WO2022000572A1 - 激活γ-珠蛋白基因表达的方法和组合物 - Google Patents
激活γ-珠蛋白基因表达的方法和组合物 Download PDFInfo
- Publication number
- WO2022000572A1 WO2022000572A1 PCT/CN2020/101936 CN2020101936W WO2022000572A1 WO 2022000572 A1 WO2022000572 A1 WO 2022000572A1 CN 2020101936 W CN2020101936 W CN 2020101936W WO 2022000572 A1 WO2022000572 A1 WO 2022000572A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- ssodn
- sequence
- seq
- sgrna
- gene
- Prior art date
Links
- 108091005886 Hemoglobin subunit gamma Proteins 0.000 title claims abstract description 48
- 230000014509 gene expression Effects 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 29
- 230000003213 activating effect Effects 0.000 title claims abstract description 14
- 239000000203 mixture Substances 0.000 title claims description 17
- 238000010362 genome editing Methods 0.000 claims abstract description 83
- 230000000692 anti-sense effect Effects 0.000 claims abstract description 21
- 210000003958 hematopoietic stem cell Anatomy 0.000 claims abstract description 14
- 230000000295 complement effect Effects 0.000 claims abstract description 13
- 101150075675 tatC gene Proteins 0.000 claims abstract description 13
- 230000001105 regulatory effect Effects 0.000 claims abstract description 10
- 208000007056 sickle cell anemia Diseases 0.000 claims abstract description 8
- 108700028146 Genetic Enhancer Elements Proteins 0.000 claims abstract description 7
- 208000005980 beta thalassemia Diseases 0.000 claims abstract description 5
- 238000011282 treatment Methods 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 58
- 102100038617 Hemoglobin subunit gamma-2 Human genes 0.000 claims description 41
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 41
- 101001031961 Homo sapiens Hemoglobin subunit gamma-2 Proteins 0.000 claims description 33
- 108020004414 DNA Proteins 0.000 claims description 27
- 230000035772 mutation Effects 0.000 claims description 25
- 102100038614 Hemoglobin subunit gamma-1 Human genes 0.000 claims description 23
- 101001031977 Homo sapiens Hemoglobin subunit gamma-1 Proteins 0.000 claims description 23
- 108090000623 proteins and genes Proteins 0.000 claims description 19
- 238000004520 electroporation Methods 0.000 claims description 15
- 102000004389 Ribonucleoproteins Human genes 0.000 claims description 14
- 108010081734 Ribonucleoproteins Proteins 0.000 claims description 14
- 238000012217 deletion Methods 0.000 claims description 13
- 230000037430 deletion Effects 0.000 claims description 13
- 239000013612 plasmid Substances 0.000 claims description 13
- 239000013598 vector Substances 0.000 claims description 12
- 238000005516 engineering process Methods 0.000 claims description 11
- 102000004169 proteins and genes Human genes 0.000 claims description 11
- 230000008685 targeting Effects 0.000 claims description 11
- 108020005004 Guide RNA Proteins 0.000 claims description 10
- 108020004999 messenger RNA Proteins 0.000 claims description 10
- 238000003780 insertion Methods 0.000 claims description 8
- 230000037431 insertion Effects 0.000 claims description 8
- 238000010459 TALEN Methods 0.000 claims description 7
- 108091033409 CRISPR Proteins 0.000 claims description 6
- 108091081021 Sense strand Proteins 0.000 claims description 5
- 238000011144 upstream manufacturing Methods 0.000 claims description 5
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 claims description 4
- 101150034267 HBG2 gene Proteins 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 3
- 101150083167 HBG1 gene Proteins 0.000 claims description 3
- 238000000338 in vitro Methods 0.000 claims description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 229940079593 drug Drugs 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 claims 1
- 210000003743 erythrocyte Anatomy 0.000 abstract description 34
- 108091034117 Oligonucleotide Proteins 0.000 abstract description 8
- 238000013518 transcription Methods 0.000 abstract description 7
- 230000035897 transcription Effects 0.000 abstract description 7
- 108010044495 Fetal Hemoglobin Proteins 0.000 abstract description 3
- 108010038853 gamma-Globins Proteins 0.000 abstract 2
- 102100036646 Glutamyl-tRNA(Gln) amidotransferase subunit A, mitochondrial Human genes 0.000 description 25
- 101001072655 Homo sapiens Glutamyl-tRNA(Gln) amidotransferase subunit A, mitochondrial Proteins 0.000 description 25
- 108010054147 Hemoglobins Proteins 0.000 description 19
- 102000001554 Hemoglobins Human genes 0.000 description 19
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 14
- 102100031690 Erythroid transcription factor Human genes 0.000 description 11
- 101710100588 Erythroid transcription factor Proteins 0.000 description 9
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 8
- 230000000875 corresponding effect Effects 0.000 description 8
- 238000001514 detection method Methods 0.000 description 7
- 230000004069 differentiation Effects 0.000 description 7
- 208000034737 hemoglobinopathy Diseases 0.000 description 7
- 208000018337 inherited hemoglobinopathy Diseases 0.000 description 7
- 230000008439 repair process Effects 0.000 description 7
- 238000012163 sequencing technique Methods 0.000 description 7
- 238000001890 transfection Methods 0.000 description 7
- 102100021519 Hemoglobin subunit beta Human genes 0.000 description 6
- 101710163270 Nuclease Proteins 0.000 description 6
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 6
- 230000034431 double-strand break repair via homologous recombination Effects 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 6
- 102000053602 DNA Human genes 0.000 description 5
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 5
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 5
- 101000891113 Homo sapiens T-cell acute lymphocytic leukemia protein 1 Proteins 0.000 description 5
- 239000012097 Lipofectamine 2000 Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000003623 enhancer Substances 0.000 description 5
- 210000005259 peripheral blood Anatomy 0.000 description 5
- 239000011886 peripheral blood Substances 0.000 description 5
- 210000000130 stem cell Anatomy 0.000 description 5
- 238000006467 substitution reaction Methods 0.000 description 5
- 102100022976 B-cell lymphoma/leukemia 11A Human genes 0.000 description 4
- 102100035102 E3 ubiquitin-protein ligase MYCBP2 Human genes 0.000 description 4
- 101150086355 HBG gene Proteins 0.000 description 4
- 101000903703 Homo sapiens B-cell lymphoma/leukemia 11A Proteins 0.000 description 4
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- 101000702553 Schistosoma mansoni Antigen Sm21.7 Proteins 0.000 description 4
- 101000714192 Schistosoma mansoni Tegument antigen Proteins 0.000 description 4
- 102100040365 T-cell acute lymphocytic leukemia protein 1 Human genes 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 230000024245 cell differentiation Effects 0.000 description 4
- 238000012761 co-transfection Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 230000005782 double-strand break Effects 0.000 description 4
- 230000000925 erythroid effect Effects 0.000 description 4
- 208000020451 hereditary persistence of fetal hemoglobin Diseases 0.000 description 4
- 238000007480 sanger sequencing Methods 0.000 description 4
- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical compound C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 description 3
- 108700011215 E-Box Elements Proteins 0.000 description 3
- 102100027685 Hemoglobin subunit alpha Human genes 0.000 description 3
- 108091005902 Hemoglobin subunit alpha Proteins 0.000 description 3
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 3
- 102000004877 Insulin Human genes 0.000 description 3
- 108090001061 Insulin Proteins 0.000 description 3
- 101100290388 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) rnp-2 gene Proteins 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 108091028113 Trans-activating crRNA Proteins 0.000 description 3
- 108700009124 Transcription Initiation Site Proteins 0.000 description 3
- 102000004338 Transferrin Human genes 0.000 description 3
- 108090000901 Transferrin Proteins 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 229960002897 heparin Drugs 0.000 description 3
- 229920000669 heparin Polymers 0.000 description 3
- 210000003917 human chromosome Anatomy 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 229940125396 insulin Drugs 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000012581 transferrin Substances 0.000 description 3
- 108010044267 Abnormal Hemoglobins Proteins 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 102100020715 Fms-related tyrosine kinase 3 ligand protein Human genes 0.000 description 2
- 101710162577 Fms-related tyrosine kinase 3 ligand protein Proteins 0.000 description 2
- 101001066268 Homo sapiens Erythroid transcription factor Proteins 0.000 description 2
- 101000899111 Homo sapiens Hemoglobin subunit beta Proteins 0.000 description 2
- 241000193996 Streptococcus pyogenes Species 0.000 description 2
- 208000002903 Thalassemia 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
- 230000002159 abnormal effect Effects 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 208000007502 anemia Diseases 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000037429 base substitution Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 102000018146 globin Human genes 0.000 description 2
- 108060003196 globin Proteins 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 150000003278 haem Chemical class 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- CJWXCNXHAIFFMH-AVZHFPDBSA-N n-[(2s,3r,4s,5s,6r)-2-[(2r,3r,4s,5r)-2-acetamido-4,5,6-trihydroxy-1-oxohexan-3-yl]oxy-3,5-dihydroxy-6-methyloxan-4-yl]acetamide Chemical compound C[C@H]1O[C@@H](O[C@@H]([C@@H](O)[C@H](O)CO)[C@@H](NC(C)=O)C=O)[C@H](O)[C@@H](NC(C)=O)[C@@H]1O CJWXCNXHAIFFMH-AVZHFPDBSA-N 0.000 description 2
- 230000006780 non-homologous end joining Effects 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 101100252357 Caenorhabditis elegans rnp-1 gene Proteins 0.000 description 1
- 238000001353 Chip-sequencing Methods 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 108010028165 GATA1 Transcription Factor Proteins 0.000 description 1
- 102000016669 GATA1 Transcription Factor Human genes 0.000 description 1
- 102000017011 Glycated Hemoglobin A Human genes 0.000 description 1
- 108010014663 Glycated Hemoglobin A Proteins 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 101100493741 Homo sapiens BCL11A gene Proteins 0.000 description 1
- 101100220044 Homo sapiens CD34 gene Proteins 0.000 description 1
- VSNHCAURESNICA-UHFFFAOYSA-N Hydroxyurea Chemical compound NC(=O)NO VSNHCAURESNICA-UHFFFAOYSA-N 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 108010002386 Interleukin-3 Proteins 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 108010053584 alpha-Globins Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000001851 biosynthetic effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 108010089558 erythroid Kruppel-like factor Proteins 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000011134 hematopoietic stem cell transplantation Methods 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 229960000890 hydrocortisone Drugs 0.000 description 1
- 229960001330 hydroxycarbamide Drugs 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 230000001483 mobilizing effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000008263 repair mechanism Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 108091006106 transcriptional activators Proteins 0.000 description 1
- 108091006107 transcriptional repressors Proteins 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000032258 transport Effects 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
-
- 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
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem 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
- A61K38/41—Porphyrin- or corrin-ring-containing peptides
- A61K38/42—Haemoglobins; Myoglobins
-
- 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
- A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/06—Antianaemics
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
- C07K14/805—Haemoglobins; Myoglobins
-
- 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
-
- 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
- C12N15/67—General methods for enhancing the expression
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- 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]
-
- 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
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
-
- 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
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
-
- 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
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
Definitions
- the present invention relates to the technical field of gene editing, and relates to a method, composition and application for activating ⁇ -globin gene expression.
- the method uses a single-stranded oligonucleotide (ssODN) containing GATA or its antisense complementary sequence TATC as guidance information, and performs gene editing in the promoter region of the ⁇ -globin gene to form an erythroid enhancer element containing GATA, which can promote The gamma-globin gene is expressed in mature red blood cells.
- ssODN single-stranded oligonucleotide
- TATC antisense complementary sequence
- Hemoglobin (hemoglobin, Hb for short) is a special protein that carries and transports oxygen in red blood cells. Hemoglobin is composed of globin and heme. Hemoglobin in adults is mainly a tetramer ( ⁇ 2 ⁇ 2) composed of 2 ⁇ -globin and 2 ⁇ -globin, which becomes adult hemoglobin (HbA). Mutations in the gene for beta-globin (HBB) can cause beta-hemoglobin disorders (also called beta-hemoglobinopathies), including sickle cell disease (SCD) and beta-thalassemia (beta for short) -thal).
- SCD sickle cell disease
- beta-thalassemia beta-thalassemia
- Sickle cell anemia is caused by point mutations in the beta-globin structural gene, resulting in the production of abnormal hemoglobin (HbS).
- Beta-thalassemia is caused by partial or complete defects in the expression of the beta-globin gene, resulting in defective or absent hemoglobin (HbA) in adults.
- the reduction or deletion of the globin chain of hemoglobin can lead to abnormal structure of hemoglobin.
- the red blood cells containing abnormal hemoglobin have reduced deformability and shortened lifespan.
- In situ hemolysis may occur in the bone marrow. After entering the peripheral blood circulation, it is advanced by the spleen and other organs. Destruction, resulting in anemia, iron deposition in the body and even abnormal development.
- Hemoglobinopathies affect millions of people around the world.
- mutations in some special sites in some populations activate the transcription of the ⁇ -globin gene, resulting in an increase in the proportion of HbF in hemoglobin, which is called hereditary persistence of fetal hemoglobin (HPFH).
- HPFH hereditary persistence of fetal hemoglobin
- some different types of mutations were found in the promoter region of ⁇ -globin gene, such as -114 to -102 13bp del c., 4bp del c.-225 to -222, c.-114C>T, c.-117G >A, c.-158C>T, c.-167C>T, c.-170G>A, c.-175T>G, c.-175T>C, c.-195C>G, c.-196C> T, c.-198T>C, c.-201C>T, etc.
- HbF HbF in total hemoglobin to varying degrees.
- Clinical studies have found that a small number of patients with ⁇ -thalassemia or sickle cell anemia have HPFH mutations in their genomes, and the expression of HbF makes up for the deficiency of HbA, reducing or relieving the symptoms of anemia or reducing the need for blood transfusions to a certain extent.
- HbF expression makes up for the deficiency of HbA, reducing or relieving the symptoms of anemia or reducing the need for blood transfusions to a certain extent.
- HbF expression for example, hydroxyurea and other drugs are currently used clinically to induce HbF expression to treat ⁇ -hemoglobinopathies.
- the level of induced HbF in most patients is low and cannot completely improve the patient's condition.
- Gene editing technology brings new hope and new methods for the treatment of genetic diseases such as hemoglobinopathies.
- the relatively mature gene editing technologies currently developed include ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system (CRISPR-Cas system).
- ZFN zinc finger nuclease
- TALEN transcription activator-like effector nuclease
- CRISPR clustered regularly interspaced short palindromic repeats
- CRISPR-Cas system CRISPR-Cas system
- HDR homology-directed repair
- the present invention provides a method for activating ⁇ -globin gene transcription by gene editing.
- the present invention utilizes the single-stranded oligonucleotide (ssODN) containing GATA or its antisense complementary sequence TATC, through gene editing technology (such as CRISPR-Cas gene editing system), to manufacture in the proper position of the ⁇ -globin gene promoter region
- gene editing technology such as CRISPR-Cas gene editing system
- the mutation (deletion, substitution, insertion, etc.) of a few bases is edited to form an erythroid enhancer element containing GATA on the sense or antisense strand of the ⁇ -globin gene promoter region, such as NTG-N (7
- the sequence structure of -8)–WGATAR or NAG-N(7-8)–WGATAR can promote the transcription of ⁇ -globin gene in mature erythrocytes and increase the expression of HbF in erythrocytes.
- W is T or A
- R is A
- the principle of the present invention is based on gene editing technology such as CRISPR-Cas, combined with the single-stranded oligonucleotide (ssODN) designed by the present invention, to the stem cells and progenitor cells with red blood cell differentiation ability such as CD34+ hematopoietic stem cells.
- Efficient gene editing making mutations in the promoters of HBG1 and HBG2 genes encoding human ⁇ -globin (located on human chromosome 11), and artificially creating an NTG-N (7-8) on the sense or antisense strand of the promoter.
- WGATAR or NAG-N(7-8)–WGATAR sequence structure which acts as an enhancer and can recruit GATA1 and other activating factors to promote ⁇ -globin expression after target cells differentiate into erythrocytes.
- the present invention provides an ssODN containing GATA or TATC sequence, which can be used for gene editing of ⁇ -globin gene.
- the ssODN structure comprises a 5' homology arm, a replacement sequence and a 3' homology arm.
- the GATA or TATC sequence can be located at any position on the ssODN, including the replacement sequence, the 5' homology arm, the 3' homology arm, and the junction of the 5' homology arm and the replacement sequence, and the junction of the 3' homology arm and the replacement sequence.
- the length of the unilateral homology arm may be 20 to 300 nt, and in the embodiment of the present invention, the length of the ssODN homology arm is about 40 nt.
- the ssODN containing the GATA or TATC sequence is selected from the sequences shown in SEQ ID NO:40 to SEQ ID NO:65.
- the 5' and 3' of the ssODN are Phosphorothioate modified.
- the invention discloses a composition for gamma-globin gene editing.
- the composition includes ssODN, sgRNA, and CRISPR-Cas nuclease.
- the site targeted by the gene editing system sgRNA is located in the sequences shown in SEQ ID NO: 32 to SEQ ID NO: 39.
- the sgRNA is the sequence shown in SEQ ID NO: 69 to SEQ ID NO: 76.
- the Cas9 protein is a Cas9 protein derived from Streptococcus pyogenes.
- the invention discloses an electrotransformation method for ⁇ -globin gene editing. This method utilizes electroporation technology to efficiently transfect the composition formed by ssODN, sgRNA, and CRISPR-Cas nuclease into human hematopoietic stem cells to mediate efficient gene editing.
- the invention also discloses a hematopoietic stem cell.
- the above-mentioned composition comprising ssODN, sgRNA and CRISPR-Cas nuclease is transferred into the hematopoietic stem cell by electroporation, and the ⁇ -globin gene promoter region in the cell contains activating GATA element that increases the expression of the ⁇ -globin gene during red blood cell differentiation.
- the present invention has the following beneficial effects:
- the ssODN of the present invention is introduced into cells together with a gene editing system (such as CRISPR-Cas, TALEN, ZFN, etc.), which can guide the gene editing of the promoter region of the ⁇ -globin gene, and mutate at a specific site to form GATA with an activating effect. element.
- a gene editing system such as CRISPR-Cas, TALEN, ZFN, etc.
- the ssODN and CRISPR-Cas gene editing systems are introduced into human hematopoietic stem cells by electroporation, and efficient gene editing is obtained in the ⁇ -globin gene promoter region.
- the erythroid enhancer element containing GATA after the hematopoietic stem cells differentiated into erythrocytes, the expression of ⁇ -globin gene was significantly increased, and the proportion of HbF in hemoglobin was significantly increased.
- FIG. 1 illustrates the technical principle of the present invention.
- Figure 2A shows that the gene editing targeting region is the wild-type sequence between -92 and -66 of HBG1 and HBG2 genes, the position of the gRNA targeting sequence used, and the mutated sequence expected after gene editing.
- Figure 2B shows that the gene editing targeting region is the wild-type sequence between -129 and -98 of the HBG1 and HBG2 genes, the position of the gRNA targeting sequence used, and the expected mutated sequence after gene editing.
- Figure 2C shows that the gene editing targeting region is the wild-type sequence between -175 and -153 of the HBG1 and HBG2 genes, the position of the gRNA targeting sequence used, and the expected mutated sequence after gene editing.
- Figure 2D shows that the gene editing targeting region is the wild-type sequence between -192 and -160 of the HBG1 and HBG2 genes, the position of the gRNA targeting sequence used, and the expected mutated sequence after gene editing.
- Figure 2E shows the gene editing targeting region is the wild-type sequence between HBG1 gene -428 to -406 and HBG2 gene -432 to -410, the position of the gRNA targeting sequence used, and the expected mutant sequence after gene editing.
- Figure 3 shows the editing efficiency data of HBG1 and HBG2 genomes in 293T cells transfected with CRISPR-Cas plasmids, which are obtained from the INDEL assay results of genomic DNA four days after CRISPR-Cas plasmids were transfected with Lipofectamine 2000 TM in 293T cells. The percentage of total indel mutations (% of total mutations) is shown.
- Figure 4 shows the editing efficiency data of HBG2 genome by simultaneous transfection of CRISPR-Cas plasmid and ssODN in 293T cells.
- the data comes from the INDEL detection results of HBG2 four days after 293T cells were transfected with CRISPR-Cas plasmid and ssODN using Lipofectamine 2000 TM.
- Figure 5 shows the editing efficiency data of HBG2 genome by simultaneous transfection of CRISPR-Cas plasmid and ssODN in K562 cells.
- the data comes from the INDEL detection results of HBG2 four days after K562 cells were transfected with CRISPR-Cas plasmid and ssODN using Lonza 4D electroporation .
- Figure 6 shows the editing efficiency data of HBG2 genome after electrotransduction into ribonucleoprotein (RNP) and ssODN in K562 cells, the data from INDEL detection of HBG2 four days after ssODN and CRISPR-Cas/sgRNA RNPs were electroporated into K562 cells result.
- RNP ribonucleoprotein
- Figures 7A-C show HBG2 gene editing efficiency data after electroporation into RNP and ssODN in mobilized peripheral blood CD34+ cells (mPBSC), the data from ssODN and CRISPR-Cas/sgRNA RNP were electroporated into mPBSC to induce erythrocyte differentiation INDEL assay results of genomic DNA after 5 days.
- Figures 7A and 7B are sanger sequencing maps, and Figure 7C is the INDEL ratio result after analysis by synthego software.
- FIG. 9 shows the HbF expression data after mobilizing peripheral blood CD34+ cells (mPBSC) after gene editing, the data comes from ssODN and CRISPR-Cas/sgRNA RNP transferred into mPBSC by electroporation, induced red blood cell differentiation 18 days after HPLC detection of hemoglobin detection results, HbF expression in the figure is its percentage of total hemoglobin.
- This paper provides the use of gene editing technology (such as CRISPR-Cas-mediated gene editing technology, combined with the single-stranded oligonucleotides (ssODN) designed in the present invention), to CD34+ hematopoietic stem cells and other stem cells and progenitor cells with red blood cell differentiation ability.
- gene editing technology such as CRISPR-Cas-mediated gene editing technology, combined with the single-stranded oligonucleotides (ssODN) designed in the present invention
- GATA-1 is a transcriptional regulator that is highly expressed in the erythrocyte system and plays an important role in the process of erythrocyte differentiation. Genes that are specifically highly expressed in erythrocytes, such as ⁇ - and ⁇ -globin and heme biosynthetic enzymes in erythrocytes, are directly activated by GATA-1 transcription. As a transcription factor, GATA-1 selectively binds to WGATAR sequence elements on chromatin DNA (wherein W stands for T or A, R stands for A or G, and it can also be represented by T/A(GATA)A/G; The sense complementary sequence is YTATCW, where Y represents T or C, and can also be represented by T/C (TATC)T/A).
- GATA sites are also found in the regulatory region of the human ⁇ -globin gene. Notably, not all genes containing GATA sequences are activated by GATA-1. This is because GATA sequences are widely distributed in the genome and often need to form a certain form of sequence combination (element combination) with upstream and downstream sequences (cis elements) to function. For example, there are cis-regulatory elements such as CACCC or GC boxes near the GATA sequence, which usually recruit GATA-1 to bind to the transcription factor EKLF. If there is an E-box element (CAGNTG, N represents any base) near the GATA sequence, or a half E-box element (NTG), GATA-1 is recruited to bind to the transcription factor TAL1.
- E-box element CAGNTG, N represents any base
- N half E-box element
- the sequence similarity of 1400 bases upstream of the transcription start site of HBG1 and HBG2 genes is very high and is considered to be the promoter region.
- the inventors of the present application found that the promoter region of the ⁇ -globin gene itself contains multiple GATA sequences (or TATC), and also contains a lot of TG sequences, but ⁇ -globin is hardly expressed in mature erythrocytes.
- the inventors of the present application have found through in-depth research that the GATA (or TATC) and TG sequences on the ⁇ -globin gene promoter do not form effective activating GATA elements in position and orientation (such as TG-N (7-8) -WGATAR), therefore cannot recruit transcription activators such as GATA-1/TAL1 that are highly expressed in mature erythrocytes to bind (co-bind) to the ⁇ -globin gene promoter region, so ⁇ -globin is silenced in mature erythrocytes, almost not express.
- the inventors of the present application believe that if a gene editing system is used to cut these sites to form DSBs, by adding a donor DNA template (such as single-stranded oligonucleotide ssODN) to guide these sites for HDR repair, a certain proportion of The genome is edited into the expected sequence structure, and the final ⁇ -globin gene promoter region can be recruited as an enhancer due to the formation of sequences such as NTG-N(7-8)–WGATAR or NAG-N(7-8)–WGATAR Transcription activators such as GATA-1/TAL1 that are highly expressed in mature erythrocytes bind (co-bind) to the promoter region of the ⁇ -globin gene, thereby promoting the high expression of ⁇ -globin in mature erythrocytes.
- a donor DNA template such as single-stranded oligonucleotide ssODN
- gene editing can be divided into two categories. One is to destroy the sequence or structure of the target site, which is called knockout. Another type of purpose is to introduce specific sequence mutations at the target site, which is called knock in. To introduce specific sequence mutations, the most common method is to introduce a DNA donor template at the same time, and cells can use the mechanism of homologous recombination to replace the sequence in the template into the genome to achieve knock-in.
- DNA templates for HDR can be plasmid DNA, linearized double-stranded DNA, AAV-delivered DNA, or single-stranded oligonucleotides (ssODN). ssODN has been used more and more in gene editing because of its high efficiency and convenient synthesis, and is especially suitable for gene editing in cultured cells in vitro.
- the ssODN structure contains a 5' homology arm, a replacement sequence, and a 3' homology arm.
- Homology arms are sequences that are homologous to the DNA regions flanking the target site to be edited, and are used to locate the target site on the chromosome.
- the corresponding sequences of the 5' homology arm and the 3' homology arm on the chromosome are usually discontinuous in position, with a certain interval in the middle, such as the interval of 1-20 nucleotides, these interval sequences are gene-edited
- the target (sequence to be edited), that is, the sequence that is desired to be replaced by gene editing.
- the "replacement sequence" between the 3' end of the 5' homology arm and the 5' end of the 3' homology arm on the ssODN is the desired result of gene editing.
- Adding ssODN at the same time of gene editing can induce homologous recombination repair during gene editing.
- the to-be-edited sequence in the cell genome is repaired into a replacement sequence.
- the length of the replacement sequence in the ssODN can be smaller than the sequence to be edited, in which case the result of gene editing is the deletion and/or replacement of the sequence to be edited in the original genome.
- the length of the replacement sequence in the ssODN can be 0 bases, in which case the sequence to be edited on the genome is deleted.
- the length of the replacement sequence in the ssODN may be larger than the sequence to be edited, in which case the result of gene editing is that the sequence to be edited is replaced or/and the sequence to be edited is inserted.
- CRISPR-Cas is a gene editing technology, including but not limited to various naturally occurring or artificially designed CRISPR-Cas systems, such as CRISPR-Cas9 system, CRISPR-Cas12 system, etc.
- the working principle of CRISPR-Cas9 is that crRNA (CRISPR-derived RNA) combines with tracrRNA (trans-activating RNA) through base pairing to form a tracrRNA/crRNA complex, which guides the nuclease Cas9 protein at the sequence target position paired with crRNA. Dot cut double-stranded DNA.
- tracrRNA and crRNA can also be replaced by an artificially synthesized sgRNA with guiding role.
- sgRNA sgRNA with guiding role.
- other CRISPR-Cas systems it is necessary to design the corresponding sgRNA or crRNA.
- systems such as TALEN or ZFN the corresponding TALEN or ZFN nuclease needs to be designed according to the editing site disclosed in the present invention.
- Example 1 Screening of ssODN and CRISPR-Cas systems for introduction of activating GATA elements into gamma-globin gene promoters
- Region 1 between -92 to -66 of HBG1 and HBG2 promoters (Fig. 2A); Region 2, HBG1 and HBG2 Promoter -129 to -98 (Fig. 2B); Region No. 3, between HBG1 and HBG2 promoters -175 to -153 (Fig. 2C); Region No. 4, between HBG1 gene and HBG2 gene -192 to -160 between -428 to -406 of HBG1 genes and -432 to -410 of HBG2 genes (FIG. 2E).
- the suitable mutation target types of the sequences in each region were analyzed, as shown in Table 1 (SEQ ID NO: 3 to SEQ ID NO: 31).
- the underline indicates the base that needs to be replaced, which is the desired result after gene editing.
- the length of the replacement base is 0-6 bases, and the effect achieved is deletion, replacement, insertion, or a combination of deletion, replacement, and insertion.
- Target site and edited sequence SEQ ID NO: 3 Zone 1, wild type TTGACCAATAGTCTTAGAGTATCCAGT SEQ ID NO: 4 Mutant_01 TTGACCAATAGT GA TAGAGTATCCAGT SEQ ID NO: 5 Mutant_02 TTGACCAATAG AGA TAGAGTATCCAGT SEQ ID NO: 6 Mutant_03 TTGACCAATA AGA TAGAGTATCCAGT
- the present invention selects the spCas9CRISPR-Cas system (derived from Streptococcus pyogenes) with higher cutting efficiency, and analyzes target sites with higher cutting efficiency.
- select site_1 to site_8 as candidate target sites, and the identified target site DNA sequences and PAM sequences (NGG) are shown in Table 2 (SEQ ID NO: 32 to SEQ ID NO: 39).
- SEQ ID site sgRNA recognition site and PAM (5′-3′) DNA strand SEQ ID NO: 32 site_1 ACTGGATACTCTAAGACTAT TGG antisense strand SEQ ID NO: 33 site_2 CTTGTCAAGGCTATTGGTCA AGG antisense strand SEQ ID NO: 34 site_3 GTTTGCCTTGTCAAGGCTAT TGG antisense strand SEQ ID NO: 35 site_4 TGGTCAAGTTTGCCTTGTCA AGG antisense strand SEQ ID NO: 36 site_5 CTTGACCAATAGCCTTGACA AGG chain of justice SEQ ID NO: 37 site_6 TATCTGTCTGAAACGGTCCC TGG chain of justice SEQ ID NO: 38 site_7 ATGCAAAATATCTGTCTGAAA CGG chain of justice SEQ ID NO: 39 site_8 TCCCTGAACTTTTCAAAAAT TGG chain of justice
- the ssODN structure comprises a 5' homology arm, a replacement sequence and a 3' homology arm.
- the GATA or TATC sequence can be located at any position on the ssODN, including the replacement sequence, the 5' homology arm, the 3' homology arm, and the junction of the 5' homology arm and the replacement sequence, and the junction of the 3' homology arm and the replacement sequence. .
- the homology arms on both sides of the ssODN can be symmetric or asymmetric (the lengths on both sides are different).
- the ssODN with symmetric homology arms is uniformly used for experiments in the present invention.
- the length of the homology arms on both sides of the ssODN can be 20-300 nt.
- the embodiment of the present invention uses ssODN with the length of the homology arms on both sides of about 40 nt for example.
- the ssODN can be the sense strand or the antisense strand of the DNA in the editing region (the homology arm sequence is the same as the corresponding sense strand or the same as the antisense strand).
- the ssODN uniformly selects the same DNA strand as the sgRNA recognition site as the ssODN.
- the sequence in ssODN can be 0, 1, 2, 3, 4, 5, or 6 bases in addition to the homologous arm sequences on both sides.
- the effect achieved is deletion, replacement, and insertion. , or a combination of delete, replace, and insert.
- the effect achieved can be to change more than 1 base on the genome, such as deletion or substitution of 1-20 bases.
- the sequences shown in Table 3 (SEQ ID NO: 40 to SEQ ID NO: 65), the underlined bases are homologous arms on both sides, the length is about 40nt, and the underlines between the homologous arms on both sides are not underlined.
- the bases are substitution bases, wherein the substitution bases between the homology arms on both sides of ssODN_16 are 0.
- the first three nucleotides at both ends of ssODN were modified with phosphorothioate to enhance the stability of ssODN and improve its activity in gene editing.
- sgRNA-spCas9 vector PX459, pSpCas9(BB)-2A-Puro
- This vector expresses the spCas9 protein and can express sgRNA with a length of 100nt, and its 5′ end is 20nt.
- the guide sequence, followed by 80 nt is the universal sgRNA backbone sequence (SEQ ID NO: 66, GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGGUGCUUUU).
- the method of cloning sgRNA is: synthesizing the corresponding guide strand sequence oligo (same as the 20nt target site sequence in Table 2) and complementary strand oligo (adding cacc to the 5'-end of the guide strand sequence, if the 5'-end of the guide strand is the first If a nucleotide is not guanine G, add caccG to the 5'-end of the guide strand; add aaac to the 5'-end of the complementary strand. If a G is added to the 5'-end of the guide strand, add a G to the 5'-end of the complementary strand. The 3'-end adds C to its complementary pairing).
- oligos were mixed in equal amounts, heat-deformed at 95°C and annealed, and then ligated into the PX459 vector digested with restriction enzyme Bbs I, and cloned successfully by colony PCR and sanger sequencing.
- PCR used SEQ ID NO: 67 (5'-3': GGACTATCATATGCTTACCGTAAC) and SEQ ID NO: 68 (5'-3': GGCGGGCCATTTACCGTAAG) as primers.
- 8 plasmids were obtained, expressing sgRNAs from SEQ ID NO: 69 to SEQ ID NO: 76 respectively (Table 4).
- the 8 plasmids expressed were transfected into 293T cells, and the method was referred to the method of Lipofectamine 2000 TM kit.
- the transfected cells were cultured for 4 days, and the cells were collected to extract genomic DNA.
- SEQ ID NO: 77 and SEQ ID NO: 78, and SEQ ID NO: 79 and SEQ ID NO: 78 respectively the HBG1 and HBG2 promoter gene fragments were amplified by KOD Plus high-fidelity enzyme PCR method, and the obtained PCR products were purified Sequencing was performed using SEQ ID NO:77 and SEQ ID NO:79, respectively.
- the files obtained from the sequencing results were analyzed for gene editing efficiency by synthego software. As shown in Figure 3, except for the relatively low editing efficiency of sgRNA-8 (about 20%), the overall editing efficiency of other groups (indels, Indels) was 40%. -50% or so.
- Example 2 Co-transfection of ssODN and sgRNA-spCas9 vector into 293T cells for gene editing
- the above eight sgRNA-spCas9 vectors were co-transfected with appropriate ssODN into 293T cells to test whether the addition of ssODN could induce gene editing to form the expected mutation type (HDR repair).
- 8 kinds of sgRNA-spCas9 vectors were combined according to the sgRNA+ssODN combination method shown in Table 6 to form 30 combinations respectively.
- ssODN was added at the same time of transfection of plasmid, and ssODN and plasmid were delivered into cells together.
- the transfected cells were cultured for 4 days, and the cells were collected to extract genomic DNA.
- HBG2 Since the gene editing efficiencies of HBG1 and HBG2 are almost positively correlated, and the gene editing efficiency of HBG2 is often slightly higher than that of HBG1 ( Figure 3), only the genome editing efficiency of HBG2 was selected for comparison in subsequent experiments.
- the HBG2 promoter gene fragment was amplified by PCR, purified and sequenced. The sequencing results were analyzed by the synthego software to analyze the gene editing efficiency.
- the addition of ssODN and the co-transfection of the sgRNA-spCas9 vector can improve the editing efficiency, and most groups induced about 10-20% of the gene editing to form the expected mutation type (Figure 4).
- Example 3 Electroporation Co-transformation of ssODN and sgRNA-spCas9 vector into K562 cells for gene editing
- liposomes such as Lipofectamine 2000 TM reagent
- electroporation is generally used for transfection of suspension cells. Take part of the combination of sgRNA and ssODN in Example 2, and introduce it into K562 cells by lonza-4D electroporator, and use the K562 cell transfection procedure that comes with the instrument. The transfected cells were cultured for 4 days, the cells were collected to extract genomic DNA, and the HBG2 promoter gene fragment was amplified by PCR for sequencing. The sequencing results were analyzed by synthego software.
- the gene editing efficiency showed that the combination of sgRNA-1, sgRNA-2, sgRNA-5 and ssODN in K562 could achieve higher editing efficiency (Figure 5), which was slightly higher than that in 293T cells.
- the -spCas9 vector induced the expected mutation type also slightly higher than 293T cells.
- Example 4 Co-transfection of ssODN and spCas9 protein/sgRNA RNP into K562 cells by electroporation for efficient gene editing
- RNP ribonucleoprotein
- the transfected cells were cultured for 4 days, the cells were collected to extract genomic DNA, and the HBG2 promoter gene fragment was amplified by PCR for sequencing.
- the sequencing results were analyzed by the synthego software to analyze the gene editing efficiency (Fig. 6).
- the addition of ssODN when electroporating RNP in K562 significantly improved the overall gene editing efficiency by about two times (Fig. 6).
- electroporation of ssODN and RNP could induce a higher proportion of gene editing to form the expected mutation type (Fig. 6).
- Example 5 Electroporation of ssODN and spCas9 protein/sgRNA RNP into human hematopoietic stem/progenitor cells to achieve efficient gene editing and significantly increase HBG expression
- mPBSC peripheral blood-derived human CD34-positive cells
- SFEM StemSpan serum-free expansion medium
- SCF human cytokines
- TPO TPO
- Flt3L human cytokines
- the EO-100 program that comes with the lonza-4D electroporator delivers ssODN and spCas9/sgRNA RNPs into mPBSC cells.
- the cells were cultured in SFEM enriched with human cytokines (SCF/TPO/Flt3L, 100 ng/ml each) for one day, and then divided into three stages to induce mPBSC to differentiate into erythrocytes.
- the first stage cultured in IMDM medium containing EPO, SCF, human AB serum, insulin, transferrin, heparin, IL-3, hydrocortisone and other additives for 7 days.
- IMDM medium containing EPO, SCF, human AB serum, insulin, transferrin, heparin, IL-3, hydrocortisone and other additives for 7 days.
- 2 x 10e5 cells were taken out to extract genomic DNA, and then the HBG2 promoter fragment was amplified by PCR and sent to sanger sequencing.
- electrotransduction of ssODN and spCas9/sgRNA RNP in mPBSC cells resulted in efficient gene editing, with partial genome editing resulting in 30% of the expected mutation types (Fig. 7A-C).
- Electroporated mPBSC cells and untreated mPBSC cells were induced in the first stage of differentiation for 7 days, and then were cultured in IMDM medium containing EPO, SCF, human AB serum, insulin, transferrin, heparin and other additives. The cells were cultured for 4 days, and finally cultured in IMDM medium containing EPO, human AB serum, insulin, transferrin, heparin and other supplements for 7 days.
- both the electrotransduced mPBSC cells and the untreated mPBSC cells were markedly dark red after centrifugation, which contained a large number of erythrocytes, indicating that the electrotransformed mPBSC cells could differentiate into erythrocytes normally.
- the hemoglobin of the electrotransduced hematopoietic stem cells differentiated into red blood cells in the RNP-2 group was detected by HPLC.
- the content of fetal hemoglobin (HbF) was significantly increased, ranging from 27% to 36%, which was more than 2-fold higher than that of the control group (13.3%) ( Figure 9).
- the present invention provides a gene editing method for enhancing ⁇ -globin gene expression which is different from the prior art.
- the ssODN disclosed in the present invention can guide the formation of expected mutations at predetermined sites in the regulatory regions of HBG1 and HBG2 genes after gene editing, thereby forming an activating GATA element, which is in the gene editing process.
- cells such as hematopoietic stem cells
- they play a positive regulatory role and activate or significantly increase the expression of ⁇ -globin gene. Therefore, the invention has potential application value in gene therapy of ⁇ -hemoglobinopathies.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Developmental Biology & Embryology (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Gastroenterology & Hepatology (AREA)
- Mycology (AREA)
- Diabetes (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
SEQ ID | 编号 | 目标位点及编辑后的序列(5′-3′) |
SEQ ID NO:3 | 一号区,野生型 | TTGACCAATAGTCTTAGAGTATCCAGT |
SEQ ID NO:4 | 突变型_01 | TTGACCAATAGT GATAGAGTATCCAGT |
SEQ ID NO:5 | 突变型_02 | TTGACCAATAG AGATAGAGTATCCAGT |
SEQ ID NO:6 | 突变型_03 | TTGACCAATA AGATAGAGTATCCAGT |
SEQ ID NO:7 | 突变型_04 | TTGACCAATA TGATAGAGTATCCAGT |
SEQ ID NO:8 | 二号区,野生型 | CAGCCTTGCCTTGACCAATAGCCTTGACAAGG |
SEQ ID NO:9 | 突变型_05 | CAGCCTTGCCTTGAC AGATAGCCTTGACAAGG |
SEQ ID NO:10 | 突变型_06 | CAGCCTTGCCTTGAC TGATAGCCTTGACAAGG |
SEQ ID NO:11 | 突变型_07 | CAGCCTTGCCTTGA GAGATAGCCTTGACAAGG |
SEQ ID NO:12 | 突变型_08 | CAGCCTTGCCTTGA GTGATAGCCTTGACAAGG |
SEQ ID NO:13 | 突变型_09 | CAGCCTTGCCTTGA AAGATAGCCTTGACAAGG |
SEQ ID NO:14 | 突变型_10 | CAGCCTTGCCTTGA ATGATAGCCTTGACAAGG |
SEQ ID NO:15 | 突变型_11 | CAGCCTTGCCTTGA TAGATAGCCTTGACAAGG |
SEQ ID NO:16 | 突变型_12 | CAGCCTTGCCTTGA TTGATAGCCTTGACAAGG |
SEQ ID NO:17 | 突变型_13 | CAGCCTTGCCTTGA TAAATAGCCTTGACAAGG |
SEQ ID NO:18 | 突变型_14 | CAGCCTTGCCTTGA TAAAATAGCCTTGACAAGG |
SEQ ID NO:19 | 突变型_15 | CAGCCTTGCCTTGA TAATAGCCTTGACAAGG |
SEQ ID NO:20 | 突变型_16 | CAGCCTTGCCTTGATAGCCTTGACAAGG |
SEQ ID NO:21 | 突变型_17 | CAGCCTTGCCTTGA TAAGG |
SEQ ID NO:22 | 突变型_18 | CAGCCTTGCCTTGA TAAAGG |
SEQ ID NO:23 | 三号区,野生型 | TATCTGTCTGAAACGGTCCCTGG |
SEQ ID NO:24 | 突变型_19 | TATCTGTCTGAAACGGT AGATAACCCTGG |
SEQ ID NO:25 | 四号区,野生型 | CACTATCTCAATGCAAATATCTGTCTGAAACGG |
SEQ ID NO:26 | 突变型_20 | CACTATCTCAATGCAA CAGAAACGG |
SEQ ID NO:27 | 五号区,野生型 | TCCCTGAACTTTTCAAAAATTGG |
SEQ ID NO:28 | 突变型_21 | TCCCTGAACTTTTCA GATAATTGG |
SEQ ID NO:29 | 突变型_22 | TCCCTGAACTTTTCA GATAAATTGG |
SEQ ID NO:30 | 突变型_23 | TCCCTGAACTTTTCA GATAAAATTGG |
SEQ ID NO:31 | 突变型_24 | TCCCTGAACTTTTCA GATAGAATTGG |
SEQ ID | 位点 | sgRNA识别位点及PAM(5′-3′) | DNA链 |
SEQ ID NO:32 | site_1 | ACTGGATACTCTAAGACTAT TGG | 反义链 |
SEQ ID NO:33 | site_2 | CTTGTCAAGGCTATTGGTCA AGG | 反义链 |
SEQ ID NO:34 | site_3 | GTTTGCCTTGTCAAGGCTAT TGG | 反义链 |
SEQ ID NO:35 | site_4 | TGGTCAAGTTTGCCTTGTCA AGG | 反义链 |
SEQ ID NO:36 | site_5 | CTTGACCAATAGCCTTGACA AGG | 正义链 |
SEQ ID NO:37 | site_6 | TATCTGTCTGAAACGGTCCC TGG | 正义链 |
SEQ ID NO:38 | site_7 | ATGCAAATATCTGTCTGAAA CGG | 正义链 |
SEQ ID NO:39 | site_8 | TCCCTGAACTTTTCAAAAAT TGG | 正义链 |
SEQ ID | 位点 | sgRNA识别位点及PAM(5′-3′) |
SEQ ID NO:77 | HBG1_F | TACTGCGCTGAAACTGTGGCTT |
SEQ ID NO:78 | HBG1/2_R | CTTCCCAGGGTTTCTCCTCCAG |
SEQ ID NO:79 | HBG2_F | TGCACTGAAACTGTTGCTTTATAGGA |
组合 | 转染复合物 | 组合 | 转染复合物 |
组合1 | sgRNA-1+ssODN_1 | 组合16 | sgRNA-2+ssODN_16 |
组合2 | sgRNA-1+ssODN_2 | 组合17 | sgRNA-2+ssODN_17 |
组合3 | sgRNA-1+ssODN_3 | 组合18 | sgRNA-2+ssODN_18 |
组合4 | sgRNA-1+ssODN_4 | 组合19 | sgRNA-3+ssODN_17 |
组合5 | sgRNA-2+ssODN_5 | 组合20 | sgRNA-3+ssODN_18 |
组合6 | sgRNA-2+ssODN_6 | 组合21 | sgRNA-4+ssODN_17 |
组合7 | sgRNA-2+ssODN_7 | 组合22 | sgRNA-4+ssODN_18 |
组合8 | sgRNA-2+ssODN_8 | 组合23 | sgRNA-5+ssODN_19 |
组合9 | sgRNA-2+ssODN_9 | 组合24 | sgRNA-5+ssODN_20 |
组合10 | sgRNA-2+ssODN_10 | 组合25 | sgRNA-6+ssODN_21 |
组合11 | sgRNA-2+ssODN_11 | 组合26 | sgRNA-7+ssODN_22 |
组合12 | sgRNA-2+ssODN_12 | 组合27 | sgRNA-8+ssODN_23 |
组合13 | sgRNA-2+ssODN_13 | 组合28 | sgRNA-8+ssODN_24 |
组合14 | sgRNA-2+ssODN_14 | 组合29 | sgRNA-8+ssODN_25 |
组合15 | sgRNA-2+ssODN_15 | 组合30 | sgRNA-8+ssODN_26 |
Claims (29)
- 一种激活γ-珠蛋白基因表达的方法,该方法包括如下操作:通过基因编辑技术在γ-珠蛋白基因调控区的正义链或反义链人工引入GATA或TATC序列,使γ-珠蛋白基因的调控区形成包含GATA基序的增强子元件。
- 如权利要求1所述的方法,其中引入GATA或TATC序列通过删除序列、插入序列、序列突变,或者以上方式的组合来实现。
- 如权利要求1所述的方法,其中GATA基序为WGATAR序列,或者其对应的反义互补序列YTATCW,其中W为T或A,R为A或G,Y为T或C。
- 如权利要求1所述的方法,其中所述的增强子元件中进一步包含NTG基序或者NAG基序,或者其对应的反义互补序列,其中N为A、G、C或T。
- 如权利要求4所述的方法,其中所述GATA基序与NTG基序或NAG基序的距离是7至8个碱基,且所述NTG基序或NAG基序位于所述GATA基序上游。
- 一种用于基因编辑的ssODN,所述ssODN中含有GATA或TATC序列。
- 如权利要求6所述的ssODN,其中所述ssODN中包含5′同源臂、替换序列、3′同源臂。
- 如权利要求7所述的ssODN,其中所述ssODN的5′同源臂和3′同源臂是对称的,或者是非对称的,5′同源臂和3′同源臂的长度为20至300nt。
- 如权利要求7所述的ssODN,其中所述ssODN的5′同源臂或3′同源臂选自拟编辑区域DNA的正义链或者是反义链。
- 如权利要求7所述的ssODN,其中所述ssODN中替换序列的碱基数量为0-6个。
- 如权利要求7所述的ssODN,其中所述ssODN中5′同源臂的3′末端与3′同源臂的5′末端相距0-20个碱基。
- 如权利要求6所述的ssODN,其中所述ssODN包含选自SEQ ID NO:4至SEQ ID NO:7,SEQ ID NO:9至SEQ ID NO:22,SEQ ID NO:24,SEQ ID NO:26,SEQ ID NO:28至SEQ ID NO:31所示序列,或者其反义互补序列。
- 如权利要求6所述的ssODN,其中所述ssODN包含选自SEQ ID NO:40至SEQ ID NO:65所示序列,或者其反义互补序列。
- 如权利要求6所述的ssODN,其中所述ssODN进行了化学修饰,其5′和3′端是硫代磷酸修饰的核苷酸。
- 一种sgRNA,所述sgRNA包含指导序列,所述指导序列与位于HBG1或HBG2调控区域内的DNA正义链或者反义链部分或者完全互补。
- 如权利要求15所述的sgRNA,所述HBG1调控区域的序列如SEQ ID NO:1所示。
- 如权利要求15所述的sgRNA,所述HBG2调控区域的序列如SEQ ID NO:2所示。
- 如权利要求15所述的sgRNA,所述sgRNA分子的指导RNA序列对应的DNA序列与选自SEQ ID NO:32至SEQ ID NO:39所示序列部分或者完全相同。
- 如权利要求15所述的sgRNA,所述sgRNA为选自SEQ ID NO:69至SEQ ID NO:76所示序列中的任一种或多种。
- 一种用于基因编辑的组合物,所述组合物包含权利要求6至14任一项所述的ssODN,以及靶向HBG1或HBG2基因调控区的基因编辑系统。
- 如权利要求20所述的组合物,所述基因编辑系统是CRISPR-Cas编辑系统、TALEN编辑系统、ZFN编辑系统。
- 如权利要求21所述的组合物,所述CRISPR-Cas编辑系统是CRISPR-Cas9、CRISPR-Cas12系统。
- 如权利要求21所述的组合物,所述组合物中CRISPR-Cas编辑系统是指导RNA以及Cas蛋白组成的核糖核蛋白复合物。
- 如权利要求21所述的组合物,所述组合物中CRISPR-Cas编辑系统是编码Cas蛋白的mRNA以及gRNA组成的RNA复合物。
- 如权利要求21所述的组合物,所述组合物中CRISPR-Cas编辑系统是表达Cas蛋白及gRNA的质粒。
- 如权利要求20所述的组合物,所述组合物中包含权利要求15至19任一项所述的sgRNA,所述sgRNA是化学合成的,或者体外转录的。
- 一种激活γ-珠蛋白基因的试剂盒,其特征在于,包括:(1)如权利要求6-14任一项所述的ssODN。(2)如权利要求15-19任一项所述的sgRNA。(3)表达Cas9或Cas12蛋白的载体,Cas9或Cas12蛋白,Cas9或Cas12蛋白对应的mRNA中的至少一种。
- 一种造血干细胞,其特征在于,其通过采用电转方法将如权利要求20-26任一项所述的组合物转入造血干细胞中而得到。
- 如权利要求6-14任一项所述的ssODN、权利要求15-19任一项所述的sgRNA、权利要求20-26任一项所述的组合物、权利要求27所述的试剂盒,权利要求28所述的造血干细胞在制备用于治疗β-地中海贫血或者镰刀型贫血症的细胞或药物中的用途。
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2020455671A AU2020455671A1 (en) | 2020-07-01 | 2020-07-14 | Method for activating expression of gamma-globin gene, and composition |
EP20942540.4A EP4177343A1 (en) | 2020-07-01 | 2020-07-14 | Method for activating expression of gamma-globin gene, and composition |
US18/010,448 US20230310506A1 (en) | 2020-07-01 | 2020-07-14 | Method for activating expression of gamma-globin gene, and composition |
JP2022577265A JP2023530969A (ja) | 2020-07-01 | 2020-07-14 | γ-グロビン遺伝子発現を活性化する方法、および組成物 |
CN202080032995.3A CN115943209A (zh) | 2020-07-01 | 2020-07-14 | 激活γ-珠蛋白基因表达的方法和组合物 |
ZA2022/13480A ZA202213480B (en) | 2020-07-01 | 2022-12-13 | Method for activating expression of gamma-globin gene, and composition |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010616648.7 | 2020-07-01 | ||
CN202010616648.7A CN111876416B (zh) | 2020-07-01 | 2020-07-01 | 激活γ-珠蛋白基因表达的方法和组合物 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2022000572A1 true WO2022000572A1 (zh) | 2022-01-06 |
Family
ID=73157578
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2020/101936 WO2022000572A1 (zh) | 2020-07-01 | 2020-07-14 | 激活γ-珠蛋白基因表达的方法和组合物 |
Country Status (7)
Country | Link |
---|---|
US (1) | US20230310506A1 (zh) |
EP (1) | EP4177343A1 (zh) |
JP (1) | JP2023530969A (zh) |
CN (2) | CN111876416B (zh) |
AU (1) | AU2020455671A1 (zh) |
WO (1) | WO2022000572A1 (zh) |
ZA (1) | ZA202213480B (zh) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP4358982A2 (en) * | 2021-06-25 | 2024-05-01 | The J. David Gladstone Institutes, A Testamentary Trust Established under The Will of J. David Gladstone | Primordial germ cells |
CN115851706A (zh) * | 2021-09-24 | 2023-03-28 | 华东师范大学 | 一种碱基编辑系统及其应用 |
CN114848851A (zh) * | 2022-04-29 | 2022-08-05 | 广州医科大学附属第三医院(广州重症孕产妇救治中心、广州柔济医院) | 治疗β-地中海贫血的药物 |
WO2024098383A1 (zh) * | 2022-11-11 | 2024-05-16 | 深圳华大生命科学研究院 | 蛋白突变体及其治疗与hbb基因突变相关疾病的应用 |
CN116790593B (zh) * | 2023-06-28 | 2024-05-10 | 贵州医科大学 | 一种激活γ珠蛋白表达的sgRNA及其CRISPR/Cas9复合体与应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017191503A1 (en) * | 2016-05-05 | 2017-11-09 | Crispr Therapeutics Ag | Materials and methods for treatment of hemoglobinopathies |
CN109153994A (zh) * | 2016-03-14 | 2019-01-04 | 爱迪塔斯医药公司 | 用于治疗β-血红蛋白病的CRISPR/CAS相关方法和组合物 |
WO2019079347A1 (en) * | 2017-10-16 | 2019-04-25 | The Broad Institute, Inc. | USES OF BASIC EDITORS ADENOSINE |
CN109735497A (zh) * | 2017-10-27 | 2019-05-10 | 博雅辑因(北京)生物科技有限公司 | 一种提高胎儿血红蛋白表达水平的方法 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110106203B (zh) * | 2019-05-24 | 2023-08-11 | 中国医学科学院血液病医院(血液学研究所) | 一种新型hbb过表达载体及其设计方法和应用 |
-
2020
- 2020-07-01 CN CN202010616648.7A patent/CN111876416B/zh active Active
- 2020-07-14 US US18/010,448 patent/US20230310506A1/en active Pending
- 2020-07-14 EP EP20942540.4A patent/EP4177343A1/en active Pending
- 2020-07-14 WO PCT/CN2020/101936 patent/WO2022000572A1/zh unknown
- 2020-07-14 CN CN202080032995.3A patent/CN115943209A/zh active Pending
- 2020-07-14 JP JP2022577265A patent/JP2023530969A/ja active Pending
- 2020-07-14 AU AU2020455671A patent/AU2020455671A1/en active Pending
-
2022
- 2022-12-13 ZA ZA2022/13480A patent/ZA202213480B/en unknown
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109153994A (zh) * | 2016-03-14 | 2019-01-04 | 爱迪塔斯医药公司 | 用于治疗β-血红蛋白病的CRISPR/CAS相关方法和组合物 |
WO2017191503A1 (en) * | 2016-05-05 | 2017-11-09 | Crispr Therapeutics Ag | Materials and methods for treatment of hemoglobinopathies |
WO2019079347A1 (en) * | 2017-10-16 | 2019-04-25 | The Broad Institute, Inc. | USES OF BASIC EDITORS ADENOSINE |
CN109735497A (zh) * | 2017-10-27 | 2019-05-10 | 博雅辑因(北京)生物科技有限公司 | 一种提高胎儿血红蛋白表达水平的方法 |
Also Published As
Publication number | Publication date |
---|---|
JP2023530969A (ja) | 2023-07-20 |
CN111876416A (zh) | 2020-11-03 |
ZA202213480B (en) | 2023-09-27 |
CN115943209A (zh) | 2023-04-07 |
EP4177343A1 (en) | 2023-05-10 |
US20230310506A1 (en) | 2023-10-05 |
CN111876416B (zh) | 2021-09-03 |
AU2020455671A1 (en) | 2023-02-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2022000572A1 (zh) | 激活γ-珠蛋白基因表达的方法和组合物 | |
US11963982B2 (en) | CRISPR/RNA-guided nuclease systems and methods | |
US20240002843A1 (en) | Compositions and methods for the treatment of hemoglobinopathies | |
KR102532663B1 (ko) | 베타 이상헤모글로빈증의 치료를 위한 crispr/cas-관련 방법 및 조성물 | |
AU2016225179C1 (en) | Materials and methods for treatment of hemoglobinopathies | |
WO2018170184A1 (en) | Systems and methods for the treatment of hemoglobinopathies | |
WO2021083183A9 (zh) | 一种造血干细胞hbb基因修复的方法及产品 | |
US20200190536A1 (en) | Recombinant lentiviral vector for stem cell-based gene therapy of sickle cell disorder | |
CN112359065A (zh) | 一种提高基因敲入效率的小分子组合物 | |
CN113430195A (zh) | 靶向β-珠蛋白基因的gRNA分子,其合成方法和修正突变类型的方法 | |
Mettananda | Genetic and epigenetic therapies for β-thalassaemia by altering the expression of α-globin gene | |
JP2024504608A (ja) | 遺伝子操作rnaを使用した内因性adarの活用による標的化rnaの編集 | |
WO2023046086A1 (zh) | 一种碱基编辑系统及其应用 | |
WO2022155458A1 (en) | Systems and methods for base editing of hbg1/2 gene promoter and fetal hemoglobin induction | |
Boontanrart et al. | Engineering of the endogenous HBD promoter increases HbA2 | |
Suwito et al. | A Review of CRISPR Cas9 for SCA: Treatment Strategies and Could Target β-globin Gene and BCL11A Gene using CRISPR Cas9 Prevent the Patient from Sickle Cell Anemia? | |
CN114072518B (zh) | 用于治疗地中海贫血或镰状细胞病的方法和组合物 | |
EP4273242A1 (en) | Crispr-based modification of human hbd gene | |
Suwito et al. | Choirotussanijjah. A Review of CRISPR Cas9 for SCA: Treatment Strategies and Could Target β-globin Gene and BCL11A Gene using CRISPR Cas9 Prevent the Patient from Sickle Cell Anemia? Open Access Maced J Med Sci. 2023 Feb 03; 11 (F): 95-106 | |
CN113512535A (zh) | 改变细胞内基因组序列的方法、基因编辑细胞及应用 |
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: 20942540 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2022577265 Country of ref document: JP Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2020455671 Country of ref document: AU Date of ref document: 20200714 Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2020942540 Country of ref document: EP Effective date: 20230201 |