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 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 the factor OCT4, any one factor selected from SOX1, SOX3 and GMNN, and the factor Klf4.

[0007] In some embodiments, the factor is a protein, a nucleic acid encoding the same, or a mixture thereof.

[0008] For example, the OCT4, SOX1, SOX3, GMNN, and Klf4 are proteins, fusion proteins combined with a membrane permeability domain, and are encoded by nucleic acids, such as DNA or RNA.

[0009] 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.

[0010] In some embodiments, the factors are present on one or more expression vectors.

[0011] In some embodiments, the factors are present on the same expression vector.

[0012] In some embodiments, the factor is in the form of mRNA.

[0013] 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.

[0014] 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 OCT4, SOX1, SOX3, GMNN, and Klf4.

[0015] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or adjuvant.

[0016] In some embodiments, the subject is a mammal, including but not limited to humans, mice, rats, cows, sheep, horses, dogs, cats, pigs, or monkeys.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] In some embodiments, the OCT4, SOX1, SOX3, GMNN, 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.

[0022] In some embodiments, the OCT4, SOX1, SOX3, GMNN, Klf4 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.

[0023] 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.

[0024] In some embodiments, the composition reverses aging of a cell, tissue, organ, or subject, thereby rejuvenating the cell, tissue, or organ.

[0025] In some embodiments, the composition can promote axonal regeneration in neuronal cells of a subject.

[0026] In some embodiments, the composition can cause fibroblasts in the subject's skin to proliferate.

[0027] In some embodiments, the composition can promote chondrocyte proliferation in a subject.

[0028] In some embodiments, the composition can promote the proliferation of muscle stem cells in a subject.

[0029] In some embodiments, the methods can reverse senescence of skin fibroblasts in a subject.

[0030] In some embodiments, the composition can reverse senescence of chondrocytes.

[0031] In some embodiments, the composition does not induce teratoma formation.

[0032] In some embodiments, the composition does not induce complete reprogramming.

[0033] In some embodiments, the composition does not reprogram the cell to an induced pluripotent stem cell (iPSC) state.

[0034] 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.

[0035] In some embodiments, the composition comprises factor OCT4, any one factor selected from SOX1, SOX3 and GMNN, and factor Klf4.

[0036] In some embodiments, the factor is a protein, a nucleic acid encoding the same, or a mixture thereof.

[0037] For example, the OCT4, SOX1, SOX3, GMNN, and Klf4 are proteins, fusion proteins combined with a membrane permeability domain, and are encoded by nucleic acids, such as DNA or RNA.

[0038] 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.

[0039] In some embodiments, the factors are present on one or more expression vectors.

[0040] In some embodiments, the factors are present on the same expression vector.

[0041] In some embodiments, the factor is in the form of mRNA.

[0042] 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.

[0043] 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 OCT4, SOX1, SOX2, SOX3, GMNN, and Klf4.

[0044] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or adjuvant.

[0045] In some embodiments, the subject is a mammal, including but not limited to humans, mice, rats, cows, sheep, horses, dogs, cats, pigs, or monkeys.

[0046] In some embodiments, the subject is a chicken, duck, goose, or bird.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] In some embodiments, the OCT4, SOX1, SOX3, GMNN, 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.

[0052] In some embodiments, the OCT4, SOX1, SOX3, GMNN, Klf4 are nucleic acids encoding human proteins, or nucleic acids encoding non-human proteins, for example, nucleic acids encoding proteins of mammals (e.g., mice, rats, cattle, sheep, horses, dogs, cats, pigs, monkeys, chickens, ducks, geese, birds), or nucleic acids that have at least 80% identity and maintain activity with corresponding nucleic acids encoding human proteins or nucleic acid sequences encoding non-human proteins. In a third aspect, the present invention provides the use of the composition of the first aspect in the preparation of a medicament for rejuvenating cells, tissues, or organs of a subject.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] In some embodiments, the subject has, is suspected of having, or is at risk of having:

[0057] Diseases, conditions or manifestations associated with impaired cell, tissue or organ vitality or aging;

[0058] 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;

[0059] The neurodegenerative disease is cerebral stroke, Huntington's disease, senile dementia, Alzheimer's disease, Parkinson's disease;

[0060] 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;

[0061] The metabolic disease is type I diabetes, type II diabetes, hyperglycemia, hyperinsulinemia, insulin resistance, obesity, gout;

[0062] The musculoskeletal disease is muscular dystrophy, myasthenia gravis, osteoporosis, osteoarthritis, osteochondrosis, osteochondritis, osteonecrosis, and osteopenia;

[0063] 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;

[0064] The skin-related aging symptoms or diseases are wrinkles, age spots, seborrheic keratosis, tinea pedis, urticaria, neurodermatitis, and pigmented nevi;

[0065] 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;

[0066] Diseases related to the reproductive system include erectile dysfunction and premature ovarian failure.

[0067] 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.

[0068] In some embodiments, the method reverses aging of a cell, tissue, organ, or subject, thereby rejuvenating the cell, tissue, or organ.

[0069] In some embodiments, the methods can promote axonal regeneration in neuronal cells of the subject.

[0070] In some embodiments, the methods can cause the subject's skin fibroblasts to proliferate.

[0071] In some embodiments, the composition can promote chondrocyte proliferation in a subject.

[0072] In some embodiments, the methods can promote proliferation of muscle stem cells in the subject.

[0073] In some embodiments, the methods can reverse senescence of skin fibroblasts in a subject.

[0074] In some embodiments, the composition can reverse senescence of chondrocytes.

[0075] In some embodiments, the method does not induce teratoma formation.

[0076] In some embodiments, the method does not induce complete reprogramming.

[0077] In some embodiments, the method does not reprogram the cells to an induced pluripotent stem cell (iPSC) state.

[0078] In 2006, scientists discovered that OCT4, SOX2, KLF4, and c-Myc are four "Yamanaka factors" capable of reprogramming cells to a pluripotent state. In 2016, scientists discovered that these four "Yamanaka factors" can also reprogram senescent cells to young cells. However, inducing the expression of four transcription factors in mice carries the safety risk of causing teratomas in vivo, and the greater the number of factors, the greater the uncertainty of in vivo regulation.

[0079] The present invention unexpectedly discovered that, in the absence of c-Myc, a combination of OCT4, any one factor selected from SOX1, SOX3 and GMNN, and Klf4 can also achieve the reversal of senescent cells.

[0080] 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, transforms aged, incapacitated cells into youthful, vigorous 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.

[0081] The factor in the present invention can be a transcription factor, including but not limited to OCT4, SOX1, SOX3, GMNN, Klf4. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] FIG1 shows that the composition of the present invention can promote axon regeneration of neuronal cells.

[0083] FIG2 shows that the composition of the present invention can promote the proliferation of skin fibroblasts.

[0084] FIG3 shows that the composition of the present invention can promote the proliferation of mouse chondrocytes.

[0085] FIG4 shows that the composition of the present invention can promote the proliferation of muscle stem cells in aged mice.

[0086] FIG5 shows that the composition of the present invention can reverse the senescence of skin fibroblasts in aged mice.

[0087] FIG6 shows that the composition of the present invention can reverse chondrocyte senescence. Example

[0088] In the following embodiments and drawings:

[0089] OCT4 was named A, SOX1 was named C1, SOX3 was named C2, GMNN was named C3, and Klf4 was named D.

[0090] The OCT4, SOX1, SOX3, GMNN, and Klf4 used in the examples are all human OCT4, human SOX1, human SOX3, human GMNN, and human Klf4.

[0091] Example 1 Composition promotes axon regeneration of neuronal cells

[0092] The main purpose of this example is to select SH-SY5Y (human neuroblastoma cell line), differentiate them into neurons, and determine the regenerative effects of different compositions on neurons. The specific methods and results are as follows:

[0093] 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.

[0094] As shown in Figure 1 , the combined use of A, D, and C1 / C2 / C3 can promote the increase in neurite area.

[0095] Example 2 Composition promotes proliferation of fibroblasts in the skin of aged mice

[0096] The main purpose of this example is to select skin cells from aged mice and observe whether the proliferation ability of skin cells from aged mice is enhanced by administering drugs. The specific method and results are as follows:

[0097] Methods: 20-month-old mice were euthanized and the dorsal skin was shaved and excised. The skin was cleaned with povidone-iodine and rinsed with cold 1× PBS before excision. Skin fibroblasts were isolated using trypsin and collagenase. Fibroblasts were resuspended and cultured in DMEM glutamax medium. 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.

[0098] As shown in Figure 2, the combined use of A, D and C1 / C2 / C3 significantly increased the proliferation capacity of skin cells.

[0099] Example 3 Composition promotes proliferation of chondrocytes in aged mice

[0100] The main purpose of this example is to select old mice, isolate chondrocytes, and observe whether the chondrocytes have stronger proliferation ability after administration. The specific method and results are as follows:

[0101] 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.

[0102] As shown in Figure 3, the combination of A, D and C1 / C2 / C3 can significantly promote the proliferation of chondrocytes.

[0103] Example 4 Composition Promotes Proliferation of Muscle Stem Cells in Aged Mice

[0104] 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:

[0105] Methods: Fifteen-month-old mice were selected and sacrificed by cervical dislocation. 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, single fibers were transferred and the myofiber suspension was passed through a 40 μm filter using a syringe with a 21G needle. The obtained stem cells were directly plated on culture dishes coated with Matrigel growth factor (GFR) basement membrane matrix (Corning) or on 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, with the medium changed every 2 days. After 6 days of treatment with the different compositions, the cell proliferation capacity was observed.

[0106] As shown in Figure 4, the combination of A, D and C1 / C2 / C3 can significantly promote the proliferation of muscle stem cells.

[0107] Example 5 Composition Reverses Senescence of Skin Fibroblasts in Aged Mice

[0108] 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:

[0109] 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%).

[0110] As shown in Figure 5, the combination of A, D and C1 / C2 / C3 significantly reversed the senescence of skin fibroblasts in aged mice.

[0111] Example 6 Composition Reversing Aging of Chondrocytes

[0112] 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:

[0113] 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%).

[0114] As shown in Figure 6, the combination of A, D and C1 / C2 / C3 significantly reversed the senescence of chondrocytes.

Claims

1. A composition for rejuvenating cells, tissues or organs of a subject, comprising the factor OCT4, any one factor selected from SOX1, SOX3 and GMNN, and the factor Klf4.

2. The composition according to claim 1, characterized in that The factors OCT4, SOX1, SOX3, GMNN, Klf4 are proteins, nucleic acids encoding them or mixtures thereof, Preferably, the factors OCT4, SOX1, SOX3, GMNN, Klf4 are proteins, fusion proteins combined with membrane permeability domains, 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 OCT4, SOX1, SOX3, GMNN, 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 OCT4, SOX1, SOX3, GMNN, 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 OCT4, SOX1, SOX3, GMNN, 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 OCT4, SOX1, SOX3, GMNN, 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 OCT4, SOX1, SOX3, GMNN, 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 OCT4, SOX1, SOX3, GMNN, 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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