WO2024012502A1 - Composition and use method therefor - Google Patents
Composition and use method therefor Download PDFInfo
- Publication number
- WO2024012502A1 WO2024012502A1 PCT/CN2023/107076 CN2023107076W WO2024012502A1 WO 2024012502 A1 WO2024012502 A1 WO 2024012502A1 CN 2023107076 W CN2023107076 W CN 2023107076W WO 2024012502 A1 WO2024012502 A1 WO 2024012502A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- subject
- disease
- composition
- oct4
- Prior art date
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 75
- 238000000034 method Methods 0.000 title claims abstract description 37
- 210000000056 organ Anatomy 0.000 claims abstract description 46
- 230000003716 rejuvenation Effects 0.000 claims abstract description 33
- 210000004027 cell Anatomy 0.000 claims description 99
- 210000001519 tissue Anatomy 0.000 claims description 48
- 230000032683 aging Effects 0.000 claims description 39
- 101710126211 POU domain, class 5, transcription factor 1 Proteins 0.000 claims description 32
- 101000687905 Homo sapiens Transcription factor SOX-2 Proteins 0.000 claims description 29
- 102100024270 Transcription factor SOX-2 Human genes 0.000 claims description 28
- 101000886596 Homo sapiens Geminin Proteins 0.000 claims description 27
- 101000652332 Homo sapiens Transcription factor SOX-1 Proteins 0.000 claims description 27
- 101000687911 Homo sapiens Transcription factor SOX-3 Proteins 0.000 claims description 27
- 102100039956 Geminin Human genes 0.000 claims description 26
- 102100030248 Transcription factor SOX-1 Human genes 0.000 claims description 26
- 102100024276 Transcription factor SOX-3 Human genes 0.000 claims description 26
- 108090000144 Human Proteins Proteins 0.000 claims description 25
- 102000003839 Human Proteins Human genes 0.000 claims description 25
- 150000007523 nucleic acids Chemical class 0.000 claims description 24
- 239000013598 vector Substances 0.000 claims description 23
- 102100035423 POU domain, class 5, transcription factor 1 Human genes 0.000 claims description 21
- 201000010099 disease Diseases 0.000 claims description 21
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 21
- 102000039446 nucleic acids Human genes 0.000 claims description 21
- 108020004707 nucleic acids Proteins 0.000 claims description 21
- 241000699670 Mus sp. Species 0.000 claims description 20
- 230000035755 proliferation Effects 0.000 claims description 20
- 210000001612 chondrocyte Anatomy 0.000 claims description 18
- 108020004999 messenger RNA Proteins 0.000 claims description 18
- 102000004169 proteins and genes Human genes 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- 210000001626 skin fibroblast Anatomy 0.000 claims description 14
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 12
- 230000001973 epigenetic effect Effects 0.000 claims description 12
- 239000013604 expression vector Substances 0.000 claims description 10
- 210000001665 muscle stem cell Anatomy 0.000 claims description 10
- 210000002569 neuron Anatomy 0.000 claims description 10
- 241000283690 Bos taurus Species 0.000 claims description 9
- 241000282693 Cercopithecidae Species 0.000 claims description 9
- 241000283073 Equus caballus Species 0.000 claims description 9
- 241000700159 Rattus Species 0.000 claims description 9
- 241000287828 Gallus gallus Species 0.000 claims description 8
- 241000124008 Mammalia Species 0.000 claims description 8
- 241000272525 Anas platyrhynchos Species 0.000 claims description 7
- 241000272814 Anser sp. Species 0.000 claims description 7
- 241000282898 Sus scrofa Species 0.000 claims description 7
- 230000006378 damage Effects 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 7
- 208000027418 Wounds and injury Diseases 0.000 claims description 6
- 208000014674 injury Diseases 0.000 claims description 6
- 230000001737 promoting effect Effects 0.000 claims description 6
- 230000008929 regeneration Effects 0.000 claims description 6
- 238000011069 regeneration method Methods 0.000 claims description 6
- 208000024891 symptom Diseases 0.000 claims description 6
- 241000282326 Felis catus Species 0.000 claims description 5
- 241001494479 Pecora Species 0.000 claims description 5
- 230000035899 viability Effects 0.000 claims description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 4
- 208000024827 Alzheimer disease Diseases 0.000 claims description 4
- 208000024172 Cardiovascular disease Diseases 0.000 claims description 4
- 108020004414 DNA Proteins 0.000 claims description 4
- 201000005569 Gout Diseases 0.000 claims description 4
- 206010019280 Heart failures Diseases 0.000 claims description 4
- 208000023178 Musculoskeletal disease Diseases 0.000 claims description 4
- 201000009859 Osteochondrosis Diseases 0.000 claims description 4
- 210000000988 bone and bone Anatomy 0.000 claims description 4
- 208000030533 eye disease Diseases 0.000 claims description 4
- 230000001771 impaired effect Effects 0.000 claims description 4
- 208000027866 inflammatory disease Diseases 0.000 claims description 4
- 208000030159 metabolic disease Diseases 0.000 claims description 4
- 210000003205 muscle Anatomy 0.000 claims description 4
- 210000004165 myocardium Anatomy 0.000 claims description 4
- 230000004770 neurodegeneration Effects 0.000 claims description 4
- 208000015122 neurodegenerative disease Diseases 0.000 claims description 4
- 201000008482 osteoarthritis Diseases 0.000 claims description 4
- 210000000496 pancreas Anatomy 0.000 claims description 4
- 210000004994 reproductive system Anatomy 0.000 claims description 4
- 208000001072 type 2 diabetes mellitus Diseases 0.000 claims description 4
- 230000037303 wrinkles Effects 0.000 claims description 4
- 241000701022 Cytomegalovirus Species 0.000 claims description 3
- 241000702421 Dependoparvovirus Species 0.000 claims description 3
- 241000711408 Murine respirovirus Species 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- 241000700618 Vaccinia virus Species 0.000 claims description 3
- 210000003050 axon Anatomy 0.000 claims description 3
- 210000004204 blood vessel Anatomy 0.000 claims description 3
- 210000004556 brain Anatomy 0.000 claims description 3
- 210000000481 breast Anatomy 0.000 claims description 3
- 210000003555 cloaca Anatomy 0.000 claims description 3
- 150000001875 compounds Chemical group 0.000 claims description 3
- 210000005069 ears Anatomy 0.000 claims description 3
- 210000003238 esophagus Anatomy 0.000 claims description 3
- 210000001508 eye Anatomy 0.000 claims description 3
- 210000002950 fibroblast Anatomy 0.000 claims description 3
- 102000037865 fusion proteins Human genes 0.000 claims description 3
- 108020001507 fusion proteins Proteins 0.000 claims description 3
- 210000000936 intestine Anatomy 0.000 claims description 3
- 210000003734 kidney Anatomy 0.000 claims description 3
- 210000004185 liver Anatomy 0.000 claims description 3
- 210000004072 lung Anatomy 0.000 claims description 3
- 210000000214 mouth Anatomy 0.000 claims description 3
- 208000010125 myocardial infarction Diseases 0.000 claims description 3
- 210000001331 nose Anatomy 0.000 claims description 3
- 210000001672 ovary Anatomy 0.000 claims description 3
- 210000003899 penis Anatomy 0.000 claims description 3
- 239000013600 plasmid vector Substances 0.000 claims description 3
- 210000002307 prostate Anatomy 0.000 claims description 3
- 210000002784 stomach Anatomy 0.000 claims description 3
- 210000001550 testis Anatomy 0.000 claims description 3
- 230000002103 transcriptional effect Effects 0.000 claims description 3
- 241000701161 unidentified adenovirus Species 0.000 claims description 3
- 239000013603 viral vector Substances 0.000 claims description 3
- 206010002383 Angina Pectoris Diseases 0.000 claims description 2
- 206010003210 Arteriosclerosis Diseases 0.000 claims description 2
- 201000001320 Atherosclerosis Diseases 0.000 claims description 2
- 206010003671 Atrioventricular Block Diseases 0.000 claims description 2
- 206010007559 Cardiac failure congestive Diseases 0.000 claims description 2
- 208000002177 Cataract Diseases 0.000 claims description 2
- 206010012689 Diabetic retinopathy Diseases 0.000 claims description 2
- 208000010228 Erectile Dysfunction Diseases 0.000 claims description 2
- 208000010412 Glaucoma Diseases 0.000 claims description 2
- 206010018634 Gouty Arthritis Diseases 0.000 claims description 2
- 208000030836 Hashimoto thyroiditis Diseases 0.000 claims description 2
- 208000023105 Huntington disease Diseases 0.000 claims description 2
- 206010060378 Hyperinsulinaemia Diseases 0.000 claims description 2
- 206010020772 Hypertension Diseases 0.000 claims description 2
- 208000001953 Hypotension Diseases 0.000 claims description 2
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims description 2
- 206010022489 Insulin Resistance Diseases 0.000 claims description 2
- 208000010038 Ischemic Optic Neuropathy Diseases 0.000 claims description 2
- 201000005505 Measles Diseases 0.000 claims description 2
- 206010027145 Melanocytic naevus Diseases 0.000 claims description 2
- 208000029725 Metabolic bone disease Diseases 0.000 claims description 2
- 206010028372 Muscular weakness Diseases 0.000 claims description 2
- 208000009525 Myocarditis Diseases 0.000 claims description 2
- 201000009053 Neurodermatitis Diseases 0.000 claims description 2
- 208000007256 Nevus Diseases 0.000 claims description 2
- 208000008589 Obesity Diseases 0.000 claims description 2
- 206010030924 Optic ischaemic neuropathy Diseases 0.000 claims description 2
- 208000030768 Optic nerve injury Diseases 0.000 claims description 2
- 206010061323 Optic neuropathy Diseases 0.000 claims description 2
- 208000002804 Osteochondritis Diseases 0.000 claims description 2
- 206010031264 Osteonecrosis Diseases 0.000 claims description 2
- 206010049088 Osteopenia Diseases 0.000 claims description 2
- 208000001132 Osteoporosis Diseases 0.000 claims description 2
- 208000018737 Parkinson disease Diseases 0.000 claims description 2
- 206010035664 Pneumonia Diseases 0.000 claims description 2
- 208000007048 Polymyalgia Rheumatica Diseases 0.000 claims description 2
- 208000002500 Primary Ovarian Insufficiency Diseases 0.000 claims description 2
- 201000007737 Retinal degeneration Diseases 0.000 claims description 2
- 206010038848 Retinal detachment Diseases 0.000 claims description 2
- 206010039796 Seborrhoeic keratosis Diseases 0.000 claims description 2
- 208000006011 Stroke Diseases 0.000 claims description 2
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 claims description 2
- 208000034699 Vitreous floaters Diseases 0.000 claims description 2
- 206010003119 arrhythmia Diseases 0.000 claims description 2
- 230000006793 arrhythmia Effects 0.000 claims description 2
- 208000011775 arteriosclerosis disease Diseases 0.000 claims description 2
- 206010003246 arthritis Diseases 0.000 claims description 2
- 201000004709 chorioretinitis Diseases 0.000 claims description 2
- 208000029078 coronary artery disease Diseases 0.000 claims description 2
- 206010014599 encephalitis Diseases 0.000 claims description 2
- 230000002708 enhancing effect Effects 0.000 claims description 2
- 208000019622 heart disease Diseases 0.000 claims description 2
- 208000006454 hepatitis Diseases 0.000 claims description 2
- 231100000283 hepatitis Toxicity 0.000 claims description 2
- 201000001421 hyperglycemia Diseases 0.000 claims description 2
- 230000003451 hyperinsulinaemic effect Effects 0.000 claims description 2
- 201000008980 hyperinsulinism Diseases 0.000 claims description 2
- 230000036543 hypotension Effects 0.000 claims description 2
- 201000001881 impotence Diseases 0.000 claims description 2
- 230000002757 inflammatory effect Effects 0.000 claims description 2
- 208000002780 macular degeneration Diseases 0.000 claims description 2
- 201000006938 muscular dystrophy Diseases 0.000 claims description 2
- 230000036473 myasthenia Effects 0.000 claims description 2
- 206010028417 myasthenia gravis Diseases 0.000 claims description 2
- 201000002761 non-arteritic anterior ischemic optic neuropathy Diseases 0.000 claims description 2
- 235000020824 obesity Nutrition 0.000 claims description 2
- 208000001749 optic atrophy Diseases 0.000 claims description 2
- 208000020911 optic nerve disease Diseases 0.000 claims description 2
- 206010036601 premature menopause Diseases 0.000 claims description 2
- 208000017942 premature ovarian failure 1 Diseases 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000002062 proliferating effect Effects 0.000 claims description 2
- 230000004258 retinal degeneration Effects 0.000 claims description 2
- 230000004264 retinal detachment Effects 0.000 claims description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 2
- 201000003385 seborrheic keratosis Diseases 0.000 claims description 2
- 201000000596 systemic lupus erythematosus Diseases 0.000 claims description 2
- 201000004647 tinea pedis Diseases 0.000 claims description 2
- 241000282472 Canis lupus familiaris Species 0.000 claims 3
- 241000283086 Equidae Species 0.000 claims 1
- 241000282412 Homo Species 0.000 claims 1
- 206010061218 Inflammation Diseases 0.000 claims 1
- 208000007014 Retinitis pigmentosa Diseases 0.000 claims 1
- 208000034189 Sclerosis Diseases 0.000 claims 1
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 208000016097 disease of metabolism Diseases 0.000 claims 1
- 230000004054 inflammatory process Effects 0.000 claims 1
- 208000017445 musculoskeletal system disease Diseases 0.000 claims 1
- 230000003362 replicative effect Effects 0.000 claims 1
- 230000000241 respiratory effect Effects 0.000 claims 1
- 238000011084 recovery Methods 0.000 abstract 1
- 210000003491 skin Anatomy 0.000 description 10
- 239000002609 medium Substances 0.000 description 8
- 241000282414 Homo sapiens Species 0.000 description 7
- 230000009758 senescence Effects 0.000 description 7
- 230000004069 differentiation Effects 0.000 description 6
- 230000008672 reprogramming Effects 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 5
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 5
- 108091023040 Transcription factor Proteins 0.000 description 5
- 102000040945 Transcription factor Human genes 0.000 description 5
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 5
- 230000001413 cellular effect Effects 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 210000004927 skin cell Anatomy 0.000 description 5
- 241000282465 Canis Species 0.000 description 4
- 241000282324 Felis Species 0.000 description 4
- 206010043276 Teratoma Diseases 0.000 description 4
- 102000005936 beta-Galactosidase Human genes 0.000 description 4
- 108010005774 beta-Galactosidase Proteins 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000011065 in-situ storage Methods 0.000 description 4
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 4
- 210000001082 somatic cell Anatomy 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 230000017423 tissue regeneration Effects 0.000 description 4
- 201000004384 Alopecia Diseases 0.000 description 3
- 102000029816 Collagenase Human genes 0.000 description 3
- 108060005980 Collagenase Proteins 0.000 description 3
- 101001139134 Homo sapiens Krueppel-like factor 4 Proteins 0.000 description 3
- 102100020677 Krueppel-like factor 4 Human genes 0.000 description 3
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 3
- 230000003376 axonal effect Effects 0.000 description 3
- 229960002424 collagenase Drugs 0.000 description 3
- 210000001671 embryonic stem cell Anatomy 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000003102 growth factor Substances 0.000 description 3
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 230000007067 DNA methylation Effects 0.000 description 2
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 2
- 108010022894 Euchromatin Proteins 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 108010034791 Heterochromatin Proteins 0.000 description 2
- 108700021430 Kruppel-Like Factor 4 Proteins 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 241000009328 Perro Species 0.000 description 2
- 101100247004 Rattus norvegicus Qsox1 gene Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 231100000360 alopecia Toxicity 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 230000033115 angiogenesis Effects 0.000 description 2
- 210000001188 articular cartilage Anatomy 0.000 description 2
- 210000002469 basement membrane Anatomy 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 description 2
- 229960005420 etoposide Drugs 0.000 description 2
- 210000000632 euchromatin Anatomy 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 210000004458 heterochromatin Anatomy 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000012758 nuclear staining Methods 0.000 description 2
- 230000033667 organ regeneration Effects 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 102000037983 regulatory factors Human genes 0.000 description 2
- 108091008025 regulatory factors Proteins 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 230000037390 scarring Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 108091035539 telomere Proteins 0.000 description 2
- 102000055501 telomere Human genes 0.000 description 2
- 210000003411 telomere Anatomy 0.000 description 2
- 230000008467 tissue growth Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- CPKVUHPKYQGHMW-UHFFFAOYSA-N 1-ethenylpyrrolidin-2-one;molecular iodine Chemical compound II.C=CN1CCCC1=O CPKVUHPKYQGHMW-UHFFFAOYSA-N 0.000 description 1
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 1
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 1
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 1
- KDXKERNSBIXSRK-RXMQYKEDSA-N D-lysine Chemical compound NCCCC[C@@H](N)C(O)=O KDXKERNSBIXSRK-RXMQYKEDSA-N 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 239000012571 GlutaMAX medium Substances 0.000 description 1
- 101100137155 Homo sapiens POU5F1 gene Proteins 0.000 description 1
- 206010061216 Infarction Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000028389 Nerve injury Diseases 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 229920000153 Povidone-iodine Polymers 0.000 description 1
- 206010068956 Respiratory tract inflammation Diseases 0.000 description 1
- 206010038910 Retinitis Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000002449 bone cell Anatomy 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 210000003321 cartilage cell Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000011748 cell maturation Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000008668 cellular reprogramming Effects 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 201000010251 cutis laxa Diseases 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 102000054910 human GMNN Human genes 0.000 description 1
- 102000044180 human SOX1 Human genes 0.000 description 1
- 102000047444 human SOX2 Human genes 0.000 description 1
- 102000047492 human SOX3 Human genes 0.000 description 1
- 230000007574 infarction Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 210000001087 myotubule Anatomy 0.000 description 1
- 230000008764 nerve damage Effects 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 210000002241 neurite Anatomy 0.000 description 1
- 230000009689 neuronal regeneration Effects 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229960001621 povidone-iodine Drugs 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 229930002330 retinoic acid Natural products 0.000 description 1
- 238000007665 sagging Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 230000003813 thin hair Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- compositions and methods have long been sought for rejuvenating cells to restore them from an aging, mature state to a younger, more vibrant state of life, to treat certain injuries and diseases, and to generally reverse and Prevents aging of the entire organism.
- iPSC induced pluripotent stem cell
- This epoch-making cell technology reprograms cells through four transcription factors (Oct4, Sox2, Klf4 and c-Myc) and uses viral vectors to combine the four transcription factors (Oct4, Sox2, Klf4 and c-Myc).
- -Myc is transferred into differentiated somatic cells to reprogram them to obtain a cell type similar to embryonic stem cells.
- This kind of cells is similar to human embryonic stem cells and has super differentiation ability. It can differentiate into human blood cells, bone cells, nerve cells, etc., and then cultivate human organs, bones, cornea, pancreas, etc. It has huge application prospects in disease simulation, drug screening and cell therapy.
- the present invention relates to a composition for rejuvenating cells, tissues or organs in a subject based on only one factor or two factors, and methods of rejuvenating cells, tissues or organs in a subject using said composition,
- the compositions and methods are capable of rejuvenating or restoring function to cells, tissues or organs in a subject.
- the compositions and methods may be used in any disease or injury requiring repair or replacement of dysfunctional tissue.
- the present invention provides a composition for rejuvenating cells, tissues or organs in a subject, said composition comprising factor OCT4.
- the composition further comprises any one factor selected from SOX1, SOX2, SOX3 and GMNN.
- the composition includes OCT4 and SOX2.
- the composition includes OCT4, and any one factor selected from SOX1, SOX3, and GMNN.
- the factor is a protein, a nucleic acid encoding the same, or a mixture thereof.
- the OCT4, SOX1, SOX2, SOX3, and GMNN are proteins, fusion proteins combined with a permeable membrane domain, and encode nucleic acids, such as DNA or RNA.
- the factors are provided in the form of an expression vector, which can be a plasmid vector, an episomal vector, a transposon, a virus-derived vector, such as an adenovirus vector, an adeno-associated virus vector (AAV) , lentiviral vectors, Sendai virus vectors, cytomegalovirus vectors or vaccinia virus vectors.
- a virus-derived vector such as an adenovirus vector, an adeno-associated virus vector (AAV) , lentiviral vectors, Sendai virus vectors, cytomegalovirus vectors or vaccinia virus vectors.
- the factors are present on one or more expression vectors.
- the factors are present on the same expression vector.
- the factor is in the form of mRNA.
- the factors are concatenated on the same mRNA molecule, or each of the factors is on a different mRNA molecule, and the factors are in the form of a compound of multiple mRNA molecules.
- the factor is a synthetic mRNA encoding a wild-type form, a mutant, or a genetically engineered form selected from the group consisting of OCT4, SOX1, SOX2, SOX3, GMNN.
- the composition further includes a pharmaceutically acceptable carrier or adjuvant.
- the subject is a mammal including, but not limited to, human, mouse, rat, bovine, ovine, equine, canine, feline, pig, or monkey.
- the subject cells, tissues or organs are from, but are not limited to, eyes, ears, nose, mouth, including gums and tooth roots; bones, lungs, breasts, pancreas, stomach, esophagus; muscles, including cardiac muscle; Liver, blood vessels; skin, including hair; heart, brain, nervous tissue, kidneys, testicles, prostate, penis, cloaca, fins, ovaries, or intestines.
- the composition for rejuvenating cells, tissues or organs of a subject is capable of restoring epigenetic information to the cells, tissues or organs of the subject.
- the composition for rejuvenating cells, tissues or organs of a subject is capable of restoring the loss of epigenetic information due to aging, injury or disease to the cells, tissues or organs of the subject.
- the expression of promoter regulatory factors can be employed to reduce and reverse epigenetic marks associated with aging, increase epigenetic marks associated with cellular rejuvenation, reduce expression of aging-associated proteins, and increase epigenetic markers associated with cellular rejuvenation.
- the expression of rejuvenation-related proteins restores the balance between euchromatin and heterochromatin, prevents the loss of cell identity, restores cell identity, and reverses aging-related DNA methylation changes, thereby rejuvenating cells.
- the OCT4, SOX1, SOX2, SOX3, GMNN is a human protein, or a non-human protein, e.g., a mammal (e.g., mouse, rat, bovine, ovine, equine, canine, feline, porcine , monkey, chicken, duck, goose, bird), or a protein that has at least 80% identity with the amino acid sequence of the corresponding human protein or non-human protein and maintains activity.
- a mammal e.g., mouse, rat, bovine, ovine, equine, canine, feline, porcine , monkey, chicken, duck, goose, bird
- a protein that has at least 80% identity with the amino acid sequence of the corresponding human protein or non-human protein and maintains activity e.g., a mammal (e.g., mouse, rat, bovine, ovine, equine, canine, feline, porcine , monkey, chicken, duck, goose, bird), or a
- the OCT4, SOX1, SOX2, SOX3, GMNN is a nucleic acid encoding a human protein, or a nucleic acid encoding a non-human protein, e.g., encoding a mammal (e.g., mouse, rat, cow, sheep, horse, dog, cat, pig, monkey, chicken, duck, goose, bird), or have at least 80% identity and maintain identity with the corresponding nucleic acid encoding a human protein, or a nucleic acid sequence encoding a non-human protein Active nucleic acids.
- a mammal e.g., mouse, rat, cow, sheep, horse, dog, cat, pig, monkey, chicken, duck, goose, bird
- the composition induces cell reprogramming, reverses aging, improves tissue function, improves organ function, promotes tissue repair, promotes tissue survival, promotes tissue regeneration, promotes tissue growth, promotes angiogenesis, and reduces scarring, Reduces the external manifestations of aging including alopecia, hair thinning, hair graying, skin laxity and skin wrinkles, promotes organ regeneration, promotes organ survival, treats disease, or any combination thereof.
- the composition reverses aging of cells, tissues, organs or subjects, thereby rejuvenating said cells, tissues, organs.
- the composition can promote axonal regeneration of neuronal cells in a subject.
- the composition can proliferate skin fibroblasts in a subject.
- the composition can promote chondrocyte proliferation in a subject.
- the composition can promote muscle stem cell proliferation in a subject.
- the methods can reverse senescence in skin fibroblasts in a subject.
- the composition can reverse chondrocyte senescence.
- the composition does not induce teratoma formation.
- the composition does not induce complete reprogramming.
- the composition does not reprogram cells to an induced pluripotent stem cell (ipsc) state.
- ipsc induced pluripotent stem cell
- the present invention provides a method for rejuvenating cells, tissues or organs in a subject, the method comprising administering the composition of the first aspect to the subject.
- the composition includes factor OCT4.
- the composition includes factor OCT4 and any one factor selected from the group consisting of SOX1, SOX2, SOX3, and GMNN.
- the composition includes OCT4 and SOX2.
- the composition includes OCT4, and any one factor selected from SOX1, SOX3, and GMNN.
- the factor is a protein, a nucleic acid encoding the same, or a mixture thereof.
- the OCT4, SOX1, SOX2, SOX3, and GMNN are proteins, fusion proteins combined with a permeable membrane domain, and encode nucleic acids, such as DNA or RNA.
- the factors are provided in the form of an expression vector, which can be a plasmid vector, an episomal vector, a transposon, a virus-derived vector, such as an adenovirus vector, an adeno-associated virus vector (AAV) , lentiviral vector, Sendai virus vector, cytomegalovirus vector, vaccinia virus vector.
- a virus-derived vector such as an adenovirus vector, an adeno-associated virus vector (AAV) , lentiviral vector, Sendai virus vector, cytomegalovirus vector, vaccinia virus vector.
- the factors are present on one or more expression vectors.
- the factors are present on the same expression vector.
- the factor is in the form of mRNA.
- the factors are concatenated on the same mRNA molecule, or each of the factors is on a different mRNA molecule, and the factors are in the form of a compound of multiple mRNA molecules.
- the factor is a synthetic mRNA encoding a wild-type form, a mutant, or a genetically engineered form selected from the group consisting of OCT4, SOX1, SOX2, SOX3, GMNN.
- the composition further includes a pharmaceutically acceptable carrier or adjuvant.
- the subject is a mammal including, but not limited to, human, mouse, rat, bovine, ovine, equine, canine, feline, pig, or monkey.
- the subject is a chicken, duck, goose or bird.
- the subject cells, tissues or organs are from, but are not limited to, eyes, ears, nose, mouth, including gums and tooth roots; bones, lungs, breasts, pancreas, stomach, esophagus; muscles, including cardiac muscle; Liver, blood vessels; skin, including hair; heart, brain, nervous tissue, kidneys, testicles, prostate, penis, cloaca, fins, ovaries, or intestines.
- the composition for rejuvenating cells, tissues or organs of a subject is capable of restoring epigenetic information to the cells, tissues or organs of the subject.
- the composition for rejuvenating cells, tissues or organs of a subject is capable of restoring the loss of epigenetic information due to aging, injury or disease to the cells, tissues or organs of the subject.
- the expression of promoter regulatory factors can be employed to reduce and reverse epigenetic marks associated with aging, increase epigenetic marks associated with cellular rejuvenation, reduce expression of aging-associated proteins, and increase epigenetic markers associated with cellular rejuvenation.
- the expression of rejuvenation-related proteins restores the balance between euchromatin and heterochromatin, prevents the loss of cell identity, restores cell identity, and reverses aging-related DNA methylation changes, thereby rejuvenating cells.
- the OCT4, SOX1, SOX2, SOX3, GMNN is a human protein, or a non-human protein, e.g., a mammal (e.g., mouse, rat, bovine, ovine, equine, canine, feline, porcine , monkey, chicken, duck, goose, bird), or a protein that has at least 80% identity with the amino acid sequence of the corresponding human protein or non-human protein and maintains activity.
- a mammal e.g., mouse, rat, bovine, ovine, equine, canine, feline, porcine , monkey, chicken, duck, goose, bird
- a protein that has at least 80% identity with the amino acid sequence of the corresponding human protein or non-human protein and maintains activity e.g., a mammal (e.g., mouse, rat, bovine, ovine, equine, canine, feline, porcine , monkey, chicken, duck, goose, bird), or a
- the OCT4, SOX1, SOX2, SOX3, GMNN is a nucleic acid encoding a human protein, or a nucleic acid encoding a non-human protein, e.g., encoding a mammal (e.g., mouse, rat, cow, sheep, horse, dog, cat, pig, monkey, chicken, duck, goose, bird), or have at least 80% identity and maintain identity with the corresponding nucleic acid encoding a human protein, or a nucleic acid sequence encoding a non-human protein Active nucleic acids.
- a mammal e.g., mouse, rat, cow, sheep, horse, dog, cat, pig, monkey, chicken, duck, goose, bird
- the present invention provides use of the composition of the first aspect in the preparation of a medicament for rejuvenating cells, tissues or organs of a subject.
- said rejuvenating a subject's cells, tissues, or organs includes restoring a transcriptional profile or expression in said at least one cell, tissue, or organ that is lost due to aging, injury, disease, or any combination thereof. Genetic information.
- rejuvenating a subject's cells, tissues or organs includes restoring the function of the cells, increasing the potential of the cells, enhancing the viability of the cells, or increasing the ability of the cells to replicate, or Lifespan, or a combination thereof.
- the rejuvenating cells, tissues or organs of the subject includes promoting axonal regeneration of neuronal cells in the subject; proliferating fibroblasts in the skin of the subject; promoting chondrocyte proliferation in the subject; Promote the proliferation of muscle stem cells in subjects; reverse the aging of skin fibroblasts in subjects; reverse the aging of cartilage cells.
- the subject has, is suspected of having, or is at risk of:
- the diseases, conditions or symptoms associated with impaired cell, tissue or organ viability or aging are, for example, neurodegenerative diseases, cardiovascular diseases, metabolic diseases, musculoskeletal diseases, inflammatory diseases, skin-related symptoms or disease, eye disease, or disease related to the reproductive system;
- the neurodegenerative diseases include stroke, Huntington's disease, Alzheimer's disease, Alzheimer's disease, and Parkinson's disease;
- cardiovascular diseases are hypertension, coronary heart disease arrhythmia, heart disease, hypotension, heart failure, angina pectoris, arteriosclerosis, myocardial infarction, myocarditis, congestive heart failure, atrioventricular block, atherosclerosis, myocardium infarction;
- the metabolic diseases are type I diabetes, type II diabetes, hyperglycemia, hyperinsulinemia, insulin resistance, obesity, and gout;
- the musculoskeletal diseases are muscular dystrophy, myasthenia, myasthenia gravis, osteoporosis, osteoarthritis, osteochondrosis, osteochondritis, osteonecrosis, and osteopenia;
- the inflammatory diseases are systemic lupus erythematosus, polymyalgia rheumatica, gouty arthritis, degenerative arthritis, rheumatoid arthritis, inflammatory arthritis, Hashimoto's thyroiditis, inflammatory bowel disease, hepatitis, Pneumonia, respiratory tract inflammation, encephalitis;
- the skin-related signs of aging or diseases include wrinkles, age spots, seborrheic keratosis, tinea pedis, measles, neurodermatitis, and nevus;
- the eye diseases are macular degeneration, retinal degeneration, retinal detachment, diabetic retinopathy, glaucoma, optic atrophy, floaters, cataracts, non-arteritic anterior ischemic optic neuropathy, chorioretinitis, pigmented retina inflammation, traumatic optic neuropathy, compressive optic neuropathy;
- the methods induce cellular reprogramming, reverse aging, improve tissue function, improve organ function, promote tissue repair, promote tissue survival, promote tissue regeneration, promote tissue growth, promote angiogenesis, reduce scarring, alleviate External manifestations of aging, including alopecia, thinning hair, graying of hair, sagging skin and skin wrinkles, promoting organ regeneration, promoting organ survival, treating disease, or any combination thereof.
- the methods reverse senescence of cells, tissues, organs, or subjects, thereby rejuvenating said cells, tissues, organs.
- the methods can promote axonal regeneration of neuronal cells in a subject.
- the methods can proliferate skin fibroblasts in a subject.
- the composition can promote chondrocyte proliferation in a subject.
- the methods can promote muscle stem cell proliferation in a subject.
- the methods can reverse senescence in a subject's skin fibroblasts.
- the composition can reverse chondrocyte senescence.
- the methods do not induce teratoma formation.
- the methods do not induce complete reprogramming.
- the methods do not reprogram the cells to an induced pluripotent stem cell (ipsc) state.
- ipsc induced pluripotent stem cell
- OCT4, SOX2, KLF4 and c-Myc are four "Yamanaka factors” capable of reprogramming cells into a pluripotent state.
- these four "Yamanaka factors” can also reprogram senescent cells into young cells.
- inducing the expression of the four transcription factors in mice carries safety risks and factors that lead to the formation of teratomas in the body. The more it is, the more likely it is to bring about uncertainty in body regulation.
- the present invention unexpectedly found that in the absence of KLF4 and c-Myc, OCT4 alone, or OCT4 only in combination with any one of SOX1, SOX2, SOX3 and GMNN, can also achieve the reversal of senescent cells.
- “Factor” in the present invention refers to a biologically active protein, or nucleic acid encoding it, such as DNA or RNA or a mixture thereof, which acts on cells to change transcription, and which, after expression, will cause aging and incapacity. Cells transform into young and energetic cells.
- the factors may be non-integrating, ie, provided to the recipient somatic cell in a form that does not result in integration of exogenous DNA into the genome of the recipient cell.
- Factors in the present invention may be transcription factors, including but not limited to OCT4, SOX1, SOX2, SOX3, and GMNN.
- Figure 1 shows that the composition of the present invention can promote axon regeneration of neuronal cells.
- Figure 2 shows that the composition of the present invention can promote the proliferation of skin fibroblasts.
- Figure 3 shows that the composition of the present invention can promote the proliferation of mouse chondrocytes.
- Figure 4 shows that the composition of the present invention can promote the proliferation of muscle stem cells in aged mice.
- Figure 5 shows that the composition of the present invention can reverse the aging of skin fibroblasts in aged mice.
- Figure 6 shows that the composition of the present invention can reverse the aging of chondrocytes.
- OCT4 is named A
- SOX2 is named B
- SOX1 is named C1
- SOX3 is named C2
- GMNN is named C3.
- the OCT4, SOX1, SOX2, SOX3, and GMNN used in the embodiments are all human OCT4, human SOX1, human SOX2, human SOX3, and human GMNN.
- Example 1 Composition promotes axon regeneration of neuronal cells
- the main purpose of this example is to select SH-SY5Y (human neuroblastoma cell line) cells, differentiate them into neuronal cells, and determine the regenerative effects of different compositions on neuronal cells.
- SH-SY5Y human neuroblastoma cell line
- EMEM/F12 medium (1:1) containing 2.5% FBS, 1 ⁇ PS and 10 ⁇ M all-trans retinoic acid (ATRA, Stemcell Technologies, 72264) (differentiation medium 1)
- ATRA all-trans retinoic acid
- Cells were induced to differentiate for 3 days and then incubated for 3 days in EMEM/F12 (1:1) (differentiation medium 2) containing 1% FBS, 1 ⁇ PS and 10 ⁇ M ATRA.
- the cells were then seeded on cell culture plates coated with poly-D-lysine (ThermoFisher Scientific, A3890401) and cultured for 1 day.
- the medium was replaced with serum-free serum containing 1 ⁇ Glutamax (ThermoFisher Scientific, 35050061), PS and BDNF.
- B27 neuronal medium differentiated medium 3
- differentiation medium 3 B27 neuronal medium
- a alone, A and B or the combination of A and C can promote the increase of neurite area.
- Example 2 Composition promotes proliferation of skin fibroblasts in aged mice
- the main purpose of this embodiment is to select skin cells of aged mice and observe whether the proliferation ability of skin cells in aged mice is enhanced by administering the drug.
- the specific methods and results are as follows:
- mice 20-month old mice were selected. After the mice were euthanized, the shaved back skin was excised. Before the skin was removed, it was washed with iodophor (povidone iodine) and rinsed with cold 1 ⁇ PBS.
- Mouse skin fibroblasts were isolated using trypsin and collagenase. Fibroblasts were resuspended and cultured in DMEM glutamax medium. The cells are treated with the mRNA drug of the target composition, and the cells are collected after 6 days to detect whether the proliferation ability of the aged skin fibroblasts is enhanced.
- a alone, A and B or a combination of A and C can significantly increase the proliferation ability of skin cells.
- Example 3 Composition promotes proliferation of chondrocytes in aged mice
- the main purpose of this embodiment is to select elderly mice, isolate chondrocytes, and observe whether the chondrocytes have stronger proliferation ability.
- the specific methods and results are as follows:
- Methods Select 18-month-old elderly mice, isolate the mouse articular cartilage, gently stir the tissue fragments to separate the soft tissue, transfer the remaining cartilage fragments to a new petri dish, and digest the articular cartilage overnight. Pass the collagenase solution through a pipette in order to disperse cell aggregates to form a suspension of separated cells, wash with PBS, resuspend with culture medium, and centrifuge. The chondrocyte proliferation ability was detected 6 days after administration.
- a alone, A and B or a combination of A and C can significantly promote the proliferation of chondrocytes.
- Example 4 Composition promotes proliferation of muscle stem cells in aged mice
- the main purpose of this example is to select aged mice, isolate muscle stem cells (MuSC), and observe whether the muscle stem cells have stronger proliferation ability after drug treatment.
- the specific methods and results are as follows:
- mice 15-month old mice were selected, sacrificed by cervical dislocation, the muscles were isolated, cut into small pieces, and cultured in DMEM medium (DMEM; ThermoFisher Scientific) containing 0.2% type I collagenase (Sigma-Aldrich). Incubate at 37°C for 2 hours. Single fibers were collected using a pipette tip and cultured in DMEM containing 1 g/l glucose (supplemented with 10% horse serum (HS, ThermoFisher Scientific), 1% penicillin/streptomycin (ThermoFisher Scientific) and 0.5% chicken embryo extract ( CEE, ThermoFisher Scientific)) was purified twice.
- DMEM DMEM
- ThermoFisher Scientific ThermoFisher Scientific
- CEE chicken embryo extract
- the obtained stem cells were plated directly on a culture dish coated with Matrigel Growth Factor (GFR) basement membrane matrix (Corning) or a culture dish containing a coverslip coated with Matrigel GFR basement membrane matrix.
- GFR Matrigel Growth Factor
- Cells were expanded in DMEM containing 1 g/l glucose and supplemented with 10% HS, 20% FBS, 1% PS and 0.5% CEE and cultured at 37°C, 5% CO with culture changes every 2 days. base. After being treated with different compositions for 6 days, the proliferation ability of the cells was observed.
- a alone, A and B or the combination of A and C can significantly promote the proliferation of muscle stem cells.
- Example 5 Composition reverses aging of skin fibroblasts in aged mice
- the main purpose of this embodiment is to select skin cells of aged mice and observe whether the activity of the aging marker ⁇ -galactosidase in the skin cells of aged mice is changed through administration.
- the specific methods and results are as follows:
- a alone, A and B, or the combination of A and C can significantly reverse the aging of skin fibroblasts in aged mice.
- Example 6 Composition reverses aging of chondrocytes
- the main purpose of this example is to select chondrocytes, use 20 ⁇ M etoposide to induce senescence, and then examine the change of the composition on the activity of the aging marker ⁇ -galactosidase in the cells.
- the specific methods and results are as follows:
- a alone, A and B or the combination of A and C can significantly reverse the aging of chondrocytes.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Physical Education & Sports Medicine (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
A composition for cell, tissue or organ rejuvenation of a subject based on two factors, and a method for cell, tissue, or organ rejuvenation of a subject using said composition. The composition and the method are able to cause cell, tissue, or organ rejuvenation or recovery of function in a subject.
Description
所有的生物都会发生伴随细胞和组织功能活力下降的衰老。衰老的特征在于分子、细胞、组织和生物体水平上逐渐发生的功能丧失。主要特征包括端粒磨损、遗传不稳定、表观遗传和转录改变以及错误蛋白质的积累等问题。在包括脊椎动物在内的许多动物中，重要器官的再生和修复能力随着衰老和疾病的发生急剧下降。与成人的体细胞不同，按时间顺序更接近受精时期的个体细胞(如胚胎和婴儿的细胞)显示出细胞的年轻状态，并具有更大的抵抗损伤和压力的能力，能够治愈、更新和再生器官和组织。因此，长期以来一直寻求用于使细胞恢复活力从而将它们从衰老的成熟状态恢复到更年轻，更有活力的生命状态的组合物和方法，用以治疗某些损伤和疾病，以及总体逆转和预防整个生物体的衰老。All living things undergo aging, which is accompanied by a decrease in the functional vitality of cells and tissues. Aging is characterized by the progressive loss of function at the molecular, cellular, tissue and organismal levels. Key features include problems such as telomere attrition, genetic instability, epigenetic and transcriptional changes, and the accumulation of faulty proteins. In many animals, including vertebrates, the ability to regenerate and repair vital organs declines dramatically with aging and disease. Unlike adult somatic cells, individual cells that are chronologically closer to the time of fertilization (such as those of embryos and infants) display a youthful state of cells and have a greater ability to resist damage and stress and to heal, renew, and regenerate. Organs and tissues. Accordingly, compositions and methods have long been sought for rejuvenating cells to restore them from an aging, mature state to a younger, more vibrant state of life, to treat certain injuries and diseases, and to generally reverse and Prevents aging of the entire organism.
2006年，日本京都大学山中伸弥教授开发的诱导多能干细胞(iPSC)技术，能够逆转细胞的端粒磨损和氧化应激。这项具有划时代意义的细胞技术，简而言之是通过4个转录因子(Oct4、Sox2、Klf4和c-Myc)重编程细胞，利用病毒载体将四个转录因子(Oct4,Sox2,Klf4和c-Myc)的组合转入分化的体细胞中，使其重编程而得到类似胚胎干细胞的一种细胞类型。从而使体细胞退化到类似胚胎干细胞的状态。这种细胞类似人类的胚胎干细胞，具有超强的分化能力，可以分化出人的血细胞，骨细胞，神经细胞等，进而培养出人的脏器，骨，眼角膜，胰腺等。在疾病模拟、药物筛选和细胞治疗中有着巨大的应用前景。In 2006, the induced pluripotent stem cell (iPSC) technology developed by Professor Shinya Yamanaka of Kyoto University, Japan, can reverse telomere attrition and oxidative stress in cells. This epoch-making cell technology, in short, reprograms cells through four transcription factors (Oct4, Sox2, Klf4 and c-Myc) and uses viral vectors to combine the four transcription factors (Oct4, Sox2, Klf4 and c-Myc). -Myc) is transferred into differentiated somatic cells to reprogram them to obtain a cell type similar to embryonic stem cells. This causes somatic cells to degenerate into a state similar to embryonic stem cells. This kind of cells is similar to human embryonic stem cells and has super differentiation ability. It can differentiate into human blood cells, bone cells, nerve cells, etc., and then cultivate human organs, bones, cornea, pancreas, etc. It has huge application prospects in disease simulation, drug screening and cell therapy.
2016年，科学家发现OCT4，SOX2，KLF4和c-Myc这四个“Yamanaka因子”，能够直接将衰老细胞重编程逆转为相对年轻的细胞。然而，在小鼠中诱导四种转录因子表达具有导致体内畸胎瘤的形成的安全性风险，并且重编程因子越多越容易带来体内调控的不确定性以及临床开发的难度。In 2016, scientists discovered that four "Yamanaka factors", OCT4, SOX2, KLF4 and c-Myc, can directly reverse the reprogramming of senescent cells into relatively young cells. However, inducing the expression of four transcription factors in mice carries the safety risk of leading to the formation of teratomas in vivo, and the more reprogramming factors, the more likely it is to bring uncertainty in in vivo regulation and difficulty in clinical development.
发明内容Contents of the invention
本发明涉及一种基于仅仅一个因子或两个因子的用于受试者细胞、组织或器官恢复活力的组合物，以及利用所述组合物使受试者细胞、组织或器官恢复活力的方法，所述组合物和方法能够使受试者细胞、组织或器官恢复活力或恢复功能。所述组合物和方法可用于需要修复或替换功能失效组织的任何疾病或损伤。The present invention relates to a composition for rejuvenating cells, tissues or organs in a subject based on only one factor or two factors, and methods of rejuvenating cells, tissues or organs in a subject using said composition, The compositions and methods are capable of rejuvenating or restoring function to cells, tissues or organs in a subject. The compositions and methods may be used in any disease or injury requiring repair or replacement of dysfunctional tissue.
第一方面，本发明提供了用于受试者细胞、组织或器官恢复活力的组合物，所述组合物包含因子OCT4。In a first aspect, the present invention provides a composition for rejuvenating cells, tissues or organs in a subject, said composition comprising factor OCT4.
在一些实施方案中，所述组合物还包含选自SOX1，SOX2，SOX3和GMNN中的任一种因子。In some embodiments, the composition further comprises any one factor selected from SOX1, SOX2, SOX3 and GMNN.
在一些实施方案中，所述组合物包含OCT4和SOX2。In some embodiments, the composition includes OCT4 and SOX2.
在一些实施方案中，所述组合物包含OCT4，和选自SOX1，SOX3和GMNN中的任一种因子。In some embodiments, the composition includes OCT4, and any one factor selected from SOX1, SOX3, and GMNN.
在一些实施方案中，所述因子为蛋白，编码其的核酸或其混合物。In some embodiments, the factor is a protein, a nucleic acid encoding the same, or a mixture thereof.
例如，所述OCT4，SOX1，SOX2，SOX3，GMNN为蛋白，与透膜结构域结合的融合蛋白，其编码核酸，例如DNA或RNA的形式。For example, the OCT4, SOX1, SOX2, SOX3, and GMNN are proteins, fusion proteins combined with a permeable membrane domain, and encode nucleic acids, such as DNA or RNA.
在一些实施方案中，所述因子以表达载体的形式提供，所述表达载体可以为质粒载体，附加型载体，转座子，病毒衍生的载体，如腺病毒载体，腺相关病毒载体(AAV)，慢病毒载体，仙台病毒载体，巨细胞病毒载体或牛痘病毒载体。In some embodiments, the factors are provided in the form of an expression vector, which can be a plasmid vector, an episomal vector, a transposon, a virus-derived vector, such as an adenovirus vector, an adeno-associated virus vector (AAV) , lentiviral vectors, Sendai virus vectors, cytomegalovirus vectors or vaccinia virus vectors.
在一些实施方案中，所述因子存在于一个或者多个表达载体上。In some embodiments, the factors are present on one or more expression vectors.
在一些实施方案中，所述因子存在于同一表达载体上。In some embodiments, the factors are present on the same expression vector.
在一些实施方案中，所述因子为mRNA的形式。In some embodiments, the factor is in the form of mRNA.
在一些实施方案中，所述因子串联在同一mRNA分子上，或者每一种所述因子在不同的mRNA分子上，所述因子为多种mRNA分子的复方形式。In some embodiments, the factors are concatenated on the same mRNA molecule, or each of the factors is on a different mRNA molecule, and the factors are in the form of a compound of multiple mRNA molecules.
在一些实施方案中，所述因子为合成的mRNA，所述合成的mRNA编码选自OCT4，SOX1，SOX2，SOX3，GMNN的野生型形式，突变体，基因工程化形式。In some embodiments, the factor is a synthetic mRNA encoding a wild-type form, a mutant, or a genetically engineered form selected from the group consisting of OCT4, SOX1, SOX2, SOX3, GMNN.
在一些实施方案中，所述组合物还包含可药用载体或佐剂。In some embodiments, the composition further includes a pharmaceutically acceptable carrier or adjuvant.
在一些实施方案中，所述受试者为哺乳动物，所述哺乳动物包括但不限于人，小鼠，大鼠，牛，羊，马，犬，猫，猪或猴。In some embodiments, the subject is a mammal including, but not limited to, human, mouse, rat, bovine, ovine, equine, canine, feline, pig, or monkey.
在一些实施方案中，所述受试者细胞、组织或器官来自但不限于眼，耳，鼻，口，包括牙龈和牙根；骨，肺，乳房，胰腺，胃，食道；肌肉，包括心肌；肝，血管；皮肤，包括毛发；心脏，脑，神经组织，肾，睾丸，前列腺，阴茎，泄殖腔，鳍，卵巢或肠。In some embodiments, the subject cells, tissues or organs are from, but are not limited to, eyes, ears, nose, mouth, including gums and tooth roots; bones, lungs, breasts, pancreas, stomach, esophagus; muscles, including cardiac muscle; Liver, blood vessels; skin, including hair; heart, brain, nervous tissue, kidneys, testicles, prostate, penis, cloaca, fins, ovaries, or intestines.
在一些实施方案中，所述用于受试者细胞、组织或器官恢复活力的组合物能够使所述受试者细胞、组织或器官恢复表观遗传信息。In some embodiments, the composition for rejuvenating cells, tissues or organs of a subject is capable of restoring epigenetic information to the cells, tissues or organs of the subject.
在一些实施方案中，所述用于受试者细胞、组织或器官恢复活力的组合物能够使所述受试者细胞、组织或器官恢复由于衰老，损伤或疾病导致表观遗传信息的丧失。In some embodiments, the composition for rejuvenating cells, tissues or organs of a subject is capable of restoring the loss of epigenetic information due to aging, injury or disease to the cells, tissues or organs of the subject.
在一些实施方案中，可以采用启动子调控因子的表达，以减少和逆转与衰老相关的表观遗传标记，增加与细胞年轻化相关的表观遗传标记，减少衰老相关蛋白的表达，增加与细胞年轻化相关的蛋白的表达，恢复常染色质和异染色质之间的平衡，防止细胞身份丧失，恢复细胞身份，逆转衰老相关的DNA甲基化变化，从而使细胞恢复活力。In some embodiments, the expression of promoter regulatory factors can be employed to reduce and reverse epigenetic marks associated with aging, increase epigenetic marks associated with cellular rejuvenation, reduce expression of aging-associated proteins, and increase epigenetic markers associated with cellular rejuvenation. The expression of rejuvenation-related proteins restores the balance between euchromatin and heterochromatin, prevents the loss of cell identity, restores cell identity, and reverses aging-related DNA methylation changes, thereby rejuvenating cells.
在一些实施方案中，所述OCT4，SOX1，SOX2，SOX3，GMNN是人蛋白，或非人蛋白，例如，哺乳动物(例如，小鼠，大鼠，牛，羊，马，犬，猫，猪，猴，鸡，鸭，鹅，鸟)的蛋白，或者是与对应的人蛋白或非人蛋白的氨基酸序列具有至少80％同一性并保持活性的蛋白。In some embodiments, the OCT4, SOX1, SOX2, SOX3, GMNN is a human protein, or a non-human protein, e.g., a mammal (e.g., mouse, rat, bovine, ovine, equine, canine, feline, porcine , monkey, chicken, duck, goose, bird), or a protein that has at least 80% identity with the amino acid sequence of the corresponding human protein or non-human protein and maintains activity.
在一些实施方案中，所述OCT4，SOX1，SOX2，SOX3，GMNN是编码人蛋白的核酸，或编码非人蛋白的核酸，例如，编码哺乳动物(例如，小鼠，大鼠，牛，羊，马，犬，猫，猪，猴，鸡，鸭，鹅，鸟)的蛋白的核酸，或者是与对应的编码人蛋白的核酸，或编码非人蛋白的核酸序列具有至少80％同一性并保持活性的核酸。In some embodiments, the OCT4, SOX1, SOX2, SOX3, GMNN is a nucleic acid encoding a human protein, or a nucleic acid encoding a non-human protein, e.g., encoding a mammal (e.g., mouse, rat, cow, sheep, horse, dog, cat, pig, monkey, chicken, duck, goose, bird), or have at least 80% identity and maintain identity with the corresponding nucleic acid encoding a human protein, or a nucleic acid sequence encoding a non-human protein Active nucleic acids.
在一些实施方案中，所述组合物诱导细胞重编程，逆转衰老，改善组织功能，改善器官功能，促进组织修复，促进组织存活，促进组织再生，促进组织生长，促进血管生成，减少疤痕形成，减轻衰老的外部表现，包括脱发症，头发稀疏，头发变白，皮肤松弛和皮肤皱纹，促进器官再生，促进器官存活，治疗疾病或其任意组合。In some embodiments, the composition induces cell reprogramming, reverses aging, improves tissue function, improves organ function, promotes tissue repair, promotes tissue survival, promotes tissue regeneration, promotes tissue growth, promotes angiogenesis, and reduces scarring, Reduces the external manifestations of aging including alopecia, hair thinning, hair graying, skin laxity and skin wrinkles, promotes organ regeneration, promotes organ survival, treats disease, or any combination thereof.
在一些实施方案中，所述组合物逆转细胞、组织、器官或受试者的衰老，从而使所述细胞、组织、器官恢复活力。In some embodiments, the composition reverses aging of cells, tissues, organs or subjects, thereby rejuvenating said cells, tissues, organs.
在一些实施方案中，所述组合物可以促进受试者神经元细胞轴突再生。In some embodiments, the composition can promote axonal regeneration of neuronal cells in a subject.
在一些实施方案中，所述组合物可以使受试者的皮肤成纤维细胞增殖。In some embodiments, the composition can proliferate skin fibroblasts in a subject.
在一些实施方案中，所述组合物可以促进受试者软骨细胞增殖。
In some embodiments, the composition can promote chondrocyte proliferation in a subject.
在一些实施方案中，所述组合物可以促进受试者肌肉干细胞增殖。In some embodiments, the composition can promote muscle stem cell proliferation in a subject.
在一些实施方案中，所述方法可以逆转受试者皮肤成纤维细胞的衰老。In some embodiments, the methods can reverse senescence in skin fibroblasts in a subject.
在一些实施方案中，所述组合物可以逆转软骨细胞的衰老。In some embodiments, the composition can reverse chondrocyte senescence.
在一些实施方案中，所述组合物不诱导畸胎瘤的形成。In some embodiments, the composition does not induce teratoma formation.
在一些实施方案中，所述组合物不诱导完全重编程。In some embodiments, the composition does not induce complete reprogramming.
在一些实施方案中，所述组合物不将细胞重新编程为诱导多能性干细胞(ipsc)状态。In some embodiments, the composition does not reprogram cells to an induced pluripotent stem cell (ipsc) state.
第二方面，本发明提供了受试者细胞、组织或器官恢复活力的方法，所述方法包括向受试者体内施用第一方面的组合物。In a second aspect, the present invention provides a method for rejuvenating cells, tissues or organs in a subject, the method comprising administering the composition of the first aspect to the subject.
在一些实施方案中，所述组合物包含因子OCT4。In some embodiments, the composition includes factor OCT4.
在一些实施方案中，所述组合物包含因子OCT4和选自SOX1，SOX2，SOX3和GMNN中的任一种因子。In some embodiments, the composition includes factor OCT4 and any one factor selected from the group consisting of SOX1, SOX2, SOX3, and GMNN.
在一些实施方案中，所述组合物包含OCT4和SOX2。In some embodiments, the composition includes OCT4 and SOX2.
在一些实施方案中，所述组合物包含OCT4，和选自SOX1，SOX3和GMNN中的任一种因子。In some embodiments, the composition includes OCT4, and any one factor selected from SOX1, SOX3, and GMNN.
在一些实施方案中，所述因子为蛋白，编码其的核酸或其混合物。In some embodiments, the factor is a protein, a nucleic acid encoding the same, or a mixture thereof.
例如，所述OCT4，SOX1，SOX2，SOX3，GMNN为蛋白，与透膜结构域结合的融合蛋白，其编码核酸，例如DNA或RNA的形式。For example, the OCT4, SOX1, SOX2, SOX3, and GMNN are proteins, fusion proteins combined with a permeable membrane domain, and encode nucleic acids, such as DNA or RNA.
在一些实施方案中，所述因子以表达载体的形式提供，所述表达载体可以为质粒载体，附加型载体，转座子，病毒衍生的载体，如腺病毒载体，腺相关病毒载体(AAV)，慢病毒载体，仙台病毒载体，巨细胞病毒载体，牛痘病毒载体。In some embodiments, the factors are provided in the form of an expression vector, which can be a plasmid vector, an episomal vector, a transposon, a virus-derived vector, such as an adenovirus vector, an adeno-associated virus vector (AAV) , lentiviral vector, Sendai virus vector, cytomegalovirus vector, vaccinia virus vector.
在一些实施方案中，所述因子存在于一种或者多种表达载体上。In some embodiments, the factors are present on one or more expression vectors.
在一些实施方案中，所述因子存在于同一表达载体上。In some embodiments, the factors are present on the same expression vector.
在一些实施方案中，所述因子为mRNA的形式。In some embodiments, the factor is in the form of mRNA.
在一些实施方案中，所述因子串联在同一mRNA分子上，或者每一种所述因子在不同的mRNA分子上，所述因子为多种mRNA分子的复方形式。In some embodiments, the factors are concatenated on the same mRNA molecule, or each of the factors is on a different mRNA molecule, and the factors are in the form of a compound of multiple mRNA molecules.
在一些实施方案中，所述因子为合成的mRNA，所述合成的mRNA编码选自OCT4，SOX1，SOX2，SOX3，GMNN的野生型形式，突变体，基因工程化形式。In some embodiments, the factor is a synthetic mRNA encoding a wild-type form, a mutant, or a genetically engineered form selected from the group consisting of OCT4, SOX1, SOX2, SOX3, GMNN.
在一些实施方案中，所述组合物还包含可药用载体或佐剂。In some embodiments, the composition further includes a pharmaceutically acceptable carrier or adjuvant.
在一些实施方案中，所述受试者为哺乳动物，所述哺乳动物包括但不限于人，小鼠，大鼠，牛，羊，马，犬，猫，猪或猴。In some embodiments, the subject is a mammal including, but not limited to, human, mouse, rat, bovine, ovine, equine, canine, feline, pig, or monkey.
在一些实施方案中，所述受试者为鸡，鸭，鹅或鸟。In some embodiments, the subject is a chicken, duck, goose or bird.
在一些实施方案中，所述受试者细胞、组织或器官来自但不限于眼，耳，鼻，口，包括牙龈和牙根；骨，肺，乳房，胰腺，胃，食道；肌肉，包括心肌；肝，血管；皮肤，包括毛发；心脏，脑，神经组织，肾，睾丸，前列腺，阴茎，泄殖腔，鳍，卵巢或肠。In some embodiments, the subject cells, tissues or organs are from, but are not limited to, eyes, ears, nose, mouth, including gums and tooth roots; bones, lungs, breasts, pancreas, stomach, esophagus; muscles, including cardiac muscle; Liver, blood vessels; skin, including hair; heart, brain, nervous tissue, kidneys, testicles, prostate, penis, cloaca, fins, ovaries, or intestines.
在一些实施方案中，所述用于受试者细胞、组织或器官恢复活力的组合物能够使所述受试者细胞、组织或器官恢复表观遗传信息。In some embodiments, the composition for rejuvenating cells, tissues or organs of a subject is capable of restoring epigenetic information to the cells, tissues or organs of the subject.
在一些实施方案中，所述用于受试者细胞、组织或器官恢复活力的组合物能够使所述受试者细胞、组织或器官恢复由于衰老，损伤或疾病导致表观遗传信息的丧失。In some embodiments, the composition for rejuvenating cells, tissues or organs of a subject is capable of restoring the loss of epigenetic information due to aging, injury or disease to the cells, tissues or organs of the subject.
在一些实施方案中，可以采用启动子调控因子的表达，以减少和逆转与衰老相关的表观遗传标记，增加与细胞年轻化相关的表观遗传标记，减少衰老相关蛋白的表达，增加与细胞年轻化相关的蛋白的表达，恢复常染色质和异染色质之间的平衡，防止细胞身份丧失，恢复细胞身份，逆转衰老相关的DNA甲基化变化，从而使细胞恢复活力。In some embodiments, the expression of promoter regulatory factors can be employed to reduce and reverse epigenetic marks associated with aging, increase epigenetic marks associated with cellular rejuvenation, reduce expression of aging-associated proteins, and increase epigenetic markers associated with cellular rejuvenation. The expression of rejuvenation-related proteins restores the balance between euchromatin and heterochromatin, prevents the loss of cell identity, restores cell identity, and reverses aging-related DNA methylation changes, thereby rejuvenating cells.
在一些实施方案中，所述OCT4，SOX1，SOX2，SOX3，GMNN是人蛋白，或非人蛋白，例如，哺乳动物(例如，小鼠，大鼠，牛，羊，马，犬，猫，猪，猴，鸡，鸭，鹅，鸟)的蛋白，或者是与对应的人蛋白或非人蛋白的氨基酸序列具有至少80％同一性并保持活性的蛋白。In some embodiments, the OCT4, SOX1, SOX2, SOX3, GMNN is a human protein, or a non-human protein, e.g., a mammal (e.g., mouse, rat, bovine, ovine, equine, canine, feline, porcine , monkey, chicken, duck, goose, bird), or a protein that has at least 80% identity with the amino acid sequence of the corresponding human protein or non-human protein and maintains activity.
在一些实施方案中，所述OCT4，SOX1，SOX2，SOX3，GMNN是编码人蛋白的核酸，或编码非人蛋白的核酸，例如，编码哺乳动物(例如，小鼠，大鼠，牛，羊，马，犬，猫，猪，猴，鸡，鸭，鹅，鸟)的蛋白的核酸，或者是与对应的编码人蛋白的核酸，或编码非人蛋白的核酸序列具有至少80％同一性并保持活性的核酸。In some embodiments, the OCT4, SOX1, SOX2, SOX3, GMNN is a nucleic acid encoding a human protein, or a nucleic acid encoding a non-human protein, e.g., encoding a mammal (e.g., mouse, rat, cow, sheep, horse, dog, cat, pig, monkey, chicken, duck, goose, bird), or have at least 80% identity and maintain identity with the corresponding nucleic acid encoding a human protein, or a nucleic acid sequence encoding a non-human protein Active nucleic acids.
第三方面，本发明提供了第一方面的组合物在制备用于使受试者细胞、组织或器官恢复活力的药物中的应用。In a third aspect, the present invention provides use of the composition of the first aspect in the preparation of a medicament for rejuvenating cells, tissues or organs of a subject.
在一些实施方案中，所述使受试者细胞、组织或器官恢复活力包括恢复所述至少一种细胞，组织或器官中由于衰老，损伤，疾病或其任何组合而丢失的转录谱或表观遗传信息。In some embodiments, said rejuvenating a subject's cells, tissues, or organs includes restoring a transcriptional profile or expression in said at least one cell, tissue, or organ that is lost due to aging, injury, disease, or any combination thereof. Genetic information.
在一些实施方案中，所述使受试者细胞、组织或器官恢复活力包括恢复所述细胞的功能、增加所述细胞的潜能、增强所述细胞的存活能力或增加所述细胞的复制能力或寿命，或其组合。In some embodiments, rejuvenating a subject's cells, tissues or organs includes restoring the function of the cells, increasing the potential of the cells, enhancing the viability of the cells, or increasing the ability of the cells to replicate, or Lifespan, or a combination thereof.
在一些实施方案中，所述使受试者细胞、组织或器官恢复活力包括促进受试者神经元细胞轴突再生；使受试者皮肤的成纤维细胞增殖；促进受试者软骨细胞增殖；促进受试者肌肉干细胞增殖；逆转受试者皮肤成纤维细胞的衰老；逆转软骨细胞的衰老。In some embodiments, the rejuvenating cells, tissues or organs of the subject includes promoting axonal regeneration of neuronal cells in the subject; proliferating fibroblasts in the skin of the subject; promoting chondrocyte proliferation in the subject; Promote the proliferation of muscle stem cells in subjects; reverse the aging of skin fibroblasts in subjects; reverse the aging of cartilage cells.
在一些实施方案中，所述受试者患有，怀疑患有，或具风险患：In some embodiments, the subject has, is suspected of having, or is at risk of:
与细胞、组织或器官活力受损或与衰老有关的疾病，病况或表征；Diseases, conditions or symptoms associated with impaired viability of cells, tissues or organs or associated with aging;
所述与细胞、组织或器官活力受损或与衰老有关的疾病，病况或表征例如为神经退行性疾病，心血管疾病，代谢性疾病，肌肉骨骼疾病，炎性疾病，与皮肤有关的表征或病症，眼部疾病，或与生殖系统相关的疾病；The diseases, conditions or symptoms associated with impaired cell, tissue or organ viability or aging are, for example, neurodegenerative diseases, cardiovascular diseases, metabolic diseases, musculoskeletal diseases, inflammatory diseases, skin-related symptoms or disease, eye disease, or disease related to the reproductive system;
所述神经退行性疾病为脑中风，亨廷顿舞蹈病，老年性痴呆，阿尔茨海默症，帕金森；The neurodegenerative diseases include stroke, Huntington's disease, Alzheimer's disease, Alzheimer's disease, and Parkinson's disease;
所述心血管疾病为高血压，冠心病心律失常，心脏病，低血压，心力衰竭，心绞痛，动脉硬化，心肌梗死，心肌炎，充血性心力衰竭，房室传导阻滞，动脉粥样硬化，心肌梗塞；The cardiovascular diseases are hypertension, coronary heart disease arrhythmia, heart disease, hypotension, heart failure, angina pectoris, arteriosclerosis, myocardial infarction, myocarditis, congestive heart failure, atrioventricular block, atherosclerosis, myocardium infarction;
所述代谢性疾病为I型糖尿病，II型糖尿病，高血糖，高胰岛素血症，胰岛素抗性，肥胖，痛风；The metabolic diseases are type I diabetes, type II diabetes, hyperglycemia, hyperinsulinemia, insulin resistance, obesity, and gout;
所述肌肉骨骼疾病为肌肉萎缩症，肌无力，重症肌无力，骨质疏松症，骨关节炎，骨软骨病，骨软骨炎，骨坏死，、骨质稀少症；The musculoskeletal diseases are muscular dystrophy, myasthenia, myasthenia gravis, osteoporosis, osteoarthritis, osteochondrosis, osteochondritis, osteonecrosis, and osteopenia;
所述炎性疾病为系统性红斑狼疮，风湿性多肌痛，痛风性关节炎，退行性关节炎，类风湿性关节炎，炎性关节炎，桥本甲状腺炎，炎性肠病，肝炎，肺炎，呼吸道炎症，脑炎；The inflammatory diseases are systemic lupus erythematosus, polymyalgia rheumatica, gouty arthritis, degenerative arthritis, rheumatoid arthritis, inflammatory arthritis, Hashimoto's thyroiditis, inflammatory bowel disease, hepatitis, Pneumonia, respiratory tract inflammation, encephalitis;
所述与皮肤有关的衰老表征或病症为皱纹，老年斑，脂溢性角化病，手脚癣，寻麻疹，神经性皮炎，色素痣；The skin-related signs of aging or diseases include wrinkles, age spots, seborrheic keratosis, tinea pedis, measles, neurodermatitis, and nevus;
所述眼部疾病为黄斑变性，视网膜变性，视网膜脱离，糖尿病性视网膜病，青光眼，视神经萎缩，飞蚊症，白内障，非动脉炎性前部缺血性视神经病变，脉络膜视网膜炎，色素性视网膜炎，外伤性视神经病变，压迫性视神经病变；The eye diseases are macular degeneration, retinal degeneration, retinal detachment, diabetic retinopathy, glaucoma, optic atrophy, floaters, cataracts, non-arteritic anterior ischemic optic neuropathy, chorioretinitis, pigmented retina inflammation, traumatic optic neuropathy, compressive optic neuropathy;
与生殖系统相关的疾病为勃起功能障碍以及卵巢早衰。Diseases related to the reproductive system are erectile dysfunction and premature ovarian failure.
在一些实施方案中，所述方法诱导细胞重编程，逆转衰老，改善组织功能，改善器官功能，促进组织修复，促进组织存活，促进组织再生，促进组织生长，促进血管生成，减少疤痕形成，减轻衰老的外部表现，包括脱发症，头发稀疏，头发变白，皮肤松弛和皮肤皱纹，促进器官再生，促进器官存活，治疗疾病或其任意组合。In some embodiments, the methods induce cellular reprogramming, reverse aging, improve tissue function, improve organ function, promote tissue repair, promote tissue survival, promote tissue regeneration, promote tissue growth, promote angiogenesis, reduce scarring, alleviate External manifestations of aging, including alopecia, thinning hair, graying of hair, sagging skin and skin wrinkles, promoting organ regeneration, promoting organ survival, treating disease, or any combination thereof.
在一些实施方案中，所述方法逆转细胞、组织、器官或受试者的衰老，从而使所述细胞、组织、器官恢复活力。In some embodiments, the methods reverse senescence of cells, tissues, organs, or subjects, thereby rejuvenating said cells, tissues, organs.
在一些实施方案中，所述方法可以促进受试者神经元细胞轴突再生。In some embodiments, the methods can promote axonal regeneration of neuronal cells in a subject.
在一些实施方案中，所述方法可以使受试者的皮肤成纤维细胞增殖。In some embodiments, the methods can proliferate skin fibroblasts in a subject.
在一些实施方案中，所述组合物可以促进受试者软骨细胞增殖。In some embodiments, the composition can promote chondrocyte proliferation in a subject.
在一些实施方案中，所述方法可以促进受试者肌肉干细胞增殖。In some embodiments, the methods can promote muscle stem cell proliferation in a subject.
在一些实施方案中，所述方法可以逆转受试者的皮肤成纤维细胞的衰老。In some embodiments, the methods can reverse senescence in a subject's skin fibroblasts.
在一些实施方案中，所述组合物可以逆转软骨细胞的衰老。In some embodiments, the composition can reverse chondrocyte senescence.
在一些实施方案中，所述方法不诱导畸胎瘤的形成。In some embodiments, the methods do not induce teratoma formation.
在一些实施方案中，所述方法不诱导完全重编程。In some embodiments, the methods do not induce complete reprogramming.
在一些实施方案中，所述方法不将细胞重新编程为诱导多能性干细胞(ipsc)状态。In some embodiments, the methods do not reprogram the cells to an induced pluripotent stem cell (ipsc) state.
2006年，科学家发现OCT4，SOX2，KLF4和c-Myc是四个“Yamanaka因子”，能够将细胞重编程为多能状态。2016年，科学家发现这四个“Yamanaka因子”也可以将衰老细胞重编程为年轻细胞，然而，在小鼠中诱导四种转录因子表达会具有导致体内畸胎瘤的形成的安全性风险并且因子越多越容易带来体内调控的不确定性。In 2006, scientists discovered that OCT4, SOX2, KLF4 and c-Myc are four "Yamanaka factors" capable of reprogramming cells into a pluripotent state. In 2016, scientists discovered that these four "Yamanaka factors" can also reprogram senescent cells into young cells. However, inducing the expression of the four transcription factors in mice carries safety risks and factors that lead to the formation of teratomas in the body. The more it is, the more likely it is to bring about uncertainty in body regulation.
本发明预料之外地发现，在不存在KLF4和c-Myc的情况下，单独的OCT4，或OCT4仅与SOX1，SOX2，SOX3和GMNN中的任一种组合也可以实现衰老细胞的逆转。The present invention unexpectedly found that in the absence of KLF4 and c-Myc, OCT4 alone, or OCT4 only in combination with any one of SOX1, SOX2, SOX3 and GMNN, can also achieve the reversal of senescent cells.
本发明中的“因子”是指一种生物活性蛋白，或编码其的核酸，例如DNA或RNA或其混合物，其作用于细胞上以改变转录，并且其在表达后，将衰老的失去能力的细胞转变为年轻活力的细胞。在一些实施方式中，因子可以是非整合的，即以不导致外源DNA整合到受体细胞的基因组的形式向受体体细胞提供。"Factor" in the present invention refers to a biologically active protein, or nucleic acid encoding it, such as DNA or RNA or a mixture thereof, which acts on cells to change transcription, and which, after expression, will cause aging and incapacity. Cells transform into young and energetic cells. In some embodiments, the factors may be non-integrating, ie, provided to the recipient somatic cell in a form that does not result in integration of exogenous DNA into the genome of the recipient cell.
本发明中的因子可以是转录因子，包括但不限于OCT4，SOX1，SOX2，SOX3，GMNN。Factors in the present invention may be transcription factors, including but not limited to OCT4, SOX1, SOX2, SOX3, and GMNN.
图1显示了本发明的组合物可以促进神经元细胞轴突再生。Figure 1 shows that the composition of the present invention can promote axon regeneration of neuronal cells.
图2显示了本发明的组合物可以促进皮肤成纤维细胞的增殖。Figure 2 shows that the composition of the present invention can promote the proliferation of skin fibroblasts.
图3显示了本发明的组合物可以促进小鼠软骨细胞的增殖。Figure 3 shows that the composition of the present invention can promote the proliferation of mouse chondrocytes.
图4显示了本发明的组合物可以促进老年小鼠肌肉干细胞的增殖。Figure 4 shows that the composition of the present invention can promote the proliferation of muscle stem cells in aged mice.
。.
图5显示了本发明的组合物可以逆转老年小鼠皮肤成纤维细胞的衰老。Figure 5 shows that the composition of the present invention can reverse the aging of skin fibroblasts in aged mice.
图6显示了本发明的组合物可以逆转软骨细胞的衰老。Figure 6 shows that the composition of the present invention can reverse the aging of chondrocytes.
以下实施例以及附图中：In the following examples and drawings:
OCT4命名为A，SOX2命名为B，SOX1命名为C1、SOX3命名为C2、GMNN命名为C3。OCT4 is named A, SOX2 is named B, SOX1 is named C1, SOX3 is named C2, and GMNN is named C3.
在实施例中所采用的OCT4，SOX1，SOX2，SOX3，GMNN均为人OCT4，人SOX1，人SOX2，人SOX3，人GMNN。The OCT4, SOX1, SOX2, SOX3, and GMNN used in the embodiments are all human OCT4, human SOX1, human SOX2, human SOX3, and human GMNN.
实施例1组合物促进神经元细胞轴突再生Example 1 Composition promotes axon regeneration of neuronal cells
本实施例主要目的在于选取SH-SY5Y(人神经母细胞瘤细胞株)细胞，使其分化为神经元细胞，判定不同组合物对神经元细胞的再生作用。具体方法和结果如下：The main purpose of this example is to select SH-SY5Y (human neuroblastoma cell line) cells, differentiate them into neuronal cells, and determine the regenerative effects of different compositions on neuronal cells. The specific methods and results are as follows:
方法：细胞铺板后1天，使用含有2.5％FBS，1×PS和10μM全反式视黄酸(ATRA，Stemcell Technologies，72264)的EMEM/F12培养基(1：1)(分化培养基1)诱导细胞分化3天，然后在含有1％FBS，1×PS和10μM ATRA的EMEM/F12(1：1)(分化培养基2)中孵育3天。然后将细胞接种在涂有聚-D-赖氨酸(ThermoFisher Scientific，A3890401)的细胞培养板培养1天后，将培养基更换为含有1×Glutamax(ThermoFisher Scientific，35050061)，PS和BDNF的无血清B27神经元培养基(分化培养基3)继续培养至少5天以促进细胞分化成熟。分化完成后，用组合物mRNA处理细胞5天，然后加入长春新碱24小时以诱导神经损伤。然后用PBS洗涤神经元一次，并将新鲜的分化培养基3加入到板中继续培养9天。统计神经元再生与维持情况。Method: One day after cell plating, use EMEM/F12 medium (1:1) containing 2.5% FBS, 1×PS and 10 μM all-trans retinoic acid (ATRA, Stemcell Technologies, 72264) (differentiation medium 1) Cells were induced to differentiate for 3 days and then incubated for 3 days in EMEM/F12 (1:1) (differentiation medium 2) containing 1% FBS, 1×PS and 10 μM ATRA. The cells were then seeded on cell culture plates coated with poly-D-lysine (ThermoFisher Scientific, A3890401) and cultured for 1 day. The medium was replaced with serum-free serum containing 1×Glutamax (ThermoFisher Scientific, 35050061), PS and BDNF. Continue culturing in B27 neuronal medium (differentiation medium 3) for at least 5 days to promote cell differentiation and maturation. After completion of differentiation, cells were treated with composition mRNA for 5 days, and then vincristine was added for 24 hours to induce nerve damage. Neurons were then washed once with PBS, and fresh differentiation medium 3 was added to the plates to continue culturing for 9 days. Statistics on neuronal regeneration and maintenance.
如图1所示，单独的A、A和B或者A和C两联联用可以促进神经突面积的增加。As shown in Figure 1, A alone, A and B or the combination of A and C can promote the increase of neurite area.
实施例2组合物促进老年小鼠皮肤成纤维细胞的增殖Example 2 Composition promotes proliferation of skin fibroblasts in aged mice
本实施例主要目的在于选取老年小鼠皮肤细胞，通过给药，观察老年小鼠皮肤细胞增殖能力是否增强。具体方法和结果如下：The main purpose of this embodiment is to select skin cells of aged mice and observe whether the proliferation ability of skin cells in aged mice is enhanced by administering the drug. The specific methods and results are as follows:
方法：选择20月龄老年小鼠，小鼠安乐死后，切除被剃光的背部皮肤，在切除皮肤前用碘伏(聚维酮碘)清洗并用冷1×PBS冲洗。用胰酶和胶原酶分离小鼠的皮肤成纤维。将成纤维细胞重悬并用DMEM glutamax培养基进行培养。用目标组合物的mRNA药物处理细胞，6天后收集细胞，检测衰老的皮肤成纤维细胞增殖能力是否增强。Methods: 20-month old mice were selected. After the mice were euthanized, the shaved back skin was excised. Before the skin was removed, it was washed with iodophor (povidone iodine) and rinsed with cold 1×PBS. Mouse skin fibroblasts were isolated using trypsin and collagenase. Fibroblasts were resuspended and cultured in DMEM glutamax medium. The cells are treated with the mRNA drug of the target composition, and the cells are collected after 6 days to detect whether the proliferation ability of the aged skin fibroblasts is enhanced.
如图2所示，单独的A、A和B或者A和C两两联可以显著增加皮肤细胞的增殖能力。As shown in Figure 2, A alone, A and B or a combination of A and C can significantly increase the proliferation ability of skin cells.
实施例3组合物促进老年小鼠软骨细胞的增殖Example 3 Composition promotes proliferation of chondrocytes in aged mice
本实施例主要目的在于选取老年小鼠，分离软骨细胞，观察软骨细胞是否具有更强的增殖能力。具体方法和结果如下：The main purpose of this embodiment is to select elderly mice, isolate chondrocytes, and observe whether the chondrocytes have stronger proliferation ability. The specific methods and results are as follows:
方法：选择18月龄的老年小鼠，分离小鼠关节软骨，轻轻搅动组织碎片来分离软组织，将留下的软骨碎片转移到新的培养皿中，消化关节软骨过夜。将胶原酶溶液依次通过移液管以分散细胞聚集物，形成分离细胞的悬浮液，用PBS洗涤后用培养基重悬，离心培养。给药6天后检测软骨细胞增殖能力。Methods: Select 18-month-old elderly mice, isolate the mouse articular cartilage, gently stir the tissue fragments to separate the soft tissue, transfer the remaining cartilage fragments to a new petri dish, and digest the articular cartilage overnight. Pass the collagenase solution through a pipette in order to disperse cell aggregates to form a suspension of separated cells, wash with PBS, resuspend with culture medium, and centrifuge. The chondrocyte proliferation ability was detected 6 days after administration.
如图3所示，单独的A、A和B或者A和C两两联用可以显著促进软骨细胞的增殖。As shown in Figure 3, A alone, A and B or a combination of A and C can significantly promote the proliferation of chondrocytes.
实施例4组合物促进老年小鼠肌肉干细胞的增殖Example 4 Composition promotes proliferation of muscle stem cells in aged mice
本实施例主要目的在于选取对老年小鼠，分离肌肉干细胞(MuSC)，观察药物处理后肌肉干细胞是否具有更强的增殖能力。具体方法和结果如下：The main purpose of this example is to select aged mice, isolate muscle stem cells (MuSC), and observe whether the muscle stem cells have stronger proliferation ability after drug treatment. The specific methods and results are as follows:
方法：选择15月龄的老年小鼠，通过颈脱位处死小鼠，分离肌肉，切成小块，并在含0.2％I型胶原酶(Sigma-Aldrich)的DMEM培养基(DMEM；ThermoFisher Scientific)中于37℃孵育2小时。使用移液器吸头收集单纤维，在含有1g/l葡萄糖的DMEM(添加10％马血清(HS，ThermoFisher Scientific)、1％青霉素/链霉素(ThermoFisher Scientific)和0.5％鸡胚提取物(CEE，ThermoFisher Scientific))中纯化两次。然后，转移单纤维并将肌纤维悬浮液通过带有21G针头的注射器过40μm过滤器。将获得的干细胞直接铺板在涂有Matrigel生长因子(GFR)基底膜基质(Corning)的培养皿或含有涂有Matrigel GFR基底膜基质的盖玻片的培养皿上。细胞在含有1g/l葡萄糖并补充有10％HS、20％FBS、1％PS和0.5％CEE的DMEM中扩增，并在37℃、5％CO2条件下培养，每2天更换一次培养基。用不同组合物处理6天后，观察细胞的增殖能力。Methods: 15-month old mice were selected, sacrificed by cervical dislocation, the muscles were isolated, cut into small pieces, and cultured in DMEM medium (DMEM; ThermoFisher Scientific) containing 0.2% type I collagenase (Sigma-Aldrich). Incubate at 37°C for 2 hours. Single fibers were collected using a pipette tip and cultured in DMEM containing 1 g/l glucose (supplemented with 10% horse serum (HS, ThermoFisher Scientific), 1% penicillin/streptomycin (ThermoFisher Scientific) and 0.5% chicken embryo extract ( CEE, ThermoFisher Scientific)) was purified twice. Then, transfer the single fibers and pass the myofiber suspension through a 40 μm filter through a syringe with a 21G needle. The obtained stem cells were plated directly on a culture dish coated with Matrigel Growth Factor (GFR) basement membrane matrix (Corning) or a culture dish containing a coverslip coated with Matrigel GFR basement membrane matrix. Cells were expanded in DMEM containing 1 g/l glucose and supplemented with 10% HS, 20% FBS, 1% PS and 0.5% CEE and cultured at 37°C, 5% CO with culture changes every 2 days. base. After being treated with different compositions for 6 days, the proliferation ability of the cells was observed.
如图4所示，单独的A、A和B或者A和C的两两联用可以显著促进肌肉干细胞的增殖。As shown in Figure 4, A alone, A and B or the combination of A and C can significantly promote the proliferation of muscle stem cells.
实施例5组合物逆转老年小鼠皮肤成纤维细胞的衰老Example 5 Composition reverses aging of skin fibroblasts in aged mice
本实施例主要目的在于选取老年小鼠皮肤细胞，通过给药，观察老年小鼠皮肤细胞中衰老标志物β-半乳糖苷酶的活性是否改变。具体方法和结果如下：The main purpose of this embodiment is to select skin cells of aged mice and observe whether the activity of the aging marker β-galactosidase in the skin cells of aged mice is changed through administration. The specific methods and results are as follows:
取老年小鼠皮肤成纤维细胞，用目标组合物的mRNA药物处理细胞，6天后，加入4％多聚甲醛固定15分钟后，使用β-半乳糖苷酶原位染色试剂盒(碧云天)进行原位染色。染色完成后，进一步加入DAPI进行细胞核染色以便统计细胞总数。之后，于荧光显微镜下观察，计算阳性细胞率(阳性细胞率＝染色阳性细胞数/总细胞数×100％)。Take aged mouse skin fibroblasts and treat the cells with the mRNA drug of the target composition. After 6 days, add 4% paraformaldehyde to fix for 15 minutes, and then use β-galactosidase in situ staining kit (Beyotime). In situ staining. After the staining is completed, DAPI is further added for nuclear staining to count the total number of cells. Afterwards, the cells were observed under a fluorescence microscope and the positive cell rate was calculated (positive cell rate = number of stained positive cells/total number of cells × 100%).
如图5所示，单独的A、A和B或者A和C的两两联用可以显著逆转老年小鼠皮肤成纤维细胞的衰老。As shown in Figure 5, A alone, A and B, or the combination of A and C can significantly reverse the aging of skin fibroblasts in aged mice.
实施例6组合物逆转软骨细胞的衰老Example 6 Composition reverses aging of chondrocytes
本实施例主要目的在于选取软骨细胞，使用20μM的依托泊苷进行衰老诱导，之后考察组合物对细胞中衰老标志物β-半乳糖苷酶活性的改变。具体方法和结果如下：The main purpose of this example is to select chondrocytes, use 20 μM etoposide to induce senescence, and then examine the change of the composition on the activity of the aging marker β-galactosidase in the cells. The specific methods and results are as follows:
取人软骨细胞系C28/I2，使用20μM的依托泊苷进行衰老诱导，诱导24小时后，用目标组合物的mRNA药物处理细胞，6天后，加入4％多聚甲醛固定15分钟后，使用β-半乳糖苷酶原位染色试剂盒(碧云天)进行原位染色。染色完成后，进一步加入DAPI进行细胞核染色以便统计细胞总数。之后，于荧光显微镜下观察，计算阳性细胞率(阳性细胞率＝染色阳性细胞数/总细胞数×100％)。Take the human chondrocyte cell line C28/I2 and use 20 μM etoposide for senescence induction. After 24 hours of induction, the cells are treated with the mRNA drug of the target composition. After 6 days, 4% paraformaldehyde is added to fix it for 15 minutes, and then use β -Galactosidase in situ staining kit (Beyotime) was used for in situ staining. After the staining is completed, DAPI is further added for nuclear staining to count the total number of cells. Afterwards, the cells were observed under a fluorescence microscope and the positive cell rate was calculated (positive cell rate = number of stained positive cells/total number of cells × 100%).
如图6所示，单独的A、A和B或者A和C的两两联用可以显著逆转软骨细胞的衰老。
As shown in Figure 6, A alone, A and B or the combination of A and C can significantly reverse the aging of chondrocytes.
Claims (13)
- 用于受试者细胞、组织或器官恢复活力的组合物，所述组合物包含OCT4。A composition for rejuvenating cells, tissues or organs in a subject, the composition comprising OCT4.
- 根据权利要求1的组合物，其特征在于，所述组合物还包含选自SOX1，SOX2，SOX3和GMNN中的任一种因子，The composition according to claim 1, characterized in that the composition further comprises any factor selected from the group consisting of SOX1, SOX2, SOX3 and GMNN,优选地，所述组合物包含OCT4和SOX2，或者Preferably, the composition comprises OCT4 and SOX2, or所述组合物包含OCT4，和选自SOX1，SOX3和GMNN中的任一种因子。The composition includes OCT4, and any one factor selected from SOX1, SOX3 and GMNN.
- 根据权利要求1至2任一项的组合物，其特征在于，所述因子OCT4，SOX1，SOX2，SOX3，GMNN为蛋白，编码其的核酸或其混合物，The composition according to any one of claims 1 to 2, characterized in that the factors OCT4, SOX1, SOX2, SOX3, and GMNN are proteins, nucleic acids encoding them, or mixtures thereof,优选地，所述因子OCT4，SOX1，SOX2，SOX3，GMNN为蛋白，与透膜结构域结合的融合蛋白，其编码核酸的形式。Preferably, the factors OCT4, SOX1, SOX2, SOX3, and GMNN are proteins, fusion proteins combined with a membrane-permeable domain, and are in the form of encoding nucleic acids.
- 根据权利要求1至2任一项的组合物，其特征在于，所述因子OCT4，SOX1，SOX2，SOX3，GMNN为表达载体的形式，所述表达载体为质粒载体，附加型载体，转座子，或病毒载体，The composition according to any one of claims 1 to 2, characterized in that the factors OCT4, SOX1, SOX2, SOX3, and GMNN are in the form of expression vectors, and the expression vectors are plasmid vectors, episomal vectors, and transposons. , or viral vector,优选地，所述病毒载体为腺病毒载体，腺相关病毒载体(AAV)，慢病毒载体，仙台病毒载体，巨细胞病毒载体或牛痘病毒载体，Preferably, the viral vector is an adenovirus vector, an adeno-associated virus vector (AAV), a lentiviral vector, a Sendai virus vector, a cytomegalovirus vector or a vaccinia virus vector,优选地，所述因子存在于同一表达载体上，或者存在于多个表达载体上。Preferably, the factors are present on the same expression vector, or on multiple expression vectors.
- 根据权利要求1至2任一项的组合物，其特征在于，所述因子OCT4，SOX1，SOX2，SOX3，GMNN为DNA或RNA的形式，The composition according to any one of claims 1 to 2, characterized in that the factors OCT4, SOX1, SOX2, SOX3, GMNN are in the form of DNA or RNA,优选地，所述因子为mRNA的形式,Preferably, the factor is in the form of mRNA,所述因子串联在同一mRNA分子上，或者所述因子OCT4，SOX1，SOX2，SOX3，GMNN为不同mRNA分子的复方形式，The factors are connected in series on the same mRNA molecule, or the factors OCT4, SOX1, SOX2, SOX3, and GMNN are compound forms of different mRNA molecules,优选地，所述因子为合成的mRNA，所述合成的mRNA编码选自OCT4，SOX1，SOX2，SOX3，GMNN的野生型形式，突变体，基因工程化形式。Preferably, the factor is a synthetic mRNA encoding a wild-type form, a mutant, or a genetically engineered form selected from the group consisting of OCT4, SOX1, SOX2, SOX3, and GMNN.
- 根据权利要求1至2任一项的组合物，其特征在于，所述受试者为哺乳动物，所述哺乳动物包括但不限于人，小鼠，大鼠，牛，羊，马，犬，猫，猪或猴，The composition according to any one of claims 1 to 2, characterized in that the subject is a mammal, and the mammal includes but is not limited to humans, mice, rats, cattle, sheep, horses, and dogs, cat, pig or monkey,所述受试者细胞、组织或器官来自但不限于眼，耳，鼻，口，包括牙龈和牙根；骨，肺，乳房，胰腺，胃，食道；肌肉，包括心肌；肝，血管；皮肤，包括毛发；心脏，脑，神经组织，肾，睾丸，前列腺，阴茎，泄殖腔，鳍，卵巢或肠。The cells, tissues or organs of the subject come from, but are not limited to, eyes, ears, nose, mouth, including gums and tooth roots; bones, lungs, breasts, pancreas, stomach, esophagus; muscles, including myocardium; liver, blood vessels; skin, Includes hair; heart, brain, nervous tissue, kidneys, testicles, prostate, penis, cloaca, fins, ovaries or intestines.
- 根据权利要求1至2任一项的组合物，其特征在于，所述OCT4，SOX1，SOX2，SOX3，GMNN是人蛋白，或非人蛋白，或与所述人蛋白或非人蛋白的氨基酸序列具有至少80％同一性并保持活性的蛋白，The composition according to any one of claims 1 to 2, characterized in that the OCT4, SOX1, SOX2, SOX3, GMNN are human proteins, or non-human proteins, or have the same amino acid sequence as the human proteins or non-human proteins. A protein that is at least 80% identical and remains active,所述非人蛋白为小鼠，大鼠，牛，羊，马，犬，猫，猪，猴，鸡，鸭，鹅，鸟的相应蛋白；或者The non-human protein is the corresponding protein of mouse, rat, cow, sheep, horse, dog, cat, pig, monkey, chicken, duck, goose, or bird; or所述OCT4，SOX1，SOX2，SOX3，GMNN是编码人蛋白的核酸，或编码非人蛋白的核酸，或者是与对应的编码人蛋白的核酸，或编码非人蛋白的核酸序列具有至少80％同一性并保持活性的核酸， The OCT4, SOX1, SOX2, SOX3, GMNN is a nucleic acid encoding a human protein, or a nucleic acid encoding a non-human protein, or has at least 80% identity with the corresponding nucleic acid encoding a human protein, or a nucleic acid sequence encoding a non-human protein. nucleic acids that remain active and所述核酸是编码小鼠，大鼠，牛，羊，马，犬，猫，猪，猴，鸡，鸭，鹅，鸟的相应蛋白的核酸。The nucleic acid is a nucleic acid encoding the corresponding protein of mouse, rat, cow, sheep, horse, dog, cat, pig, monkey, chicken, duck, goose, and bird.
- 使受试者细胞、组织或器官恢复活力的方法，所述方法包括向受试者体内施用权利要求1-7任一项的组合物。A method of rejuvenating cells, tissues or organs of a subject, the method comprising administering to the subject a composition according to any one of claims 1-7.
- 权利要求1-7任一项的组合物在制备用于使受试者细胞、组织或器官恢复活力的药物中的应用。Use of the composition of any one of claims 1 to 7 in the preparation of a medicament for rejuvenating cells, tissues or organs of a subject.
- 根据权利要求8的方法或权利要求9的应用，所述使受试者细胞、组织或器官恢复活力包括恢复所述至少一种细胞，组织或器官中由于衰老，损伤，疾病或其任何组合而丢失的转录谱或表观遗传信息。The method according to claim 8 or the use of claim 9, said rejuvenating cells, tissues or organs of the subject comprising restoring the at least one cell, tissue or organ that is damaged by aging, injury, disease or any combination thereof. Lost transcriptional profiles or epigenetic information.
- 根据权利要求8的方法或权利要求9的应用，所述使受试者细胞、组织或器官恢复活力包括恢复所述细胞的功能、增加所述细胞的潜能、增强所述细胞的存活能力或增加所述细胞的复制能力或寿命，或其组合。The method according to claim 8 or the use of claim 9, said rejuvenating the cells, tissues or organs of the subject comprising restoring the function of the cells, increasing the potential of the cells, enhancing the viability of the cells or increasing The replicative capacity or longevity of the cell, or a combination thereof.
- 根据权利要求8的方法或权利要求9的应用，所述使受试者细胞、组织或器官恢复活力包括促进受试者神经元细胞轴突再生；使受试者皮肤的成纤维细胞增殖；促进受试者软骨细胞增殖；促进受试者肌肉干细胞增殖；逆转受试者皮肤成纤维细胞的衰老；逆转软骨细胞的衰老。The method according to claim 8 or the application of claim 9, wherein rejuvenating cells, tissues or organs of the subject includes promoting axon regeneration of neuronal cells in the subject; proliferating fibroblasts in the skin of the subject; promoting Proliferate the subjects' chondrocytes; promote the proliferation of the subjects' muscle stem cells; reverse the aging of the subjects' skin fibroblasts; reverse the aging of the chondrocytes.
- 权利要求1-7任一项的组合物，权利要求8的方法，权利要求9的应用，或权利要求10-12任一项的方法或应用，所述受试者患有，怀疑患有，或具风险患：The composition of any one of claims 1-7, the method of claim 8, the use of claim 9, or the method or use of any one of claims 10-12, the subject suffering from, suspected of suffering from, May be at risk of:与细胞、组织或器官活力受损或与衰老有关的疾病，病况或表征；Diseases, conditions or symptoms associated with impaired viability of cells, tissues or organs or associated with aging;所述与细胞、组织或器官活力受损或与衰老有关的疾病，病况或表征为神经退行性疾病，心血管疾病，代谢性疾病，肌肉骨骼疾病，炎性疾病，与皮肤有关的表征或病症，眼部疾病，或与生殖系统相关的疾病；The diseases, conditions or symptoms associated with impaired cell, tissue or organ vitality or aging are neurodegenerative diseases, cardiovascular diseases, metabolic diseases, musculoskeletal diseases, inflammatory diseases, skin-related symptoms or conditions , eye diseases, or diseases related to the reproductive system;优选地，所述神经退行性疾病为脑中风，亨廷顿舞蹈病，老年性痴呆，阿尔茨海默症，帕金森；Preferably, the neurodegenerative disease is stroke, Huntington's disease, Alzheimer's disease, Alzheimer's disease, and Parkinson's disease;优选地，所述心血管疾病为高血压，冠心病心律失常，心脏病，低血压，心力衰竭，心绞痛，动脉硬化，心肌梗死，心肌炎，充血性心力衰竭，房室传导阻滞，动脉粥样硬化，心肌梗塞；Preferably, the cardiovascular disease is hypertension, coronary heart disease arrhythmia, heart disease, hypotension, heart failure, angina pectoris, arteriosclerosis, myocardial infarction, myocarditis, congestive heart failure, atrioventricular block, atherosclerosis Sclerosis, myocardial infarction;优选地，所述代谢性疾病为I型糖尿病，I I型糖尿病，高血糖，高胰岛素血症，胰岛素抗性，肥胖，痛风；Preferably, the metabolic disease is type I diabetes, type II diabetes, hyperglycemia, hyperinsulinemia, insulin resistance, obesity, and gout;优选地，所述肌肉骨骼疾病为肌肉萎缩症，肌无力，重症肌无力，骨质疏松症，骨关节炎，骨软骨病，骨软骨炎，骨坏死，骨质稀少症；Preferably, the musculoskeletal disease is muscular dystrophy, myasthenia, myasthenia gravis, osteoporosis, osteoarthritis, osteochondrosis, osteochondritis, osteonecrosis, and osteopenia;优选地，所述炎性疾病为系统性红斑狼疮，风湿性多肌痛，痛风性关节炎，退行性关节炎，类风湿性关节炎，炎性关节炎，桥本甲状腺炎，炎性肠病，肝炎，肺炎，呼吸道炎症，脑炎；Preferably, the inflammatory disease is systemic lupus erythematosus, polymyalgia rheumatica, gouty arthritis, degenerative arthritis, rheumatoid arthritis, inflammatory arthritis, Hashimoto's thyroiditis, inflammatory bowel disease , hepatitis, pneumonia, respiratory inflammation, encephalitis;优选地，所述与皮肤有关的衰老表征或病症为皱纹，老年斑，脂溢性角化病，手脚癣，寻麻疹，神经性皮炎，色素痣；Preferably, the skin-related signs of aging or diseases are wrinkles, age spots, seborrheic keratosis, tinea pedis, measles, neurodermatitis, and nevus;优选地，所述眼部疾病为黄斑变性，视网膜变性，视网膜脱离，糖尿病性视网膜病，青光眼，视神经萎缩，飞蚊症，白内障，非动脉炎性前部缺血性视神经病变，脉络膜视网膜炎，色素性视网膜炎，外伤性视神经病变，压迫性视神经病变；Preferably, the eye disease is macular degeneration, retinal degeneration, retinal detachment, diabetic retinopathy, glaucoma, optic atrophy, floaters, cataracts, non-arteritic anterior ischemic optic neuropathy, chorioretinitis, Retinitis pigmentosa, traumatic optic neuropathy, compressive optic neuropathy;优选地，与生殖系统相关的疾病为勃起功能障碍以及卵巢早衰。 Preferably, the diseases related to the reproductive system are erectile dysfunction and premature ovarian failure.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210815986.2 | 2022-07-12 | ||
| CN202210815986 | 2022-07-12 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2024012502A1 true WO2024012502A1 (en) | 2024-01-18 |
Family
ID=89535633
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2023/107076 WO2024012502A1 (en) | 2022-07-12 | 2023-07-12 | Composition and use method therefor |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2024012502A1 (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016043410A1 (en) * | 2014-09-18 | 2016-03-24 | 서울대학교산학협력단 | Mesenchymal stem cells overexpressing oct4 and sox2 and use thereof |
| CN107810268A (en) * | 2015-05-19 | 2018-03-16 | 思特科技公司 | By directly reprogramming the method for inducing oligodendrocyte precursors by the human body cell for introducing Oct4 |
| US20180346933A1 (en) * | 2015-11-23 | 2018-12-06 | The Regents Of The University Of Colorado, A Body Corporate | Methods and compositions for reprogramming cells |
| CN112154210A (en) * | 2018-03-13 | 2020-12-29 | 利兰斯坦福初级大学董事会 | Transient cell reprogramming for reversing cell aging |
| CN113453702A (en) * | 2018-09-28 | 2021-09-28 | 哈佛大学的校长及成员们 | Cell reprogramming to reverse senescence and promote tissue and tissue regeneration |
| CN115247152A (en) * | 2022-09-21 | 2022-10-28 | 呈诺再生医学科技(北京)有限公司 | Method for preparing hematopoietic stem cells or hematopoietic stem and progenitor cells and method for culturing long-term regenerative hematopoietic stem cells |
-
2023
- 2023-07-12 WO PCT/CN2023/107076 patent/WO2024012502A1/en unknown
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016043410A1 (en) * | 2014-09-18 | 2016-03-24 | 서울대학교산학협력단 | Mesenchymal stem cells overexpressing oct4 and sox2 and use thereof |
| CN107810268A (en) * | 2015-05-19 | 2018-03-16 | 思特科技公司 | By directly reprogramming the method for inducing oligodendrocyte precursors by the human body cell for introducing Oct4 |
| US20180346933A1 (en) * | 2015-11-23 | 2018-12-06 | The Regents Of The University Of Colorado, A Body Corporate | Methods and compositions for reprogramming cells |
| CN112154210A (en) * | 2018-03-13 | 2020-12-29 | 利兰斯坦福初级大学董事会 | Transient cell reprogramming for reversing cell aging |
| CN113453702A (en) * | 2018-09-28 | 2021-09-28 | 哈佛大学的校长及成员们 | Cell reprogramming to reverse senescence and promote tissue and tissue regeneration |
| CN115247152A (en) * | 2022-09-21 | 2022-10-28 | 呈诺再生医学科技(北京)有限公司 | Method for preparing hematopoietic stem cells or hematopoietic stem and progenitor cells and method for culturing long-term regenerative hematopoietic stem cells |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Taghipour et al. | Transplantation of undifferentiated and induced human exfoliated deciduous teeth-derived stem cells promote functional recovery of rat spinal cord contusion injury model | |
| Guo et al. | Cotransplant of neural stem cells and NT-3 gene modified Schwann cells promote the recovery of transected spinal cord injury | |
| US20060247195A1 (en) | Method of altering cell properties by administering rna | |
| Wijeyekoon et al. | Cell replacement therapy for Parkinson's disease | |
| US20020098584A1 (en) | Postmortem stem cells | |
| KR20060037346A (en) | Oligodendrocyte precursor cells and methods of obtaining and culturing the same | |
| US20220213502A1 (en) | Methods and Compositions for Selective Generation of Dopaminergic Precursors | |
| US20160011177A1 (en) | Screening method for therapeutic agents for charcot-marie-tooth disease and self-differentiation motor neurons used therefor | |
| JP2004511266A (en) | Therapeutic uses for mesenchymal stromal cells | |
| Cantini et al. | Gene transfer into satellite cell from regenerating muscle: bupivacaine allows β-gal transfection and expression in vitro and in vivo | |
| Mellott et al. | Nonviral reprogramming of human wharton's jelly cells reveals differences between ATOH1 homologues | |
| Upadhyay | Role of regeneration in tissue repairing and therapies | |
| WO2024012502A1 (en) | Composition and use method therefor | |
| Yuan et al. | Small molecule epigenetic modulators in pure chemical cell fate conversion | |
| WO2024012504A1 (en) | Composition and method for using same | |
| WO2024012503A1 (en) | Composition and use method therefor | |
| WO2019014553A1 (en) | Induction of neural progenitor cells, oligodendrocyte progenitor cells, and oligodendrocytes by stem cell differentiation using landmark transcription factors | |
| CN111484977B (en) | Method of reprogramming to produce functional noradrenergic neurons | |
| BR112021003791A2 (en) | composition and method for direct reprogramming of urinary cells into neural stem cells, and pharmaceutical composition. | |
| US10421944B2 (en) | Method for producing neural stem cells using synthetic peptide | |
| CA2595528A1 (en) | Method of treatment by administration of rna | |
| WO2024071382A1 (en) | Pluripotential stem cell and cell differentiated and induced from stem cell having glp-1 secretion function | |
| KR102594254B1 (en) | Factor for direct Conversion into motor neuron | |
| JP7300764B2 (en) | Gene and cell therapy agents using cell fusion technology, and uses thereof | |
| US20060263876A1 (en) | Neural crest stem cells and uses thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 23838992 Country of ref document: EP Kind code of ref document: A1 |