CN116064402A - Method for preparing insulin-secreting cells by combining CRISPRa and chemical induction - Google Patents
Method for preparing insulin-secreting cells by combining CRISPRa and chemical induction Download PDFInfo
- Publication number
- CN116064402A CN116064402A CN202211021415.8A CN202211021415A CN116064402A CN 116064402 A CN116064402 A CN 116064402A CN 202211021415 A CN202211021415 A CN 202211021415A CN 116064402 A CN116064402 A CN 116064402A
- Authority
- CN
- China
- Prior art keywords
- vector
- insulin
- sgrna
- seq
- cells
- 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
- 238000000034 method Methods 0.000 title claims abstract description 48
- 210000002660 insulin-secreting cell Anatomy 0.000 title claims abstract description 26
- 230000006698 induction Effects 0.000 title abstract description 47
- 239000000126 substance Substances 0.000 title abstract description 24
- 239000013598 vector Substances 0.000 claims abstract description 87
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 29
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims abstract description 26
- 239000000203 mixture Substances 0.000 claims abstract description 23
- 230000014509 gene expression Effects 0.000 claims abstract description 19
- 230000001939 inductive effect Effects 0.000 claims abstract description 13
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 39
- 239000002609 medium Substances 0.000 claims description 27
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 23
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 18
- 239000012091 fetal bovine serum Substances 0.000 claims description 17
- 108020004414 DNA Proteins 0.000 claims description 11
- 101800003838 Epidermal growth factor Proteins 0.000 claims description 11
- 102100033237 Pro-epidermal growth factor Human genes 0.000 claims description 11
- 229940116977 epidermal growth factor Drugs 0.000 claims description 11
- 102000004169 proteins and genes Human genes 0.000 claims description 11
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 claims description 11
- 102100024364 Disintegrin and metalloproteinase domain-containing protein 8 Human genes 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 10
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 10
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 9
- 210000003954 umbilical cord Anatomy 0.000 claims description 9
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 claims description 8
- 102400000322 Glucagon-like peptide 1 Human genes 0.000 claims description 8
- 101800000224 Glucagon-like peptide 1 Proteins 0.000 claims description 8
- 108050000588 Neurogenic differentiation factor 1 Proteins 0.000 claims description 8
- 230000004069 differentiation Effects 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 8
- 108020001507 fusion proteins Proteins 0.000 claims description 5
- 102000037865 fusion proteins Human genes 0.000 claims description 5
- 239000013603 viral vector Substances 0.000 claims description 5
- 102000053602 DNA Human genes 0.000 claims description 4
- 210000001185 bone marrow Anatomy 0.000 claims description 4
- 210000004700 fetal blood Anatomy 0.000 claims description 3
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 abstract description 23
- 230000003914 insulin secretion Effects 0.000 abstract description 14
- 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 18
- 108090001061 Insulin Proteins 0.000 description 13
- 102000004877 Insulin Human genes 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 9
- 229940125396 insulin Drugs 0.000 description 9
- 239000013612 plasmid Substances 0.000 description 9
- 239000012634 fragment Substances 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 8
- 230000004913 activation Effects 0.000 description 7
- 238000003776 cleavage reaction Methods 0.000 description 7
- 230000007017 scission Effects 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- 241000282326 Felis catus Species 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 102100032063 Neurogenic differentiation factor 1 Human genes 0.000 description 4
- 238000011529 RT qPCR Methods 0.000 description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 230000024245 cell differentiation Effects 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 206010012601 diabetes mellitus Diseases 0.000 description 4
- 229950010131 puromycin Drugs 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- -1 B27 Chemical compound 0.000 description 3
- 108091033409 CRISPR Proteins 0.000 description 3
- 102100031780 Endonuclease Human genes 0.000 description 3
- 108010042407 Endonucleases Proteins 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 241000713666 Lentivirus Species 0.000 description 3
- 102000003960 Ligases Human genes 0.000 description 3
- 108090000364 Ligases Proteins 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000000411 inducer Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 2
- 241000701822 Bovine papillomavirus Species 0.000 description 2
- OBMZMSLWNNWEJA-XNCRXQDQSA-N C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 Chemical compound C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 OBMZMSLWNNWEJA-XNCRXQDQSA-N 0.000 description 2
- 241000282836 Camelus dromedarius Species 0.000 description 2
- 241000700199 Cavia porcellus Species 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 241000699800 Cricetinae Species 0.000 description 2
- 241000283074 Equus asinus Species 0.000 description 2
- 241000283073 Equus caballus Species 0.000 description 2
- 101150110640 Fdx1 gene Proteins 0.000 description 2
- 241000699694 Gerbillinae Species 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 108050008339 Heat Shock Transcription Factor Proteins 0.000 description 2
- 101000638044 Homo sapiens Neurogenic differentiation factor 1 Proteins 0.000 description 2
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 2
- 241000282342 Martes americana Species 0.000 description 2
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 2
- 241001482564 Nyctereutes procyonoides Species 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 101710176384 Peptide 1 Proteins 0.000 description 2
- 241000009328 Perro Species 0.000 description 2
- 238000010806 PrimeScriptTM RT Reagent kit Methods 0.000 description 2
- 238000010240 RT-PCR analysis Methods 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 241000555745 Sciuridae Species 0.000 description 2
- 241000282898 Sus scrofa Species 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 241000282485 Vulpes vulpes Species 0.000 description 2
- 239000000443 aerosol Substances 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000013256 coordination polymer Substances 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 241001493065 dsRNA viruses Species 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000012528 insulin ELISA Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 201000006417 multiple sclerosis Diseases 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 239000006187 pill Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 210000002955 secretory cell Anatomy 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 241000701447 unidentified baculovirus Species 0.000 description 2
- 241001529453 unidentified herpesvirus Species 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 101150028074 2 gene Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 101800001382 Betacellulin Proteins 0.000 description 1
- 108010005054 Deoxyribonuclease BamHI Proteins 0.000 description 1
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 1
- 101100118093 Drosophila melanogaster eEF1alpha2 gene Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 108020005004 Guide RNA Proteins 0.000 description 1
- 101150019359 HSF1 gene Proteins 0.000 description 1
- 102000000039 Heat Shock Transcription Factor Human genes 0.000 description 1
- 101000596769 Homo sapiens Transcription factor p65 Proteins 0.000 description 1
- 102000003746 Insulin Receptor Human genes 0.000 description 1
- 108010001127 Insulin Receptor Proteins 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 101150035573 MS2 gene Proteins 0.000 description 1
- 108010057466 NF-kappa B Proteins 0.000 description 1
- 102000003945 NF-kappa B Human genes 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 1
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 1
- 102100029837 Probetacellulin Human genes 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 206010041047 Slow virus infection Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 208000016097 disease of metabolism Diseases 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 239000006196 drop Substances 0.000 description 1
- 239000007938 effervescent tablet Substances 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000003889 eye drop Substances 0.000 description 1
- 229940012356 eye drops Drugs 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000011223 gene expression profiling Methods 0.000 description 1
- 230000004153 glucose metabolism Effects 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000007923 nasal drop Substances 0.000 description 1
- 229940100662 nasal drops Drugs 0.000 description 1
- 230000005709 nerve cell growth Effects 0.000 description 1
- 229960003966 nicotinamide Drugs 0.000 description 1
- 235000005152 nicotinamide Nutrition 0.000 description 1
- 239000011570 nicotinamide Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000006201 parenteral dosage form Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000006190 sub-lingual tablet Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000000080 wetting agent Substances 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
- 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
- C12N5/0676—Pancreatic 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
- A61K35/37—Digestive system
- A61K35/39—Pancreas; Islets of Langerhans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/44—Thiols, e.g. mercaptoethanol
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/335—Glucagon; Glucagon-like peptide [GLP]; Exendin
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1392—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from mesenchymal stem cells from other natural sources
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Diabetes (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Pharmacology & Pharmacy (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Virology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Physiology (AREA)
- Emergency Medicine (AREA)
- Epidemiology (AREA)
- Developmental Biology & Embryology (AREA)
- Toxicology (AREA)
- Nutrition Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Endocrinology (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Chemical Kinetics & Catalysis (AREA)
Abstract
The invention provides a method for inducing mesenchymal stem cells to differentiate into insulin-secreting cells, which comprises the steps of introducing a vector composition to activate the expression of a neuroD1 gene, and carrying out chemical induction and combined induction to obtain the insulin-secreting cells. The insulin secretion amount of the insulin secretion cells obtained by the method is obviously high.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a method for preparing insulin-secreting cells by combining CRISPRa and chemical induction.
Background
Diabetes is a metabolic disease of disturbed glucose metabolism in the human body, the root cause of which is the relative or absolute deficiency of insulin secretion. Insulin secreting cells are transplanted as an alternative therapy to make up for the shortage of pancreas and islet organs, have been widely used in preclinical and clinical studies, and have very desirable therapeutic effects. Thus, researchers have invented various methods for preparing insulin-secreting cells. In terms of cell sources, each induction scheme involves embryonic stem cells, induced pluripotent stem cells, adipose-derived mesenchymal stem cells, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, and the like.
The method for inducing the insulin-secreting cells can be classified into two types, namely a chemical induction method, namely, a method for inducing stem cells to differentiate into insulin-secreting cells by using a chemical reagent, wherein the method is perfected and optimized by a plurality of researchers, but the induction procedure is still relatively complex, and the induction efficiency is relatively low; and secondly, a genetic induction method, namely directly inputting related genes for regulating insulin secretion cell differentiation into stem cells, and inducing cell differentiation under the condition of chemical induction or no chemical induction. Although this method can simplify the induction procedure and enhance the cell differentiation efficiency, it has a disadvantage that the inputted gene is generally only one of the gene transcription variants, which limits the regulation of the cell differentiation by the inputted gene.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a method for preparing insulin-secreting cells by combining CRISPRa and chemical induction, which comprises the steps of utilizing Cas9 protein (dCAS 9) without enzyme activity, accurately positioning a transcription activation element in a promoter region of a gene under the guidance of specific selected gRNA, activating the expression of the gene, and combining the chemical induction of a specific inducer to effectively activate and up-regulate the expression of transcription factors related to the differentiation of the insulin-secreting cells. The method has obviously higher induction efficiency than the method of singly using chemical induction, and the method has higher efficiency than singly using one sgRNA by adopting a plurality of specific selected sgRNAs, and the expression of endogenous genes is activated most efficiently. And simultaneously avoids the problem of gene transcription variant deletion caused by direct gene input. Furthermore, the method adopts the activated transcription factor neuroD1 instead of Fdx1, and also avoids the problem of hepatitis caused by the over-expression of Fdx1 alone.
In a first aspect of the present invention, there is provided a method of inducing differentiation of mesenchymal stem cells into insulin-secreting cells, said method comprising introducing a vector composition to activate expression of the NeuroD1 gene, and inducing the differentiation of the mesenchymal stem cells into insulin-secreting cells.
Preferably, the mesenchymal stem cells are derived from a human or non-human animal.
Preferably, the mesenchymal stem cells are derived from umbilical cord, bone marrow, cord blood or fat.
In one embodiment of the invention, the mesenchymal stem cells are derived from umbilical cord of a human or non-human animal.
Preferably, the mesenchymal stem cells can be obtained by screening and extracting umbilical cord, bone marrow, cord blood or fat of human or non-human animals, or purchasing the finished product.
Preferably, the non-human animal is a non-human mammal, including but not limited to wild animals, zoo animals, economic animals, pets, laboratory animals, and the like. Preferably, the non-human mammal includes, but is not limited to, a pig, cow, sheep, horse, donkey, fox, raccoon dog, marten, camel, dog, cat, rabbit, mouse (e.g., rat, mouse, guinea pig, hamster, gerbil, dragon cat, squirrel) or monkey, and the like.
In one embodiment of the present invention, the umbilical cord mesenchymal stem cells are the best material for inducing insulin-secreting cells due to their relatively abundant sources, proliferative and differentiation potential, low immunogenicity, genetic stability, and unexplained disputes.
Preferably, the carrier composition comprises:
a) A vector expressing one or more sgrnas and MS2 binding sites, or one or more vectors expressing sgrnas and MS2 binding sites; wherein the sgrnas may be the same or different.
B) A vector expressing fusion protein MS2-P65-HSF 1; the method comprises the steps of,
c) Vector expressing dCas9 protein.
Preferably, the vector composition further comprises other vectors required for packaging the virus.
Preferably, the vector is a bacterial vector, a fungal vector or a viral vector.
In one embodiment of the present invention, the vector is any vector that can deliver genetic material into cells in the prior art, such as phage vectors, baculovirus vectors, herpesvirus vectors, poxvirus vectors, RNA virus vectors, bovine papillomavirus vectors, epstein barr virus vectors, adenovirus vectors, lentiviral vectors, or retrovirus vectors.
Preferably, the target site sequence of the sgRNA comprises SEQ ID NO: 25. 26, 27 or 28, or a combination of two or more thereof.
Preferably, the double stranded DNA sequence encoding the sgRNA is selected from any one or a combination of two or more of the following:
A)SEQ ID NO：1、2；
B)SEQ ID NO：3、4；
C)SEQ ID NO：5、6；
D)SEQ ID NO：7、8。
in one embodiment of the invention, after the vector composition is introduced into a mesenchymal stem cell, the vector expressing the sgRNA and the MS2 binding site transcribes into the sgRNA and the RNA with a stem-loop structure capable of specifically binding to the MS2 protein, and forms a complex of the "genomic DNA-dCAS9-sgRNA-MS2 binding site-MS2-P65-HSF1" structure with the other two vectors (the vector expressing the fusion protein MS2-P65-HSF1, the vector expressing the dCAS9 protein). Further, transcription is initiated and NeuroD1 gene expression is activated.
Preferably, the method further comprises the step of culturing the mesenchymal stem cells introduced into the carrier composition.
Preferably, the culture of mesenchymal stem cells employs a culture medium comprising fetal bovine serum, more preferably a DMEM culture medium comprising fetal bovine serum, wherein the culture medium preferably comprises fetal bovine serum of 1% -20%, more preferably 2% -10%, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).
Preferably, the method further comprises a step of chemical induction, and preferably, the chemical induction uses one or a combination of more than two of epidermal growth factor, B27, nicotinamide, betacallin, glucagon-like peptide-1 or beta-mercaptoethanol as an inducer.
Preferably, the medium further comprises a label (e.g., hygromycin) and an antibiotic (e.g., puromycin).
Adding one or more than two of fetal bovine serum, epidermal growth factor, B27, nicotinamide, betacalulin, glucagon-like peptide-1 or beta-mercaptoethanol into a culture medium used for culture; preferably, the culture medium is a DMEM culture medium.
In a specific embodiment of the invention, the fetal bovine serum is added in an amount of 1% -20%, preferably 2% -10%, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20% of the culture medium.
In one embodiment of the invention, the epidermal growth factor is added in an amount of 80-150ng/mL, preferably 80-100ng/mL or 100-120ng/mL, e.g., 80, 90, 100, 11, 120, 130, 140, 150ng/mL EGF.
In one embodiment of the invention, the amount of B27 added is 1% -10% of the medium, preferably 1-5%, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10%.
In one embodiment of the invention, the nicotinamide is added in an amount of 5-20nM, preferably 8-12nM, e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.
In one embodiment of the invention, the betacallin is added in an amount of 2-20. Mu.g/mL, preferably 5-12. Mu.g/mL, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. Mu.g/mL.
In one embodiment of the invention, the glucagon-like peptide-1 is added in an amount of 5-20nM, preferably 8-12nM, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.
In one embodiment of the invention, the beta-mercaptoethanol is added in an amount of 0.001 to 1mM, preferably 0.01 to 0.2mM, such as 0.001, 0.01, 0.02, 0.05, 0.08, 0.1, 0.15, 0.2, 0.5, 1mM, etc.
In one embodiment of the invention, the method comprises transfecting the vector composition into mesenchymal stem cells (preferably 8-20h, e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20), culturing with DMEM medium comprising fetal bovine serum (5% -20%) for 2-5 days (preferably 3 days), exchanging DMEM medium comprising fetal bovine serum, epidermal growth factor and B27 for 2-5 days (preferably 3 days), exchanging DMEM medium comprising nicotinamide, B27, betacalin, glucagon-like peptide-1 and beta-mercaptoethanol for 2-5 days (preferably 3 days), and then exchanging DMEM medium comprising nicotinamide, B27, betacalin-lin, glucagon-like peptide-1 and beta-mercaptoethanol every 2-5 days (preferably 3 days).
In one embodiment of the invention, the method comprises:
umbilical cord mesenchymal stem cells are inoculated into DMEM medium (10% fetal bovine serum is added), and when the cell density grows to 50% -90% (preferably 60% -80%, e.g. 65%, 70%, 75%), the vector composition is transfected into the cells for 10-25 hours (preferably 12-24 hours, e.g. 15, 17, 20 hours), and the antibiotic-containing DMEM medium (10% fetal bovine serum, 400 μg/mL Hygromycin (Hygromycin) and 5 μg/mL Puromycin (Puromycin)) is replaced for three days. The DMEM medium containing inducer (2% fetal calf serum, 100ng/mL EGF (Epidermal Growth Factor) and 2% B27) was then changed for 3 days, and on day 4 the DMEM medium containing 10nM nicotinamide, 2% B27, 10. Mu.g/mL betacallin, 10nM GLP-1 (glucoon-like peptide-1) and 0.1mM beta-mercaptoethanol was changed for each 3 days.
In a second aspect of the present invention, there is provided an insulin-secreting cell obtained by the above-described method of inducing differentiation of mesenchymal stem cells into insulin-secreting cells.
In a third aspect of the present invention, there is provided a carrier composition comprising:
a) A vector expressing one or more sgrnas and MS2 binding sites, or one or more vectors expressing sgrnas and MS2 binding sites; wherein the sgrnas may be the same or different.
B) A vector expressing fusion protein MS2-P65-HSF 1; the method comprises the steps of,
c) A vector expressing dCas9 protein;
preferably, the vector composition further comprises other vectors required for packaging the virus.
Preferably, the vector is a viral vector;
optionally, the vector is a bacterial vector, a fungal vector or a viral vector.
In one embodiment of the present invention, the vector is any vector that can deliver genetic material into cells in the prior art, such as phage vectors, baculovirus vectors, herpesvirus vectors, poxvirus vectors, RNA virus vectors, bovine papillomavirus vectors, epstein barr virus vectors, adenovirus vectors, lentiviral vectors, or retrovirus vectors.
Preferably, the target site sequence of the sgRNA comprises SEQ ID NO: 25. 26, 27 or 28, or a combination of two or more thereof.
Preferably, the double stranded DNA sequence encoding the sgRNA is selected from any one or a combination of two or more of the following:
A)SEQ ID NO：1、2；
B)SEQ ID NO：3、4；
C)SEQ ID NO：5、6；
D)SEQ ID NO：7、8。
in a fourth aspect of the invention, there is provided an sgRNA whose target site sequence comprises the sequence set forth in SEQ ID NO: 25. 26, 27 or 28, or a combination of two or more thereof.
Preferably, the double stranded DNA sequence encoding the sgRNA is selected from any one or a combination of two or more of the following:
A)SEQ ID NO：1、2；
B)SEQ ID NO：3、4；
C)SEQ ID NO：5、6；
D)SEQ ID NO：7、8。
in a fifth aspect of the invention there is provided a cell comprising the vector composition described above or the sgRNA described above.
Preferably, the cells are microbial cells (e.g., bacteria, fungi, viruses), and may be plant cells, animal cells, or human cells.
In one embodiment of the invention, the cells include, but are not limited to, E.coli, mesenchymal stem cells, HEK293.
In a sixth aspect, the invention provides the use of the vector composition, the sgRNA or the cell described above for inducing differentiation of mesenchymal stem cells into insulin-secreting cells.
In a seventh aspect, the present invention provides a vector composition as defined above, an sgRNA as defined above, a cell as defined above or an insulin secreting cell as defined above, for use in a method of inducing insulin secretion, the method comprising:
a) Lowering blood sugar;
b) Treating and/or preventing diabetes;
c) Preparing medicine for treating and/or preventing diabetes.
According to an eighth aspect of the present invention, there is provided a medicament comprising the above-described carrier composition, the above-described sgrnas, the above-described cells or the above-described induced insulin secreting cells, and pharmaceutically acceptable excipients.
In a ninth aspect of the invention, there is provided a method of treating diabetes comprising administering to an individual the above-described vector composition, sgRNA, cells, insulin secreting cells obtained by the above-described induction, insulin secreted by the secretory cells obtained by the above-described induction, or the above-described medicament.
In a tenth aspect of the present invention, there is provided a method for lowering blood glucose comprising administering to an individual the above-described vector composition, sgRNA, cells, insulin secreting cells obtained by the above-described induction, insulin secreted by the secretory cells obtained by the above-described induction, or the above-described medicament.
The "neuroD1 gene" described herein represents Neuronal Differentiation 1, the neuronal differentiation 1 gene.
"MS2 gene" stands for Multiple sclerosis, susceptibility to,2, multiple sclerosis-sensitive 2 gene.
"P65 gene" means RELA pro-to-oncogene, NF-kB debug, RELA protooncogene, NF-kB subunit gene.
"HSF1 gene" represents heat shock transcription factor 1, heat shock transcription factor 1 gene.
The "HEK293" described herein represents human embryonic kidney cells 293.
The "dCas9" described in the present invention is a protein formed by mutating the Cas9 cleavage site, which has only the ability to bind to genome and sgRNA, but does not have the ability to cleave DNA.
The number of the "plurality" is more than 2, including but not limited to 2 to 50, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.
The "activation" of the present invention may be to activate a gene which is not expressed originally, to be expressed, or to increase the expression level of a gene which is expressed in a low amount originally.
The term "treatment" as used herein means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.
The "pharmaceutically acceptable auxiliary materials" in the present invention include, but are not limited to, one or more of carriers, excipients, diluents, wetting agents, fillers, binders, lubricants, disintegrants, antioxidants, buffers, suspending agents, solubilizers, thickeners, stabilizers, flavoring agents, preservatives, etc.
The "drug" as described herein may be administered by any suitable route, such as by gastrointestinal (e.g., oral) or parenteral (e.g., intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.) route.
The "drug" according to the present invention may be any suitable dosage form, such as a parenteral or parenteral dosage form, and preferably includes, but is not limited to, tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder mists, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, drop pills, gels and the like.
The various dosage forms of the medicament can be prepared according to the conventional production method in the pharmaceutical field.
The "individual" as used herein may be a human or non-human mammal, which may be a wild animal, zoo animal, economic animal, pet animal, laboratory animal, etc. Preferably, the non-human mammal includes, but is not limited to, a pig, cow, sheep, horse, donkey, fox, raccoon dog, marten, camel, dog, cat, rabbit, mouse (e.g., rat, mouse, guinea pig, hamster, gerbil, dragon cat, squirrel) or monkey, and the like.
All combinations of items to which the term "and/or" is attached "in this description shall be taken to mean that the respective combinations have been individually listed herein. For example, "a and/or B" includes "a", "a and B", and "B". Also for example, "A, B and/or C" include "a", "B", "C", "a and B", "a and C", "B and C" and "a and B and C".
The term "comprising" or "including" as used herein is an open reading frame, and when used to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.
Drawings
Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:
fig. 1: the structure diagram of the modified lentiviral vector is shown in the specification, wherein the lentiviral vectors are Lenti-dCAs9-VP64-T2A-BlastR, lenti-MS2-P65-HSF1-T2A-hygroR and Lenti-sgRNA2.0-PuroR respectively.
Fig. 2: bsmBI cleavage site and the sticky ends formed by single cleavage of Lenti-sgRNA2.0-PuroR by BsmBI.
Fig. 3: efficiency of gene activation following independent transfection and co-transfection of the NeuroD1 gene sgRNA into HEK293 cells. A: RT-PCR analysis of different sgRNA activation efficiencies. B: qPCR analysis of the activation efficiency of the NeuroD1 gene by different sgrnas. Wherein CN is a negative control, sgRNA1-4 is mixed by equal molar ratio of sgRNA1, sgRNA2, sgRNA3 and sgRNA4, and CP is a positive control.
Fig. 4A: at 7d post-chemical induction, the insulin-related gene expression levels of each group.
Fig. 4B: at 14d post-chemical induction, insulin-related gene expression levels were measured for each group.
Fig. 4C: at 21d post-chemical induction, the insulin-related gene expression levels of each group.
Fig. 5: at chemical induction of 7d, 14d and 21d, insulin secretion was induced in the group and in the combination.
Fig. 6: insulin secretion in the induction group and the combination group after sugar induction.
Detailed Description
The method and application of the present invention will now be described with reference to the accompanying drawings, which are given by way of illustration only and are not intended to limit the scope of the invention.
The lentiviral vectors used in the examples, lenti-dCAs9-VP64_blast (# 61425), lenti-MS2-P65-HSF1_hygro (# 61426) and Lenti-sgRNA2.0 (# 61427) were all from ADDGNE.
Examples were obtained using PGK-Puror from SBI (System Biosciences), catalog number: CD810A-1.
Example 1: lenti-dCAS9-SAM/sgRNA2.0 vector construction
1. The EF1a promoter in the Lenti-dCAs9-VP64_blast and the Lenti-MS2-P65-HSF1_hygro vector was replaced by a CMV strong promoter.
CMV promoter primers with NheI and BsiWI endonuclease sites were designed, and the sequences of the primers were
NheI-CMV-F：5’-GCTAGCCCCGTTACATAACTTACGG-3’(SEQ ID NO：9)；
CMV-BsiWI-R：5’-CGTACGGATCTGACGGTTCACTAAA-3’(SEQ ID NO：10)；
Amplifying NheI-CMV-BsiWI DNA fragment from plasmid carrying CMV promoter, cloning the DNA fragment onto pEasy vector to construct pEasy-CMV vector, sequencing and screening positive monoclonal, and extracting plasmid for standby. The NheI-BsiWI double cleavage was performed with the Lenti-dCAs9-VP64_blast, lenti-MS2-P65-HSF1_hygro and pEasy-CMV vectors, and the cleavage system was shown in Table 1.
Table 1: nheI-BsiWI cleavage system
| Plasmid(s) | 1μg |
| NheI | 1μL |
| BsiWI | 1μL |
| 10×CutSmart Buffer | 5μL |
| ddH 2 O | Constant volume to 50. Mu.L |
The digested products were separated by 1% agarose gel electrophoresis at 37℃overnight, and DNA fragments of 12820bp (Lenti-dCAS 9-VP 64_blast), 10471bp (Lenti-MS 2-P65-HSF1_hygro) and 521bp (CMV) were recovered and purified, respectively. The 12820bp and 10471bp fragments were ligated with the 521bp fragment with T4 ligase, respectively. The reaction product is transformed into stbl3 escherichia coli competent, coated on LB solid plates containing Amp, cultured overnight at 37 ℃, and positive clones are screened, thus being modified lentiviral vectors Lenti-dCAs9-VP64-T2A-BlastR and Lenti-MS2-P65-HSF1-T2A-hygroR, as shown in figure 1.
2. Lenti-sgRNA2.0 vector modification, wherein EF1a-BleoR was modified to PGK-PuroR.
The PGK-PuroR primer with BamHI and EcoRI endonuclease sites was designed and the sequence was:
BamHI-PGK-F:5'-GGATCCGGGTAGGGGAGGCGCTTTTC-3' (SEQ ID NO: 11), and PuroR-EcoRI-R:5'-GAATTCTCAGGCACCGGGCTTGCGGG-3' (SEQ ID NO: 12),
the plasmid with PGK-PuroR is used as a template, the BamHI-PGK-PuroR-EcoRI fragment is obtained by DNA high-fidelity polymerase amplification and is connected to a blunt end vector to form a pEasy-PGK-PuroR vector, and correct cloning is selected and plasmid is extracted for standby. BamHI-EcoRI double digested Lenti-sgRNA2.0 and pEasy-PGK-PuroR, the digestion system is shown in Table 2.
Table 2: enzyme cutting system
| Plasmid(s) | 1μg |
| BamHI | 1μL |
| EcoRI | 1μL |
| 10×CutSmart Buffer | 5μL |
| ddH 2 O | Make up to 50 mu L |
The enzyme-digested product is separated by 1% agarose gel electrophoresis after being incubated overnight at 37 ℃, DNA fragments with the sizes of 8351bp (Lenti-sgRNA2.0) and 1146bp (PGK-PuroR) are purified and recovered, T4 ligase is connected, stbl3 escherichia coli is converted by the ligation product, positive clones are screened, and the modified lentiviral plasmid Lenti-sgRNA2.0-PuroR is obtained, and the legend is shown in figure 1.
3. sgRNA design and vector construction
The efficient and specific target sequence selection and design are the precondition for constructing the sgRNA expression vector, and also determine the targeting specificity and the efficiency for inducing Cas9 to cut the target gene. The sgRNA design of the NeuroD1 gene the sgRNA sequences in table 3 were specifically selected in this application, out of several of the sgRNA sequences designed using the CHOPCHOP (version 3) online sgRNA design tool.
Table 3: sgRNA sequences and positions
Based on the cohesive ends obtained by single cleavage of Lenti-sgRNA2.0-Puror by the endonuclease BsmBI (FIG. 2), cohesive ends of the sgRNA insert vector (FIG. 2) were designed and synthesized by commercial company.
Single-stranded oligodeoxyribonucleic acid synthesized by commercial companies is synthesized into double strands. The method comprises the following steps: dissolving the synthesized freeze-dried powder into 10 mu M, taking 5 mu L of complementary strands, adding into a 0.2mL PCR tube, and then placing into a PCR instrument, wherein the reaction procedure is that the temperature is 37 ℃ for 10min;95 ℃ for 5min; the temperature was lowered by 1.5℃per minute to room temperature.
Double-stranded sgRNA phosphorylation. T4 Polynucleotide kinase the synthesized double stranded sgRNA was phosphorylated, and the reaction system is shown in Table 4.
Table 4: double-strand sgRNA phosphorylation reaction system
Storing at 37 deg.C for 1h at-20 deg.C.
Construction of sgRNA vector. The endonuclease BsmBI single enzyme cuts the Lenti-sgRNA2.0-PuroR, the enzyme cutting system is shown in Table 5.
Table 5: construction of enzyme cutting system for sgRNA vector
| Lenti-sgRNA2.0- | 1μg | |
| 10×CutSmart Buffer | 5μL | |
| BsmBI | 0.5μL | |
| ddH 2 O | Make up to 50 mu L | |
| Final volume | 50μL |
37℃for 20min. The digested product was separated by 1% agarose gel, and the large fragment was recovered by purification. T4 ligase connects double-stranded sgRNA and Lenti-sgRNA2.0-PuroR, the connection product is converted into stbl3 escherichia coli competent, and positive clones are screened by sequencing.
Example 2: dCAS9-SAM/sgRNA activation of NeuroD1 expression
HEK293 was inoculated into 6 well cell culture platesIn the medium, DMEM added with 10% of fetal bovine serum and 4mM of L-glutamine, 37 ℃ and 5% CO 2 The incubator was cultured until the cell density was 70%. Transfection reagent Lipofectamine 3000 cotransfection of Lenti-dCAs9-VP64-T2A-BlastR, lenti-MS2-P65-HSF1-T2A-hygroR and Lenti-sgRNA2.0-Puror prepared in example 1, transfection systems are shown in Table 6.
Table 6: transfection system
| Opti-MEM medium | 125μL |
| Total plasmid DNA (molar ratio 1:1:1) | 2.5μg |
| P3000 | 5μL |
| Lipofectamine 3000 | 3μL |
Standing at room temperature for 15min at 37deg.C with 5% CO 2 Culturing in an incubator for 24 hours, replacing fresh culture medium, culturing for 48 hours, and collecting cells. Invitrogen TRIzol RNA the extraction kit is used for extracting total RNA, and the extraction process is described in the product specification. The quality of the extracted RNA samples was identified by 1% agarose gel electrophoresis.
TakaRa PrimeScript RT reagent Kit reverse transcription kit cDNA was synthesized (see product description for details).
cDNA samples were subjected to qPCR and RT-PCR analysis using ABI-7500. Untransfected HEK293 cells served as negative control, labeled CN, islet cells served as positive control, labeled CP, GAPDH served as internal reference, and primer sequences are shown in table 7.
Table 7: primer sequences
| Primer name | Sequence (5 '-3') | Number (SEQ ID NO: x) | |
| GAPDH-F | AGAAGGCTGGGGCTCATTTG | 13 | |
| GAPDH- | AGGGGCCATCCACAGTCTTC | 14 | |
| Pdx1-F | GGATGAAGTCTACCAAAGCTCACGC | 15 | |
| Pdx1-R | CCAGATCTTGATGTGTCTCTCGGTC | 16 | |
| insulin-F | AACCAACACCTGTGCGGCTCA | 17 | |
| insulin-R | TGCCTGCGGGCTGCGTCTA | 18 | |
| Ngn3-F | GTAGAAAGGATGACGCCTCAACC | 19 | |
| Ngn3- | TCAGTGCCAACTCGCTCTTAGG | 20 | |
| NKX6.1- | CTGGAGAAGACTTTCGAACAA | 21 | |
| NKX6.1-R | AGAGGCTTATTGTAGTCGTCG | 22 | |
| NeuroD1-F | AGTTCTCAGGACGAGGAGCA | 23 | |
| NeuroD1-R | TCCGACAGAGCCCAGATGTA | 24 |
The relative expression of the gene is 2 -ΔΔC And (5) analyzing by a method.
The results showed that the efficiency of 4 sgrnas synergistic to activate NeuroD1 was significantly higher than that of activation alone (fig. 3).
Example 3: preparation and verification of lentivirus package and insulin secreting cells
The lentivirus package adopts a four-plasmid system, PEI is used as a transfection reagent, HEK293 cells are transfected together, liquid is changed after 12 hours, the supernatant is collected after 72 hours, and the concentrated virus is purified by a purification column. The virus titer was determined by qPCR. Wherein, the sgrnas are 4 sgrnas to act synergistically.
1. The combination of dCAS9-SAM/NeuroD1_sgRNA and chemical induction for preparing insulin-secreting cells comprises the following steps:
p3-generation umbilical cord mesenchymal stem cells (Umbilical Cord Mesenchymal Stem Cell, UC-MSC) were inoculated into DMEM medium (10% fetal bovine serum was added), when the cell density was grown to 70%, lentivirus Lenti-dCAs9-VP64-T2A-BlastR, lenti-MS2-P65-HSF1-T2A-hygroR and Lenti-sgRNA2.0-Puror prepared in example 1 were co-infected cells, and after 12 hours of infection, chemical induction was started after culturing for three days by exchanging the DMEM medium (10% fetal bovine serum, 400. Mu.g/mL Hygromycin (Hygromycin) and 5. Mu.g/mL Puromycin) containing antibiotics. When in chemical induction, two parallel experimental groups are additionally arranged, and the three experimental groups are respectively a slow virus infection and chemical induction combined group, an induction group and a non-infection non-induction UC-MSC group. At the beginning of the chemical induction, the combination and induction groups were first incubated in DMEM medium supplemented with 2% fetal bovine serum, 100ng/mL EGF (Epidermal Growth Factor) and 2% nerve cell growth agent (B27) for 3d, 4d, and replaced with DMEM medium supplemented with 10nM nicotinamide, 2% B27, 10. Mu.g/mL betacellulin, 10nM GLP-1 (glucoon-like peptide-1) and 0.1mM beta-mercaptoethanol, and the medium was replaced every 3 days. UC-MSC group was directly cultured in DMEM medium containing 10% fetal bovine serum, and the medium was changed every three days.
2. Insulin-related gene expression profiling
Cells 7d, 14d and 21d after the start of chemical induction were collected, total RNA was extracted by Invitrogen TRIzol RNA extraction kit, and the quality of the extracted RNA was identified by 1% agarose gel electrophoresis. TakaRa PrimeScript RT reagent Kit reverse transcription kit cDNA was synthesized. cDNA samples were subjected to qPCR analysis using ABI-7500. GAPDH was used as an internal reference and the primer sequences are shown in Table 7. The relative expression of the gene is 2 -ΔΔC And (5) analyzing by a method.
The results showed that the insulin-related gene expression levels in the combination groups were significantly higher than those in the induction groups (P < 0.05) at the induction groups 7d, 14d and 21d, and that the expression levels of the genes were gradually increased from the first 14d to 21d except for the insulin gene. At 7d, no expression of the insulin gene was detected in both experimental groups, and at 14d, the insulin gene was detected to be expressed initially, and at 21d, the insulin expression level was maximized (FIG. 4).
3. Insulin secretion analysis
Supernatants were collected at 7d, 14d, and 21d following chemical induction and assayed for insulin secretion by the Abcam insulin ELISA kit. The results showed that the change in insulin content in the cell culture supernatant was consistent with the change in the expression level of the insulin gene in the cells (FIGS. 4 and 5), and that the insulin secretion amount in the combination group was significantly higher than that in the induction group (P < 0.05).
4. Influence of sugar induction on insulin secretion
At chemical induction 21d, the induction medium was removed, after washing with PBS, DMEM low-sugar medium (5.5 mM glucose) was added for 3 hours, the supernatant was collected, after washing with PBS twice, and after culturing with DMEM high-sugar medium (25 mM glucose) for 3 hours, the supernatant was collected for comparison. Abcam insulin ELISA kit for measuring insulin secretion. The results showed that the insulin secretion was higher in both the induction group and the combination group under high sugar conditions than in the low sugar and that the insulin secretion was significantly higher in both the combination group under low sugar and high sugar conditions than in the induction group under the same conditions (P < 0.05) (fig. 6).
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (10)
1. A method for inducing differentiation of mesenchymal stem cells into insulin-secreting cells, said method comprising introducing a vector composition to activate expression of the NeuroD1 gene, and inducing the differentiation of the mesenchymal stem cells into insulin-secreting cells.
2. The method of claim 1, wherein the carrier composition comprises:
a) A vector expressing one or more sgrnas and MS2 binding sites, or one or more vectors expressing sgrnas and MS2 binding sites;
b) A vector expressing fusion protein MS2-P65-HSF 1; the method comprises the steps of,
c) A vector expressing dCas9 protein;
preferably, the vector is a viral vector.
3. The method of claim 1 or 2, wherein the target site sequence of the sgRNA comprises the sequence of SEQ ID NO: 25. 26, 27 or 28, or a combination of two or more thereof.
4. A method according to claim 3, characterized in that the double stranded DNA sequence encoding the sgRNA is selected from any one of the group or a combination of two or more of the following:
A)SEQ ID NO：1、2；
B)SEQ ID NO：3、4；
C)SEQ ID NO：5、6；
D)SEQ ID NO：7、8。
5. the method of any one of claims 1-4, wherein the mesenchymal stem cells are derived from umbilical cord, bone marrow, cord blood or fat of a human or non-human animal.
6. The method of any one of claims 1-5, further comprising culturing mesenchymal stem cells introduced into the vector composition, wherein the culturing is performed using a medium comprising one or more of fetal bovine serum, epidermal growth factor, B27, nicotinamide, betacallin, glucagon-like peptide-1, or beta-mercaptoethanol; preferably, the culture medium is a DMEM culture medium.
7. The method of any one of claims 1-6, comprising transfecting the vector composition into mesenchymal stem cells, culturing with DMEM medium comprising fetal bovine serum for 2-5 days, exchanging DMEM medium comprising fetal bovine serum, epidermal growth factor and B27 for 2-5 days, exchanging DMEM medium comprising nicotinamide, B27, betacallin, glucagon-like peptide-1 and β -mercaptoethanol for 2-5 days, and then exchanging DMEM medium comprising nicotinamide, B27, betacallin, glucagon-like peptide-1 and β -mercaptoethanol every 2-5 days.
8. A carrier composition, said carrier composition comprising:
a) A vector expressing one or more sgrnas and MS2 binding sites, or one or more vectors expressing sgrnas and MS2 binding sites;
b) A vector expressing fusion protein MS2-P65-HSF 1; the method comprises the steps of,
c) A vector expressing dCas9 protein;
preferably, the vector is a viral vector;
the target site sequence of the sgRNA comprises SEQ ID NO: 25. 26, 27 or 28, or a combination of two or more thereof.
9. An sgRNA, wherein the target site sequence of the sgRNA comprises the sequence set forth in SEQ ID NO: 25. 26, 27 or 28, or a combination of two or more thereof.
10. A medicament comprising insulin secreting cells induced by the method of any of claims 1-7, the vector composition of claim 8, the sgRNA of claim 9, and pharmaceutically acceptable excipients.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211021415.8A CN116064402A (en) | 2022-08-24 | 2022-08-24 | Method for preparing insulin-secreting cells by combining CRISPRa and chemical induction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211021415.8A CN116064402A (en) | 2022-08-24 | 2022-08-24 | Method for preparing insulin-secreting cells by combining CRISPRa and chemical induction |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116064402A true CN116064402A (en) | 2023-05-05 |
Family
ID=86170565
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211021415.8A Pending CN116064402A (en) | 2022-08-24 | 2022-08-24 | Method for preparing insulin-secreting cells by combining CRISPRa and chemical induction |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116064402A (en) |
-
2022
- 2022-08-24 CN CN202211021415.8A patent/CN116064402A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7263327B2 (en) | Non-Integrating DNA Vectors for Genetic Modification of Cells | |
| US20230060518A1 (en) | Leaper technology based method for treating mps ih and composition | |
| JP7197363B2 (en) | Genome editing of human neural stem cells using nucleases | |
| US20200155606A1 (en) | Crispr/rna-guided nuclease systems and methods | |
| JP2019517281A (en) | Gene therapy for neurotheloid lipofuscinosis | |
| WO2019178416A1 (en) | Systems and methods for the treatment of hemoglobinopathies | |
| CN113164624B (en) | Compositions and methods for restoring paternal UBE3A gene expression in human Happy puppet syndrome | |
| Wang | Application of modified mRNA in somatic reprogramming to pluripotency and directed conversion of cell fate | |
| CN117015605A (en) | Targeted RNA editing using engineered RNAs by utilizing endogenous ADAR | |
| CN110760480B (en) | Anti-tumor NK (Natural killer) cell and preparation method thereof | |
| CA3206298A1 (en) | Functional nucleic acid molecule and method | |
| WO2019173654A2 (en) | Systems and methods for the treatment of hemoglobinopathies | |
| CN117126855A (en) | Method for constructing Igf2bp1 gene knockout mouse embryonic stem cell line | |
| CN109055433B (en) | Method for activating expression of endogenous Ngn3 and MAFA genes | |
| Goncz et al. | Small fragment homologous replacement-mediated modification of genomic β-globin sequences in human hematopoietic stem/progenitor cells | |
| CN116064402A (en) | Method for preparing insulin-secreting cells by combining CRISPRa and chemical induction | |
| CA2991056A1 (en) | Humanized heart muscle | |
| CN116218906A (en) | RNA editor expression plasmid, exosome aptamer fusion expression plasmid and targeted gene RNA editing method | |
| US20230167458A1 (en) | Forskolin-inducible promoters and hypoxia-inducible promoters | |
| CN116064676A (en) | Method for preparing insulin secretion cells by CRISPRa mediated mesenchymal stem cells | |
| US20240026311A1 (en) | ARTIFICIALLY SYNTHESIZED mRNA AND USE OF SAME | |
| CN116064370A (en) | Method for improving islet beta cell function by gene editing UC-MSC | |
| CN114941013A (en) | Recombinant mesenchymal stem cells for treating diabetes pneumonia | |
| CN115851706A (en) | Base editing system and application thereof | |
| CA3126886A1 (en) | Liver-specific inducible promoters and methods of use thereof |
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 |