序列如SEQ ID NO.1所示;所述dCas9的核苷酸序列如SEQ ID NO.2所示。
2.一种靶向巨噬细胞特定基因的CRISPR/Cas9表观编辑系统,其特征在于,所述系统包括以下组分:PCP-EZH2、sgRNA-PP7和dCas9;所述PCP-EZH2为PCP基因和EZH2基因的融合基因,所述PCP基因的核苷酸序列如SEQ ID NO.3所示;所述EZH2基因的核苷酸序列如SEQ IDNO.4所示;所述sgRNA-PP7为插入能靶向巨噬细胞特定基因的sgRNA的含PP7适配子茎环的基因,还未插入靶向巨噬细胞特定基因的sgRNA的含PP7适配子茎环的基因的核苷酸序列如SEQ ID NO.1所示;所述dCas9的核苷酸序列如SEQ ID NO.2所示。
3.一种靶向巨噬细胞HIF1α启动子区的CRISPR/Cas9表观编辑系统,其特征在于,所述系统包括以下组分:PCP-EZH2、sgRNA-HIF1α-PP7和dCas9;所述PCP-EZH2为PCP基因和EZH2基因的融合基因,所述PCP基因的核苷酸序列如SEQ ID NO.3所示;所述EZH2基因的核苷酸序列如SEQ ID NO.4所示;所述sgRNA-HIF1α-PP7为插入了靶向巨噬细胞HIF1α的sgRNA的含PP7适配子茎环的基因,核苷酸序列如SEQ ID NO.5所示;所述dCas9的核苷酸序列如SEQ IDNO.2所示。
4.基于权利要求1~3任一项所述系统的巨噬细胞的CRISPR/Cas9表观编辑载体系统,所述载体系统包括:sgRNA-X-PP7表达载体或sgRNA-PP7表达载体或sgRNA-HIF1α-PP7表达载体和PCP-EZH2表达载体、dCas9表达载体。
5.基于权利要求4所述载体系统的巨噬细胞的编辑方法,包括以下步骤:将sgRNA-X-PP7表达载体或sgRNA-PP7表达载体或sgRNA-HIF1α-PP7表达载体和PCP-EZH2表达载体、dCas9表达载体转染到巨噬细胞中。
6.利用权利要求5所述编辑方法制备得到的巨噬细胞。
7.权利要求1~3任一项所述系统或权利要求4所述载体系统在制备促进巨噬细胞向M1极化和/或增强巨噬细胞吞噬能力和/或恢复巨噬细胞杀伤肿瘤能力的药物中的应用。
8.权利要求1~3任一项所述系统或权利要求4所述载体系统或权利要求6所述巨噬细胞在制备抗肿瘤药物中的应用。
9.权利要求1~3任一项所述系统或权利要求4所述载体系统或权利要求6所述巨噬细胞在制备抑制肿瘤生长和/或减少肿瘤血管生成的药物中的应用。
10.权利要求1~3任一项所述系统或权利要求4所述载体系统或权利要求6所述巨噬细胞在制备解除肿瘤免疫抑制和/或促进免疫细胞对肿瘤的杀伤作用的药物中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110312960.1A CN113322275A (zh) | 2021-03-24 | 2021-03-24 | 一种巨噬细胞的CRISPR/Cas9表观编辑系统、方法、编辑后的巨噬细胞和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110312960.1A CN113322275A (zh) | 2021-03-24 | 2021-03-24 | 一种巨噬细胞的CRISPR/Cas9表观编辑系统、方法、编辑后的巨噬细胞和应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN113322275A true CN113322275A (zh) | 2021-08-31 |
Family
ID=77414604
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110312960.1A Pending CN113322275A (zh) | 2021-03-24 | 2021-03-24 | 一种巨噬细胞的CRISPR/Cas9表观编辑系统、方法、编辑后的巨噬细胞和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113322275A (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120276010A1 (en) * | 2003-06-18 | 2012-11-01 | Szalay Aladar A | Microorganisms for therapy |
CN109999038A (zh) * | 2019-04-15 | 2019-07-12 | 中山大学 | 一种基于阻断巨噬细胞效应的抗肿瘤药物组合物及其应用 |
CN110993030A (zh) * | 2019-11-21 | 2020-04-10 | 复旦大学 | 一种单个活细胞内rna出核流量的监测方法和系统 |
-
2021
- 2021-03-24 CN CN202110312960.1A patent/CN113322275A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120276010A1 (en) * | 2003-06-18 | 2012-11-01 | Szalay Aladar A | Microorganisms for therapy |
CN109999038A (zh) * | 2019-04-15 | 2019-07-12 | 中山大学 | 一种基于阻断巨噬细胞效应的抗肿瘤药物组合物及其应用 |
CN110993030A (zh) * | 2019-11-21 | 2020-04-10 | 复旦大学 | 一种单个活细胞内rna出核流量的监测方法和系统 |
Non-Patent Citations (1)
Title |
---|
YAN DONG ET AL.,: "HIF1α epigenetically repressed macrophages via CRISPR/Cas9-EZH2 system for enhanced cancer immunotherapy", 《BIOACT MATER》 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Ma et al. | An oncolytic virus expressing IL15/IL15Rα combined with off-the-shelf EGFR-CAR NK cells targets glioblastoma | |
JP6580555B2 (ja) | 増強された養子細胞療法 | |
US11896616B2 (en) | Stimulatory cell lines for ex vivo expansion and activation of natural killer cells | |
US11497774B2 (en) | Coordinating gene expression using RNA destabilizing elements | |
JP2021523110A (ja) | 遺伝子発現のためのナノ粒子及びその使用 | |
JP2021536435A (ja) | 核酸とcar修飾免疫細胞とを含む治療薬およびその使用 | |
JP2021527694A (ja) | 腫瘍溶解性ウイルスを用いた処置 | |
CN111094358A (zh) | 一种分离的嵌合抗原受体以及包含其的修饰t细胞及用途 | |
Swan et al. | IL7 and IL7 Flt3L co-expressing CAR T cells improve therapeutic efficacy in mouse EGFRvIII heterogeneous glioblastoma | |
CN110393791B (zh) | hnRNPA2B1的抗感染作用及其应用 | |
WO2024055339A1 (zh) | 用于制备和扩增通用型人源化抗cd19 car-nk细胞的方法及其用途 | |
CN113322275A (zh) | 一种巨噬细胞的CRISPR/Cas9表观编辑系统、方法、编辑后的巨噬细胞和应用 | |
Wang et al. | A cell-specific nuclear factor-kappa B–activating gene expression strategy for delivering cancer immunotherapy | |
House et al. | CRISPR-Cas9 screening identifies an IRF1-SOCS1-mediated negative feedback loop that limits CXCL9 expression and antitumor immunity | |
WO2021218802A1 (zh) | 可受微小rna调控的分离的重组溶瘤痘病毒及其应用 | |
CN111499766B (zh) | 针对慢性淋巴细胞白血病的免疫效应细胞、其制备方法和应用 | |
WO2021133775A1 (en) | Immunotherapy for direct reprogramming of cancer cells into immune cells/antigen presenting cells/dendritic cells | |
CN111154757B (zh) | Aggf1基因的3’-UTR序列及其应用 | |
CN117304343B (zh) | Gpc3靶向的car-nk细胞的制备及其应用 | |
CN111407891B (zh) | 新型自噬受体ccdc50作为靶点在制备治疗病原体感染或癌症的药物中的应用 | |
KR20220095228A (ko) | 암의 t 세포 치료를 위한 세포독성 효과기 기억 t 세포를 생산하는 방법 | |
CN117402261A (zh) | 一种基于重组腺病毒的car-nk细胞制备方法及其应用 | |
CN113711991A (zh) | 一种以pap为靶点的药物筛选动物模型的构建方法和应用 | |
CN116284409A (zh) | Gpc3 car-t细胞及其在制备治疗癌症的药物中的用途 | |
CN115804853A (zh) | 包含rna分子的组合物及其在制备瘤内注射剂中的用途 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210831 |
|
RJ01 | Rejection of invention patent application after publication |