Compositions and methods of use thereof

Abstract

The present invention provides a composition for vitality restoration of cells, tissues or organs of a subject based on three factors, and a method for vitality restoration of cells, tissues or organs of a subject using the composition, the composition and method being capable of vitality restoration or function restoration of cells, tissues or organs of a subject. The compositions and methods are useful for any disease or injury requiring repair or replacement of functional failure tissue.

Description

Compositions and methods of use thereof Background Art

[0001] All living things will experience aging, which is accompanied by a decline in the vitality of cell and tissue function. Aging is characterized by a gradual loss of function at the molecular, cellular, tissue and organismal levels. The main characteristics include problems such as telomere attrition, genetic instability, epigenetic and transcriptional changes, and the accumulation of erroneous proteins. In many animals, including vertebrates, the regeneration and repair capacity of important organs decreases sharply with aging and the occurrence of disease. Unlike adult somatic cells, individual cells that are chronologically closer to the fertilization period (such as cells of embryos and infants) show a youthful state of cells and have a greater ability to resist damage and stress, and are able to heal, renew and regenerate organs and tissues. Therefore, compositions and methods for rejuvenating cells to restore them from an aging mature state to a younger, more energetic state of life have long been sought for the treatment of certain injuries and diseases, as well as for the overall reversal and prevention of aging of the entire organism.

[0002] In 2006, Professor Shinya Yamanaka of Kyoto University in Japan developed induced pluripotent stem cell (iPSC) technology, which can reverse telomere attrition and oxidative stress in cells. This groundbreaking cell technology, in short, reprograms cells by inducing four transcription factors (Oct4, Sox2, Klf4, and c-Myc). Using a viral vector, this combination of transcription factors (Oct4, Sox2, Klf4, and c-Myc) is transferred into differentiated somatic cells, reprogramming them to a cell type similar to embryonic stem cells. This reprograms the somatic cells to a state similar to embryonic stem cells. These cells, similar to human embryonic stem cells, possess exceptional differentiation capacity, capable of giving rise to human blood cells, bone cells, neurons, and other cells, and can subsequently be used to culture human organs, including bones, corneas, and pancreas. It holds enormous promise for applications in disease modeling, drug screening, and cell therapy.

[0003] Since 2016, scientists have discovered that four transcription factors (Oct4, Sox2, Klf4, and c-My) can also reprogram senescent cells into younger ones. However, the current combinations of reprogramming factors that can achieve anti-aging or cell rejuvenation are limited, and their safety needs to be further improved. Therefore, the field urgently needs more and safer reprogramming factors that can rejuvenate cells, tissues, or organs in the subject.

[0004] Summary of the Invention

[0005] The present invention relates to a composition for rejuvenating cells, tissues, or organs in a subject, and a method for rejuvenating cells, tissues, or organs in a subject using the composition. The composition and method are capable of rejuvenating or restoring the function of cells, tissues, or organs in a subject. The composition and method can be used for any disease or injury requiring the repair or replacement of dysfunctional tissue.

[0006] In a first aspect, the present invention provides a composition for rejuvenating cells, tissues or organs of a subject, the composition comprising any one factor selected from SOX1, SOX2, SOX3 and GMNN, any one factor selected from GATA3 and GATA6, and factor Klf4.

[0007] In some embodiments, the composition comprises GATA6, SOX2, and Klf4.

[0008] In some embodiments, the composition comprises GATA6, SOX1, and Klf4.

[0009] In some embodiments, the composition comprises GATA3, SOX2, and Klf4.

[0010] In some embodiments, the composition comprises GATA3, SOX1, and Klf4.

[0011] In some embodiments, the composition comprises GATA6, SOX3, and Klf4.

[0012] In some embodiments, the composition comprises GATA6, GMNN, and Klf4.

[0013] In some embodiments, the composition comprises GATA3, SOX3, and Klf4.

[0014] In some embodiments, the composition comprises GATA3, GMNN, and Klf4.

[0015] In some embodiments, the factor is a protein, a nucleic acid encoding the same, or a mixture thereof.

[0016] For example, the SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, and Klf4 are proteins, fusion proteins combined with a membrane-permeable domain, and are encoded by nucleic acids, such as DNA or RNA.

[0017] In some embodiments, the factor is 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), a lentivirus vector, a Sendai virus vector, a cytomegalovirus vector or a vaccinia virus vector.

[0018] In some embodiments, the factors are present on one or more expression vectors.

[0019] In some embodiments, the factors are present on the same expression vector.

[0020] In some embodiments, the factor is in the form of mRNA.

[0021] In some embodiments, the factors are tandemly linked to the same mRNA molecule, or each of the factors is linked to a different mRNA molecule, and the factors are in the form of a complex of multiple mRNA molecules.

[0022] In some embodiments, the factor is a synthetic mRNA encoding a wild-type form, a mutant form, or a genetically engineered form of a gene selected from the group consisting of SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, and Klf4.

[0023] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or adjuvant.

[0024] In some embodiments, the subject is a mammal, including but not limited to humans, mice, rats, cows, sheep, horses, dogs, cats, pigs, or monkeys.

[0025] In some embodiments, the subject is a chicken, duck, goose, or bird.

[0026] In some embodiments, the subject cell, tissue or organ is from, but is not limited to, the eye, ear, nose, mouth, including gums and tooth roots; bone, lung, breast, pancreas, stomach, esophagus; muscle, including myocardium; liver, blood vessels; skin, including hair; heart, brain, nervous tissue, kidney, testicle, prostate, penis, cloaca, fin, ovary or intestine.

[0027] 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.

[0028] In some embodiments, the composition for rejuvenating cells, tissues, or organs in a subject is capable of restoring epigenetic information lost in the cells, tissues, or organs of the subject due to aging, injury, or disease.

[0029] In some embodiments, promoter expression can be used to reduce and reverse epigenetic marks associated with aging, increase epigenetic marks associated with cell rejuvenation, reduce the expression of aging-associated proteins, increase the expression of proteins associated with cell rejuvenation, restore the balance between euchromatin and heterochromatin, prevent the loss of cell identity, restore cell identity, reverse aging-associated DNA methylation changes, and thus rejuvenate cells.

[0030] In some embodiments, the SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, Klf4 is a human protein, or a non-human protein, for example, a mammalian (e.g., mouse, rat, cow, sheep, horse, dog, cat, pig, monkey, chicken, duck, goose, bird) protein, 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.

[0031] In some embodiments, 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, for example, a nucleic acid encoding a protein of a mammal (e.g., mouse, rat, cow, sheep, horse, dog, cat, pig, monkey, chicken, duck, goose, bird), or a nucleic acid that has at least 80% identity with the corresponding nucleic acid encoding a human protein, or a nucleic acid sequence encoding a non-human protein and maintains activity.

[0032] In some embodiments, rejuvenating a cell, tissue, or organ of a subject comprises restoring a transcriptional profile or epigenetic information lost in the at least one cell, tissue, or organ due to aging, injury, disease, or any combination thereof.

[0033] In some embodiments, rejuvenating the subject's cells, tissues, or organs comprises restoring the function of the cells, increasing the potency of the cells, enhancing the viability of the cells, or increasing the replicative capacity or lifespan of the cells, or a combination thereof.

[0034] In some embodiments, the rejuvenation of the subject's cells, tissues, or organs comprises promoting axon regeneration in the subject's neuronal cells; promoting proliferation of the subject's skin fibroblasts; promoting proliferation of the subject's chondrocytes; promoting proliferation of the subject's muscle stem cells; reversing aging of the subject's skin fibroblasts; and reversing aging of chondrocytes.

[0035] In some embodiments, the subject has, is suspected of having, or is at risk of having:

[0036] Diseases, conditions or manifestations associated with impaired cell, tissue or organ vitality or aging;

[0037] The diseases, conditions or symptoms associated with impaired cell, tissue or organ vitality or aging are, for example, neurodegenerative diseases, cardiovascular diseases, metabolic diseases, musculoskeletal diseases, inflammatory diseases, symptoms or disorders associated with the skin, eye diseases, or diseases associated with the reproductive system;

[0038] The neurodegenerative disease is cerebral stroke, Huntington's disease, senile dementia, Alzheimer's disease, Parkinson's disease;

[0039] 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, myocardial infarction;

[0040] The metabolic disease is type I diabetes, type II diabetes, hyperglycemia, hyperinsulinemia, insulin resistance, obesity, gout;

[0041] The musculoskeletal disease is muscular dystrophy, myasthenia gravis, osteoporosis, osteoarthritis, osteochondrosis, osteochondritis, osteonecrosis, and osteopenia;

[0042] 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 tract inflammation, encephalitis;

[0043] The skin-related aging symptoms or diseases are wrinkles, age spots, seborrheic keratosis, tinea pedis, urticaria, neurodermatitis, and pigmented nevi;

[0044] The eye disease is macular degeneration, retinal degeneration, retinal detachment, diabetic retinopathy, glaucoma, optic atrophy, floaters, cataract, non-arteritic anterior ischemic optic neuropathy, chorioretinitis, retinitis pigmentosa, traumatic optic neuropathy, compressive optic neuropathy;

[0045] Diseases related to the reproductive system include erectile dysfunction and premature ovarian failure.

[0046] 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, reduces scar formation, alleviates external manifestations of aging including alopecia, hair thinning, hair graying, skin sagging and skin wrinkling, promotes organ regeneration, promotes organ survival, treats disease, or any combination thereof.

[0047] In some embodiments, the composition reverses aging of a cell, tissue, organ, or subject, thereby rejuvenating the cell, tissue, or organ.

[0048] In some embodiments, the composition can promote neuronal regeneration in a subject.

[0049] In some embodiments, the composition can cause fibroblasts in the subject's skin to proliferate.

[0050] In some embodiments, the composition can promote chondrocyte proliferation in a subject.

[0051] In some embodiments, the composition can promote the proliferation of muscle stem cells in a subject.

[0052] In some embodiments, the methods can reverse senescence of skin fibroblasts in a subject.

[0053] In some embodiments, the composition can reverse senescence of chondrocytes.

[0054] In some embodiments, the composition does not induce teratoma formation or does not induce tumor growth or tumor formation.

[0055] In some embodiments, the composition does not induce complete reprogramming.

[0056] In some embodiments, the composition does not reprogram cells into induced pluripotent stem cells (iPSCs).

[0057] 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.

[0058] The composition comprises any one factor selected from SOX1, SOX2, SOX3 and GMNN, any one factor selected from GATA3 and GATA6, and factor Klf4.

[0059] In some embodiments, the composition comprises GATA6, SOX2, and Klf4.

[0060] In some embodiments, the composition comprises GATA6, SOX1, and Klf4.

[0061] In some embodiments, the composition comprises GATA3, SOX2, and Klf4.

[0062] In some embodiments, the composition comprises GATA3, SOX1, and Klf4.

[0063] In some embodiments, the composition comprises GATA6, SOX3, and Klf4.

[0064] In some embodiments, the composition comprises GATA6, GMNN, and Klf4.

[0065] In some embodiments, the composition comprises GATA3, SOX3, and Klf4.

[0066] In some embodiments, the composition comprises GATA3, GMNN, and Klf4.

[0067] In some embodiments, the factor is a polypeptide, a nucleic acid encoding the same, or a mixture thereof.

[0068] For example, the SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, and Klf4 are proteins, or fusion proteins bound to their membrane-permeable domains, which encode nucleic acids, such as DNA or RNA.

[0069] In some embodiments, the factor is 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), a lentivirus vector, a Sendai virus vector, a cytomegalovirus vector or a vaccinia virus vector.

[0070] In some embodiments, the factors are present on one or more expression vectors.

[0071] In some embodiments, the factors are present on the same expression vector.

[0072] In some embodiments, the factor is in the form of mRNA.

[0073] In some embodiments, the factors are tandemly linked to the same mRNA molecule, or each of the factors is linked to a different mRNA molecule, and the factors are in the form of a complex of multiple mRNA molecules.

[0074] In some embodiments, the factor is a synthetic mRNA encoding a wild-type form, a mutant form, or a genetically engineered form of a gene selected from the group consisting of SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, and Klf4.

[0075] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or adjuvant.

[0076] In some embodiments, the subject is a mammal, including but not limited to humans, mice, rats, cows, sheep, horses, dogs, cats, pigs, or monkeys.

[0077] In some embodiments, the subject is a chicken, duck, goose, or bird.

[0078] In some embodiments, the subject cell, tissue or organ is from, but is not limited to, the eye, ear, nose, mouth, including gums and tooth roots; bone, lung, breast, pancreas, stomach, esophagus; muscle, including myocardium; liver, blood vessels; skin, including hair; heart, brain, nervous tissue, kidney, testicle, prostate, penis, cloaca, fin, ovary or intestine.

[0079] 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.

[0080] In some embodiments, the composition for rejuvenating cells, tissues, or organs in a subject is capable of restoring epigenetic information lost in the cells, tissues, or organs of the subject due to aging, injury, or disease.

[0081] In some embodiments, promoter expression can be used to reduce and reverse epigenetic marks associated with aging, increase epigenetic marks associated with cell rejuvenation, reduce the expression of aging-associated proteins, increase the expression of proteins associated with cell rejuvenation, restore the balance between euchromatin and heterochromatin, prevent the loss of cell identity, restore cell identity, reverse aging-associated DNA methylation changes, and thus rejuvenate cells.

[0082] In some embodiments, the SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, Klf4 is a human protein, or a non-human protein, for example, a mammalian (e.g., mouse, rat, cow, sheep, horse, dog, cat, pig, monkey, chicken, duck, goose, bird) protein, 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.

[0083] In some embodiments, 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, for example, a nucleic acid encoding a protein of a mammal (e.g., mouse, rat, cow, sheep, horse, dog, cat, pig, monkey, chicken, duck, goose, bird), or a nucleic acid that has at least 80% identity with the corresponding nucleic acid encoding a human protein, or a nucleic acid sequence encoding a non-human protein and maintains activity.

[0084] 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.

[0085] In some embodiments, rejuvenating a cell, tissue, or organ of a subject comprises restoring a transcriptional profile or epigenetic information lost in the at least one cell, tissue, or organ due to aging, injury, disease, or any combination thereof.

[0086] In some embodiments, rejuvenating the subject's cells, tissues, or organs comprises restoring the function of the cells, increasing the potency of the cells, enhancing the viability of the cells, or increasing the replicative capacity or lifespan of the cells, or a combination thereof.

[0087] In some embodiments, the rejuvenation of the subject's cells, tissues, or organs comprises promoting axon regeneration in the subject's neuronal cells; proliferation of the subject's skin fibroblasts; proliferation of the subject's chondrocytes; proliferation of the subject's muscle stem cells; reversing aging of the subject's skin fibroblasts; and reversing aging of chondrocytes.

[0088] In some embodiments, the subject has, is suspected of having, or is at risk of having:

[0089] Diseases, conditions or manifestations associated with impaired cell, tissue or organ vitality or aging;

[0090] The diseases, conditions or symptoms associated with impaired cell, tissue or organ vitality or aging are, for example, neurodegenerative diseases, cardiovascular diseases, metabolic diseases, musculoskeletal diseases, inflammatory diseases, symptoms or disorders associated with the skin, eye diseases, or diseases associated with the reproductive system;

[0091] The neurodegenerative disease is cerebral stroke, Huntington's disease, senile dementia, Alzheimer's disease, Parkinson's disease;

[0092] 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, myocardial infarction;

[0093] The metabolic disease is type I diabetes, type II diabetes, hyperglycemia, hyperinsulinemia, insulin resistance, obesity, gout;

[0094] The musculoskeletal disease is muscular dystrophy, myasthenia gravis, osteoporosis, osteoarthritis, osteochondrosis, osteochondritis, osteonecrosis, and osteopenia;

[0095] 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 tract inflammation, encephalitis;

[0096] The skin-related aging symptoms or diseases are wrinkles, age spots, seborrheic keratosis, tinea pedis, urticaria, neurodermatitis, and pigmented nevi;

[0097] The eye disease is macular degeneration, retinal degeneration, retinal detachment, diabetic retinopathy, glaucoma, optic atrophy, floaters, cataract, non-arteritic anterior ischemic optic neuropathy, chorioretinitis, retinitis pigmentosa, traumatic optic neuropathy, compressive optic neuropathy;

[0098] Diseases related to the reproductive system include erectile dysfunction and premature ovarian failure.

[0099] .

[0100] In some embodiments, the method 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, reduces scar formation, alleviates external manifestations of aging including alopecia, hair thinning, hair graying, skin laxity and skin wrinkling, promotes organ regeneration, promotes organ survival, treats disease, or any combination thereof.

[0101] In some embodiments, the method reverses aging of a cell, tissue, organ, or subject, thereby rejuvenating the cell, tissue, or organ.

[0102] In some embodiments, the methods can promote axonal regeneration in neuronal cells of the subject.

[0103] In some embodiments, the methods can cause proliferation of fibroblasts from the subject's skin.

[0104] In some embodiments, the composition can promote chondrocyte proliferation in a subject.

[0105] In some embodiments, the methods can promote proliferation of muscle stem cells in the subject.

[0106] In some embodiments, the methods can reverse senescence of skin fibroblasts in a subject.

[0107] In some embodiments, the composition can reverse senescence of chondrocytes.

[0108] In some embodiments, the method does not induce teratoma formation, or does not induce tumor growth or tumor formation.

[0109] In some embodiments, the method does not induce complete reprogramming.

[0110] In some embodiments, the method does not reprogram the cell to a pluripotent state, or the method does not reprogram the cell to an "induced pluripotent stem cell" or "iPSC."

[0111] OCT4, SOX2, KLF4, and c-Myc are four "Yamanaka factors" that can reprogram cells to a pluripotent state. However, inducing the expression of these four transcription factors in transgenic mice leads to teratoma formation in vivo and other acute toxicities.

[0112] The present invention unexpectedly discovered that in the absence of OCT4 and c-Myc, any one factor selected from SOX1, SOX2, SOX3 and GMNN, any one factor selected from GATA3 and GATA6, and KLF4, that is, these three factors can achieve the reversal of senescent cells.

[0113] In the present invention, a "factor" refers to a biologically active protein, or a nucleic acid encoding the same, such as DNA or RNA, or a mixture thereof, which acts on cells to alter transcription and, upon expression, converts senescent cells into young cells. In some embodiments, the factor can be non-integrating, i.e., provided to the recipient somatic cell in a form that does not result in integration of the exogenous DNA into the recipient cell's genome.

[0114] The factor in the present invention can be a transcription factor, including but not limited to SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, and Klf4. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] FIG1 shows that the composition of the present invention can promote neuronal cell regeneration.

[0116] FIG2 shows that the composition of the present invention can promote the proliferation of senescent skin fibroblasts.

[0117] FIG3 shows that the composition of the present invention can promote the proliferation of mouse chondrocytes.

[0118] FIG4 shows that the composition of the present invention can promote the proliferation of muscle stem cells in aged mice.

[0119] .

[0120] FIG5 shows that the composition of the present invention can reverse the senescence of skin fibroblasts in aged mice.

[0121] FIG6 shows that the composition of the present invention can reverse chondrocyte senescence. Example

[0122] In the following embodiments and drawings:

[0123] GATA3 was named A1, GATA6 was named A2; SOX1 was named B1, SOX2 was named B2, SOX3 was named B3, GMNN was named B4; Klf4 was named C.

[0124] 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.

[0125] Example 1 Composition promotes axon regeneration of neuronal cells

[0126] 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.

[0127] The specific methods and results are as follows:

[0128] Methods: One day after cell plating, cells were induced to differentiate for 3 days using 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), and then incubated for 3 days in EMEM / F12 (1:1) containing 1% FBS, 1× PS, and 10 μM ATRA (Differentiation Medium 2). Cells were then plated on cell culture plates coated with poly-D-lysine (ThermoFisher Scientific, A3890401) and cultured for 1 day. The culture medium was then replaced with serum-free B27 neuronal medium (Differentiation Medium 3) containing 1× Glutamax (ThermoFisher Scientific, 35050061), PS, and BDNF and cultured for at least 5 days to promote cell differentiation and maturation. After differentiation was complete, cells were treated with the mRNA combination for 5 days, followed by the addition of vincristine (100 nM; Sigma, V8879) for 24 hours to induce neuronal injury. Neurons were then washed once with PBS, and fresh differentiation medium 3 was added to the plates for an additional 9 days. Neuronal regeneration and maintenance were assessed.

[0129] As shown in Figure 1, the combination of A1 / A2 with B1 / B2 / B3 / B4 and C can promote the increase of neurite area. Example 2 Composition promotes the proliferation of skin fibroblasts in aged mice

[0130] The main purpose of this example is to select skin cells from aged mice and observe whether the proliferation ability of the skin cells from aged mice is enhanced by administering drugs.

[0131] The specific methods and results are as follows:

[0132] Methods: 20-month-old mice were euthanized and their dorsal skin was shaved and excised. The skin was cleaned with povidone-iodine and rinsed with cold 1X PBS before excision. Skin fibroblasts were isolated using trypsin and collagenase. Fibroblasts were resuspended and cultured in DMEM glutamax medium. The cells were treated with the mRNA drug compound of interest and harvested 6 days later to assess whether the proliferation of aged skin fibroblasts was enhanced.

[0133] As shown in Figure 2 , the combination of A1 / A2 with B1 / B2 / B3 / B4 and C can promote the proliferation of senescent skin fibroblasts.

[0134] Example 3 Composition promotes proliferation of mouse chondrocytes

[0135] The main purpose of this example is to select aged mice, isolate chondrocytes, and observe whether the chondrocytes have stronger proliferation ability.

[0136] The specific methods and results are as follows:

[0137] Methods: Articular cartilage was isolated from 18-month-old mice. The soft tissue was separated by gently agitating the tissue fragments. The remaining cartilage fragments were transferred to a fresh culture dish and digested overnight. Collagenase solution was pipetted sequentially to disperse cell aggregates to form a suspension of isolated cells. The cells were washed with PBS, resuspended in culture medium, and cultured by centrifugation. Chondrocyte proliferation was measured 6 days after administration.

[0138] As shown in Figure 3 , the combination of A1 / A2 with B1 / B2 / B3 / B4 and C can promote the proliferation of senescent chondrocytes.

[0139] Example 4 Composition Promotes Proliferation of Muscle Stem Cells in Aged Mice

[0140] The main purpose of this example is to isolate muscle stem cells (MuSCs) from aged mice and observe whether the MuSCs have a stronger proliferation capacity after drug treatment. The specific methods and results are as follows:

[0141] Methods: Fifteen-month-old mice were selected and sacrificed by cervical dislocation. The muscles were isolated, cut into small pieces, and incubated in DMEM medium (DMEM; ThermoFisher Scientific) containing 0.2% type I collagenase (Sigma-Aldrich) at 37°C for 2 hours. Single fibers were collected using a pipette tip and purified twice 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). Then, the single fibers were transferred and the myofiber suspension was passed through a syringe with a 21G needle through a 40 μm filter. The obtained stem cells were directly plated on culture dishes coated with Matrigel growth factor (GFR) basement membrane matrix (Corning) or culture dishes containing coverslips coated with Matrigel GFR basement membrane matrix. The 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% CO2. The culture medium was changed every 2 days. After 6 days of treatment with different compositions, the proliferation capacity of the cells was observed.

[0142] As shown in Figure 4 , the combination of A1 / A2 with B1 / B2 / B3 / B4 and C can promote the proliferation of aged muscle stem cells.

[0143] Example 5 Composition Reverses Senescence of Skin Fibroblasts in Aged Mice

[0144] The main purpose of this example is to select skin cells from aged mice and observe whether the activity of β-galactosidase, a marker of aging, changes in the skin cells by administering drugs. The specific method and results are as follows:

[0145] Skin fibroblasts from aged mice were treated with the mRNA drug of interest. Six days later, the cells were fixed with 4% paraformaldehyde for 15 minutes and then stained in situ using a β-galactosidase in situ staining kit (Biyuntian). Following staining, DAPI was added to stain the nuclei for cell count. The cells were then observed under a fluorescence microscope, and the percentage of positive cells was calculated (positive cell percentage = number of positively stained cells / total number of cells × 100%).

[0146] As shown in Figure 5, the combination of A1 / A2 with B1 / B2 / B3 / B4 and C can significantly reverse the senescence of skin fibroblasts in aged mice.

[0147] Example 6 Composition Reversing Aging of Chondrocytes

[0148] The main purpose of this example is to select chondrocytes, use 20μM etoposide to induce senescence, and then examine the effect of the composition on the activity of β-galactosidase, a senescence marker in the cells. The specific method and results are as follows:

[0149] Human chondrocyte cell line C28 / I2 was induced to senescence using 20 μM etoposide. Twenty-four hours after induction, the cells were treated with the mRNA drug of the target composition. Six days later, the cells were fixed with 4% paraformaldehyde for 15 minutes and then stained in situ using a β-galactosidase in situ staining kit (Biyuntian). After staining, DAPI was added to stain the nuclei for cell count. Afterwards, the cells were observed under a fluorescence microscope, and the percentage of positive cells was calculated (positive cell percentage = number of positively stained cells / total number of cells × 100%).

[0150] As shown in Figure 6, the combination of A1 / A2 with B1 / B2 / B3 / B4 and C can significantly reverse the senescence of chondrocytes.

Claims

1. A composition for rejuvenating cells, tissues or organs of a subject, comprising any one factor selected from SOX1, SOX2, SOX3 and GMNN, any one factor selected from GATA3 and GATA6, and factor Klf4.

2. The composition according to claim 1, characterized in that The factors SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, Klf4 are proteins, nucleic acids encoding them or mixtures thereof, Preferably, the factors SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, and Klf4 are proteins, fusion proteins bound to the membrane permeability domain, and are in the form of nucleic acid encoding them.

3. The composition according to any one of claims 1 to 2, characterized in that The factors SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, Klf4 are in the form of expression vectors, which are plasmid vectors, episomal vectors, transposons, or viral vectors. Preferably, the viral vector is an adenoviral vector, an adeno-associated viral vector (AAV), a lentiviral vector, a Sendai viral vector, a cytomegalovirus vector or a vaccinia viral vector. Preferably, the factors are present on the same expression vector, or on multiple expression vectors.

4. The composition according to any one of claims 1 to 2, characterized in that The factors SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, and Klf4 are in the form of DNA or RNA. Preferably, the factor is in the form of mRNA, The factors are tandemly linked to the same mRNA molecule, or the factors SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, and Klf4 are in the form of a compound of different mRNA molecules. Preferably, the factor is a synthetic mRNA encoding a wild-type form, a mutant form, or a genetically engineered form of a gene selected from the group consisting of SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, and Klf4.

5. The composition according to any one of claims 1 to 2, characterized in that The subject is a mammal, including but not limited to humans, mice, rats, cattle, sheep, horses, dogs, cats, pigs or monkeys, The subject's cells, tissues or organs are from, but 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, including hair; heart, brain, nervous tissue, kidneys, testicles, prostate, penis, cloaca, fins, ovaries, or intestines.

6. The composition according to any one of claims 1 to 2, characterized in that The factors SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, Klf4 are human proteins, or non-human proteins, or proteins that have at least 80% identity with the amino acid sequence of the human or non-human proteins and maintain activity, The non-human protein is a corresponding protein of mouse, rat, cow, sheep, horse, dog, cat, pig, monkey, chicken, duck, goose or bird; or The factors SOX1, SOX2, SOX3, GMNN, GATA3, GATA6, and Klf4 are nucleic acids encoding human proteins, or nucleic acids encoding non-human proteins, or nucleic acids that have at least 80% identity with corresponding nucleic acids encoding human proteins, or nucleic acids encoding non-human proteins and maintain activity, The nucleic acid is a nucleic acid encoding the corresponding protein of mouse, rat, cattle, sheep, horse, dog, cat, pig, monkey, chicken, duck, goose, or bird.

7. A method for rejuvenating cells, tissues or organs in a subject, the method comprising administering to the subject a composition according to any one of claims 1 to 6.

8. Use of the composition of any one of claims 1 to 6 in the preparation of a medicament for rejuvenating cells, tissues or organs of a subject.

9. The method of claim 7 or the use of claim 8, wherein rejuvenating the subject's cells, tissues or organs comprises restoring transcriptional profiles or epigenetic information lost in the at least one cell, tissue or organ due to aging, injury, disease or any combination thereof.

10. The method according to claim 7 or the use according to claim 8, wherein rejuvenating the subject's cells, tissues or organs comprises restoring the function of the cells, increasing the potential of the cells, enhancing the survival ability of the cells or increasing the replication ability or lifespan of the cells, or a combination thereof.

11. The method according to claim 7 or the use according to claim 8, wherein rejuvenating the subject's cells, tissues or organs comprises promoting axon regeneration of the subject's neurons; proliferation of the subject's skin fibroblasts; proliferation of the subject's chondrocytes; proliferation of the subject's muscle stem cells; reversing aging of the subject's skin fibroblasts; and reversing aging of chondrocytes.

12. The composition of any one of claims 1 to 6, the method of claim 7, the use of claim 8, or the method or use of any one of claims 9 to 11, wherein the subject has, is suspected of having, or is at risk of having: Diseases, conditions or manifestations associated with impaired cell, tissue or organ vitality or aging; The disease, condition or manifestation associated with impaired cell, tissue or organ vitality or aging is a neurodegenerative disease, cardiovascular disease, metabolic disease, musculoskeletal disease, inflammatory disease, manifestation or condition related to the skin, eye disease, or disease related to the reproductive system; Preferably, the neurodegenerative disease is cerebral stroke, Huntington's disease, senile dementia, Alzheimer's disease, Parkinson's disease; 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, myocardial infarction; Preferably, the metabolic disease is type I diabetes, type II diabetes, hyperglycemia, hyperinsulinemia, insulin resistance, obesity, or gout; Preferably, the musculoskeletal disease is muscular dystrophy, myasthenia gravis, osteoporosis, osteoarthritis, osteochondrosis, osteochondritis, osteonecrosis, or osteopenia; 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 tract inflammation, encephalitis; Preferably, the skin-related aging symptoms or conditions are wrinkles, age spots, seborrheic keratosis, tinea pedis, urticaria, neurodermatitis, and pigmented nevi; Preferably, the eye disease is macular degeneration, retinal degeneration, retinal detachment, diabetic retinopathy, glaucoma, optic atrophy, floaters, cataract, non-arteritic anterior ischemic optic neuropathy, chorioretinitis, retinitis pigmentosa, traumatic optic neuropathy, compressive optic neuropathy; Preferably, the diseases related to the reproductive system are erectile dysfunction and premature ovarian failure.

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