WO2024012503A1 - 组合物及其使用方法 - Google Patents
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- WO2024012503A1 WO2024012503A1 PCT/CN2023/107077 CN2023107077W WO2024012503A1 WO 2024012503 A1 WO2024012503 A1 WO 2024012503A1 CN 2023107077 W CN2023107077 W CN 2023107077W WO 2024012503 A1 WO2024012503 A1 WO 2024012503A1
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- 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
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- 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 of a subject, and a method for using the composition to rejuvenate the cells, tissues or organs of a subject.
- the composition and the method can rejuvenate the cells, tissues or organs of a subject. Rejuvenate or restore function to the subject's cells, tissues or organs.
- 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 of a subject, the composition comprising any factor selected from the group consisting of SOX1, SOX2, SOX3 and GMNN, and selected from the group consisting of GATA3 and GATA6 Any of the factors, and factor Klf4.
- the composition includes GATA6, SOX2 and Klf4.
- the composition includes GATA6, SOX1 and Klf4.
- the composition includes GATA3, SOX2 and Klf4.
- the composition includes GATA3, SOX1 and Klf4.
- the composition includes GATA6, SOX3 and Klf4.
- the composition includes GATA6, GMNN and Klf4.
- the composition includes GATA3, SOX3 and Klf4.
- the composition includes GATA3, GMNN and Klf4.
- the factor is a protein, a nucleic acid encoding the same, or a mixture thereof.
- the SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, and Klf4 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 SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, Klf4.
- 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 SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, Klf4 is a human protein, or a non-human protein, e.g., a mammal (e.g., mouse, rat, cow, sheep, horse, canine , cat, pig, 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, cow, sheep, horse, canine , cat, pig, monkey, chicken, duck, goose, bird
- the SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, Klf4, or any combination thereof is a nucleic acid encoding a human protein, or a nucleic acid encoding a non-human protein, e.g., encoding a mammalian (e.g., mouse , rat, cow, sheep, horse, dog, cat, pig, monkey, chicken, duck, goose, bird), or a corresponding nucleic acid encoding a human protein, or a nucleic acid sequence encoding a non-human protein Nucleic acids that are at least 80% identical and remain active.
- a mammalian e.g., mouse , rat, cow, sheep, horse, dog, cat, pig, monkey, chicken, duck, goose, bird
- 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 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 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 neuronal regeneration in a subject.
- the composition can proliferate fibroblasts in the subject's skin.
- 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 a subject's skin fibroblasts.
- the composition can reverse chondrocyte senescence.
- the composition does not induce teratoma formation or does not induce tumor growth. or tumor formation.
- the composition does not induce complete reprogramming.
- the composition does not reprogram cells into induced pluripotent stem cells (ipsc).
- 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.
- composition includes any one factor selected from SOX1, SOX2, SOX3 and GMNN, any one factor selected from GATA3 and GATA6, and factor Klf4.
- the composition includes GATA6, SOX2 and Klf4.
- the composition includes GATA6, SOX1 and Klf4.
- the composition includes GATA3, SOX2 and Klf4.
- the composition includes GATA3, SOX1 and Klf4.
- the composition includes GATA6, SOX3 and Klf4.
- the composition includes GATA6, GMNN and Klf4.
- the composition includes GATA3, SOX3 and Klf4.
- the composition includes GATA3, GMNN and Klf4.
- the factor is a polypeptide, a nucleic acid encoding the same, or a mixture thereof.
- SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, and Klf4 are proteins, fusion proteins combined with their membrane-permeable domains, 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 SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, Klf4.
- 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.
- promoter-regulated expression of factors associated with aging can be reduced and reversed.
- Related epigenetic marks increase epigenetic marks related to cell rejuvenation, reduce the expression of senescence-related proteins, increase the expression of proteins related to cell rejuvenation, restore the balance between euchromatin and heterochromatin, Prevents loss of cell identity, restores cell identity, and reverses aging-related DNA methylation changes, thereby rejuvenating cells.
- the SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, Klf4 is a human protein, or a non-human protein, e.g., a mammal (e.g., mouse, rat, cow, sheep, horse, canine , cat, pig, 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, cow, sheep, horse, canine , cat, pig, monkey, chicken, duck, goose, bird
- the SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, Klf4, or any combination thereof is a nucleic acid encoding a human protein, or a nucleic acid encoding a non-human protein, e.g., encoding a mammalian (e.g., mouse , rat, cow, sheep, horse, dog, cat, pig, monkey, chicken, duck, goose, bird), or a corresponding nucleic acid encoding a human protein, or a nucleic acid sequence encoding a non-human protein Nucleic acids that are at least 80% identical and remain active.
- a mammalian 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 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 fibroblasts in the subject's skin.
- 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 method does not induce teratoma formation, or does not induce tumor growth or tumor formation.
- the methods do not induce complete reprogramming.
- the methods do not reprogram the cells to a pluripotent state, or the methods do not reprogram the cells into "induced pluripotent stem cells" or "iPSCs.”
- OCT4, SOX2, KLF4 and c-Myc are four "Yamanaka factors" capable of reprogramming cells into a pluripotent state.
- induction of expression of the four transcription factors in transgenic mice resulted in teratoma formation in vivo, among other acute toxicities.
- the present invention unexpectedly found that in the absence of OCT4 and c-Myc, any factor selected from SOX1, SOX2, SOX3 and GMNN, any factor selected from GATA3 and GATA6, and KLF4, that is, These three factors can 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, transforms senescent cells into young ones. cell.
- 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 SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, and Klf4.
- Figure 1 shows that the composition of the present invention can promote neuronal cell regeneration.
- Figure 2 shows that the composition of the present invention can promote the proliferation of aging 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.
- GATA3 is named A1, GATA6 is named A2; SOX1 is named B1, SOX2 is named B2, SOX3 is named B3, GMNN is named B4; Klf4 is named C.
- the SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, and Klf4 used in the examples are all human SOX1, human SOX2, human SOX3, human GMNN, human GATA3, human GATA6, and human Klf4.
- 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.
- 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 then 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
- 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.
- mice 20-month-old mice were selected. After the mice were euthanized, the shaved back skin was removed. Before the skin was removed, it was washed with iodophor (povidone-iodine) and rinsed with cold 1XPBS.
- 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.
- the combination of A1/A2 with B1/B2/B3/B4 and C can promote the proliferation of aging skin fibroblasts.
- Example 3 Composition promotes proliferation of mouse chondrocytes
- the main purpose of this embodiment is to select elderly mice, isolate chondrocytes, and observe whether the chondrocytes have stronger proliferation ability.
- 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 proliferation ability of chondrocytes was detected 6 days after administration.
- the combination of A1/A2 with B1/B2/B3/B4 and C can promote the proliferation of aging 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).
- DMEM DMEM
- ThermoFisher Scientific ThermoFisher Scientific
- CEE chicken embryo extract
- the obtained stem cells were directly plated on Matrigel growth factor (GFR) basement membrane matrix (Corning) on Petri dishes or Petri dishes containing coverslips coated with Matrigel GFR basement membrane matrix.
- GFR Matrigel growth factor
- Cells were grown in agar containing 1 g/l glucose supplemented with 10% HS, 20% FBS, 1% ps, and 0.5% CEE. Expand in DMEM and culture at 37°C and 5% CO2 . The culture medium is replaced every 2 days. After 6 days of treatment with different compositions, the proliferation ability of the cells is observed.
- the combination of A1/A2 with B1/B2/B3/B4 and C can promote the proliferation of aging muscle stem cells.
- Example 5 Composition reverses aging of skin fibroblasts in aged mice
- the main purpose of this example 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 changes.
- the specific methods and results are as follows:
- the combination of A1/A2 with B1/B2/B3/B4 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:
- the combination of A1/A2 with B1/B2/B3/B4 and C can significantly reverse the aging of chondrocytes.
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Abstract
本发明提供了基于三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。
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。
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。
2016年之后，科学家发现4个转录因子(Oct4、Sox2、Klf4和c-My)也能够使得衰老细胞重编程为年轻细胞。然而，现有可实现抗衰老或恢复细胞活力的重编程因子组合比较局限，安全性有待进一步提高。因此，本领域亟需更多、更安全的使受试者细胞、组织或器官恢复活力的重编程因子。
发明内容
本发明涉及一种用于受试者细胞、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。
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，所述组合物包含选自SOX1，SOX2，SOX3和GMNN中的任一种因子，选自GATA3和GATA6中的任一种因子，和因子Klf4。
在一些实施方案中，所述组合物包含GATA6，SOX2和Klf4。
在一些实施方案中，所述组合物包含GATA6，SOX1和Klf4。
在一些实施方案中，所述组合物包含GATA3，SOX2和Klf4。
在一些实施方案中，所述组合物包含GATA3，SOX1和Klf4。
在一些实施方案中，所述组合物包含GATA6，SOX3和Klf4。
在一些实施方案中，所述组合物包含GATA6，GMNN和Klf4。
在一些实施方案中，所述组合物包含GATA3，SOX3和Klf4。
在一些实施方案中，所述组合物包含GATA3，GMNN和Klf4。
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。
例如，所述SOX1，SOX2，SOX3，GMNN，GATA3，GATA6，Klf4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。
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。
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。
在一些实施方案中，所述因子存在于同一表达载体上。
在一些实施方案中，所述因子为mRNA的形式。
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。
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SOX1，SOX2，SOX3，GMNN，GATA3，GATA6，Klf4的野生型形式，突变体，基因工程化形式。
在一些实施方案中，所述组合物还包含可药用载体或佐剂。
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。
在一些实施方案中，所述受试者为鸡，鸭，鹅或鸟。
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。
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。
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。
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。
在一些实施方案中，所述SOX1，SOX2，SOX3，GMNN，GATA3，GATA6，Klf4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。
在一些实施方案中，所述SOX1，SOX2，SOX3，GMNN，GATA3，GATA6，Klf4或其任何组合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。
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。
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。
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；逆转受试者的皮肤成纤维细胞的衰老；逆转软骨细胞的衰老。
在一些实施方案中，所述受试者患有，怀疑患有，或具风险患：
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；
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；
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；
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；
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；
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；
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；
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；
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；
与生殖系统相关的疾病为勃起功能障碍以及卵巢早衰。
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。
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。
在一些实施方案中，所述组合物可以促进受试者神经元再生。
在一些实施方案中，所述组合物可以使受试者皮肤的成纤维细胞增殖。
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。
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。
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。
在一些实施方案中，所述组合物可以逆转软骨细胞的衰老。
在一些实施方案中，所述组合物不诱导畸胎瘤的形成或者不诱导肿瘤的生长
或肿瘤的形成。
在一些实施方案中，所述组合物不诱导完全重编程。
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)。
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。
所述组合物包含选自SOX1，SOX2，SOX3和GMNN中的任一种因子，选自GATA3和GATA6中的任一种因子，和因子Klf4。
在一些实施方案中，所述组合物包含GATA6，SOX2和Klf4。
在一些实施方案中，所述组合物包含GATA6，SOX1和Klf4。
在一些实施方案中，所述组合物包含GATA3，SOX2和Klf4。
在一些实施方案中，所述组合物包含GATA3，SOX1和Klf4。
在一些实施方案中，所述组合物包含GATA6，SOX3和Klf4。
在一些实施方案中，所述组合物包含GATA6，GMNN和Klf4。
在一些实施方案中，所述组合物包含GATA3，SOX3和Klf4。
在一些实施方案中，所述组合物包含GATA3，GMNN和Klf4。
在一些实施方案中，所述因子为多肽，编码其的核酸或其混合物。
例如，所述SOX1，SOX2，SOX3，GMNN，GATA3，GATA6，Klf4为蛋白，与其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。
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。
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。
在一些实施方案中，所述因子存在于同一表达载体上。
在一些实施方案中，所述因子为mRNA的形式。
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。
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SOX1，SOX2，SOX3，GMNN，GATA3，GATA6，Klf4的野生型形式，突变体，基因工程化形式。
在一些实施方案中，所述组合物还包含可药用载体或佐剂。
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。
在一些实施方案中，所述受试者为鸡，鸭，鹅或鸟。
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。
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。
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。
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。
在一些实施方案中，所述SOX1，SOX2，SOX3，GMNN，GATA3，GATA6，Klf4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。
在一些实施方案中，所述SOX1，SOX2，SOX3，GMNN，GATA3，GATA6，Klf4或其任何组合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。
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。
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。
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。
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。
在一些实施方案中，所述受试者患有，怀疑患有，或具风险患：
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；
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；
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；
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；
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；
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；
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；
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；
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；
与生殖系统相关的疾病为勃起功能障碍以及卵巢早衰。
。
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。
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。
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。
在一些实施方案中，所述方法可以使受试者皮肤的成纤维细胞增殖。
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。
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。
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。
在一些实施方案中，所述组合物可以逆转软骨细胞的衰老。
在一些实施方案中，所述方法不诱导畸胎瘤的形成，或者不诱导肿瘤的生长或肿瘤的形成。
在一些实施方案中，所述方法不诱导完全重编程。
在一些实施方案中，所述方法不将细胞重新编程为多能状态，或者是所述方法不将细胞重新编程为“经诱导的多潜能干细胞”或“iPSC”。
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。然而，在转基因小鼠中诱导四种转录因子表达会导致体内畸胎瘤的形成，以及其他急性毒性。
本发明预料之外地发现，在不存在OCT4和c-Myc的情况下，选自SOX1，SOX2，SOX3和GMNN的任一种因子，选自GATA3和GATA6的任一种因子，以及KLF4，也就是这三种因子就可以实现衰老细胞的逆转。
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，将衰老细胞转变为年轻细胞。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。
本发明中的因子可以是转录因子，包括但不限于SOX1，SOX2，SOX3，GMNN，GATA3，GATA6，Klf4。
图1显示了本发明的组合物可以促进神经元细胞再生。
图2显示了本发明的组合物可以促进衰老皮肤成纤维细胞增殖。
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。
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。
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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。
图6显示了本发明的组合物可以逆转软骨细胞的衰老。
以下实施例以及附图中：
GATA3命名为A1，GATA6命名为A2；SOX1命名为B1，SOX2命名为B2，SOX3命名为B3，GMNN命名为B4；Klf4命名为C。
在实施例中所采用的SOX1，SOX2，SOX3，GMNN，GATA3，GATA6，Klf4均为人SOX1，人SOX2，人SOX3，人GMNN，人GATA3，人GATA6，人Klf4。
实施例1组合物促进神经元细胞轴突再生
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：
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天以促进细胞的分化成熟。分化完成后，用组合物mRNA处理细胞5天，然后加入长春新碱(100nM；Sigma，V8879)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。
如图1所示，A1/A2与B1/B2/B3/B4和C组合可以促进神经突面积的增加。实施例2组合物促进老年小鼠皮肤成纤维细胞的增殖
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：
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1XPBS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。
如图2所示，A1/A2与B1/B2/B3/B4和C组合可以促进衰老皮肤成纤维细胞的增殖。
实施例3组合物促进小鼠软骨细胞的增殖
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：
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天后检测软骨细胞的增殖能力。
如图3所示，A1/A2与B1/B2/B3/B4和C组合可以促进衰老软骨细胞的增殖。
实施例4组合物促进老年小鼠肌肉干细胞的增殖
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：
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。
如图4所示，A1/A2与B1/B2/B3/B4和C组合可以促进衰老肌肉干细胞的增殖。
实施例5组合物逆转老年小鼠皮肤成纤维细胞的衰老
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：
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％)。
如图5所示，A1/A2与B1/B2/B3/B4和C组合可以显著逆转老年小鼠皮肤成纤维细胞的衰老。
实施例6组合物逆转软骨细胞的的衰老
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：
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％)。
如图6所示，A1/A2与B1/B2/B3/B4和C组合可以显著逆转软骨细胞的衰老。
Claims (12)
- 用于受试者细胞、组织或器官恢复活力的组合物，所述组合物包含选自SOX1，SOX2，SOX3和GMNN中的任一种因子，选自GATA3和GATA6中的任一种因子，和因子Klf4。
- 根据权利要求1的组合物，其特征在于，所述因子SOX1，SOX2，SOX3，GMNN，GATA3，GATA6，Klf4为蛋白，编码其的核酸或其混合物，优选地，所述因子SOX1，SOX2，SOX3，GMNN，GATA3，GATA6，Klf4为蛋白，与透膜结构域结合的融合蛋白，其编码核酸的形式。
- 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SOX1，SOX2，SOX3，GMNN，GATA3，GATA6，Klf4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，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，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。
- 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SOX1，SOX2，SOX3，GMNN，GATA3，GATA6，Klf4为DNA或RNA的形式，优选地，所述因子为mRNA的形式,所述因子串联在同一mRNA分子上，或者所述因子SOX1，SOX2，SOX3，GMNN，GATA3，GATA6，Klf4为不同mRNA分子的复方形式，优选地，所述因子为合成的mRNA，所述合成的mRNA编码选自SOX1，SOX2，SOX3，GMNN，GATA3，GATA6，Klf4的野生型形式，突变体，基因工程化形式。
- 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，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。
- 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SOX1，SOX2，SOX3，GMNN，GATA3，GATA6，Klf4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，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；或者所述因子SOX1，SOX2，SOX3，GMNN，GATA3，GATA6，Klf4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，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。
- 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1-6任一项的组合物。
- 权利要求1-6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。
- 根据权利要求7的方法或权利要求8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。
- 根据权利要求7的方法或权利要求8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。
- 根据权利要求7的方法或权利要求8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。
- 权利要求1-6任一项的组合物，权利要求7的方法，权利要求8的应用，或权利要求9-11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：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；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；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；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；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；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；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；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；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；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。
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| WO2020069373A1 (en) * | 2018-09-28 | 2020-04-02 | President And Fellows Of Harvard College | Cellular reprogramming to reverse aging and promote organ and tissue regeneration |
| CN113453702A (zh) * | 2018-09-28 | 2021-09-28 | 哈佛大学的校长及成员们 | 细胞重编程以逆转衰老并促进组织和组织再生 |
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