CN118374548A - Reprogramming factor anti-aging expression system, biological material and application - Google Patents
Reprogramming factor anti-aging expression system, biological material and application Download PDFInfo
- Publication number
- CN118374548A CN118374548A CN202410448498.1A CN202410448498A CN118374548A CN 118374548 A CN118374548 A CN 118374548A CN 202410448498 A CN202410448498 A CN 202410448498A CN 118374548 A CN118374548 A CN 118374548A
- Authority
- CN
- China
- Prior art keywords
- transcription factor
- nucleic acid
- acid molecule
- expression system
- molecule encoding
- 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
- 230000014509 gene expression Effects 0.000 title claims abstract description 137
- 230000008672 reprogramming Effects 0.000 title claims abstract description 49
- 230000003712 anti-aging effect Effects 0.000 title claims abstract description 32
- 239000012620 biological material Substances 0.000 title claims abstract description 14
- 102000040945 Transcription factor Human genes 0.000 claims abstract description 132
- 108091023040 Transcription factor Proteins 0.000 claims abstract description 132
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 79
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 79
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 79
- 108010068425 Octamer Transcription Factor-3 Proteins 0.000 claims abstract description 53
- 102000002584 Octamer Transcription Factor-3 Human genes 0.000 claims abstract description 53
- 102000005635 SOXB1 Transcription Factors Human genes 0.000 claims abstract description 43
- 108010045179 SOXB1 Transcription Factors Proteins 0.000 claims abstract description 43
- 108700021430 Kruppel-Like Factor 4 Proteins 0.000 claims abstract description 37
- 101150111214 lin-28 gene Proteins 0.000 claims abstract description 33
- 210000000056 organ Anatomy 0.000 claims abstract description 7
- 108020004999 messenger RNA Proteins 0.000 claims description 90
- 239000013604 expression vector Substances 0.000 claims description 70
- 239000013598 vector Substances 0.000 claims description 66
- 239000002773 nucleotide Substances 0.000 claims description 30
- 125000003729 nucleotide group Chemical group 0.000 claims description 30
- 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 claims description 28
- 238000002360 preparation method Methods 0.000 claims description 24
- 241000131390 Glis Species 0.000 claims description 15
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 15
- 229950010131 puromycin Drugs 0.000 claims description 14
- 230000008439 repair process Effects 0.000 claims description 7
- 230000006378 damage Effects 0.000 claims description 6
- 108010042634 F2A4-K-NS peptide Proteins 0.000 claims description 5
- 101800001494 Protease 2A Proteins 0.000 claims description 5
- 101800001066 Protein 2A Proteins 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 208000027418 Wounds and injury Diseases 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 208000014674 injury Diseases 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 230000003612 virological effect Effects 0.000 claims description 3
- 208000030159 metabolic disease Diseases 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims 1
- 102200157658 rs1555229948 Human genes 0.000 claims 1
- 230000001973 epigenetic effect Effects 0.000 abstract description 38
- 230000000694 effects Effects 0.000 abstract description 15
- 210000001672 ovary Anatomy 0.000 abstract description 11
- 210000000845 cartilage Anatomy 0.000 abstract description 10
- 230000002441 reversible effect Effects 0.000 abstract description 7
- 230000032683 aging Effects 0.000 abstract description 6
- 206010016654 Fibrosis Diseases 0.000 abstract description 5
- 101150037203 Sox2 gene Proteins 0.000 abstract description 5
- 230000004761 fibrosis Effects 0.000 abstract description 5
- 210000002216 heart Anatomy 0.000 abstract description 5
- 210000003491 skin Anatomy 0.000 abstract description 4
- 210000001550 testis Anatomy 0.000 abstract description 4
- 210000003292 kidney cell Anatomy 0.000 abstract description 3
- 230000008929 regeneration Effects 0.000 abstract description 3
- 238000011069 regeneration method Methods 0.000 abstract description 3
- 230000004060 metabolic process Effects 0.000 abstract description 2
- 231100001083 no cytotoxicity Toxicity 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 89
- 241000702421 Dependoparvovirus Species 0.000 description 80
- 108020004414 DNA Proteins 0.000 description 60
- 241000701161 unidentified adenovirus Species 0.000 description 58
- 210000004027 cell Anatomy 0.000 description 57
- 239000013612 plasmid Substances 0.000 description 55
- 229960003722 doxycycline Drugs 0.000 description 47
- SGKRLCUYIXIAHR-AKNGSSGZSA-N (4s,4ar,5s,5ar,6r,12ar)-4-(dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1=CC=C2[C@H](C)[C@@H]([C@H](O)[C@@H]3[C@](C(O)=C(C(N)=O)C(=O)[C@H]3N(C)C)(O)C3=O)C3=C(O)C2=C1O SGKRLCUYIXIAHR-AKNGSSGZSA-N 0.000 description 39
- 241000699670 Mus sp. Species 0.000 description 30
- 108091028043 Nucleic acid sequence Proteins 0.000 description 25
- 239000007924 injection Substances 0.000 description 22
- 238000002347 injection Methods 0.000 description 22
- 238000001890 transfection Methods 0.000 description 22
- 241000700605 Viruses Species 0.000 description 19
- 230000006698 induction Effects 0.000 description 19
- 241000699666 Mus <mouse, genus> Species 0.000 description 16
- 108700008625 Reporter Genes Proteins 0.000 description 15
- 210000004185 liver Anatomy 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 15
- 101150108076 lin28a gene Proteins 0.000 description 12
- 108010026228 mRNA guanylyltransferase Proteins 0.000 description 12
- 230000011987 methylation Effects 0.000 description 12
- 238000007069 methylation reaction Methods 0.000 description 12
- 230000002611 ovarian Effects 0.000 description 12
- 210000002503 granulosa cell Anatomy 0.000 description 11
- 239000000047 product Substances 0.000 description 11
- 239000012634 fragment Substances 0.000 description 10
- 238000012408 PCR amplification Methods 0.000 description 9
- 101710126211 POU domain, class 5, transcription factor 1 Proteins 0.000 description 9
- 108020005202 Viral DNA Proteins 0.000 description 9
- 238000004364 calculation method Methods 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 9
- 210000004072 lung Anatomy 0.000 description 9
- 239000002953 phosphate buffered saline Substances 0.000 description 9
- 238000012163 sequencing technique Methods 0.000 description 9
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 8
- 210000001612 chondrocyte Anatomy 0.000 description 8
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 102000034356 gene-regulatory proteins Human genes 0.000 description 8
- 108091006104 gene-regulatory proteins Proteins 0.000 description 8
- 229960002591 hydroxyproline Drugs 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 8
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 7
- 238000004113 cell culture Methods 0.000 description 7
- 208000005069 pulmonary fibrosis Diseases 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 241000710929 Alphavirus Species 0.000 description 6
- 238000013218 HFD mouse model Methods 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 108010005774 beta-Galactosidase Proteins 0.000 description 6
- 208000019425 cirrhosis of liver Diseases 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 210000002950 fibroblast Anatomy 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 238000010172 mouse model Methods 0.000 description 6
- 230000009758 senescence Effects 0.000 description 6
- 108060004795 Methyltransferase Proteins 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000000090 biomarker Substances 0.000 description 5
- 238000001369 bisulfite sequencing Methods 0.000 description 5
- 239000003651 drinking water Substances 0.000 description 5
- 235000020188 drinking water Nutrition 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 238000001990 intravenous administration Methods 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 108091026890 Coding region Proteins 0.000 description 4
- 238000007400 DNA extraction Methods 0.000 description 4
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 4
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 4
- 108010076504 Protein Sorting Signals Proteins 0.000 description 4
- 101100247004 Rattus norvegicus Qsox1 gene Proteins 0.000 description 4
- 108700009124 Transcription Initiation Site Proteins 0.000 description 4
- 102100038313 Transcription factor E2-alpha Human genes 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 4
- 231100000135 cytotoxicity Toxicity 0.000 description 4
- 230000003013 cytotoxicity Effects 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000012173 estrus Effects 0.000 description 4
- 235000019197 fats Nutrition 0.000 description 4
- 230000003176 fibrotic effect Effects 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 229910052754 neon Inorganic materials 0.000 description 4
- GKAOGPIIYCISHV-UHFFFAOYSA-N neon atom Chemical compound [Ne] GKAOGPIIYCISHV-UHFFFAOYSA-N 0.000 description 4
- 230000002035 prolonged effect Effects 0.000 description 4
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 3
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 3
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 3
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 3
- 101800000980 Protease nsP2 Proteins 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 101710137500 T7 RNA polymerase Proteins 0.000 description 3
- 108091036066 Three prime untranslated region Proteins 0.000 description 3
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000007405 data analysis Methods 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 101150024821 tetO gene Proteins 0.000 description 3
- 239000012096 transfection reagent Substances 0.000 description 3
- 108020005345 3' Untranslated Regions Proteins 0.000 description 2
- 108020003589 5' Untranslated Regions Proteins 0.000 description 2
- RYVNIFSIEDRLSJ-UHFFFAOYSA-N 5-(hydroxymethyl)cytosine Chemical compound NC=1NC(=O)N=CC=1CO RYVNIFSIEDRLSJ-UHFFFAOYSA-N 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 2
- 102000018120 Recombinases Human genes 0.000 description 2
- 108010091086 Recombinases Proteins 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 230000037213 diet Effects 0.000 description 2
- 235000009200 high fat diet Nutrition 0.000 description 2
- 238000012165 high-throughput sequencing Methods 0.000 description 2
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 210000001778 pluripotent stem cell Anatomy 0.000 description 2
- 239000000276 potassium ferrocyanide Substances 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000012372 quality testing Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 description 2
- 231100000027 toxicology Toxicity 0.000 description 2
- 230000010474 transient expression Effects 0.000 description 2
- UVBYMVOUBXYSFV-XUTVFYLZSA-N 1-methylpseudouridine Chemical compound O=C1NC(=O)N(C)C=C1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 UVBYMVOUBXYSFV-XUTVFYLZSA-N 0.000 description 1
- BGNGWHSBYQYVRX-UHFFFAOYSA-N 4-(dimethylamino)benzaldehyde Chemical compound CN(C)C1=CC=C(C=O)C=C1 BGNGWHSBYQYVRX-UHFFFAOYSA-N 0.000 description 1
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 1
- 241001655883 Adeno-associated virus - 1 Species 0.000 description 1
- 241000702423 Adeno-associated virus - 2 Species 0.000 description 1
- 241000202702 Adeno-associated virus - 3 Species 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 108091029430 CpG site Proteins 0.000 description 1
- 101100239628 Danio rerio myca gene Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101800000515 Non-structural protein 3 Proteins 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 208000002500 Primary Ovarian Insufficiency Diseases 0.000 description 1
- 101800001758 RNA-directed RNA polymerase nsP4 Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 210000003321 cartilage cell Anatomy 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 210000003756 cervix mucus Anatomy 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 235000019864 coconut oil Nutrition 0.000 description 1
- 239000003240 coconut oil Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002431 foraging effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 210000002064 heart cell Anatomy 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 238000013227 male C57BL/6J mice Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000009245 menopause Effects 0.000 description 1
- XBCXJKGHPABGSD-UHFFFAOYSA-N methyluracil Natural products CN1C=CC(=O)NC1=O XBCXJKGHPABGSD-UHFFFAOYSA-N 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 206010036601 premature menopause Diseases 0.000 description 1
- 208000017942 premature ovarian failure 1 Diseases 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- -1 pseudouracil Chemical compound 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000004927 skin cell Anatomy 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000002381 testicular Effects 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 229940072172 tetracycline antibiotic Drugs 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 238000011121 vaginal smear Methods 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- 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
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—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)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- General Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Toxicology (AREA)
- General Engineering & Computer Science (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Marine Sciences & Fisheries (AREA)
- Immunology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Cosmetics (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The reprogramming factor anti-aging expression system, the biological material and the application provided by the embodiment of the application comprise a nucleic acid molecule for encoding an Oct4 transcription factor, a nucleic acid molecule for encoding a Sox2 transcription factor, a nucleic acid molecule for encoding a Klf4 transcription factor, a nucleic acid molecule for encoding a Glis1 transcription factor and a nucleic acid molecule for encoding a Lin28 transcription factor; through the mode, the OSKGL system (Oct 4, sox2, klf4, glis1 and Lin 28) has a good epigenetic reprogramming effect, can promote skin, cartilage, heart, ovary, testis and kidney cells to start epigenetic reprogramming, reverse aging, improve metabolism, enhance regeneration capacity, reverse organ fibrosis and generate no cytotoxicity.
Description
Technical Field
The application relates to the technical field of biological medicine, in particular to a reprogramming factor anti-aging expression system, a biological material and application.
Background
The epigenetic information loss is one of main driving forces for aging occurrence, development and prognosis, and researches prove that reprogramming factors in cells can recover the lost epigenetic information through epigenetic reprogramming, so that the aging of organs such as skin, heart, kidney, brain, osteoporosis, ovary, testis and the like is partially reversed, the capacity of repairing organ injury is improved, the service life of experimental animals is prolonged, and the method is a major conceptual breakthrough of biology.
In the background technology, in vivo and in vitro transient expression maintains the multipotent transcription factor, and the lost epigenetic information can be recovered through reprogramming of the epigenetic part, so that the service life of experimental animals is prolonged. However, the transient expression of the pluripotent transcription factor in the background art still has the problem of cytotoxicity and the problem of being unable to drive the initiation of epigenetic reprogramming of heart or chondrocytes.
Disclosure of Invention
In view of the above problems, embodiments of the present application provide a reprogramming factor anti-aging expression system, a biomaterial and use thereof, so as to solve the above technical problems.
In a first aspect, embodiments of the present application provide a reprogramming factor anti-aging expression system comprising a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Sox2 transcription factor, a nucleic acid molecule encoding a Klf4 transcription factor, a nucleic acid molecule encoding a Glis1 transcription factor, and a nucleic acid molecule encoding a Lin28 transcription factor.
Alternatively, the expression system is a recombinant vector having puromycin resistance.
Alternatively, the recombinant vector comprises a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a P2A peptide, a nucleic acid molecule encoding a Glis a transcription factor, a nucleic acid molecule encoding an F2A peptide, a nucleic acid molecule encoding a Klf4 transcription factor, a nucleic acid molecule encoding a T2A peptide, a nucleic acid molecule encoding a Lin28 transcription factor, a nucleic acid molecule encoding an E2A peptide, and a nucleic acid molecule encoding a Sox2 transcription factor, which are sequentially connected.
Optionally, the recombinant vector comprises piggybac transposons.
Alternatively, the nucleotide sequence of the recombinant vector is shown as SEQ ID NO. 1.
Alternatively, the expression system is an adeno-associated viral expression vector or a lentiviral expression vector.
Alternatively, the expression system comprises an expression cassette comprising a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Sox2 transcription factor, a nucleic acid molecule encoding a Klf4 transcription factor, a nucleic acid molecule encoding a Glis1 transcription factor, and a nucleic acid molecule encoding a Lin28 transcription factor.
Alternatively, the expression system includes an mRNA encoding an Oct4 transcription factor, an mRNA encoding a Sox2 transcription factor, an mRNA encoding a Klf4 transcription factor, an mRNA encoding a Glis1 transcription factor, and an mRNA encoding a Lin28 transcription factor.
In a second aspect, embodiments of the present application provide a biological material that is a host cell comprising the reprogramming factor anti-aging expression system described above.
In a third aspect, embodiments of the present application provide an application of the reprogramming factor anti-aging expression system or the biomaterial in preparing a cell part cell reprogramming reagent, an application in preparing a preparation for improving metabolic disorder, an application in preparing a repairing agent after organ injury, or an application in preparing an anti-aging drug.
The reprogramming factor anti-aging expression system, the biological material and the application provided by the embodiment of the application comprise a nucleic acid molecule for encoding an Oct4 transcription factor, a nucleic acid molecule for encoding a Sox2 transcription factor, a nucleic acid molecule for encoding a Klf4 transcription factor, a nucleic acid molecule for encoding a Glis1 transcription factor and a nucleic acid molecule for encoding a Lin28 transcription factor; through the mode, the OSKGL system (Oct 4, sox2, klf4, glis1 and Lin 28) has a good epigenetic reprogramming effect, can promote tissues and cells such as skin, cartilage, heart, ovary, testis and kidney, liver, lung and the like to start epigenetic reprogramming, improves regeneration capacity, provides repair capacity after loss, and does not complete cell reprogramming to generate cytotoxicity.
These and other aspects of the application will be more readily apparent from the following description of the embodiments.
Drawings
Fig. 1 shows a schematic structural diagram of a reprogramming factor anti-aging expression system provided by an embodiment of the present application.
FIG. 2 is a photograph showing human fibroblasts transfected in application example 1 of the present application.
Fig. 3 shows a comparative graph of the results of application example 2, comparative example 1 and comparative example 2 of the present application.
FIG. 4 shows a comparative graph of the results of application example 3, comparative example 9 and comparative example 10 of the present application;
FIG. 5 is a graph showing the comparison of the results of application example 4 of the present application;
FIG. 6 is a graph showing the comparison of the results of application example 5 of the present application;
FIG. 7 is a graph showing the comparison of the results of application example 6 of the present application;
FIG. 8 is a graph showing the comparison of the results of application example 7 of the present application;
FIG. 9 is a graph showing the comparison of the results of application example 8 of the present application;
FIG. 10 shows a schematic diagram of the structure of a first adenovirus expression vector in a reprogramming factor anti-aging expression system provided by an embodiment of the application;
FIG. 11 shows a schematic diagram of the structure of a second adenovirus expression vector in a reprogramming factor anti-aging expression system provided by an embodiment of the application;
fig. 12 shows a schematic structural diagram of a third adenovirus expression vector in a reprogramming factor anti-aging expression system according to an embodiment of the present application.
Detailed Description
The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment may be included in at least one embodiment of the application. The appearances of such phrases in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. Those of skill in the art will explicitly and implicitly appreciate that the embodiments described herein may be combined with other embodiments.
The experimental methods in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
An embodiment of the application provides a reprogramming factor anti-aging expression system comprising a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Sox2 transcription factor, a nucleic acid molecule encoding a Klf4 transcription factor, a nucleic acid molecule encoding a Glis1 transcription factor, and a nucleic acid molecule encoding a Lin28 transcription factor.
In this example, the OSKGL system (Oct 4, sox2, klf4, glis1, and Lin 28) formed has a good epigenetic reprogramming effect, and can push skin, cartilage, heart, ovary, testis, and kidney cells to initiate epigenetic reprogramming, reverse aging, improve metabolism, enhance regeneration capacity, reverse organ fibrosis, and not generate cytotoxicity.
As one embodiment, each of the nucleic acid molecules described above may be DNA. The expression system comprises a DNA nucleic acid molecule encoding an Oct4 transcription factor, a DNA nucleic acid molecule encoding a Sox2 transcription factor, a DNA nucleic acid molecule encoding a Klf4 transcription factor, a DNA nucleic acid molecule encoding a Glis1 transcription factor, and a DNA nucleic acid molecule encoding a Lin28 transcription factor.
As one embodiment, each of the nucleic acid molecules described above may be RNA. The expression system includes an RNA nucleic acid molecule encoding an Oct4 transcription factor, an RNA nucleic acid molecule encoding a Sox2 transcription factor, an RNA nucleic acid molecule encoding a Klf4 transcription factor, an RNA nucleic acid molecule encoding a Glis1 transcription factor, and an RNA nucleic acid molecule encoding a Lin28 transcription factor.
As one embodiment, the expression system may be an expression cassette. In this embodiment, the expression cassette contains a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Sox2 transcription factor, a nucleic acid molecule encoding a Klf4 transcription factor, a nucleic acid molecule encoding a Glis1 transcription factor, and a nucleic acid molecule encoding a Lin28 transcription factor.
As one embodiment, the expression system may be an adeno-associated viral expression vector or a lentiviral expression vector.
As one embodiment, the expression level of the Oct4 transcription factor is greater than twice the expression level of the Sox2 transcription factor, the expression level of the Oct4 transcription factor is greater than twice the expression level of the Klf4 transcription factor, the expression level of the Oct4 transcription factor is greater than twice the expression level of the Glis1 transcription factor, and the expression level of the Oct4 transcription factor is greater than twice the expression level of the Lin28 transcription factor. In this embodiment, the expression level of each target transcription factor is controlled within the above range, which is advantageous for further improving the partial reprogramming effect.
In some embodiments, the expression system includes a first adenoviral expression vector and a second adenoviral expression vector. Wherein the first adenovirus expression vector comprises a DNA nucleic acid molecule encoding an Oct4 transcription factor, a DNA nucleic acid molecule encoding a Sox2 transcription factor, and a DNA nucleic acid molecule encoding a Klf4 transcription factor, and the second adenovirus expression vector comprises a DNA nucleic acid molecule encoding an Oct4 transcription factor, a DNA nucleic acid molecule encoding a Glis1 transcription factor, and a DNA nucleic acid molecule encoding a Lin28 transcription factor.
In some embodiments, the first adenovirus expression vector may be an AAV9 viral DNA vector, the structure of which is shown in fig. 10, the first adenovirus expression vector may be a recombinant plasmid, and the nucleotide sequence of the first adenovirus expression vector is shown in SEQ ID No. 2. Of the nucleotide sequences shown in SEQ ID No.2, nucleotide sequences encoding Oct4 transcription factors are at positions 454 to 1533, nucleotide sequences encoding Sox2 transcription factors are at positions 1591 to 2541, and nucleotide sequences encoding Klf4 transcription factors are at positions 2596 to 4008.
In some embodiments, the second adenovirus expression vector may be an AAV9 viral DNA vector, the structure of which is shown in fig. 11, the first adenovirus expression vector may be a recombinant plasmid, and the nucleotide sequence of the second adenovirus expression vector is shown in SEQ ID No. 3. Of the nucleotide sequences shown in SEQ ID No.3, nucleotide sequences encoding Oct4 transcription factor are at positions 454 to 1533, nucleotide sequence encoding Glis.sup.1 transcription factor is at positions 1591 to 3450, and nucleotide sequence encoding lin28 transcription factor is at positions 3505 to 4134.
In some embodiments, the expression system includes, in addition to the first and second adenoviral expression vectors, a third adenoviral expression vector comprising a DNA nucleic acid molecule encoding a regulatory protein for regulating expression of each of the above-described transcription factors of interest and for controlling the expression profile of each of the transcription factors of interest. The regulatory protein may be, for example, an antibiotic-inducible regulatory protein, and in particular, an rtTA (REVERSE TETRACYCLINE-controlled transactivator) protein, which is not normally bound to a tetO site, and when the rtTA protein is bound to a tetracycline antibiotic or doxycycline, the conformation of the rtTA protein changes so that it binds to the tetO site, and a complex formed after the tetO site binds to the tTA activates a nearby promoter region, thereby promoting transcription of the gene encoding the target transcription factor. For example, the regulatory protein is Tet-3G, which can be induced by doxycycline. Simultaneously, the first adenovirus vector and the second adenovirus vector respectively express OCT4 transcription factors, so that the expression quantity of the Oct4 transcription factors is more than 2 times of that of other target transcription factors, and the effect of the application is further improved.
In some embodiments, the third adenovirus expression vector may be an AAV9 viral DNA vector, the structure of which is shown in fig. 12, the third adenovirus expression vector may be a recombinant plasmid, and the nucleotide sequence of the third adenovirus expression vector is shown in SEQ ID No. 4. In the nucleotide sequence shown as SEQ ID NO.4, positions 739 to 1482 encode a regulatory protein (Tet-3G) Is a nucleotide sequence of (a). As an embodiment, the expression system may be a recombinant vector.
In some embodiments, the expression system is a recombinant vector having puromycin resistance.
In some embodiments, the recombinant vector comprises, in sequence, a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a P2A peptide, a nucleic acid molecule encoding a Glis a transcription factor, a nucleic acid molecule encoding an F2A peptide, a nucleic acid molecule encoding a Klf4 transcription factor, a nucleic acid molecule encoding a T2A peptide, a nucleic acid molecule encoding a Lin28 transcription factor, a nucleic acid molecule encoding an E2A peptide, and a nucleic acid molecule encoding a Sox2 transcription factor. Among these, the P2A peptide, T2A peptide, E2A peptide and F2A peptide are each 2A peptide, specifically, 2A peptide is a short peptide (generally 18 to 25 amino acids) derived from a virus, which is commonly called "self-cleaving" peptide, and enables one transcript to produce various proteins. The 2A peptide does not completely "self-cleave" but rather works by allowing the ribosome to skip synthesis of glycine and proline peptide bonds at the C-terminus of the 2A element, ultimately resulting in separation of the 2A sequence end and downstream products. Wherein, the C-terminal of the upstream protein will add some additional 2A residues, while the N-terminal of the downstream protein will have additional proline. The P2A peptide, T2A peptide, E2A peptide and F2A peptide are derived from four different viruses, respectively.
In some embodiments, the recombinant vector is a doxycycline-inducible transposon, e.g., the recombinant vector may be optimized for piggybac transposons.
In some embodiments, the structure of the recombinant vector is shown in FIG. 1, the nucleotide sequence of the recombinant vector is shown in SEQ ID NO.1, and the recombinant vector may be a recombinant plasmid. Of the nucleotide sequences shown in SEQ ID No.1, nucleotide sequences encoding Oct4 transcription factors are located at positions 1772 to 2851, nucleotide sequences encoding Glis1 transcription factors are located at positions 2909 to 4772, nucleotide sequences encoding Klf4 transcription factors are located at positions 4839 to 6248, nucleotide sequences encoding Lin28 transcription factors are located at positions 6303 to 6929, and nucleotide sequences encoding Sox2 transcription factors are located at positions 6990 to 7943.
In this embodiment, the recombinant vector may be constructed according to the Gibson method, and the Gibson enzyme is used to join the recombinant vector to form the target vector sequence shown in FIG. 1, which is characterized by having puromycin resistance, and being capable of expressing the Oct4 transcription factor, sox2 transcription factor, klf4 transcription factor, glis transcription factor and lin28 transcription factor simultaneously under the induction of doxycycline.
As one embodiment, the expression system includes mRNA encoding Oct4 transcription factor, sox2 transcription factor, klf4 transcription factor, glis1 transcription factor, and Lin28 transcription factor.
In some embodiments, some or all of the nucleotides in the mRNA encoding the Oct4 transcription factor, the mRNA encoding the Sox2 transcription factor, the mRNA encoding the Klf4 transcription factor, the mRNA encoding the Glis1 transcription factor, and the mRNA encoding the Lin28 transcription factor are chemically modified to increase the stability of the mRNA in vivo.
In some embodiments, the ratio of the molar amount of the mRNA encoding Oct4 transcription factor to the sum of the molar amounts of the mRNA encoding Sox2 transcription factor, the mRNA encoding Klf4 transcription factor, the mRNA encoding Glis1 transcription factor, and the mRNA encoding Lin28 transcription factor is1 to 8:1.
In some embodiments, the chemical modification comprises replacing 100% of uracil in the mRNA encoding the Oct4 transcription factor, the mRNA encoding Sox2 transcription factor, the mRNA encoding Klf4 transcription factor, the mRNA encoding Glis transcription factor, and the mRNA encoding Lin28 transcription factor with N1-methyl pseudouridine, pseudouracil, or methyl uracil, and replacing 100% of cytosine in the mRNA encoding Oct4 transcription factor, the mRNA encoding Sox2 transcription factor, the mRNA encoding Klf4 transcription factor, the mRNA encoding Glis1 transcription factor, and the mRNA encoding Lin28 transcription factor with 5-methylcytosine or 5-hydroxymethylcytosine.
In some embodiments, the reprogramming factor anti-aging expression system of the present example further comprises: and a third mRNA encoding RNA dependent RNA polymerase (RdRp), wherein the molar amount of the third mRNA is1 to 8 times of the sum of the molar amounts of the mRNA encoding the Oct4 transcription factor, the mRNA encoding the Sox2 transcription factor, the mRNA encoding the Klf4 transcription factor, the mRNA encoding the Glis transcription factor and the mRNA encoding the Lin28 transcription factor.
In this embodiment, the replication of each transcription factor can be achieved by an RNA-dependent RNA polymerase encoded by the third mRNA.
In some embodiments, the RNA-dependent RNA polymerase encoded by the third mRNA is an alphavirus mutant replicase that produces a mutation at position 259 of the nsP2 region and a mutation at position 650 of the nsP2 region. Specifically, the mutant replicase comprises a nsP1 region (537 amino acids), a nsP2 region (799 amino acids), a nsP3 region (482 amino acids) and a nsP4 region (1254 amino acids) which are sequentially connected, the amino acid sequence of the mutant replicase is shown as SEQ ID NO.2, and two mutation points of the mutant replicase respectively generate 796 sites shown as SEQ ID NO.2 (serine S is mutated to proline P) and 1187 sites shown as SEQ ID NO.2 (arginine R is mutated to aspartic acid D).
In this embodiment, the activity of the alphavirus replicase is reduced by specific mutation of the alphavirus replicase, and limited self-replication of each transcription factor can be achieved by the RNA-dependent RNA polymerase encoded by the third mRNA, thereby avoiding cytotoxicity.
An embodiment of the present application provides a biological material that is a host cell comprising the reprogramming factor anti-aging expression system described above.
As an embodiment, the host cell may be one of a skin cell, a cartilage cell, a heart cell, an ovarian cell, a testicular cell, or a kidney cell, and the expression system may effect a partial reversal of host cell senescence.
An embodiment of the present application provides the use of the reprogramming factor anti-aging expression system or the biomaterial in preparation of a cell reprogramming agent, the use of the reprogramming factor anti-aging expression system or the biomaterial in preparation of a repair agent after organ injury, or the use of the reprogramming factor anti-aging expression system or the biomaterial in preparation of an anti-aging drug.
Example 1: preparation of the plasmid of interest
In this example, the recombinant vector is a target plasmid, which is a doxycycline-induced transposon-type plasmid; the prepared target plasmid has puromycin resistance, and can simultaneously express a transcription factor Oct4 transcription factor, a Sox2 transcription factor, a Klf4 transcription factor, a Glis1 transcription factor and a lin28 transcription factor under the induction of doxycycline. The target plasmid can be prepared according to the following steps:
S11, obtaining target DNA fragments: the target DNA fragment sequences may be obtained from plasmid products or other sources, i.e., broken down into different DNA sequences according to the specific sequences shown in FIG. 1, and ordered directly from commercial companies (e.g., IDT company).
S12, designing a primer: the primer is designed for the target DNA fragment, the end of the primer needs to be added with a sequence matched with the target plasmid, and the length of the primer is generally 30-40 bp.
S13, PCR amplification: and (3) carrying out PCR amplification on the target DNA fragment by using the designed primer, wherein the PCR conditions are adjusted according to the size of the target DNA fragment and the design of the primer.
S14, purifying a PCR product: the PCR products were purified using commercial PCR purification kits to remove other components.
S15, measuring DNA concentration: the DNA concentration of the PCR product was measured using Nanodrop et al.
S16, linearizing plasmids: the empty target plasmid was subjected to restriction enzyme digestion and linearized.
S17, adding Gibson homologous recombinase: the mixture containing the linear empty target plasmid and each target DNA fragment is added to the homologous recombinase, which automatically recombines each target DNA fragment into the complete plasmid in vitro.
S18, reaction conditions: the reaction temperature of the Gibson assembly reaction enzyme is 50 ℃ and the reaction time is 1-2 hours.
S19, transformed cells: the Gibson assembled reaction product is transformed into appropriate cells, such as E.coli, and cultured.
S110, screening positive clones: the transformed colonies were screened to find positive clones containing the plasmid of interest.
In this embodiment, the target plasmid is the recombinant vector shown in FIG. 1, and the nucleotide sequence of the target plasmid is shown as SEQ ID NO. 1.
Example 2: preparation of the target adenovirus expression vector
AAV (adeno-associated virus) vectors encoding Oct4 transcription factor, sox2 transcription factor, klf4 transcription factor, glis transcription factor, and Lin28 transcription factor were prepared as target adenovirus expression vectors. Example 3: preparation of target mRNA expression System
Preparation of target mRNA expression System
In this example, the target mRNA expression system includes a first mRNA encoding an Oct4 transcription factor, a second mRNA encoding a Sox2 transcription factor, a second mRNA encoding a Klf4 transcription factor, a second mRNA encoding a Lin28 transcription factor, a second mRNA encoding a Glis1 transcription factor, and a third mRNA encoding an alphavirus mutant replicase.
Wherein the molar amounts of the five second mRNAs are the same, and the ratio of the molar amount of the first mRNAs to the sum of the molar amounts of the five second mRNAs is 2 to 6.
Wherein the molar amount of the third mRNA is1 to 8 times of the sum of the molar amounts of mRNA encoding the Oct4 transcription factor, mRNA encoding the Sox2 transcription factor, mRNA encoding the Klf4 transcription factor, mRNA encoding the Glis1 transcription factor and mRNA encoding the Lin28 transcription factor.
The first mRNA comprises the following elements in the 5 '. Fwdarw.3' direction: a 5'UTR sequence, a signal peptide sequence, an Oct4 transcription factor coding sequence, and a 3' UTR sequence; each second mRNA comprises the following elements in the 5 '. Fwdarw.3' direction: the 5'utr sequence, the reprogramming factor coding sequence, and the 3' utr sequence, each second mRNA further comprises a signal peptide sequence.
The third mRNA can be prepared as follows:
i. Primer design: the first step in DNA template construction is to design primers. The primer should contain a promoter, transcription initiation site and T7RNA polymerase binding sequence. The design of these primers needs to take into account the following factors: promoter: the promoter should be located at the 5' end of the DNA template and be capable of recognition and binding by RNA polymerase.
Transcription initiation site: the transcription initiation site is the location where RNA polymerase begins transcription, and is located downstream of the promoter, typically 20-30 bases in length.
T7 polymerase binding sequence, i.e. 5 'or 3' untranslated region (UTR): the T7 RNA polymerase binding sequence is the site where RNA polymerase binds and begins transcription. This sequence is usually located downstream of the transcription initiation site and is 20-30 bases in length.
PCR amplification: PCR amplification was performed using the designed primers. The desired template DNA, primers, polymerase and nucleotides should be included in the PCR reaction. PCR reaction conditions are optimized according to the characteristics of the primer and template DNA.
Purifying PCR products: the PCR products are separated and purified by agarose gel electrophoresis or other methods. This step can remove impurities and non-amplified DNA in the PCR reaction.
Mutant mRNA replicase mRNA synthesis in vitro: the linearly amplified DNA template was used for in vitro transcription reactions using T7 RNA polymerase.
Removing DNA: DNA in the reaction mixture is removed by DNase or the like to avoid interference with downstream applications.
Purifying mRNA: mRNA was purified using HPLC purification methods.
Example 4: preparation of the expression System of interest (AAV-OSKGL)
The target expression system of this embodiment includes a first adenovirus expression vector, a second adenovirus expression vector, and a third adenovirus expression vector.
The first adenovirus expression vector (AAV-TET-OSK, hereinafter referred to as AAV 1-OSK) is AAV9 virus DNA vector, the structure of the recombinant vector is shown in FIG. 10, the recombinant vector can be recombinant plasmid, and the nucleotide sequence of the recombinant vector is shown as SEQ ID NO. 2. Of the nucleotide sequences shown in SEQ ID No.2, nucleotide sequences encoding Oct4 transcription factors are at positions 454 to 1533, nucleotide sequences encoding Sox2 transcription factors are at positions 1591 to 2541, and nucleotide sequences encoding Klf4 transcription factors are at positions 2596 to 4008.
The second adenovirus expression vector (AAV-TET-OGL, hereinafter referred to as AAV 2-OGL) is AAV9 virus DNA vector, the structure of the recombinant vector is shown in FIG. 11, the recombinant vector can be recombinant plasmid, and the nucleotide sequence of the recombinant vector is shown as SEQ ID NO. 3. Of the nucleotide sequences shown in SEQ ID No.3, nucleotide sequences encoding Oct4 transcription factor are at positions 454 to 1533, nucleotide sequence encoding Glis.sup.1 transcription factor is at positions 1591 to 3450, and nucleotide sequence encoding lin28 transcription factor is at positions 3505 to 4134.
The third adenovirus expression vector (pAAV-EF 1a-rtTA, hereinafter referred to as AAV 3-doxycycline induction switch) is an AAV9 virus DNA vector, the structure of the recombinant vector is shown in FIG. 12, the recombinant vector can be a recombinant plasmid, and the nucleotide sequence of the recombinant vector is shown as SEQ ID NO. 4. In the nucleotide sequence shown as SEQ ID NO.4, positions 739 to 1482 encode a regulatory protein (Tet-3G) Is a nucleotide sequence of (a).
The target expression system of this embodiment can simultaneously express the transcription factors Oct4 transcription factor, sox2 transcription factor, klf4 transcription factor, glis1 transcription factor and lin28 transcription factor under the induction of doxycycline.
Application example 1: target plasmid transfection human fibroblast experiment of example 1
First stage I: transfection of human fibroblasts with the plasmid of interest described above was accomplished using the Siemens Neon TM transfection System
1. Cell culture: the cell line to be transfected is selected and cultured under appropriate culture conditions to achieve the appropriate growth state prior to transfection.
2. Plasmid DNA preparation: the target plasmid to be transfected was dissolved in sterile PBS buffer and stored at room temperature.
3. Cell collection: the cells are collected in a centrifuge tube in a suitable manner. For example, cells are dissociated from the culture plate using digestive enzymes, collected and washed once with sterile PBS buffer to remove enzymes from the cells.
4. Cell count: cells were counted using a microscope and a cell counting plate to determine the number of cells required for transfection.
5. Cell precipitation: cells were pelleted at the bottom of the centrifuge tube, PBS buffer was removed, and appropriate amount of sterile PBS buffer was added to suspend the cells.
6. Transfection parameter settings: the Neon TM transfection system software was turned on, transfection parameters were selected in the menu, and were set according to the cell fibroblast type and the amount of DNA to be transfected.
7. Transfection: the cells and plasmid DNA were mixed and electrotransfected on the Neon TM transfection system according to the transfection parameters. The transfection mixture was transferred to a pre-prepared electrotransfection tube and electrotransfected using the Neon TM transfection system.
8. Cell recovery: the electrotransfection tube was returned to the dish containing the cell culture medium and incubated in a CO 2 incubator at 37℃for the desired period of time. Cells will begin to express transfected genes between hours and days.
9. Cell culture: the stably transfected cell lines are cultured under suitable culture conditions to a density of 80% to 90%.
10. Puromycin treatment: the puromycin solution is added to the medium to achieve the desired final concentration, typically 0.5 to 5. Mu.g/ml.
11. Cell treatment: cells were moved to medium containing puromycin and allowed to contact puromycin sufficiently so that cells not transfected with plasmid were selectively killed.
12. Cell culture: the treated cells were returned to the incubator and cultured under appropriate culture conditions. Cells treated with puromycin will die gradually, while cells transfected with plasmid will continue to survive and grow.
Second stage II: doxycycline induction completes cell reprogramming:
After the puromycin resists cell formation, a proper amount of cells are placed in a human pluripotent stem cell culture solution (15% KOSR+DMEF/F12+10 ng/mlbFGF) with mitomycin C treated Mouse Embryonic Fibroblasts (MEFs) as feeder cells, and cultured for 7-21 days, and the solution is changed every day until typical human pluripotent stem cell clones appear. For example, the culture may be carried out for 7 to 10 days, 14 to 21 days or 7 to 21 days.
Experimental results: the objective plasmid prepared in example 1 forms Oct4 transcription factor, sox2 transcription factor, klf4 transcription factor, glis transcription factor and lin28 transcription factor in human fibroblasts, and doxycycline is induced for 7 to 21 days, successfully forming induced pluripotent stem cells (iPS), as shown in fig. 2.
Application example 2: experiments with target plasmid of example 1 transfected human chondrocytes
First stage I: the above objective plasmid was used to transfect human chondrocytes (using osteoarticular human cartilage primary cells, available from Promocell Corp., cat. No. C-12710) by electroporation, and after three days of selection with Puromycin mg/ml, cell DNA was extracted after 7-10 days of doxycycline addition, and Whole genome bisulfite sequencing (white-Genome Bisulfite Sequencing, WGBS).
Second stage II: WGBS bisulfite (biosulfite) chemical treatment of DNA followed by PCR amplification and sequencing can yield information on methylated and unmethylated C. The following are the detailed steps of WGBS:
1. Bisulphite treatment: DNA is added to a reaction system containing bisulfite, which converts unmethylated C to U, and methylated C is unaffected. This step disrupts the integrity of the DNA and requires repair and purification of the DNA in a later step.
2. End adhesion repair and a tail: the 5 'and 3' ends of the DNA fragments were repaired and A-tailed to facilitate ligation of adaptors in the next step.
3. And (3) joint connection: the DNA fragments were ligated to sequencing adaptors, which contained sequence tags and targeting sequences for subsequent sequencing.
PCR amplification: and (3) carrying out PCR amplification on the DNA connected with the connector, wherein the amplified fragment is the DNA subjected to bisulphite treatment of the DNA to be detected.
5. Purifying and quality testing: and purifying and quality testing the PCR amplified products to confirm the size and concentration of the amplified products.
6. High throughput sequencing: the PCR amplified products were subjected to high throughput sequencing using an Illumina high throughput sequencer. The data obtained by sequencing contains all the C sites in the genome, as well as their methylation status.
7. Data analysis: the data from the sequencing is analyzed using bioinformatics tools, including steps of removing linker sequences, quality control, alignment to the genome, identification and annotation of methylation sites, and the like. Methylation status information for all C sites in genomic DNA can be obtained.
Comparative example 1
Preparing an OSK plasmid:
The OSK plasmid differs from the target plasmid of example 1 in that: the OSK plasmid comprises a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Sox2 transcription factor and a nucleic acid molecule encoding a Klf4 transcription factor, the OSK plasmid does not comprise a nucleic acid molecule encoding a Glis1 transcription factor and a nucleic acid molecule encoding a Lin28 transcription factor, other parts of the OSK plasmid are identical to the target plasmid, and the OSK plasmid can simultaneously express the Oct4 transcription factor, the Sox2 transcription factor and the Klf4 transcription factor.
OSK plasmid transfection of human chondrocytes:
First stage I: the OSK plasmid described above was used to transfect human chondrocytes (using osteoarticular human cartilage primary cells, available from Promocell Corp., cat. No. C-12710) by electroporation, and after three days of selection with Puromycin mg/ml, the cell DNA was extracted 7-10 days after the addition of doxycycline, and Whole genome bisulfite sequencing (white-Genome Bisulfite Sequencing, WGBS).
Second stage II: WGBS bisulfite (biosulfite) chemical treatment of DNA followed by PCR amplification and sequencing can yield information on methylated and unmethylated C. The second stage II is specifically described in application example 1, and will not be described in detail herein.
Comparative example 2
Preparing an empty plasmid:
The empty plasmid differs from the target plasmid of example 1 in that: the empty plasmid does not include a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Sox2 transcription factor, a nucleic acid molecule encoding a Klf4 transcription factor, a nucleic acid molecule encoding a Glis1 transcription factor, and a nucleic acid molecule encoding a Lin28 transcription factor, and the empty plasmid cannot form any of the transcription factors described above, and the other parts of the empty plasmid are identical to the target plasmid.
Air plasmid transfection of human chondrocytes:
First stage I: the above empty plasmid was transfected into human chondrocytes (using osteoarticular human cartilage primary cells, available from Promocell Co., inc. under the trade designation C-12710) by electroporation, and three days after selection with Puromycin mg/ml, cell DNA was extracted after 7 to 10 days of addition of doxycycline, and Whole genome bisulfite was sequenced (white-Genome Bisulfite Sequencing, WGBS).
Second stage II: WGBS bisulfite (biosulfite) chemical treatment of DNA followed by PCR amplification and sequencing can yield information on methylated and unmethylated C. The second stage II is specifically described in application example 1, and will not be described in detail herein.
Experimental results of application example 1, comparative example 1, and comparative example 2:
Referring to FIG. 3, the objective plasmid prepared in example 1 forms Oct4 transcription factor, sox2 transcription factor, klf4 transcription factor, glis transcription factor and lin28 transcription factor in human chondrocytes, doxycycline is induced for 7-10 days, the number of cartilage methylation CpG sites is obviously increased, the apparent genetic age of cartilage is reduced, and the damage repair capability is enhanced, so that osteoarthritis is prevented, in contrast to the apparent genetic age of cartilage which is not reversed by 3 encoding cell multipotent Oct4 transcription factors, sox2 transcription factor and Klf4 transcription factor in comparative example 1; comparative example 2 failed to form the above transcription factor and did not reverse the age of cartilage epigenetic.
Comparative example 3: preparation of OSKM adenovirus expression vectors
AAV (adeno-associated virus) vectors encoding Oct4 transcription factor, sox2 transcription factor, klf4 transcription factor, and c-Myc transcription factor were prepared as OSKM adenovirus expression vectors.
Comparative example 4: preparation of OSK adenovirus expression vector
AAV (adeno-associated virus) viral vectors encoding Oct4 transcription factor, sox2 transcription factor and Klf4 transcription factor were prepared as OSK adenovirus expression vectors.
Comparative example 5: preparation of OSKML adenovirus expression vectors
AAV (adeno-associated virus) vectors encoding Oct4 transcription factor, sox2 transcription factor, klf4 transcription factor, c-Myc transcription factor and lin28 transcription factor were prepared as OSKML adenovirus expression vectors.
Comparative example 6: preparation of OSKGML adenovirus expression vectors
AAV (adeno-associated virus) vectors encoding Oct4 transcription factor, sox2 transcription factor, klf4 transcription factor, glis transcription factor, c-Myc transcription factor and lin28 transcription factor were prepared as OSKGML adenovirus expression vectors.
Comparative example 7: preparation of OSKG adenovirus expression vectors
AAV (adeno-associated virus) vectors encoding Oct4 transcription factor, sox2 transcription factor, klf4 transcription factor, glis transcription factor were prepared as OSKG adenovirus expression vectors.
Comparative example 8: preparation of OSKL adenovirus expression vectors
AAV (adeno-associated virus) vectors encoding Oct4 transcription factor, sox2 transcription factor, klf4 transcription factor, and lin28 transcription factor were prepared as OSKL adenovirus expression vectors.
Toxicology experiments
The target adenovirus expression vector prepared in example 2, OSKM adenovirus expression vector prepared in comparative example 3, and OSK adenovirus expression vector prepared in comparative example 4 were each intravenously injected into mice, and observations were made for 4 weeks, and the results are shown in table 1.
TABLE 1 results of toxicology experiments
As shown in table 1, cMyc expression with other multipotent transcription factors (corresponding to comparative example 3, comparative example 5 and comparative example 6) caused significant toxicity, resulting in death of mice, OSKGL (corresponding to example 2 and example 4), OSK (corresponding to comparative example 4), OSKG (corresponding to comparative example 7) and OSKL (corresponding to comparative example 8) were not significantly toxic.
Application example 3: example 3 experiments on transfection of target mRNA into human ovarian granulosa cells
Human ovary granulosa cell culture: human ovarian granulosa cells were selected from Creatvie Bioarray company under the accession number CSC-I9239L and cultured as described.
MRNA transfection: purified target mRNA (including mutant replicase mRNA, OSKGL) is added to cells, and the mRNA is introduced into the cells using appropriate transfection reagents (e.g., liposomes, polymers, etc.), 1-10 times per 1-2 days of transfection.
Post-transfection methylation analysis and epigenetic age calculation were then performed on the human ovarian granulosa cells partially reprogrammed.
Methylation analysis step:
a. Sample preparation: the OSKMGLmRNA treated cells were collected.
Dna extraction: DNA in the samples was extracted using a commercial DNA extraction kit.
C. methylase treatment: DNA was treated with commercial methylase reagents to distinguish between methylated Cytosine and unmethylated Cytosine.
D. methylation detection: sequencing was performed using the WGBS sequencing method described above.
E. data analysis: the data is entered into the R package in the bioinformatics software. The data analysis comprises quality control, data cleaning, data preprocessing, feature extraction and statistical analysis.
2, Epigenetic age calculation
A. the epigenetic age calculator https:// dnamage. Genetics. Ucla. Edu/home on the methylation data input line was used.
B. The Horvath clock and Hannum clocks are selected.
C. after running the analysis, an epigenetic age was generated.
Comparative example 9: OSK-mRNA expression system
The OSK-mRNA expression system includes a first mRNA encoding an Oct4 transcription factor, a second mRNA encoding a Sox2 transcription factor, a second mRNA encoding a Klf4 transcription factor, and a third mRNA encoding an alphavirus mutant replicase.
Wherein the molar amounts of the two second mRNAs are the same, and the ratio of the molar amount of the first mRNA to the sum of the molar amounts of the two second mRNAs is 2 to 6.
Wherein the molar amount of the third mRNA is 1 to 8 times the sum of the molar amounts of mRNA encoding the Oct4 transcription factor, mRNA encoding the Sox2 transcription factor and mRNA encoding the Klf4 transcription factor.
The first mRNA comprises the following elements in the 5 '. Fwdarw.3' direction: a 5'UTR sequence, a signal peptide sequence, an Oct4 transcription factor coding sequence, and a 3' UTR sequence; each second mRNA comprises the following elements in the 5 '. Fwdarw.3' direction: the 5'utr sequence, the reprogramming factor coding sequence, and the 3' utr sequence, each second mRNA further comprises a signal peptide sequence.
Human ovary granulosa cell culture: human ovarian granulosa cells were selected from Creatvie Bioarray company under the accession number CSC-I9239L and cultured as described.
MRNA transfection: purified OSK-mRNA (including mutant replicase mRNA, OSK) is added to the cells and the mRNA is introduced into the cells using appropriate transfection reagents (e.g., liposomes, polymers, etc.), 1-10 times per 1-2 days.
Methylation analysis and epigenetic age calculation were then performed after partial reprogramming of transfected human ovarian granulosa cells, see in particular application example 3.
Comparative example 10: empty-mRNA expression system
The empty-mRNA expression system includes a third mRNA encoding an alphavirus mutant replicase.
Human ovary granulosa cell culture: human ovarian granulosa cells were selected from Creatvie Bioarray company under the accession number CSC-I9239L and cultured as described.
MRNA transfection: purified empty-mRNA (including mutant replicase mRNA) is added to the cells, and the mRNA is introduced into the cells using appropriate transfection reagents (e.g., liposomes, polymers, etc.), 1-10 times per 1-2 days of transfection.
Methylation analysis and epigenetic age calculation were then performed after partial reprogramming of transfected human ovarian granulosa cells, see in particular application example 3.
Experimental results of application example 3, comparative example 9 and comparative example 10
Referring to FIG. 4, in human ovarian granulosa cells, the target mRNA expression system encodes the cellular multipotent transcription factors Oct4, sox2, klf4, glis1 and Lin28, and the treatment is carried out for 7-10 days, so that the apparent genetic age of the ovaries is obviously reduced, the damage repair capacity of the ovaries is enhanced, and the premature ovarian failure is prevented and treated, and compared with the 3 cellular multipotent transcription factors Oct4, sox2 and Klf4 in comparative example 9, the apparent genetic age of the ovaries is not reversed; comparative example 10 the hollow-mRNA expression system did not reverse the epigenetic age of the ovaries.
Comparative example 11: preparation of AAV-OSK expression System
The expression system of this comparative example includes a first adenoviral expression vector and a third adenoviral expression vector.
The first adenovirus expression vector (AAV-TET-OSK, hereinafter referred to as AAV 1-OSK) is AAV9 virus DNA vector, the structure of the recombinant vector is shown in FIG. 10, the recombinant vector can be recombinant plasmid, and the nucleotide sequence of the recombinant vector is shown as SEQ ID NO. 2. Of the nucleotide sequences shown in SEQ ID No.2, nucleotide sequences encoding Oct4 transcription factors are at positions 454 to 1533, nucleotide sequences encoding Sox2 transcription factors are at positions 1591 to 2541, and nucleotide sequences encoding Klf4 transcription factors are at positions 2596 to 4008.
The third adenovirus expression vector (pAAV-EF 1a-rtTA, hereinafter referred to as AAV 3-doxycycline induction switch) is an AAV9 virus DNA vector, the structure of the recombinant vector is shown in FIG. 12, the recombinant vector can be a recombinant plasmid, and the nucleotide sequence of the recombinant vector is shown as SEQ ID NO. 4. In the nucleotide sequence shown as SEQ ID NO.4, positions 739 to 1482 encode a regulatory protein (Tet-3G) Is a nucleotide sequence of (a).
The expression system of this comparative example was capable of simultaneously expressing the transcription factor Oct4 transcription factor, sox2 transcription factor and Klf4 transcription factor under the induction of doxycycline.
Comparative example 12: preparation of AAV-OGL expression System
The expression system of this comparative example includes a second adenoviral expression vector and a third adenoviral expression vector.
The second adenovirus expression vector (AAV-TET-OGL, hereinafter referred to as AAV 2-OGL) is AAV9 virus DNA vector, the structure of the recombinant vector is shown in FIG. 11, the recombinant vector can be recombinant plasmid, and the nucleotide sequence of the recombinant vector is shown as SEQ ID NO. 3. Of the nucleotide sequences shown in SEQ ID No.3, nucleotide sequences encoding Oct4 transcription factor are at positions 454 to 1533, nucleotide sequence encoding Glis.sup.1 transcription factor is at positions 1591 to 3450, and nucleotide sequence encoding lin28 transcription factor is at positions 3505 to 4134.
The third adenovirus expression vector (pAAV-EF 1a-rtTA, hereinafter referred to as AAV 3-doxycycline induction switch) is an AAV9 virus DNA vector, the structure of the recombinant vector is shown in FIG. 12, the recombinant vector can be a recombinant plasmid, and the nucleotide sequence of the recombinant vector is shown as SEQ ID NO. 4. In the nucleotide sequence shown as SEQ ID NO.4, positions 739 to 1482 encode a regulatory protein (Tet-3G) Is a nucleotide sequence of (a).
The expression system of the comparative example can simultaneously express the transcription factor Oct4 transcription factor, glis transcription factor and lin28 transcription factor under the induction of doxycycline.
Comparative example 13: preparation of AAV-reporter gene expression systems
The expression system of this comparative example includes a DNA nucleic acid molecule encoding GFP protein.
The expression system of this comparative example is an AAV9 viral DNA vector capable of expressing GFP protein.
Application example 4: experimental study for prolonging residual life of aged mice
AAV9 viral DNA vector sequences were delivered to Bio Inc. (SignaGen) to synthesize AAV9 viruses, which gave a first adenovirus expression vector (1.5536E 13 vg/mL, AAV 1-OSK), a second adenovirus expression vector (1.5536E 13 vg/mL, AAV 2-OGL), a third adenovirus expression vector (AAV 3-doxycycline-induced switch) and a reporter gene GFP virus (1.5536E 13 vg/mL, AAV-reporter gene), respectively, all of which were expressed under the induction of doxycycline.
The expression system (AAV OSKGL) of example 4, the expression system (AAV OSK) of comparative example 11, the expression system (AAV OGL) of comparative example 12 and the expression system (AAV reporter gene) of comparative example 13 were prepared, respectively.
In the geriatric mouse model (124 week male C57BL/6J mice, purchased from Jaxson Lab), 4 treatment groups were injected by the retrobulbar intravenous route: group A: injection of the expression system of example 4 (AAV OSKGL), group B: injection of the expression system of comparative example 11 (AAV OSK), group C: injection of the expression system of comparative example 12 (AAV OGL), group D: the expression system of comparative example 13 (AAV reporter gene) was injected.
Wherein, each mouse in group A, group B and group C is injected with 1.5E12 vg (150 μl volume), and each mouse in group D is injected with 150 μl volume.
After injection, group a, group B, group C and group D induced the use of doxycycline by doxycycline for one week and for one week with the target transcription factor gene expression turned off during the study period, and then by doxycycline for one week and for one week with the target transcription factor gene expression turned off, and so on, wherein doxycycline induction was achieved by providing doxycycline-mixed drinking water at a concentration of 2 mg/mL.
All mice were tested for remaining life. Specifically, the aging marker detection and old mice remaining life calculation method is as follows:
After groups A, B, C and D were treated for 8 weeks, the livers of the mice were collected and tested as a kit Genomic DNA extraction and purification kit (NEW ENGLAND Labs, NEB#T 3050) and instructions, liver genomic DNA was extracted and purified, genomic methylation site spectra were scanned using an Illumina RRBS chip, and the senescence biomarkers (epigenetic age) for each mouse in groups A, B, C and D were calculated according to reference PMID24138928 and corresponding calculation method biological age clock (https:// dnamage. Genetics. Ucla. Edu/home).
Referring to fig. 5, group a (example 4) treated 124 weeks old rats showed reduced epigenetic biological age and prolonged residual life in the old rats. Of these, group a (example 4) significantly reduced the epigenetic biological age of geriatric mice, whereas group B (comparative example 11), group C (comparative example 12) had no significant effect in fig. 5. In fig. 5, group B (example 4) showed a significant increase in residual life in geriatric mice, while group B (comparative example 11) and group C (comparative example 12) had no significant effect. * P <0.001.
Experimental results suggest that after 8 weeks of treatment with AAV1+ AAV2+ AAV3 in vivo (group a, OSKGL of example 4), the epigenetic age of mice corresponding to the control group (group B, group C and group D) is significantly reduced and the remaining life is significantly prolonged.
Application example 5: experimental study on reducing epigenetic biological age of high-fat diet mice
AAV9 viral DNA vector sequences were delivered to Bio Inc. (SignaGen) to synthesize AAV9 viruses, which gave a first adenovirus expression vector (1.5536E 13 vg/mL, AAV 1-OSK), a second adenovirus expression vector (1.5536E 13 vg/mL, AAV 2-OGL), a third adenovirus expression vector (AAV 3-doxycycline-induced switch) and a reporter gene GFP virus (1.5536E 13 vg/mL, AAV-reporter gene), respectively, all of which were expressed under the induction of doxycycline.
The expression system (AAV OSKGL) of example 4, the expression system (AAV OSK) of comparative example 11, the expression system (AAV OGL) of comparative example 12 and the expression system (AAV reporter gene) of comparative example 13 were prepared, respectively.
Establishment of high fat diet mice model: male C57BL/6J wild-type (WT, purchased from Jaxson Lab) mice, three to four per cage, were free to feed water. Mice were first switched to the first experimental diet (LabDiet 5053, available from Labdiet corporation) for 4 months; subsequently, mice were subjected to high fat feeding (mice were switched to a high fat AIN-93G diet, i.e., by adding hydrogenated coconut oil to provide 60% calories from fat to establish a high fat diet mouse model); after 5 months of high fat feeding, a high fat diet mouse model was obtained. Livers of high fat diet mice models were collected for senescence biomarker (epigenetic age) detection.
On the high fat diet mouse model, 4 treatment groups were injected by retrobulbar intravenous route: group A: injection of the expression system of example 4 (AAV OSKGL), group B: injection of the expression system of comparative example 11 (AAV OSK), group C: injection of the expression system of comparative example 12 (AAV OGL), group D: the expression system of comparative example 13 (AAV reporter gene) was injected.
Wherein, each mouse in group A, group B and group C is injected with 1.5E12 vg (150 μl volume), and each mouse in group D is injected with 150 μl volume.
After injection, group a, group B, group C and group D induced the use of doxycycline by doxycycline for one week and for one week with the target transcription factor gene expression turned off during the study period, and then by doxycycline for one week and for one week with the target transcription factor gene expression turned off, and so on, wherein doxycycline induction was achieved by providing doxycycline-mixed drinking water at a concentration of 2 mg/mL.
All mice were tested for remaining life. Specifically, the aging marker detection and old mice remaining life calculation method is as follows:
After groups A, B, C and D were treated for 8 weeks, the livers of the mice were collected and tested as a kit Genomic DNA extraction and purification kit (NEW ENGLAND Labs, NEB#T 3050) and instructions, liver genomic DNA was extracted and purified, genomic methylation site spectra were scanned using an Illumina RRBS chip, and the senescence biomarkers (epigenetic age) for each mouse in groups A, B, C and D were calculated according to reference PMID24138928 and corresponding calculation method biological age clock (https:// dnamage. Genetics. Ucla. Edu/home).
Referring to fig. 6, group a (example 4) reduced liver epigenetic student age following a high fat diet. Group a (example 4) significantly reduced liver epigenetic student age after a high fat diet, while group B (comparative example 11), group C (comparative example 12) had no significant effect. * P <0.001.
Application example 6: experimental study for reducing epigenetic biological age of liver fibrosis mice and reversing liver fibrosis of mice
AAV9 viral DNA vector sequences were delivered to Bio Inc. (SignaGen) to synthesize AAV9 viruses, which gave a first adenovirus expression vector (1.5536E 13 vg/mL, AAV 1-OSK), a second adenovirus expression vector (1.5536E 13 vg/mL, AAV 2-OGL), a third adenovirus expression vector (AAV 3-doxycycline-induced switch) and a reporter gene GFP virus (1.5536E 13 vg/mL, AAV-reporter gene), respectively, all of which were expressed under the induction of doxycycline.
The expression system (AAV OSKGL) of example 4, the expression system (AAV OSK) of comparative example 11, the expression system (AAV OGL) of comparative example 12 and the expression system (AAV reporter gene) of comparative example 13 were prepared, respectively.
Construction of liver fibrosis mouse model: c57BL/6 mice at 6-8 weeks were given 1ml kg -1 carbon tetrachloride (CCl 4) by intraperitoneal injection twice a week, 12 consecutive injections, and liver fibrosis was induced.
On the liver fibrosis mouse model, 4 treatment groups were injected by retrobulbar intravenous route: group A: injection of the expression system of example 4 (AAV OSKGL), group B: injection of the expression system of comparative example 11 (AAV OSK), group C: injection of the expression system of comparative example 12 (AAV OGL), group D: the expression system of comparative example 13 (AAV reporter gene) was injected.
Wherein, each mouse in group A, group B and group C is injected with 1.5E12 vg (150 μl volume), and each mouse in group D is injected with 150 μl volume.
After injection, group a, group B, group C and group D induced the use of doxycycline by doxycycline for one week and for one week with the target transcription factor gene expression turned off during the study period, and then by doxycycline for one week and for one week with the target transcription factor gene expression turned off, and so on, wherein doxycycline induction was achieved by providing doxycycline-mixed drinking water at a concentration of 2 mg/mL. Meanwhile, during the above experiments, 1ml kg -1 carbon tetrachloride (CCl 4) was injected intraperitoneally twice a week.
After 8 weeks of treatment with each expression system, after the last cci 4 injection, the livers of mice were collected at 48-72h treatment, and in groups a, B, C and D, all of the livers were collected from part of the mice, and senescence biomarker (epigenetic age) detection was performed (see above step), and part of the mice were stained for liver SA- β -gal. The liver SA- β -gal staining procedure was as follows: SA- β -gal staining was performed as described previously (PMID: 30573629), fresh frozen tissue sections at pH 5.5, fixed with Phosphate Buffered Saline (PBS) containing 0.5% glutaraldehyde for 15min, washed with PBS containing 1mM MgCl 2, and stained in PBS containing 1mM MgCl 2、1mg ml-1 X- β -gal, 5mM potassium ferrocyanide and 5mM potassium ferrocyanide for 5-8h. Tissue sections were eosin stained. Five high power fields per well or section were counted and averaged to quantify the percentage of SA- β -gal positive cells. A group of mouse liver tissue specimens were fixed overnight in 10% formalin, then paraffin-embedded and cut into 5 μm thick sections, sections were hematoxylin-eosin (H & E) stained, and Sirius Red stained for detection of fibrosis, at least three whole sections per mouse were scanned for quantitative analysis of fibrosis, and images were then quantified using NIH ImageJ software. The amount of fibrotic tissue was calculated relative to the total analyzed liver area.
Referring to FIG. 7, FIG. 7A shows that group A (example 4) significantly reduced the percentage of SA- β -gal positive cells in the rat fibrotic liver, while group B (comparative example 11) and group C (comparative example 12) did not significantly affect each other. Group a (example 4) significantly reduced the percentage of red-stained positive cells in murine fibrotic liver Sirus, whereas group B (comparative example 11), group C (comparative example 12) had no significant effect, as shown in fig. 7. * P <0.001.
The experimental results suggest that liver fibrosis of mice is significantly improved after AAV OSKGL weeks of treatment in vivo.
Application example 7: experimental study for reducing epigenetic biological age and preventing ovarian aging of female mice
AAV9 viral DNA vector sequences were delivered to Bio Inc. (SignaGen) to synthesize AAV9 viruses, which gave a first adenovirus expression vector (1.5536E 13 vg/mL, AAV 1-OSK), a second adenovirus expression vector (1.5536E 13 vg/mL, AAV 2-OGL), a third adenovirus expression vector (AAV 3-doxycycline-induced switch) and a reporter gene GFP virus (1.5536E 13 vg/mL, AAV-reporter gene), respectively, all of which were expressed under the induction of doxycycline.
The expression system (AAV OSKGL) of example 4, the expression system (AAV OSK) of comparative example 11, the expression system (AAV OGL) of comparative example 12 and the expression system (AAV reporter gene) of comparative example 13 were prepared, respectively.
Female C57BL/6 mice (purchased from Jaxson Lab) of 27 weeks old and freely fed for one week to adapt to the environment, were injected by retrobulbar intravenous route into 4 treatment groups: group A: injection of the expression system of example 4 (AAV OSKGL), group B: injection of the expression system of comparative example 11 (AAV OSK), group C: injection of the expression system of comparative example 12 (AAV OGL), group D: the expression system of comparative example 13 (AAV reporter gene) was injected.
Wherein, each mouse in group A, group B and group C is injected with 1.5E12 vg (150 μl volume), and each mouse in group D is injected with 150 μl volume.
After injection, group a, group B, group C and group D induced the use of doxycycline by doxycycline for one week and for one week with the target transcription factor gene expression turned off during the study period, and then by doxycycline for one week and for one week with the target transcription factor gene expression turned off, and so on, wherein doxycycline induction was achieved by providing doxycycline-mixed drinking water at a concentration of 2 mg/mL.
During the experiment, vaginal smears were collected at about 9 am each day for 3 months and 14 consecutive days after 6 months feeding, respectively, to assess oestrus cycle status. Vaginal secretions were collected with normal saline, smeared onto glass slides, and HE stained after waiting for drying. Then, we determined the stage of the estrus cycle by cytology under a microscope and calculated the proportion of mice in the normal estrus cycle.
Referring to fig. 8, fig. 8 a shows that group a (example 4) significantly reduced the age of the ovarian epigenetic organism, while group B (comparative example 11), group C (comparative example 12) had no significant effect. In fig. 8, group B (example 4) showed a significant increase in the proportion of normal estrus cycles in female mice after 6 months of treatment, delayed menopause, while group B (comparative example 11) had no significant effect on group C (comparative example 12). * P <0.001.
Experimental results show that AAV OSKGL reduces epigenetic student age and resistance; the ovarian function is weakened.
Application example 8: experimental study for reducing epigenetic biological age of mouse lung and reducing pulmonary fibrosis
AAV9 viral DNA vector sequences were delivered to Bio Inc. (SignaGen) to synthesize AAV9 viruses, which gave a first adenovirus expression vector (1.5536E 13 vg/mL, AAV 1-OSK), a second adenovirus expression vector (1.5536E 13 vg/mL, AAV 2-OGL), a third adenovirus expression vector (AAV 3-doxycycline-induced switch) and a reporter gene GFP virus (1.5536E 13 vg/mL, AAV-reporter gene), respectively, all of which were expressed under the induction of doxycycline.
The expression system (AAV OSKGL) of example 4, the expression system (AAV OSK) of comparative example 11, the expression system (AAV OGL) of comparative example 12 and the expression system (AAV reporter gene) of comparative example 13 were prepared, respectively.
Construction of a pulmonary fibrosis mouse model: pulmonary fibrosis was induced in 6-8 week C57BL/6 mice by direct instillation of bleomycin hydrochloride BAXTER (1 mg/kg) in 50 μl of saline (0.9%) or vehicle [50 μl of saline (0.9%) ] on day 0 and 2 times 4 via tracheal catheter, resulting in a pulmonary fibrosis mouse model.
On the pulmonary fibrosis mouse model, 4 treatment groups were injected by retrobulbar intravenous route: group A: injection of the expression system of example 4 (AAV OSKGL), group B: injection of the expression system of comparative example 11 (AAV OSK), group C: injection of the expression system of comparative example 12 (AAV OGL), group D: the expression system of comparative example 13 (AAV reporter gene) was injected.
Wherein, each mouse in group A, group B and group C is injected with 1.5E12 vg (150 μl volume), and each mouse in group D is injected with 150 μl volume.
After injection, group a, group B, group C and group D induced the use of doxycycline by doxycycline for one week and for one week with the target transcription factor gene expression turned off during the study period, and then by doxycycline for one week and for one week with the target transcription factor gene expression turned off, and so on, wherein doxycycline induction was achieved by providing doxycycline-mixed drinking water at a concentration of 2 mg/mL.
After each expression system treatment, lung was collected from a portion of mice, and senescence biomarkers (epigenetic age) were detected (see step above), and hydroxyproline was biochemically quantified from a portion of mice in groups a, B, C and D. The biochemical quantitative process of hydroxyproline is as follows: the hydroxyproline content in the right lung was determined according to the manufacturer's protocol using a commercial kit from Sigma-Aldrich. Briefly, right lung lobes were homogenized in PBS and a portion was taken to hydrolyze in 6N HCl at 100 ℃ for 24 hours. The hydroxyproline concentration was then determined by the reaction of oxidized hydroxyproline with 4- (dimethylamino) benzaldehyde (DMAB), the resulting colorimetric product (560 nm) being proportional to the hydroxyproline content. The total hydroxyproline of the right lung lobe was calculated from its 560nm colorimetric calculation.
Referring to fig. 9, AAV OSKGL reduces the age of the lung epigenetic organisms and reduces pulmonary fibrosis. In fig. 9, group a (example 4) showed significant reduction in lung epigenetic biological age, while group B (comparative example 11), group C (comparative example 12) had no significant effect. In fig. 9, group B shows that group a (example 4) significantly reduced pulmonary fibrosis and reduced fibrotic pulmonary proline content after 6 months of treatment, while group B (comparative example 11) and group C (comparative example 12) had no significant effect. * P <0.001.
The experimental result shows that after the mice are treated for 8 weeks in vivo by AAV OSKGL, the lung hydroxyproline of the mice is obviously reduced and the fibrosis is obviously improved.
While the application has been described with respect to the above embodiments, it should be noted that modifications can be made by those skilled in the art without departing from the inventive concept, and these are all within the scope of the application.
Claims (10)
1. A reprogramming factor anti-aging expression system, comprising a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Sox2 transcription factor, a nucleic acid molecule encoding a Klf4 transcription factor, a nucleic acid molecule encoding a Glis1 transcription factor, and a nucleic acid molecule encoding a Lin28 transcription factor.
2. The reprogramming factor anti-aging expression system of claim 1, wherein the expression system is a recombinant vector having puromycin resistance.
3. The reprogramming factor anti-aging expression system of claim 2, wherein the recombinant vector comprises, in order, a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a P2A peptide, a nucleic acid molecule encoding a Glis a transcription factor, a nucleic acid molecule encoding an F2A peptide, a nucleic acid molecule encoding a Klf4 transcription factor, a nucleic acid molecule encoding a T2A peptide, a nucleic acid molecule encoding a Lin28 transcription factor, a nucleic acid molecule encoding an E2A peptide, and a nucleic acid molecule encoding a Sox2 transcription factor.
4. The reprogramming factor anti-aging expression system of claim 3, wherein the recombinant vector comprises piggybac transposons.
5. The reprogramming factor anti-aging expression system of claim 3, wherein the nucleotide sequence of the recombinant vector is shown in SEQ ID No. 1.
6. The reprogramming factor anti-aging expression system of claim 1, wherein the expression system is an adeno-associated viral expression vector or a lentiviral expression vector.
7. The reprogramming factor anti-aging expression system of claim 1, wherein the expression system comprises an expression cassette comprising a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Sox2 transcription factor, a nucleic acid molecule encoding a Klf4 transcription factor, a nucleic acid molecule encoding a Glis1 transcription factor, and a nucleic acid molecule encoding a Lin28 transcription factor.
8. The reprogramming factor anti-aging expression system of claim 1, wherein the expression system comprises mRNA encoding Oct4 transcription factor, sox2 transcription factor, klf4 transcription factor, glis1 transcription factor, and Lin28 transcription factor.
9. A biological material, characterized in that it is a host cell comprising the reprogramming factor anti-aging expression system of any one of claims 1 to 8.
10. Use of the reprogramming factor anti-aging expression system of any one of claims 1 to 8 or the biomaterial of claim 9 in the preparation of a cell fraction cell reprogramming agent, in the preparation of a formulation for ameliorating metabolic disorders, in the preparation of a repair agent after organ injury, or in the preparation of an anti-aging drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2024/096096 WO2024213176A1 (en) | 2023-04-14 | 2024-05-29 | Anti-aging expression system of reprogramming factor, biological material and use |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2023104013211 | 2023-04-14 | ||
| CN202310401321.1A CN116426573A (en) | 2023-04-14 | 2023-04-14 | Reprogramming factor anti-aging expression system, biological material and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118374548A true CN118374548A (en) | 2024-07-23 |
Family
ID=87082854
Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310401321.1A Pending CN116426573A (en) | 2023-04-14 | 2023-04-14 | Reprogramming factor anti-aging expression system, biological material and application |
| CN202410440650.1A Pending CN118360252A (en) | 2023-04-14 | 2024-04-12 | Fifth base resetting reagent, preparation method and application thereof |
| CN202410448498.1A Pending CN118374548A (en) | 2023-04-14 | 2024-04-12 | Reprogramming factor anti-aging expression system, biological material and application |
Family Applications Before (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310401321.1A Pending CN116426573A (en) | 2023-04-14 | 2023-04-14 | Reprogramming factor anti-aging expression system, biological material and application |
| CN202410440650.1A Pending CN118360252A (en) | 2023-04-14 | 2024-04-12 | Fifth base resetting reagent, preparation method and application thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (3) | CN116426573A (en) |
| WO (3) | WO2024212336A1 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116474120A (en) * | 2023-04-03 | 2023-07-25 | 臻赫医药(杭州)有限公司 | Reprogramming factor anti-aging mRNA composition, preparation method and application |
| CN116426573A (en) * | 2023-04-14 | 2023-07-14 | 臻赫医药(杭州)有限公司 | Reprogramming factor anti-aging expression system, biological material and application |
| CN116898988A (en) * | 2023-08-15 | 2023-10-20 | 臻赫医药(杭州)有限公司 | Skin reprogramming factor preparation, biological material and application |
| CN116870197A (en) * | 2023-08-15 | 2023-10-13 | 臻赫医药(杭州)有限公司 | Protissue cell proliferation reprogramming factor preparation and application |
Family Cites Families (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SG183315A1 (en) * | 2010-02-16 | 2012-09-27 | Univ Kyoto | Method of efficiently establishing induced pluripotent stem cells |
| WO2013022022A1 (en) * | 2011-08-08 | 2013-02-14 | 国立大学法人京都大学 | Method for efficient establishment of artificial pluripotent stem cells |
| EP3344755A4 (en) * | 2015-08-31 | 2019-05-22 | I Peace, Inc. | Pluripotent stem cell manufacturing system and method for producing induced pluripotent stem cells |
| JP7097050B2 (en) * | 2018-03-19 | 2022-07-07 | 国立大学法人京都大学 | Efficient method for establishing induced pluripotent stem cells |
| WO2020213725A1 (en) * | 2019-04-17 | 2020-10-22 | 学校法人慶應義塾 | Production method and kit of induced pluripotent stem cells |
| JPWO2021251466A1 (en) * | 2020-06-11 | 2021-12-16 | ||
| US20230044997A1 (en) * | 2021-07-15 | 2023-02-09 | Turn Biotechnologies, Inc. | Synthetic, persistent rna constructs and methods of use for cell rejuvenation and for treatment |
| US20230042860A1 (en) * | 2021-07-15 | 2023-02-09 | Turn Biotechnologies, Inc. | Polycistronic expression vectors |
| CN113846113B (en) * | 2021-09-09 | 2022-09-13 | 臻赫医药(杭州)有限公司 | Limited self-replication mRNA molecular system, preparation method and application |
| CN114703143A (en) * | 2022-04-08 | 2022-07-05 | 深圳医爱健康管理有限公司 | Multifunctional intimal stem cell hematopoietic molecular material and method |
| CN116474120A (en) * | 2023-04-03 | 2023-07-25 | 臻赫医药(杭州)有限公司 | Reprogramming factor anti-aging mRNA composition, preparation method and application |
| CN116426573A (en) * | 2023-04-14 | 2023-07-14 | 臻赫医药(杭州)有限公司 | Reprogramming factor anti-aging expression system, biological material and application |
| CN116898988A (en) * | 2023-08-15 | 2023-10-20 | 臻赫医药(杭州)有限公司 | Skin reprogramming factor preparation, biological material and application |
| CN116870197A (en) * | 2023-08-15 | 2023-10-13 | 臻赫医药(杭州)有限公司 | Protissue cell proliferation reprogramming factor preparation and application |
| CN117106726A (en) * | 2023-08-15 | 2023-11-24 | 臻赫医药(杭州)有限公司 | Adoptive immune cell, preparation method and application thereof |
| CN117281925A (en) * | 2023-08-15 | 2023-12-26 | 臻赫医药(杭州)有限公司 | Application of skin reprogramming factor preparation in preparation of skin wound healing promoting medicine |
| CN117045824A (en) * | 2023-09-13 | 2023-11-14 | 臻赫医药(杭州)有限公司 | Reprogramming factor preparation, biological material and application |
-
2023
- 2023-04-14 CN CN202310401321.1A patent/CN116426573A/en active Pending
- 2023-06-09 WO PCT/CN2023/099478 patent/WO2024212336A1/en unknown
-
2024
- 2024-04-12 CN CN202410440650.1A patent/CN118360252A/en active Pending
- 2024-04-12 CN CN202410448498.1A patent/CN118374548A/en active Pending
- 2024-05-29 WO PCT/CN2024/096096 patent/WO2024213176A1/en unknown
- 2024-05-29 WO PCT/CN2024/096095 patent/WO2024213175A1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| WO2024213176A1 (en) | 2024-10-17 |
| CN118360252A (en) | 2024-07-19 |
| WO2024212336A1 (en) | 2024-10-17 |
| CN116426573A (en) | 2023-07-14 |
| WO2024213175A1 (en) | 2024-10-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN118374548A (en) | Reprogramming factor anti-aging expression system, biological material and application | |
| EP3177726B1 (en) | Genome editing for the treatment of huntington's disease | |
| CN110628766B (en) | LncRNA coding gene related to sheep skeletal muscle development and application thereof | |
| CN116096885A (en) | Compositions and methods for targeting C9orf72 | |
| JP2011019525A (en) | NOVEL beta-ACTIN AND RPS21 PROMOTER AND USES THEREOF | |
| Bacman et al. | Manipulation of mitochondrial genes and mtDNA heteroplasmy | |
| EP3974524A1 (en) | Dna vectors, transposons and transposases for eukaryotic genome modification | |
| US11492614B2 (en) | Stem loop RNA mediated transport of mitochondria genome editing molecules (endonucleases) into the mitochondria | |
| EP3712272A1 (en) | Method for modulating rna splicing by inducing base mutation at splice site or base substitution in polypyrimidine region | |
| EP3730616A1 (en) | Split single-base gene editing systems and application thereof | |
| CA3116739A1 (en) | Compositions and methods for treating alpha-1 antitrypsin deficiencey | |
| KR20230129230A (en) | Compositions and methods for targeting BCL11A | |
| EP4282968A1 (en) | Gene transcription framework, vector system, genome sequence editing method and application | |
| US20230212615A1 (en) | Grna targeting ctgf gene and use thereof | |
| KR20230137399A (en) | Functional nucleic acid molecules and methods | |
| WO2024198961A1 (en) | Cas protein and mutant thereof, and corresponding gene editing system and use thereof | |
| Sun et al. | Study on sex-linked region and sex determination candidate gene using a high-quality genome assembly in yellow drum | |
| CN115927331A (en) | DNA framework for promoting circRNA cyclization and overexpression and construction method and application thereof | |
| CN115044583A (en) | RNA framework for gene editing and gene editing method | |
| KR20190115717A (en) | Composition and kit for reducing methylation of target DNA and induction of expression of target gene in animal cell, and method using the same | |
| US20240093185A1 (en) | Circular rnas and preparation methods thereof | |
| WO2024193704A1 (en) | Guide nucleic acids targeting dmd and uses thereof | |
| WO2024041653A1 (en) | Crispr-cas13 system and use thereof | |
| WO2023206088A1 (en) | Rna base editor for treating dmd-associated diseases | |
| CN117165557A (en) | Cas protein, corresponding gene editing system and application 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 |