Transient cellular reprogramming for the reversal of cellular aging

Abstract

To provide a composition for rejuvenating aged cells and tissues to restore functionality.SOLUTION: A composition comprises a therapeutically effective amount of cells comprising one or more nonintegrative messenger RNAs encoding one or more cellular reprogramming factors. The composition is used to treat an age-related disease or condition, a cartilage degeneration disorder, a neurodegenerative disorder, and / or a musculoskeletal dysfunction in a subject.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 642,538, filed on Mar. 13, 2018, the entire disclosure of which is hereby incorporated by reference for all purposes.

Background Art

[0002] Aging is characterized by a progressive loss of function occurring at the molecular, cellular, tissue, and organismal levels. At the chromatin level, aging is associated with a progressive accumulation of epigenetic errors that ultimately lead to abnormal gene regulation, stem cell depletion, senescence, and deregulated cell / tissue homeostasis. Techniques of nuclear reprogramming to pluripotency by overexpression of a few transcription factors can revert both the age and identity of any cell back to that of embryonic cells by driving epigenetic reprogramming. The unwanted erasure of cell identity is a problem for the development of rejuvenation therapies because of the resulting disruption of structure, function, and cell type distribution in tissues and organs.

Summary of the Invention

Problems to be Solved by the Invention

[0003] (Summary of the Invention) In view of the foregoing, there is a need for an improved method of rejuvenating cells that avoids dedifferentiation and loss of cell identity. The present disclosure addresses such a need and, moreover, provides further benefits.

Means for Solving the Problems

[0004] The present disclosure generally relates to cell rejuvenation, tissue engineering, and regenerative medicine. In particular, the present disclosure relates to compositions and methods for rejuvenating aged cells and tissues and restoring functionality by transient exposure to non-integrating mRNAs encoding reprogramming factors that keep cells in a differentiated state while rejuvenating the cells.

[0005] The present disclosure relates to cell-based therapies using rejuvenated cells. In particular, the present disclosure relates to methods for rejuvenating aged cells and tissues and restoring functionality by transient exposure to non-integrating mRNA encoding reprogramming factors that maintain the cells in a differentiated state while rejuvenating the cells.

[0006] In one aspect, provided herein is a method of rejuvenating a cell, the method comprising transfecting a cell with one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors for no more than 5 consecutive days, thereby producing a rejuvenated cell.

[0007] In one aspect, provided herein is a method for treating an age-related disease or condition, a cartilage degenerative disorder, a neurodegenerative disorder, and / or a musculoskeletal dysfunction in a subject. The method comprises administering a therapeutically effective amount of cells comprising one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors.

[0008] In one aspect, provided herein is a method for treating an age-related disease or condition, a cartilage degenerative disorder, and / or for treating a subject having a musculoskeletal dysfunction in a subject. The method comprises administering a therapeutically effective amount of one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors.

[0009] In one aspect, provided herein is a method of rejuvenating an engineered tissue ex vivo. The method comprises transfecting the tissue with one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors for no more than 5 consecutive days, thereby producing a rejuvenated engineered tissue.

[0010] In one aspect, provided herein is a pharmaceutical composition comprising a rejuvenated cell obtained by transfecting a cell with one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors for no more than 5 consecutive days.

[0011] Thus, in one aspect, the present disclosure provides a method for rejuvenating cells, comprising: a) transfecting one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors into the cells, wherein the transfecting step is performed once a day for at least 2 days and up to 4 days, and b) translating the one or more non-integrating messenger RNAs to produce one or more cell reprogramming factors in the cells, resulting in transient reprogramming of the cells, wherein the cells rejuvenate without dedifferentiating into stem cells. The method can be performed in cells in vitro, ex vivo or in vivo.

[0012] In certain embodiments, transfection with one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors is performed once a day for 2, 3 or 4 days.

[0013] In certain embodiments, the one or more cell reprogramming factors are selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG. In one embodiment, the one or more cell reprogramming factors include OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

[0014] The method can be performed in any type of cell. In some embodiments, the cells are mammalian cells (e.g., human, non-human primate, rodent, cat, dog, cow, horse, pig, goat, etc.). For example, the method can be performed in fibroblasts, endothelial cells, chondrocytes or skeletal muscle stem cells. In another embodiment, the cells are derived from an aged subject.

[0015] In certain embodiments, transient reprogramming results in increased expression of HP1γ, H3K9me3, lamin - associated protein LAP2α, and SIRT1, decreased expression of GMSCF, IL18, and TNFα, decreased nuclear folding, decreased vesicle formation, increased cellular autophagosome formation, increased chymotrypsin - like proteasome activity, increased mitochondrial membrane potential, or decreased reactive oxygen species (ROS).

[0016] In certain embodiments, the cells are in a tissue or an organ. Transient reprogramming according to the methods described herein can restore the function of cells in a tissue or an organ, increase the differentiation potential of cells in a tissue or an organ, reduce the number of senescent cells in a tissue or an organ, enhance the replicative ability of cells in a tissue or an organ, or extend the lifespan of cells in a tissue or an organ.

[0017] In another aspect, the disclosure includes a method for treating an age - related disease or condition in a subject, the method comprising: a) transfecting one or more non - integrating messenger RNAs encoding one or more cell reprogramming factors into the cells of the subject, wherein the transfecting step is performed once a day for at least 2 days and no more than 4 days, and b) expressing one or more cell reprogramming factors in the cells of the subject, resulting in transient reprogramming of the cells, wherein the cells rejuvenate without dedifferentiating into stem cells. The cells can be transfected ex vivo or in vivo.

[0018] In certain embodiments, the one or more cell reprogramming factors are selected from the group consisting of OCT4, SOX2, KLF4, c - MYC, LIN28, and NANOG. In one embodiment, the one or more cell reprogramming factors include OCT4, SOX2, KLF4, c - MYC, LIN28, and NANOG.

[0019] In certain embodiments, the age-related disease or condition is a degenerative disease, neurodegenerative disease, cardiovascular disease, peripheral vascular disease, skin disease, eye disease, autoimmune disease, endocrine disorder, metabolic disorder, musculoskeletal disorder, gastrointestinal disease, or respiratory disease.

[0020] In another embodiment, the present disclosure provides a method for treating a disease or disorder involving cartilage degeneration in a subject, the method comprising: a) transfecting one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors into chondrocytes of the subject, wherein the transfecting step is performed once a day for at least 2 days and up to 4 days; and b) expressing one or more cell reprogramming factors in the chondrocytes to effect transient reprogramming of the chondrocytes, wherein the chondrocytes rejuvenate without dedifferentiating into stem cells. The rejuvenated chondrocytes can be transplanted, for example, into the joints of the subject with arthritis.

[0021] The method can be performed ex vivo, in vitro, or in vivo. In one embodiment, chondrocytes are isolated from a cartilage sample obtained from the subject, transfected ex vivo, and then transplanted back into the subject.

[0022] In certain embodiments, the disease or disorder involving cartilage degeneration is arthritis (e.g., osteoarthritis or rheumatoid arthritis).

[0023] In certain embodiments, the treatment reduces inflammation in the subject.

[0024] In certain embodiments, the treatment reduces the expression of RANKL, iNOS, IL6, IL8, BDNF, IFNα, IFNγ, and LIF and increases the expression of COL2A1 in chondrocytes.

[0025] In another aspect, the present disclosure provides a method for treating a disease or disorder involving muscle degeneration in a subject, the method comprising: a) transfecting one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors into the subject's skeletal muscle stem cells, wherein the transfecting step is performed once a day for at least 2 days and up to 4 days; and b) expressing one or more cell reprogramming factors in the skeletal muscle stem cells to effect transient reprogramming of the skeletal muscle stem cells, such that the skeletal muscle stem cells rejuvenate without losing their ability to differentiate into muscle cells.

[0026] The method can be performed ex vivo, in vitro, or in vivo. In one embodiment, the skeletal muscle stem cells are isolated from a muscle tissue sample obtained from the subject, transfected ex vivo, and then transplanted into the muscle in the subject that requires repair or regeneration.

[0027] In certain embodiments, the one or more cell reprogramming factors are selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. In one embodiment, the one or more cell reprogramming factors include OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0028] In certain embodiments, the treatment restores the differentiation capacity of the skeletal muscle stem cells. In certain embodiments, the treatment results in the regeneration of muscle fibers.

[0029] The methods of the present disclosure can be performed in any subject. In certain embodiments, the subject is a mammal, such as a human, non-human primate, rodent, cat, dog, cow, horse, pig, or goat. In some embodiments, the subject is an elderly individual.

[0030] The above and other embodiments of the subject disclosure will be readily envisioned by those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0031]

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Mode for Carrying Out the Invention

[0032] The practice of the techniques described herein, unless otherwise indicated, uses conventional methods of medicine, cell biology, pharmacology, chemistry, biochemistry, molecular biology and recombinant DNA techniques, and immunology, within the skill of the art. Such techniques are well described in the literature. See, for example, G. Vunjak-Novakovic and R. I. Freshney Culture of Cells for Tissue Engineering (Wiley-Liss, 1st ed., 2006); Arthritis Research: Methods and Protocols, volumes 1 and 2: (Methods in Molecular Medicine, edited by Cope, Humana Press, 2007); Cartilage and Osteoarthritis (Methods in Molecular Medicine, edited by M. Sabatini, P. Pastoureau and F. De Ceuninck, Humana Press; 2004); Handbook of Experimental Immunology, volumes I-IV (edited by D. M. Weir and C. C. Blackwell, Blackwell Scientific Publications); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current edition); and Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., 2001).

[0033] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0034] I. Definitions In the description of the present disclosure, the following terms are used and are intended to be defined as set forth below.

[0035] As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural referents. Thus, for example, reference to "a cell" includes mixtures of two or more cells, and the like.

[0036] Throughout this specification, for example, references to "one embodiment", "an embodiment", "another embodiment", "certain embodiments", "related embodiments", "certain specific embodiments", "additional embodiments", or "further embodiments", or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0037] As used herein, the term "about" means a range of values that includes the specified value and that are reasonably similar to the specified value as considered by one of ordinary skill in the art. In embodiments, the term "about" means within one standard deviation using measurements generally accepted in the art. In embodiments, about means a range extending from + / −10% of the specified value. In embodiments, about means the specified value.

[0038] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" are to be interpreted as implying the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means including and limited to whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or essential and that no other elements may be present. "Consisting essentially of" means including any elements listed after this phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in this disclosure with respect to the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but that other elements are optional and may or may not be present depending on whether or not they affect the activity or action of the listed elements.

[0039] As used herein, the term "biocompatible" generally refers to materials and any of their metabolites or degradation products that are generally non-toxic to a recipient and do not cause any significant adverse effects to the subject.

[0040] As used herein, the term "cell" refers to intact living cells, whether of natural origin or modified. Cells in culture or mixed with other cells in a tissue (partial or intact) or organism can be isolated from other cells. The methods described herein can be performed, for example, on single cells, populations of cells, or samples containing tissues or organs containing cells.

[0041] As used herein, the term "non-integrated" with reference to messenger RNA (mRNA) refers to mRNA molecules that are not chromosomally or extrachromosomally integrated into the host genome and are not integrated into a vector.

[0042] As used herein, the term "transfection" refers to the uptake of exogenous DNA or RNA by a cell. When exogenous DNA or RNA is introduced inside the cell membrane, the cell is "transfected". A number of transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd ed., Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd ed., McGraw-Hill and Chu et al. (1981) Gene 13:197. Such techniques can be used for the introduction of one or more exogenous DNA or RNA molecules into cells. This term refers to both stable and transient uptake of DNA or RNA molecules. For example, transfection can be used for the transient uptake of mRNA encoding a cellular reprogramming factor into a cell that requires rejuvenation.

[0043] As used herein, the term "transient reprogramming" refers to the exposure of a cell to a cellular reprogramming factor for a period of time that is sufficient to rejuvenate the cell (i.e., eliminate all or some of the features of aging), but not long enough to cause dedifferentiation into a stem cell. Such transient reprogramming results in rejuvenated cells that retain their identity (i.e., differentiated cell type).

[0044] As used herein, the term "rejuvenated cell(s)" refers to an aged cell that has been treated or transiently reprogrammed by one or more cellular reprogramming factors such that the cell has a transcriptome profile of a younger cell while still retaining one or more cell identity markers.

[0045] As used herein, the term "mammalian cell" refers to any cell derived from a mammalian subject that is suitable for transplantation into the same or a different subject. The cells may be xenogeneic, autologous, or allogeneic. The cells may be primary cells obtained directly from a mammalian subject. The cells may be cells derived from the culture and expansion of cells obtained from a subject. In some embodiments, the cells are genetically engineered to express a recombinant protein and / or nucleic acid.

[0046] As used herein, the term "stem cell" refers to a cell that retains the ability to self-renew by mitotic cell division and can differentiate into a diverse range of specialized cell types. Mammalian stem cells can be divided into three broad categories: embryonic stem cells derived from the blastocyst, adult stem cells found in adult tissues, and umbilical cord blood stem cells found in the umbilical cord. In the developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In the adult organism, stem cells and progenitor cells act as a repair system for the body by replenishing specialized cells. Totipotent stem cells are produced from the fusion of an egg and a sperm cell. The cells produced by the first few divisions of a fertilized egg are also totipotent. These cells can differentiate into embryonic and extraembryonic cell types. Pluripotent stem cells are descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers. Multipotent stem cells can produce only cells of a closely related family of cells (e.g., hematopoietic stem cells can differentiate into red blood cells, white blood cells, platelets, etc.). Unipotent cells can produce only one cell type but have the property of self-renewal, which distinguishes them from non-stem cells. Induced pluripotent stem cells are a type of pluripotent stem cell derived from adult cells that have been reprogrammed into an embryonic-like pluripotent state. Induced pluripotent stem cells can be obtained from adult somatic cells such as, for example, skin or blood cells.

[0047] As used herein, the term "transcriptome profile" refers to the set of all RNA molecules in a single cell or cell population. This may be used to refer to total RNA or simply mRNA, depending on the particular experiment. It differs from an exosome in that it includes only the RNA molecules found in the designated cell population and typically includes the amount or concentration of each RNA molecule in addition to molecular identity. Methods for obtaining a transcriptome profile include DNA microarrays and next-generation sequencing techniques such as RNA-Seq. Transcription can be assayed at the level of individual cells by single-cell transcriptomics. There are two general methods for inferring transcriptome sequences. One approach maps sequence read data to either the reference genome of the organism itself (whose transcriptome is being assayed) or a closely related species. The other approach, de novo transcriptome assembly, uses software to directly infer transcripts from short sequence read data.

[0048] As used herein, the term "root mean square error" or "RMSE" refers to the standard deviation of the residuals (prediction errors). The residuals are a measure of the distance of the data points from the regression line. RMSE is a measure of the spread of these residuals. In other words, it indicates how closely the data is concentrated around the best-fit line.

[0049] As used herein, the term "cell viability" refers to a measure of the number of cells that are alive or dead, based on a total cell sample. High cell viability as defined herein refers to a cell population in which more than 85% of all cells are viable, preferably more than 90 - 95% are viable, and more preferably a population characterized by a high cell viability containing more than 99% viable cells.

[0050] As used herein, the term "autophagosome" refers to a spherical structure having a double membrane. This is a key structure in macroautophagy, which is also an intracellular degradation system for cytoplasmic contents (e.g., abnormal intracellular proteins, excessive or damaged organelles), as well as invading microorganisms. After formation, the autophagosome delivers cytoplasmic components to the lysosome. The outer membrane of the autophagosome fuses with the lysosome to form an autolysosome. The hydrolases of the lysosome degrade the contents delivered to the autophagosome and its inner membrane.

[0051] As used herein, the term "proteasome activity" refers to the degradation of unnecessary or damaged proteins by the proteasome, a protein complex, through proteolysis, which is a chemical reaction that cleaves peptide bonds. The term "chymotrypsin-like proteasome activity" refers to a distinct catalytic activity of the proteasome.

[0052] As used herein, the term "mitochondrial membrane potential" refers to the potential and proton gradient that is caused by redox conversions associated with the activity of the Krebs cycle and functions as an intermediate form of energy storage for the production of ATP. This is generated by proton pumps and is an essential process for energy storage in oxidative phosphorylation. This plays a key role in mitochondrial homeostasis through the selective elimination of dysfunctional mitochondria.

[0053] As used herein, the term "pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the present disclosure and that does not cause a significant adverse toxicological effect in a patient.

[0054] As used herein, the term "reactive oxygen species" or "ROS" refers to chemically reactive chemical species that contain oxygen. Examples include peroxides, superoxides, hydroxyl radicals, singlet oxygen, and alpha-oxygen. In a biological context, ROS are formed as natural by-products of normal metabolism of oxygen and have important roles in cell signaling and homeostasis.

[0055] As used herein, the term "senescence-associated secretory phenotype" or "SASP" refers to a variety of diverse cytokines, chemokines, growth factors, and proteases that are characteristic features of senescent cells. Senescent cells are still metabolically active and are stable non-dividing cells that exhibit upregulation of a wide range of genes, including genes encoding secreted proteins such as inflammatory cytokines, chemokines, extracellular matrix remodeling factors, and growth factors. These secreted proteins function physiologically in the tissue microenvironment, and according to this, the secreted proteins can propagate a stress response and communicate with neighboring cells. This phenotype, named senescence-associated secretory phenotype (SASP), reveals the paracrine function of senescent cells and is an important feature that distinguishes senescent cells from non-senescent cell cycle-arrested cells such as quiescent cells and terminally differentiated cells. "SASP cytokines" specifically refer to cytokines produced by senescent cells that create the senescence-associated secretory phenotype. Examples of cytokines include, but are not limited to, IL18, IL1A, GROA, IL22, and IL9.

[0056] As used herein, the term "methylation landscape" refers to the DNA methylation pattern of a cell or cell population.

[0057] As used herein, the term "epigenetic clock" refers to a biochemical test that can be used to measure age. This test is based on DNA methylation levels. The first multi-tissue epigenetic clock, the Horvath epigenetic clock or "Horvath clock", was developed by Steve Horvath (Horvath 2013).

[0058] As used herein, the term "cell reprogramming factor" refers to a set of transcription factors that can convert adult or differentiated cells into pluripotent stem cells. In embodiments herein, this factor includes OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0059] "Pharmaceutically acceptable salts" include salts prepared with inorganic acids such as amino acid salts, chlorides, sulfates, phosphates, diphosphates, bromides and nitrates, or salts prepared from any corresponding inorganic acid form of the precursor, for example, hydrochloride, etc., or salts prepared with organic acids such as malates, maleates, fumarates, tartrates, succinates, ethylsuccinates, citrates, acetates, lactates, methanesulfonates, benzoates, ascorbates, para-toluenesulfonates, pamoates, salicylates and stearates, and also include, but are not limited to, estolates, gluceptates and lactobionates. Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium and ammonium (including substituted ammonium).

[0060] As used herein, the term "transplantation" refers to the movement of cells, tissues or organs from another source to a subject. This term is not limited to a specific mechanism of movement. The cells can be transplanted by any suitable method such as injection or surgical implantation.

[0061] As used herein, the term "arthritis" includes, but is not limited to, osteoarthritis, rheumatoid arthritis, lupus-related arthritis, juvenile idiopathic arthritis, reactive arthritis, inflammatory bowel disease-related arthritis and psoriatic arthritis.

[0062] As used herein, the term "age-related disease or condition" refers to any disease, condition or disorder associated with aging, including but not limited to neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, dementia and stroke), cardiovascular and peripheral vascular diseases (such as atherosclerosis, peripheral artery disease (PAD), hematoma, calcification, thrombosis, embolism and aneurysm), eye diseases (such as age-related macular degeneration, glaucoma, cataract, dry eye, diabetic retinopathy, vision loss), skin diseases (atrophy and thinning of the skin, elastolysis and skin wrinkles, sebaceous gland hyperplasia or hypoplasia, senile lentigines and other pigmentation abnormalities, gray hair, hair loss or thinning, and chronic skin ulcers), autoimmune diseases (such as polymyalgia rheumatica (PMR), giant cell arteritis (GCA), rheumatoid arthritis (RA), crystalline arthritis and spondyloarthritis (SPA)), endocrine and metabolic dysfunctions (such as adult hypopituitarism, hypothyroidism, apathetic hyperthyroidism, osteoporosis, type 2 diabetes, adrenal insufficiency, various forms of hypogonadism and endocrine malignancies), musculoskeletal disorders (such as arthritis, osteoporosis, myeloma, gout, Paget's disease, fractures, myelodysplastic syndromes, ankylosis, diffuse idiopathic skeletal hyperostosis, hematogenous osteomyelitis, muscle atrophy, peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, primary lateral sclerosis and myasthenia gravis), digestive system diseases (such as cirrhosis, liver fibrosis, Barrett's esophagus), respiratory diseases (such as pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, chronic bronchitis, pulmonary embolism (PE), lung cancer and infections), and any other disease and disorder associated with aging.

[0063] As used herein, the term "disease or disorder involving cartilage degeneration" refers to any disease or disorder involving cartilage and / or joint degeneration. The term "disease or disorder involving cartilage degeneration" includes conditions, disorders, syndromes, diseases and injuries affecting the vertebral discs or joints (such as joint junctions) in animals including humans, and examples of such include, but are not limited to, arthritis, chondrophasia, spondyloarthritis, ankylosing spondylitis, lupus erythematosus, relapsing polychondritis and Sjogren's syndrome.

[0064] As used herein, the term "muscle degenerative disease or disorder" refers to any disease or disorder involving muscle degeneration. This term includes conditions, disorders, syndromes, diseases and injuries affecting muscle tissue, such as muscle atrophy, disuse muscle, muscle rupture, burns, surgery, peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, primary lateral sclerosis, myasthenia gravis, cancer, AIDS, congestive heart failure, chronic obstructive pulmonary disease (COPD), liver disease, renal failure, eating disorders, malnutrition, starvation, infections, or treatment with glucocorticoids, but is not limited thereto.

[0065] "Therapeutically effective dose or amount" is intended to mean an amount of rejuvenated cells or non-integrating messenger RNA that produces a positive therapeutic response in a subject in need of tissue repair or regeneration, such as an amount that restores function at the site of treatment and / or results in the generation of new tissue. Rejuvenated cells can be produced by transfection in vitro, ex vivo or in vivo with one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors, as described herein. Thus, for example, a "positive therapeutic response" is an improvement in an age-related disease or condition associated with a therapy, and / or an improvement in one or more symptoms of an age-related disease or condition associated with a therapy, such as restored tissue functionality, reduced pain, improved stamina, increased strength, increased mobility and / or improved cognitive function. The exact amount required (of cells or mRNA) will vary between subjects depending on the species, age and general condition of the subject, the severity of the condition being treated, the route of administration and others. The appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation, based on the information provided herein.

[0066] For example, a therapeutically effective dose or amount of rejuvenated chondrocytes, when administered as described herein, such as an amount that results in the generation of new cartilage at the treatment site (e.g., an injured joint), is intended to provide a positive therapeutic response in a subject having cartilage damage or loss. For example, the therapeutically effective dose or amount can be used in the treatment of cartilage damage or loss resulting from traumatic injury, or other degenerative diseases such as those involving arthritis or cartilage degeneration. Preferably, the therapeutically effective amount restores function and / or reduces pain and inflammation associated with cartilage damage or loss.

[0067] In another example, a therapeutically effective dose or amount of rejuvenated skeletal muscle stem cells, when administered as described herein, such as an amount that results in the generation of new muscle fibers at the treatment site (e.g., an injured muscle), is intended to provide a positive therapeutic response in a subject having muscle damage or loss. For example, the therapeutically effective dose or amount can be used in the treatment of muscle damage or loss resulting from traumatic injury, or diseases or disorders involving muscle degeneration. Preferably, the therapeutically effective amount improves muscle strength and muscle function.

[0068] As used herein, the terms "subject," "individual," and "patient" are used interchangeably herein and refer to any vertebrate subject, including humans, and other primates including non-human primates such as chimpanzees and other apes and monkey species; livestock such as cows, sheep, pigs, goats, and horses; domesticated mammals such as dogs and cats; rodents such as mice, rats, rabbits, hamsters, and guinea pigs; and birds including domesticated, wild, and game birds such as chickens, turkeys, and other poultry, ducks, geese, and the like, without limitation. In some instances, the methods of the disclosure find use in experimental animals, veterinary applications, and the development of animal models for diseases. The term does not denote a particular age. Thus, both adult and neonatal individuals are intended to be covered.

[0069] II. Methods Before describing the present disclosure in detail, it should be understood that, as certain formulations or process parameters will of course vary, the present disclosure is not limited to such specific formulations or process parameters. It should also be understood that the terminology used herein is for the purpose of describing only particular embodiments of the present disclosure and is not intended to be limiting.

[0070] A number of methods and materials similar or equivalent to those described herein may be used in the practice of the present disclosure, but the preferred materials and methods are described herein.

[0071] The present disclosure relates to a method of rejuvenating aged cells and tissues and restoring functionality by transient overexpression of mRNA that affects, for example, mitochondrial function, proteolytic activity, heterochromatin levels, histone methylation, nuclear lamina polypeptides, cytokine secretion or aging. In particular, the inventors have shown that mRNAs encoding OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG can be used to rejuvenate various cell types including fibroblasts, endothelial cells, chondrocytes and skeletal muscle stem cells while maintaining the cells in a differentiated cell state.

[0072] For a further understanding of the present disclosure, a more detailed description of methods of rejuvenating cells by transient reprogramming with mRNA and cell-based therapies using such rejuvenated cells is provided below.

[0073] a. Rejuvenation of cells In one aspect, provided herein is a method of rejuvenating a cell, the method comprising transfecting the cell with one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors for a period of 5 days or less in succession, thereby producing a rejuvenated cell.

[0074] In an embodiment, the rejuvenated cells have a phenotype or activity profile similar to that of young cells. The phenotype or activity profile includes one or more of a transcriptome profile, gene expression of one or more nuclear and / or epigenetic markers, proteolytic activity, mitochondrial health and function, SASP cytokine expression, and methylation landscape.

[0075] In embodiments, the rejuvenated cells have a transcriptomic profile similar to that of young cells. In embodiments, the transcriptomic profile of the rejuvenated cells includes an increase in gene expression of one or more genes selected from RPL37, RHOA, SRSF3, EPHB4, ARHGAP18, RPL31, FKBP2, MAP1LC3B2, Elf1, Phf8, Pol2s2, Taf1, and Sin3a. In embodiments, the transcriptomic profile of the rejuvenated cells includes an increase in gene expression of RPL37. In embodiments, the transcriptomic profile of the rejuvenated cells includes an increase in gene expression of RHOA. In embodiments, the transcriptomic profile of the rejuvenated cells includes an increase in gene expression of SRSF3. In embodiments, the transcriptomic profile of the rejuvenated cells includes an increase in gene expression of EPHB4. In embodiments, the transcriptomic profile of the rejuvenated cells includes an increase in gene expression of ARHGAP18. In embodiments, the transcriptomic profile of the rejuvenated cells includes an increase in gene expression of RPL31. In embodiments, the transcriptomic profile of the rejuvenated cells includes an increase in gene expression of FKBP2. In embodiments, the transcriptomic profile of the rejuvenated cells includes an increase in gene expression of MAP1LC3B2. In embodiments, the transcriptomic profile of the rejuvenated cells includes an increase in gene expression of Elf1. In embodiments, the transcriptomic profile of the rejuvenated cells includes an increase in gene expression of Phf8. In embodiments, the transcriptomic profile of the rejuvenated cells includes an increase in gene expression of Pol2s2. In embodiments, the transcriptomic profile of the rejuvenated cells includes an increase in gene expression of Taf1. In embodiments, the transcriptomic profile of the rejuvenated cells includes an increase in gene expression of Sin3a. In embodiments, the transcriptomic profile of the rejuvenated cells includes an increase in gene expression of RPL37, RHOA, SRSF3, EPHB4, ARHGAP18, RPL31, FKBP2, MAP1LC3B2, Elf1, Phf8, Pol2s2, Taf1, and Sin3a.

[0076] In embodiments, the rejuvenated cells exhibit increased gene expression of one or more nuclear and / or epigenetic markers as compared to a reference value. In embodiments, the one or more nuclear and / or epigenetic markers are selected from HP1 gamma, H3K9me3, lamin - associated protein LAP2 alpha, and SIRT1 protein. In embodiments, the rejuvenated cells exhibit increased gene expression of HP1 gamma. In embodiments, the rejuvenated cells exhibit increased gene expression of H3K9me3. In embodiments, the rejuvenated cells exhibit increased gene expression of lamin - associated protein LAP2 alpha. In embodiments, the rejuvenated cells exhibit increased gene expression of SIRT1 protein. In embodiments, the rejuvenated cells exhibit increased gene expression of HP1 gamma, H3K9me3, lamin - associated protein LAP2 alpha, and SIRT1 protein.

[0077] In embodiments, the rejuvenated cells have a proteolytic activity similar to that of young cells. In embodiments, the proteolytic activity is measured as increased cellular autophagosome formation, increased chymotrypsin - like proteasome activity, or a combination thereof. In embodiments, the proteolytic activity is measured as increased cellular autophagosome formation. In embodiments, the proteolytic activity is measured as increased chymotrypsin - like proteasome activity. In embodiments, the proteolytic activity is measured as increased cellular autophagosome formation and increased chymotrypsin - like proteasome activity.

[0078] In embodiments, the rejuvenated cells exhibit improved mitochondrial health and function as compared to a reference value. In embodiments, the improved mitochondrial health and function are measured as an increased mitochondrial membrane potential, a decreased reactive oxygen species (ROS), or a combination thereof. In embodiments, the improved mitochondrial health and function are measured as an increased mitochondrial membrane potential. In embodiments, the improved mitochondrial health and function are measured as a decreased reactive oxygen species (ROS). In embodiments, the improved mitochondrial health and function are measured as an increased mitochondrial membrane potential and a decreased reactive oxygen species (ROS).

[0079] In embodiments, the rejuvenated cells exhibit a decreased expression of one or more SASP cytokines as compared to a reference value. In embodiments, the one or more SASP cytokines include IL18, IL1A, GROA, IL22, and IL9. In embodiments, the rejuvenated cells exhibit a decreased expression of IL18. In embodiments, the rejuvenated cells exhibit a decreased expression of IL1A. In embodiments, the rejuvenated cells exhibit a decreased expression of GROA. In embodiments, the rejuvenated cells exhibit a decreased expression of IL22. In embodiments, the rejuvenated cells exhibit a decreased expression of IL9. In embodiments, the rejuvenated cells exhibit a decreased expression of IL18, IL1A, GROA, IL22, and IL9.

[0080] In embodiments, the rejuvenated cells exhibit a reversal of the methylation landscape. In embodiments, the reversal of the methylation landscape is measured by estimation with the Horvath clock.

[0081] In embodiments, the reference value is obtained from aged cells.

[0082] In an embodiment, cells are rejuvenated by transient reprogramming with mRNA encoding one or more cell reprogramming factors. Transient reprogramming is achieved by transfecting non-integrating mRNA into cells once a day for at least 2 days and up to 5 days. "Non-integrating" means that the mRNA molecules are not integrated chromosomally or extrachromosomally into the host genome and not integrated into a vector, such that the reprogramming is transient and does not disrupt the identity of the rejuvenated cells (i.e., the cells retain their ability to differentiate into their adult cell type). In an embodiment, transient reprogramming of cells eliminates various features of aging while avoiding complete dedifferentiation of the cells into stem cells.

[0083] In an embodiment, the step of transfecting messenger RNA into cells can be achieved by a transfection method selected from Lipofectamine and LT-1-mediated transfection, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, electroporation, encapsulation of mRNA in liposomes, and direct microinjection. In an embodiment, the step of transfecting messenger RNA into cells can be achieved by Lipofectamine and LT-1-mediated transfection. In an embodiment, the step of transfecting messenger RNA into cells can be achieved by dextran-mediated transfection. In an embodiment, the step of transfecting messenger RNA into cells can be achieved by calcium phosphate precipitation. In an embodiment, the step of transfecting messenger RNA into cells can be achieved by polybrene-mediated transfection. In an embodiment, the step of transfecting messenger RNA into cells can be achieved by electroporation. In an embodiment, the step of transfecting messenger RNA into cells can be achieved by encapsulation of mRNA in liposomes. In an embodiment, the step of transfecting messenger RNA into cells can be achieved by direct microinjection.

[0084] Reversal or rejuvenation of cells is achieved by transient overexpression of one or more mRNAs encoding cell reprogramming factors. Such cell reprogramming factors can include transcription factors, epigenetic remodelers, or small molecules that affect mitochondrial function, proteolytic activity, heterochromatin levels, histone methylation, nuclear lamina polypeptides, cytokine secretion, or aging. In embodiments, the cell reprogramming factors include one or more of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. In another embodiment, the cell reprogramming factors include OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. In certain embodiments, the cell reprogramming factors consist of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0085] In embodiments, the methods provided herein can be applied to any type of cell in need of rejuvenation. Cells in culture or mixed with other cells in a tissue (partial or intact) or a living organism can be isolated from the other cells. The methods described herein can be performed, for example, on samples including single cells, populations of cells, or tissues or organs containing cells. The cells selected for rejuvenation depend on the desired therapeutic effect for the treatment of an age-related disease or condition.

[0086] In embodiments, the cells are mammalian cells. In embodiments, the cells are human cells. In embodiments, the cells are derived from an aged subject.

[0087] In embodiments, the methods provided herein can be performed in cells, tissues or organs of the nervous, muscular, respiratory, cardiovascular, skeletal, genital, integumentary, lymphatic, excretory, endocrine (e.g., endocrine and exocrine) or digestive systems. Any type of cell, including but not limited to epithelial cells (e.g., squamous, cuboidal, columnar and pseudostratified epithelial cells), endothelial cells (e.g., venous, arterial and lymphatic endothelial cells), and cells of connective tissue, muscle and nervous systems, can potentially rejuvenate as described herein. Such cells include epidermal cells, fibroblasts, chondrocytes, skeletal muscle cells, satellite cells, cardiomyocytes, smooth muscle cells, keratinocytes, basal cells, ameloblasts, exocrine secretory cells, myoepithelial cells, osteoblasts, osteoclasts, neurons (e.g., sensory neurons, motor neurons and interneurons), glial cells (e.g., oligodendrocytes, astrocytes, ependymal cells, microglia, Schwann cells and satellite cells), pillar cells, adipocytes, pericytes, stellate cells, lung cells, blood and immune system cells (e.g., erythrocytes, monocytes, dendritic cells, macrophages, neutrophils, eosinophils, mast cells, T cells, B cells, natural killer cells), hormone-secreting cells, germ cells, interstitial cells, lens cells, photoreceptor cells, taste receptor cells and olfactory cells; and cells and / or tissues derived from the kidney, liver, pancreas, stomach, spleen, gallbladder, intestine, bladder, lung, prostate, breast, urogenital tract, pituitary cells, oral cavity, esophagus, skin, hair, nails, thyroid, parathyroid, adrenal, eye, nose or brain, but are not limited thereto.

[0088] In some embodiments, the cell is selected from fibroblasts, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells and corneal epithelial cells. In an embodiment, the cell is a fibroblast. In an embodiment, the cell is an endothelial cell. In an embodiment, the cell is a chondrocyte. In an embodiment, the cell is a skeletal muscle stem cell. In an embodiment, the cell is a keratinocyte. In an embodiment, the cell is a mesenchymal stem cell. In an embodiment, the cell is a corneal epithelial cell.

[0089] In an embodiment, the rejuvenated fibroblasts exhibit a transcriptome profile similar to that of young fibroblasts. In an embodiment, the rejuvenated fibroblasts show increased gene expression of one or more nuclear and / or epigenetic markers as compared to the reference values described above. In an embodiment, the rejuvenated fibroblasts have a proteolytic activity similar to that of the young cells described above. In an embodiment, the rejuvenated fibroblasts show improved mitochondrial health and function as compared to the reference values described above. In an embodiment, the rejuvenated fibroblasts show a reversal of the methylation landscape.

[0090] In an embodiment, the rejuvenated endothelial cells exhibit a transcriptome profile similar to that of young endothelial cells. In an embodiment, the rejuvenated endothelial cells show increased gene expression of one or more nuclear and / or epigenetic markers as compared to the reference values described above. In an embodiment, the rejuvenated endothelial cells have a proteolytic activity similar to that of the young cells described above. In an embodiment, the rejuvenated endothelial cells show improved mitochondrial health and function as compared to the reference values described above. In an embodiment, the rejuvenated endothelial cells show a reversal of the methylation landscape.

[0091] In embodiments, rejuvenated chondrocytes exhibit decreased expression of inflammatory factors and / or increased ATP and collagen metabolism. In embodiments, the inflammatory factors include RANKL, iNOS2, IL6, IFNα, MCP3, and MIP1A. In embodiments, rejuvenated chondrocytes exhibit decreased expression of RANKL. In embodiments, rejuvenated chondrocytes exhibit decreased expression of iNOS2. In embodiments, rejuvenated chondrocytes exhibit decreased expression of IL6. In embodiments, rejuvenated chondrocytes exhibit decreased expression of IFNα. In embodiments, rejuvenated chondrocytes exhibit decreased expression of MCP3. In embodiments, rejuvenated chondrocytes exhibit decreased expression of MIP1A. In embodiments, rejuvenated chondrocytes exhibit decreased expression of RANKL, iNOS2, IL6, IFNα, MCP3, and MIP1A. In embodiments, rejuvenated chondrocytes exhibit increased ATP and collagen metabolism. In embodiments, ATP and collagen metabolism are measured by one or more of increased ATP levels, decreased ROS, increased SOD2 expression, increased COL2A1 expression, and overall proliferation by chondrocytes. In embodiments, ATP and collagen metabolism are measured by increased ATP levels. In embodiments, ATP and collagen metabolism are measured by decreased ROS and increased SOD2 expression. In embodiments, ATP and collagen metabolism are measured by increased COL2A1 expression and overall proliferation by chondrocytes.

[0092] In embodiments, rejuvenated skeletal muscle stem cells exhibit higher proliferative capacity, enhanced ability to differentiate into myoblasts and muscle fibers, lower reactivation kinetics from a quiescent state, ability to rejuvenate the muscle microniche, restoration of youthful strength in muscle, or a combination thereof.

[0093] In embodiments, rejuvenated keratinocytes exhibit higher proliferative capacity, a decreased inflammatory phenotype, lower RNAKL and INOS2 expression, decreased expression of the cytokines MIP1A, IL6, IFNa, MCP3, increased ATP, increased levels of SOD2, and COL2A1 expression.

[0094] In embodiments, rejuvenated mesenchymal stem cells exhibit a decrease in senescence parameters, increased cell proliferation, and / or a decrease in ROS levels. In embodiments, rejuvenated mesenchymal stem cells exhibit a decrease in senescence parameters. In embodiments, senescence parameters include p16 expression, p21 expression, and positive SAβGal staining. In embodiments, rejuvenated mesenchymal stem cells exhibit increased cell proliferation. In embodiments, rejuvenated mesenchymal stem cells exhibit a decrease in ROS levels. In embodiments, rejuvenated mesenchymal stem cells exhibit a decrease in senescence parameters, increased cell proliferation, and a decrease in ROS levels.

[0095] In embodiments, rejuvenated corneal epithelial cells exhibit a decrease in senescence parameters. In embodiments, senescence parameters include one or more of the expression of p21, the expression of p16, mitochondrial biogenesis PGC1α, and the expression of inflammatory factor IL8. In embodiments, senescence parameters include p21. In embodiments, senescence parameters include the expression of p16. In embodiments, senescence parameters include mitochondrial biogenesis PGC1α. In embodiments, senescence parameters include the expression of inflammatory factor IL8. In embodiments, senescence parameters include one or more of the expression of p21, the expression of p16, mitochondrial biogenesis PGC1α, and the expression of inflammatory factor IL8.

[0096] The methods of the present disclosure can be used to rejuvenate cells in culture (e.g., ex vivo or in vitro) to improve function and differentiation potential for use in cell therapy. The cells used in the treatment of a patient may be autologous or allogeneic. Preferably, the cells are derived from the patient or a matched donor. For example, in ex vivo therapy, cells are obtained directly from the patient to be treated, transfected with mRNA encoding the cell reprogramming factors described herein, and re-transplanted into the patient. Such cells can be obtained, for example, from a biopsy or surgical procedure performed on the patient. Alternatively, the cells in need of rejuvenation may be directly transfected in vivo with mRNA encoding the cell reprogramming factors.

[0097] Transfection can be performed using any suitable method known in the art that results in transient uptake of mRNA encoding cell reprogramming factors into cells that require rejuvenation (i.e., for transient reprogramming). In embodiments, methods for ex vivo, in vitro, or in vivo delivery of mRNA to target cells can include methods selected from lipofectamine and LT-1-mediated transfection, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, electroporation, encapsulation of mRNA in liposomes, direct microinjection of mRNA into cells, or combinations thereof.

[0098] b. Compositions In one aspect, provided herein is a pharmaceutical composition comprising rejuvenated cells obtained by transfecting a cell with one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors for a period of up to 5 consecutive days.

[0099] In embodiments, the rejuvenated cells are autologous. In embodiments, the rejuvenated cells are allogeneic.

[0100] In embodiments, the one or more cell reprogramming factors are selected from OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. In embodiments, the cell reprogramming factors are OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0101] In embodiments, the rejuvenated cells exhibit one or more of the following: increased expression of HP1 gamma, H3K9me3, LAP2 alpha, SIRT1, increased mitochondrial membrane potential and decreased reactive oxygen species, and decreased expression of SASP cytokines. In embodiments, the SASP cytokines include one or more of IL18, IL1A, GROA, IL22, and IL9.

[0102] In certain embodiments, a composition comprising rejuvenated cells for use in cell therapy can further comprise one or more additional factors such as nutrients, cytokines, growth factors, extracellular matrix (ECM) components, antibiotics, antioxidants, or immunosuppressive agents to improve cell function or viability. The composition can also further comprise a pharmaceutically acceptable carrier.

[0103] Examples of growth factors include, but are not limited to, fibroblast growth factor (FGF), insulin-like growth factor (IGF), transforming growth factor beta (TGF-β), epiregulin, epidermal growth factor (“EGF”), endothelial cell growth factor (“ECGF”), nerve growth factor (“NGF”), leukemia inhibitory factor (“LIF”), bone morphogenetic protein-4 (“BMP-4”), hepatocyte growth factor (“HGF”), vascular endothelial growth factor-A (“VEGF-A”), and cholecystokinin octapeptide.

[0104] Examples of ECM components include, but are not limited to, proteoglycans (e.g., chondroitin sulfate, heparan sulfate, and keratan sulfate), non-proteoglycan polysaccharides (e.g., hyaluronic acid), fibers (e.g., collagen and elastin), and other ECM components (e.g., fibronectin and laminin).

[0105] Examples of immunosuppressive agents include, but are not limited to, steroids (e.g., prednisone) or non-steroids (e.g., sirolimus (Rapamune, Wyeth-Ayerst Canada), tacrolimus (Prograf, Fujisawa Canada), and anti-IL2R daclizumab (Zenapax, Roche Canada)). Other immunosuppressive agents include 15-deoxyspergualin, cyclosporine, methotrexate, rapamycin, Rapamune (sirolimus / rapamycin), FK506, or lysophilin (LSF).

[0106] One or more pharmaceutically acceptable excipients may be included. Examples include, but are not limited to, carbohydrates, inorganic salts, antibacterial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.

[0107] For example, an antibacterial agent for preventing or inhibiting the growth of microorganisms may be included. Non-limiting examples of antibacterial agents suitable for the present disclosure include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof. The antibacterial agent also includes antibiotics that can also be used for preventing bacterial infections. Examples of antibiotics include amoxicillin, penicillin, sulfonamides, cephalosporins, erythromycin, streptomycin, gentamicin, tetracycline, clarithromycin, ciprofloxacin, azithromycin, and others. Antifungal agents such as miconazole and terconazole are also included.

[0108] Various antioxidants may be included, such as reduced glutathione (GSH) or its precursors, glutathione or glutathione analogs, glutathione monoesters, and molecules having a thiol group such as N-acetylcysteine. Other suitable antioxidants include superoxide dismutase, catalase, vitamin E, trolox, lipoic acid, lazaroid, butylhydroxyanisole (BHA), vitamin K, and others.

[0109] Excipients suitable for the injectable composition include water, alcohol, polyol, glycerin, vegetable oil, phospholipid, and surfactant. Carbohydrates such as sugar, alditol, derivatized sugar such as aldonic acid, esterified sugar, and / or sugar polymer can be present as excipients. Specific carbohydrate excipients include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and others; disaccharides such as lactose, sucrose, trehalose, cellobiose, and others; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, and others; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myo-inositol, and others. The excipient can also include inorganic salts or buffers such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, monobasic sodium phosphate, dibasic sodium phosphate, and combinations thereof.

[0110] An acid or a base can also be present as an excipient. Non-limiting examples of acids that can be used include acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.

[0111] Typically, the optimal amount of any individual excipient is determined by routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from small to large amounts), testing for stability and other parameters, and then determining the range within which optimal performance is achieved without significant adverse effects. However, generally, the excipient(s) is / are present in the composition in an amount of from about 1% to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about 15 to about 95% by weight of excipient, with a concentration of less than 30% by weight being most preferred. These aforementioned pharmaceutical excipients are described, along with other excipients, in Remington: The Science & Practice of Pharmacy, 19th Edition, Williams & Williams (1995), Physician’s Desk Reference, 52nd Edition, Medical Economics, Montvale, NJ (1998) and Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, D.C., 2000.

[0112] c. Administration The methods of the present disclosure can be used for the treatment of a subject with an age-related disease or condition. For example, cell therapies (e.g., in vitro, ex vivo or in vivo) involving transient reprogramming of cells by transfection with non-integrating mRNA encoding reprogramming factors can be used for the treatment of neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, dementia and stroke), cardiovascular and peripheral vascular diseases (e.g., atherosclerosis, peripheral artery disease (PAD), hematoma, calcification, thrombosis, embolism and aneurysm), eye diseases (e.g., age-related macular degeneration, glaucoma, cataract, dry eye, diabetic retinopathy, vision loss), skin diseases (skin atrophy and thinning, elastolysis and skin wrinkles, seborrhea hyperplasia or hypoplasia, senile lentigines and other pigmentation abnormalities, gray hair, alopecia or hair thinning, and chronic skin ulcers), autoimmune diseases (e.g., polymyalgia rheumatica (PMR), giant cell arteritis (GCA), rheumatoid arthritis (RA), crystalline arthritis and spondyloarthritis (SPA)), endocrine and metabolic dysfunctions (e.g., adult hypopituitarism, hypothyroidism, apathetic thyrotoxicosis, osteoporosis, type 2 diabetes, adrenal insufficiency, various forms of hypogonadism and endocrine malignancies), musculoskeletal disorders (e.g., arthritis, osteoporosis, myeloma, gout, Paget's disease, fractures, myelodysplastic syndromes, ankylosis, diffuse idiopathic skeletal hyperostosis, hematogenous osteomyelitis, muscle atrophy, peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, primary lateral sclerosis and myasthenia gravis), gastrointestinal diseases (e.g., cirrhosis, hepatic fibrosis, Barrett's esophagus), respiratory diseases (e.g., pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, chronic bronchitis, pulmonary embolism (PE), lung cancer and infections), and any other diseases and disorders associated with aging, including but not limited to, can be used for the treatment of a subject with various age-related diseases and conditions.

[0113] In one aspect, provided herein is a method for treating an age-related disease or condition, a cartilage degenerative disorder, a neurodegenerative disorder, and / or a musculoskeletal dysfunction in a subject. The method includes administering to the subject a therapeutically effective amount of cells comprising one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors.

[0114] At least one therapeutically effective treatment cycle by transfection with one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors can be administered to a subject for the treatment of an age-related disease or condition.

[0115] In embodiments, the age-related disease or condition is selected from an eye, skin, or musculoskeletal dysfunction.

[0116] In embodiments, the subject has a cartilage degenerative disorder. In embodiments, the disorder is selected from arthritis, chondrophasia, spondyloarthritis, ankylosing spondylitis, lupus erythematosus, relapsing polychondritis, and Sjogren's syndrome. In embodiments, the disorder is arthritis. In embodiments, the disorder is chondrophasia. In embodiments, the disorder is spondyloarthritis. In embodiments, the disorder is ankylosing spondylitis. In embodiments, the disorder is lupus erythematosus. In embodiments, the disorder is relapsing polychondritis. In embodiments, the disorder is Sjogren's syndrome.

[0117] In embodiments, the treatment decreases the expression of one or more inflammatory factors and / or increases ATP and collagen metabolism. In embodiments, the inflammatory factor is selected from RANKL, iNOS2, IL6, IFNα, MCP3, and MIP1A. In embodiments, ATP and collagen metabolism are measured by one or more of increased ATP levels, decreased ROS, increased SOD2, increased COL2A1, and overall proliferation by chondrocytes.

[0118] In embodiments, the treatment of a subject with rejuvenated cells by ex vivo or in vitro transfection in cell culture, or with a composition for transplanting rejuvenated cells, is typically administered by injection or surgical implantation into areas requiring tissue regeneration or repair, but not necessarily so.

[0119] In embodiments, a therapeutically effective amount of rejuvenated cells is selected from fibroblasts, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells. In embodiments, a therapeutically effective amount of rejuvenated cells is fibroblasts. In embodiments, a therapeutically effective amount of rejuvenated cells is endothelial cells. In embodiments, a therapeutically effective amount of rejuvenated cells is chondrocytes. In embodiments, a therapeutically effective amount of rejuvenated cells is skeletal muscle stem cells. In embodiments, a therapeutically effective amount of rejuvenated cells is keratinocytes. In embodiments, a therapeutically effective amount of rejuvenated cells is mesenchymal stem cells. In embodiments, a therapeutically effective amount of rejuvenated cells is corneal epithelial cells.

[0120] In embodiments, rejuvenated corneal epithelium exhibits a decrease in aging parameters. In embodiments, the aging parameters include one or more of the expression of p21 and p16, mitochondrial neogenesis PGC1α, and the expression of the inflammatory factor IL8.

[0121] In one embodiment, chondrocytes in the area of cartilage damage or loss are transfected in vivo with an effective amount of one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors sufficient to effect rejuvenation of chondrocytes and generation of new cartilage at the treatment site. Alternatively, rejuvenated chondrocytes produced by ex vivo or in vitro transfection can be locally administered to an area of cartilage damage or loss such as the damaged joint of a subject or other suitable treatment site. The therapeutically effective dose or amount of rejuvenated chondrocytes is intended to be an amount that provides a positive therapeutic response in a subject having cartilage damage or loss, such as an amount that results in the generation of new cartilage at the treatment site (e.g., the damaged joint). For example, the therapeutically effective dose or amount can be used for the treatment of cartilage damage or loss resulting from traumatic injury or other degenerative diseases such as arthritis or other diseases involving cartilage degeneration. Preferably, the therapeutically effective amount restores function and / or reduces pain and inflammation associated with cartilage damage or loss.

[0122] In another embodiment, skeletal muscle stem cells are transfected in vivo with an effective amount of one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors sufficient to effect rejuvenation (i.e., restoration of differentiation potential) of skeletal muscle stem cells and generation of new muscle fibers at the treatment site (e.g., the damaged muscle). Alternatively, rejuvenated skeletal muscle stem cells produced by ex vivo or in vitro transfection can be locally administered to the damaged muscle that requires repair or regeneration. For example, the therapeutically effective dose or amount can be used for the treatment of muscle damage or loss resulting from traumatic injury, muscle atrophy, or diseases or disorders involving muscle degeneration. The therapeutically effective dose or amount of rejuvenated skeletal muscle stem cells is intended to be an amount that provides a positive therapeutic response in a subject having muscle damage or loss, such as an amount that results in the generation of new muscle fibers at the treatment site (e.g., the damaged muscle). Preferably, the therapeutically effective amount improves muscle strength and muscle function, reduces pain, improves stamina, and / or increases mobility.

[0123] In one aspect, as described above in this specification, methods are provided herein for treating an age-related disease or condition of interest, a cartilage degenerative disorder, and / or for treating a subject having a musculoskeletal dysfunction. The method includes administering, as described above in this specification, a therapeutically effective amount of one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors.

[0124] In embodiments, cells in a subject can be rejuvenated by in vivo transfection with an effective amount of one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors, as described herein.

[0125] In one aspect, methods are provided herein for rejuvenating an engineered tissue ex vivo. The method includes transfecting the tissue with one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors for a period of 5 days or less in succession, thereby producing a rejuvenated engineered tissue.

[0126] In embodiments, the engineered tissue exhibits aging parameters, a decrease in inflammation-promoting factors, an improvement in histological score, or a combination thereof. In embodiments, the engineered tissue exhibits a decrease in one or more aging parameters. In embodiments, the aging parameters are selected from p16 expression, positive SAβGal staining, and the expression of the inflammation-promoting factors IL8 and MMP1. In embodiments, the engineered tissue exhibits a decrease in p16 expression. In embodiments, the engineered tissue exhibits a decrease in positive SAβGal staining. In embodiments, the engineered tissue exhibits a decrease in the expression of the inflammation-promoting factors IL8 and MMP1. In embodiments, the engineered tissue exhibits an improvement in histological score. In embodiments, the histological score includes morphology, organization, and / or quality.

[0127] In embodiments, the engineered tissue is an engineered skin tissue and an organoid.

[0128] d. Kit The present disclosure also provides a kit comprising one or more containers holding a composition comprising one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors for transient reprogramming of cells. The kit can further comprise a transfection agent, a medium for culturing cells, and optionally, one or more other factors such as growth factors, ECM components, antibiotics and the like. The mRNA encoding the cell reprogramming factor and / or other compositions may be in liquid form or lyophilized state. Such a kit can also comprise components that preserve or maintain the mRNA, protecting it from degradation. Such components may be RNase-free or capable of protecting against RNases. Suitable containers for the composition can include, for example, bottles, vials, syringes and test tubes. The containers can be made of various materials including glass or plastic. The containers can have a sterile access port (for example, the container can be an intravenous solution bag or vial having a stopper that can be penetrated by a hypodermic needle).

[0129] The kit can further comprise a second container containing a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution or dextrose solution. This can also contain other materials useful to the end user, including other pharmaceutically acceptable formulation solutions such as buffers, diluents, filters, needles and syringes or other delivery devices. The delivery device may be pre-filled with the composition.

[0130] The kit can also include a package insert containing written instructions for a method of treating an age-related disease or condition. The package insert may be an unapproved draft package insert or a package insert approved by the Food and Drug Administration (FDA) or other regulatory agency.

[0131] In certain embodiments, the kit comprises mRNA encoding one or more cell reprogramming factors selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. In one embodiment, the kit comprises mRNA encoding the OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG cell reprogramming factors.

[0132] III. Experiments Examples of specific embodiments for carrying out the present disclosure are shown below. The examples are provided for illustrative purposes only and are in no way intended to limit the scope of the present disclosure.

[0133] Attempts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but of course some experimental error and deviation should be tolerated.

[0134] It is understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes suggested thereby to those skilled in the art should be included within the spirit and scope of the present application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Examples

[0135] [Example 1]: Transient and non-integrating cell reprogramming promotes a multifaceted reversal of aging The experiments described herein depict the extent of the aging reversal effect that can be achieved by a transient reprogramming protocol that halts before cell identity is irreversibly lost. Recent evidence has also shown that partial transgenic reprogramming can alleviate age-associated features and extend lifespan in progeroid mice. However, it is unclear how this form of "epigenetic rejuvenation" can be widely applied to natural aging and, importantly, how it can be safely bridged to human cells. The data herein show that transient reprogramming based on mRNA technology reverses the features of physiological aging, reduces the age-related disease phenotype, and restores the regenerative response that declines with age in somatic and stem cells obtained from human clinical samples. The non-integrating method of transient cell reprogramming described herein opens the way to new, more translatable strategies for ex vivo cell rejuvenation therapy aimed at regenerative medicine and for in vivo tissue rejuvenation therapy that delays or reverses the physiological decline of natural aging and the pathogenesis of age-related diseases.

[0136] To examine whether substantial and measurable reprogramming of any cell age can be achieved before the point of no return and whether this can lead to any alleviation of cell function and physiological function, the effect of transient reprogramming on the aged physiological functions of two distinct cell types, fibroblasts and endothelial cells, derived from otherwise healthy human subjects was evaluated and compared to the same cell types harvested from young donors. Fibroblasts were derived from punch arm and abdominal skin biopsies (young controls 25 - 35 years old, n = 3, and aged group 60 - 70 years old, n = 3), while endothelial cells were extracted from collagenase digestion of the iliac vein and artery (young controls 15 - 25 years old, n = 3, and aged group 45 - 50 years old, n = 3).

[0137] A non-integrating reprogramming protocol was utilized. The protocol was optimized based on a cocktail of mRNAs expressing OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG (OSKMLN). Exploration of multiple reprogramming durations revealed that both cell types exhibited rapid changes in multiple aging parameters as early as R2X2 (two reprogramming transfections and a 2-day relaxation back to the basal state), although the most prominent effects were seen at R4X2 (Figures 1A and 2A). The protocol consistently produces induced pluripotent stem cell (iPSC) colonies regardless of donor age after 12 - 15 daily transfections; the Applicants concluded that PNR in their platform occurs at approximately day 5 of reprogramming based on the observation that the first detectable expression of endogenous pluripotency-related lncRNAs occurs on day 5. Accordingly, a transient reprogramming protocol was employed where OSKMLN was transfected daily for 4 consecutive days, and gene expression analysis was performed 2 days after the interruption.

[0138] Paired-end bulk RNA sequencing was performed in both cell types for the same three cohorts: young (Y), untreated aged (UA), and treated aged (TA). First, the quantile-normalized transcriptomes of young and untreated aged cells for each cell type (“Y vs UA”) were compared. The data showed that 961 genes (5.85%) in fibroblasts (678 upregulated, 289 downregulated) and 748 genes (4.80%) in endothelial cells (389 upregulated, 377 downregulated) were different between young and aged cells at a significance criterion of p < 0.05 and a log fold change cutoff of + / - 0.5 (Figure 6). These gene sets were enriched in many of the known aging pathways identified in the Molecular Signatures Database hallmark gene set collection. When the directionality of expression above or below the mean of each gene was mapped, a clear similarity between treated and young cells was observed for both fibroblasts and endothelial cells, in contrast to aged cells. Performing principal component analysis (PCA) in this gene set space, it was determined that the young and aged populations were separable along the first principal component (PC1), which explained 64.8% of the variance in fibroblasts and 60.9% of the variance in endothelial cells. Intriguingly, the treated cells also clustered closer to the younger population along PC1 (Figures 1K and 2J).

[0139] Using the same significance criteria defined above, untreated and treated - aged populations (the "UA vs. TA") were compared, and it was found that 1,042 genes in fibroblasts (734 up - regulated, 308 down - regulated) and 992 genes in endothelial cells (461 up - regulated, 531 down - regulated) were differentially expressed. Interestingly, within these gene sets, the Applicants found an enrichment of aging pathways in the molecular signature database. When comparing the profiles young vs. untreated - aged ("Y vs. UA") and untreated - aged vs. treated - aged ("UA vs. TA") in each cell type, 24.7% overlap in fibroblasts (odds ratio 4.53, p < 0.05) and 16.7% overlap in endothelial cells (odds ratio 3.84, p < 0.05) were observed, and the directionality of the changes in gene expression matched that of the young (i.e., if higher in youth, then higher in treated - aged); less than 0.5% moved in the opposite direction in either cell type.

[0140] Next, these transcriptome profiles were used to verify the retention of cell identity after transient reprogramming. For this purpose, using established cell identity markers, the Applicants verified that there was no significant change due to treatment (Figure 10). In addition, the Applicants were unable to detect the expression of any pluripotency - related markers (other than the transfected OSKMLN mRNA) (Figure 10). In short, the analysis of the transcriptome signature revealed that transient reprogramming induces a younger gene expression profile while retaining cell identity.

[0141] Epigenetic clocks based on DNA methylation levels are the most accurate molecular biomarkers of age across tissues and cell types and predict many age-related phenotypes, including lifespan. Exogenous expression of the reprogramming factors (OSKM) is known to revert the epigenetic age of primary cells to a prenatal state. To test whether transient expression of OSKMLN can reverse the epigenetic clock, two epigenetic clocks applied to human fibroblasts and endothelial cells were used: Horvath's original pan-tissue epigenetic clock (based on 353 cytosine-phosphate-guanine pairs.) and the Skin & Blood clock (based on 391 CpGs.).

[0142] According to the pan-tissue epigenetic clock, transient OSKMLN significantly (two-sided mixed effects model P value = 0.023) reverted the DNA methylation age (mean age difference = -3.40 years, standard error 1.17). The rejuvenation effect was more pronounced in endothelial cells (mean age difference = -4.94 years, SE = 1.63, Figure 6H) than in fibroblasts (mean age difference = -1.84, SE = 1.46, Figure 6G). Using the Skin and Blood epigenetic clock, qualitatively similar but less significant results were obtained (overall rejuvenation effect -1.35 years, SE = 0.67, one-sided mixed effects model P value = 0.042, mean rejuvenation in endothelial cells and fibroblasts was -1.62 years and -1.07, respectively.).

[0143] Motivated by these results, we analyzed the effect of transient reprogramming on various features of cellular physiological aging. Using a panel of 11 established assays covering features of aging (Figure 11), we performed most of the analyses using single-cell high-throughput imaging to capture quantitative changes and distribution shifts of single cells across the entire cell population. We performed the full analysis separately for each individual cell line (total 19 fibroblast cell lines: 3 young, 8 aged, and 8 treated-aged; total 17 endothelial cell lines: 3 young, 7 aged, and 7 treated-aged). We performed statistical analyses for each pair of sample sets; for ease of presentation, we then pooled the data by age category (see Materials and Methods for a detailed description of the statistical methods used). We performed control experiments by adopting the same transfection scheme using mRNA encoding GFP.

[0144] To expand our epigenetic findings, we performed experiments to quantitatively measure the epigenetic repression mark H3K9me3, the heterochromatin-related protein HP1γ, and the nuclear lamina support protein LAP2α by immunofluorescence (IF) (Figure 1B, Figure 1C, Figure 2B, Figure 2C, Figure 5A–Figure 5C). Aged fibroblasts and endothelial cells showed a decrease in nuclear signals for all three markers compared to young cells. Treatment of aged cells resulted in an increase in these markers in both cell types. Next, we tested both pathways involved in cellular proteolytic activity by measuring autophagosome formation and chymotrypsin-like proteasome activity, which decrease with aging. Treatment increased both pathways to levels similar to or even higher than those in young cells, suggesting that the initial steps in reprogramming promote the active clearance of degraded biomolecules (Figure 1D, Figure 2D, Figure 5G–Figure 5H).

[0145] From an energy metabolism perspective, aged cells exhibit decreased mitochondrial activity, accumulation of reactive oxygen species (ROS), and deregulated nutrient sensing. Therefore, the effects of treatments in aged cells were examined by measuring mitochondrial membrane potential, mitochondrial ROS, and the level of sirtuin 1 protein (SIRT1) in cells. Transient reprogramming increased mitochondrial membrane potential in both cell types (left panels of Figures 1E, 2E, and 5E), which, as in young cells (Figures 1F, 2F, and 5D), decreased ROS in fibroblasts (right panels of Figures 1E, 2E, and 5F) and increased SIRT1 protein levels. Senescence-associated beta-galactosidase staining showed a significant decrease in the number of senescent cells in aged endothelial cells (Figures 1H, 2H, and 5I). This decrease was also accompanied by a decrease in pro-inflammatory senescence-associated secretory phenotype (SASP) cytokines in endothelial cells (Figure 5J). Finally, in both cell types, telomere length measured by quantitative fluorescence in situ hybridization did not show a significant elongation upon treatment (Figures 1G and 2G), suggesting that the cells did not dedifferentiate into a stem cell-like state in which telomerase activity was reactivated.

[0146] Next, when evaluating the persistence of these effects, it was found that most were significantly retained 4 and 6 days after the interruption of reprogramming. By repeating the same set of experiments in fibroblasts and endothelial cells transfected for only 2 consecutive days, it was investigated how rapidly these physiological rejuvenation changes became apparent. Notably, the data showed that most of the rejuvenation effects, although mostly more modest, could already be seen after 2 days of treatment.

[0147] In summary, this data demonstrates that transient expression of OSKMLN can induce a rapid and persistent reversal of cellular age at the transcriptomic, epigenetic, and cellular levels in human cells. Importantly, these data demonstrate that the process of "cellular rejuvenation," termed epigenetic reprogramming of aging or "ERA" herein, is involved in the iPSC reprogramming process very early and rapidly. These epigenetic and transcriptional changes occur before any epigenetic reprogramming of cellular identity takes place.

[0148] These signs of the beneficial effects of ERA in cellular aging led to experiments to investigate whether ERA could also reverse the age-related inflammatory phenotype. After obtaining preliminary evidence of this reversal in endothelial cells (Figure 5J), the analysis was extended to osteoarthritis, a disease strongly associated with aging and characterized by a prominent inflammatory spectrum affecting chondrocytes within joints. Chondrocytes were isolated from cartilage of 60 - 70-year-old patients undergoing total joint replacement surgery for advanced OA and their treatment outcomes were compared with chondrocytes isolated from young individuals. Transient reprogramming was performed for 2 or 3 days and analysis was carried out 2 days after the interruption of reprogramming. A more consistent effect across patients was brought about by longer treatment. The treatment showed a significant decrease in the intracellular mRNA levels of pro-inflammatory cytokines (Figure 7I), RANKL and iNOS2, and the levels of inflammatory factors secreted by the cells (Figures 3H - 3I and 7I). In addition, ERA promoted cell proliferation (Figures 3A and 7D), increased ATP production (Figures 3C and 7A), and decreased oxidative stress as revealed by decreased mitochondrial ROS and increased RNA levels of the antioxidant SOD2, a gene shown to be downregulated in OA (Figures 3D, 3E, 7B and 7D). ERA did not affect the expression level of SOX9, a transcription factor central to chondrocyte identity and function, but significantly increased the level of expression of COL2A1, the major collagen in articular cartilage (qRT-PCR in Figures 3B, 7E and 7F), suggesting the maintenance of chondrogenic cell identity. Collectively, these results indicate that transient expression of OSKMLN can promote partial reversal of gene expression and cellular physiological functions in aged OA chondrocytes towards a healthier state.

[0149] Loss of stem cells with function and regenerative capacity represents another important feature of aging. Experiments were conducted to evaluate the effect of transient reprogramming in the aging changes of somatic stem cells that impair regeneration. First, the effect of transient reprogramming was examined in mouse-derived skeletal muscle stem cells (MuSCs). Using an artificial niche, MuSCs were treated for 2 days while maintaining them in a quiescent state. Initial experiments were performed using young (3 months) and aged (20 - 24 months) mouse MuSCs isolated by FACS. Treatment of aged MuSCs decreased both the time to the first division, which was closer to the faster activation kinetics of quiescent young MuSCs, and the mitochondrial mass. Furthermore, the treatment partially rescued the decreased ability of single MuSCs to form colonies. When such cells were further cultured, the data showed that the treatment did not change the expression of the myogenic marker MyoD, but instead improved its ability to differentiate into myotubes, suggesting that transient reprogramming does not disrupt myogenic fate but can enhance myogenic potential.

[0150] Next, the MuSC function and differentiation ability to regenerate new tissue in vivo were examined. To do this, young, aged, or transiently reprogrammed aged MuSCs were transduced with lentiviruses expressing luciferase and green fluorescent protein (GFP), and then these cells were transplanted into the injured tibialis anterior (TA) muscle of immunodeficient mice. Longitudinal bioluminescence imaging (BLI) initially showed that the muscle transplanted with treated - aged MuSCs exhibited the highest signal (day 4, Figure 4B), but by day 11 post - transplantation, it became comparable to that of the muscle with young MuSCs; conversely, the muscle with untreated - aged MuSCs showed a lower signal at all time points post - transplantation (Figure 4B). Immunofluorescence analysis further revealed a greater number of donor - derived (GFP + ) myofibers in the TA transplanted with treated MuSCs compared to that with untreated - aged MuSCs (Figure 4C). Furthermore, GFP from treated - aged cells +The myofibers showed an increased cross-sectional area compared to their untreated counterparts and were actually even larger than those of young controls (Figure 4D). Collectively, these results suggest an improved tissue regenerative capacity of transiently reprogrammed aged MuSCs. Three months later, all mice were sacrificed and no neoplastic lesions or teratomas were found.

[0151] To examine the potential long-term benefits of the treatment, a second injury was induced 60 days after cell transplantation, and again, the data showed that TA muscles transplanted with transiently reprogrammed aged MuSCs yielded higher BLI signals (Figure 4E).

[0152] Sarcopenia is an age-related condition characterized by loss of muscle mass and force production. Similarly, in mice, muscle function shows progressive degeneration with aging. To examine whether transient reprogramming of aged MuSCs improves cell-based treatment in the restoration of muscle physiological function in older mice, electrophysiological tests were performed to measure the production of twitch force in TA muscles isolated from young (4 months) or aged (27 months) immunodeficient mice. The data showed that TA muscles from aged mice had lower twitch forces compared to young mice, suggesting an age-related loss of force production (Figure 4F). Next, MuSCs were isolated from aged mice (20 - 24 months). After treating the aged MuSCs, the cells were transplanted into TA muscles with cardiotoxin injury in aged (27 months) immunodeficient mice. Thirty (30) days were allowed for the transplanted muscles to sufficiently regenerate. Electrophysiological tests were performed to measure the production of twitch force. Muscles transplanted with untreated aged MuSCs showed forces comparable to those of non-transplanted muscles from aged control mice (Figure 4H). In contrast, muscles that received treated aged MuSCs showed twitch forces comparable to those of non-transplanted muscles from young control mice. These results support that transient reprogramming combined with MuSC-based therapies can restore the physiological function of aged muscles to that of youthful muscles.

[0153] Finally, these results were translated to human MuSCs. Using surgical samples obtained from patients in different age ranges (10 - 80 years old), these were transduced with GFP and luciferase-expressing lentiviral vectors, and the experiments were repeated. Similar to the mice, the transplanted transiently reprogrammed aged human MuSCs resulted in increased BLI signals compared to untreated MuSCs from the same individual, comparable to those observed in young MuSCs (Figure 8D). Interestingly, the BLI signal ratio between the treated and untreated MuSCs in the contralateral muscle was higher in the older age group (60 - 80 years old) than in the younger age groups (10 - 30 or 30 - 55 years old), suggesting that ERA restores the functions lost in aged cells to a younger level (Figure 8E). Collectively, these results suggest that transient reprogramming partially restores the differentiation potential of aged MuSCs to a similar extent as that of young MuSCs without compromising their fate, thus having potential as a cell therapy in regenerative medicine.

[0154] Fibroblasts and keratinocytes from patients over 65 years old were combined to reconstitute three-dimensional (3D) in vitro engineered skin, which was transfected by adding a cocktail of reprogramming factors to the culture medium. Histological analysis for quality assessment was performed and numerical scores were assigned (Figure 8A). Rejuvenation was observed with reprogramming factors as measured by increased numerical scores compared to control untreated and retinoic acid-treated samples.

[0155] Retinal epithelial cells were cultured ex vivo and transiently reprogrammed with OSKMN for 2 or 3 days. The results showed a significant decrease in the expression of p16 (Figure 9A), p21 (Figure 9B), IL8 (Figure 9C), and PGC1a (Figure 9D).

[0156] Nuclear reprogramming into induced pluripotent stem cells (iPSCs) is a multiphase process that includes induction, maturation, and stabilization. After completion of such dynamic and complex "epigenetic reprogramming," iPSCs are not only pluripotent but also youthful. The data herein demonstrate that a non-integrating, mRNA-based platform for transient cell reprogramming can very rapidly reverse the characteristics of aging in the induction phase where cell-autonomous epigenetic erasure has not yet occurred. The data indicate that a rejuvenation process occurs in aged human cells by restoring lost functionality in diseased and aged stem cells while preserving cell identity.

[0157] [Example 2]: Method mRNA transfection : Cells were transfected using either mRNA-In (mTI Global Stem) for fibroblasts and chondrocytes to reduce cytotoxicity and Lipofectamine MessengerMax (Thermo Fisher) for endothelial cells and MuSCs, which are more difficult to transfect, according to the manufacturer's protocol. The culture medium was changed for fibroblasts and endothelial cells 4 hours after transfection, but not for chondrocytes or MuSCs because overnight incubation is required for the production of significant mRNA uptake. Delivery efficiency was confirmed by both immunostaining of GFP mRNA and individual factors in the OSKMNL cocktail. mRNA synthesis and transfection optimization were performed by the facility at ESI BIO, where Jens Durruthy-Durruthy, who is also a member of the Sebastiano Lab, serves as an advisor.

[0158] Fibroblast isolation and culture: Isolation was performed on healthy patients, and biopsies were taken from the proximal aspect of the mid-upper arm or the abdomen using 2 mm punch biopsies from a mixture of male and female patients in their 60s to 70s (aged) and 30s to 40s (young). Cells were cultured from these explants and maintained in Eagle's minimum essential medium containing Earle's salts supplemented with non-essential amino acids, 10% fetal bovine serum, and 1% penicillin / streptomycin.

[0159] Endothelial cell isolation and culture : Isolation was performed at the Coriell Institute from the iliac arteries and veins removed prior to death from donors in their 45s to 50s (aged) and 10s (young) who had died from sudden head trauma but were otherwise healthy. The tissue was digested with collagenase and culture was initiated using the cells released from the lumen. Cells were maintained in Medium 199 supplemented with 2 mM L-glutamine, 15% fetal bovine serum, 0.02 mg / ml endothelial growth supplement, 0.05 mg / ml heparin, and 1% penicillin / streptomycin.

[0160] Nuclear immunocytochemistry : Cells were washed with HBSS and then fixed with 15% paraformaldehyde in PBS for 15 minutes. Next, the cells were blocked with a blocking solution in PBS containing 1% BSA and 0.3% Triton X-100 for 30 minutes. Next, the primary antibody was applied in 1% BSA and 0.3% Triton X-100 in PBS and incubated overnight at 4°C. The next day, the cells were washed with HBSS and incubated for 2 hours before switching to the corresponding Alexa Flour-labeled secondary antibody. The cells were then washed again and then stained with DAPI for 30 minutes. Finally, the cells were switched to HBSS for imaging.

[0161] Autophagosome formation staining: The cells were washed with HBSS and switched to a staining solution containing an LC3-based fluorescent autophagosome marker (Sigma). Next, the cells were incubated at 37 °C with 5% CO2 for 20 minutes. Next, the cells were washed twice with HBSS / Ca / Mg. Next, the cells were stained for 15 minutes using the cell labeling dye CellTracker Deep Red. Next, the cells were switched to HBSS / Ca / Mg for single cell imaging by Operetta.

[0162] Proteasome activity measurement : The wells were first stained with PrestoBlue (Thermo), a cell viability dye, for 10 minutes. The well signals were read using a TECAN fluorescence plate reader. Next, the cells were washed with HBSS / Ca / Mg before switching to the original medium containing the LLVY-R110 fluorogenic substrate (Sigma) cleaved by chymotrypsin-like 20S proteasome activity. Next, the cells were incubated at 37 °C with 5% CO2 for 2 hours before reading again in a TECAN fluorescence plate reader.

[0163] Mitochondrial membrane potential staining: Tetramethylrhodamine, methyl ester, perchlorate (Thermo) was added to the cell culture medium. This dye is sequestered by mitochondria based on its membrane potential. Next, the cells were incubated at 37 °C with 5% CO2 for 30 minutes. Next, the cells were washed twice with HBSS / Ca / Mg before staining for 15 minutes using CellTracker Deep Red. Finally, the cells were imaged with Operetta in fresh HBSS / Ca / Mg.

[0164] Mitochondrial ROS measurement: The cells were washed with HBSS / Ca / Mg and then switched to HBSS / Ca / Mg containing MitoSOX, a fluorescent generating dye oxidized by superoxide in mitochondria. The cells were incubated at 37 °C for 10 minutes with 5% CO2. Next, the cells were washed twice with HBSS / Ca / Mg and then stained with CellTracker Deep Red for 15 minutes. Finally, the cells were imaged with an Operetta in fresh HBSS / Ca / Mg.

[0165] SaβGal histochemistry : The cells were washed twice with HBSS / Ca / Mg and then fixed with 15% paraformaldehyde in PBS for 6 minutes. Next, the cells were rinsed three times with HBSS / Ca / Mg before staining with an X-gal chromogenic substrate cleaved by endogenous β-galactosidase. The cells were maintained in the staining solution and incubated overnight at 37 °C with ambient CO2. The next day, the cells were washed again with HBSS / Ca / Mg before switching to a 70% glycerol solution for imaging under a Leica brightfield microscope.

[0166] Cytokine profiling: This work was carried out in collaboration with the Human Immune Monitoring Center at Stanford University. Cell culture media were collected and spun at 400 rcf for 10 minutes at RT. Next, the supernatant was snap frozen in liquid nitrogen until analysis. Analysis was performed using the human 63-plex kit (eBiosciences / Affymetrix). Beads were added to 96-well plates and washed in a Biotek ELx405 washer. Samples were added to plates containing mixed antibody-conjugated beads, incubated for 1 hour at room temperature, followed by overnight incubation with shaking at 4°C. Low temperature and room temperature incubation steps were performed on an orbital shaker at 500 - 600 rpm. After overnight incubation, the plates were washed in a Biotek ELx405 washer, and then biotinylated detection antibody was added while shaking at room temperature for 75 minutes. The plates were washed as described above and streptavidin-PE was added. After incubation at room temperature for 30 minutes, washing was performed as described above and read buffer was added to the wells. Each sample was measured in duplicate twice. Plates were read using a Luminex 200 instrument with 50 beads with lower binding per cytokine per sample. Custom assay control beads from Radix Biosolutions were added to all wells.

[0167] Antibody : Five primary antibodies were used for nuclear measurements: rabbit anti-histone H3K9me3 histone methylation (1:4000), mouse anti-HP1γ heterochromatin marker (1:200), rabbit anti-LAP2α (1:500) nuclear organizing protein, mouse anti-lamin A / C nuclear membrane marker, and rabbit anti-SIRT1 (1:200).

[0168] RNA sequencing and data analysis The cells were washed and digested with TRIzol (Thermo). Total RNA was isolated using a Total RNA Purification Kit (Norgen Biotek Corp), the RNA quality was evaluated using an RNA Analysis screentape (R6K screentape, Agilent), and RNA with RIN>9 was reverse transcribed into cDNA. Using the TruSeq RNA Sample Preparation Kit v2 (Illumina), a cDNA library was prepared using 1 μg of total RNA. The RNA quality was evaluated by an Agilent Bioanalyzer 2100, and RNA with RIN>9 was reverse transcribed into cDNA. Using the TruSeq RNA Sample Preparation Kit v2 (Illumina) with the added benefit of molecular indexing, a cDNA library was prepared using 500 ng of total RNA. Prior to any PCR amplification step, all cDNA fragment ends were randomly ligated to an adapter pair containing an 8-bp unique molecular index. Next, the molecular-indexed cDNA library was PCR amplified (15 cycles), and then QC was performed using a Bioanalzyer and Qubit. After successful QC, this was sequenced on an Illumina Nextseq platform to obtain 80-bp single-end read data. The read data was trimmed by 2 nucleotides at each end to remove low-quality portions and improve mapping to the genome. The resulting 78-nucleotide read data was compressed by removing duplicates, but the number of times each sequence occurred in each sample in the database was tracked. Next, the unique read data was mapped to the human genome using exact matches. This misreads read data with exon-exon boundary crossings, as well as errors and SNPs / mutations, but has no substantial impact on the estimation of the expression level of each gene. Next, the underlying genomic-derived annotation was assigned to each of the mapped read data. In the case of multiple annotations (e.g., miRNAs occurring in gene introns), a hierarchy based on heuristics was used to give each read data a unique identity.Next, this was used to identify the read data belonging to each transcript and to establish the coverage across each position of the transcript. Since this coverage was non-uniform and peaked, the applicants used the median of this coverage as an estimate of the gene expression value. Quantile normalization was used to compare the expression in different samples. Further data analysis was performed in MATLAB®. Next, the ratio of the expression levels was calculated to estimate the logarithm (base 2) of the fold change. The Student's t-test was used to determine significance with a p < 0.05 cutoff. ENCODE gene analysis, developed and made publicly available by the Butte Lab at the Stanford Center for Biomedical Informatics Research, was used for transcription factor identification.

[0169] Mouse : C57BL / 6 male mice and NSG mice were obtained from the Jackson Laboratory. NOD / MrkBomTac-Prkdcscid female mice were obtained from Taconic Biosciences. Mice were housed and maintained in the Veterinary Medical Unit of the Veterans Affairs Palo Alto Health Care Systems. The animal protocol was approved by the Administrative Panel of Laboratory Animal Care at Stanford University.

[0170] Human skeletal muscle specimen : The subjects ranged in age from 10 to 78 years. Human muscle biopsy specimens were obtained after obtaining patient informed consent as part of a human test research protocol approved by the Stanford University Institutional Review Board. All experiments were performed using fresh muscle specimens according to the availability of clinical procedures. Sample processing for cell analysis was started within 1 to 12 hours of sample isolation. In all tests, the standard deviation reflected the variability in the data from the tests using true biological replicates (i.e., distinct donors). The data were not correlated with donor identity.

[0171] MuSC isolation and purification: Muscle was harvested from the hindlimbs, mechanically dissociated to obtain a fragmented muscle suspension. Subsequently, digestion was performed for 45 - 50 minutes in a solution of Collagenase II-Ham's F10 (500 units per ml; Invitrogen). After washing, a second digestion was performed for 30 minutes with Collagenase II (100 units per ml) and Dispase (2 units per ml; ThermoFisher). The resulting cell suspension was washed, filtered, and stained with antibodies of VCAM-biotin (clone 429; BD Bioscience) diluted 1:100, CD31-FITC (clone MEC13.3; BD Bioscience), CD45-APC (clone 30-F11; BD Bioscience), and Sca-1-Pacific-Blue (clone D7; Biolegend). Human MuSCs were purified from fresh surgical samples 50, 51. The surgical samples were carefully dissected from adipose and fibrous tissues, and a disassociated muscle suspension was prepared as described for mouse tissues. Next, the resulting cell suspension was washed, filtered, and stained with anti-CD31-Alexa Fluor488 (clone WM59; BioLegend; #303110, 1:75), anti-CD45-Alexa Fluor 488 (clone HI30; Invitrogen; #MHCD4520, 1:75), anti-CD34-FITC (clone 581; BioLegend; #343503, 1:75), anti-CD29-APC (clone TS2 / 16; BioLegend; #303008, 1:75), and anti-NCAM-biotin (clone HCD56; BioLegend; #318319, 1:75). Next, unbound primary antibodies were washed, and the cells were incubated with streptavidin-PE / Cy7 (BioLegend) for 15 minutes at 4°C to detect NCAM-biotin. Cell sorting was performed on a calibrated BD-FACS Aria II (registered trademark) or BD FACSAria III flow cytometer equipped with 488-nm, 633-nm, and 405-nm lasers to obtain a MuSC population. A small portion of the sorted cells was seeded and stained for Pax7 and MyoD to evaluate the purity of the sorted population. See Supplementary Information for the FACS gating strategy.

[0172] Bioluminescence imaging :Bioluminescent imaging was performed using a Xenogen IVIS-Spectrum System (Caliper Life Sciences). Mice were anesthetized with 2% isoflurane at a flow rate of 2.5 l / min (n = 4). An intraperitoneal injection of D-luciferin (50 mg / ml, Biosynth International Inc.) dissolved in sterile PBS was administered. Immediately after the injection, mice were imaged for 30 seconds at maximum sensitivity (f-stop 1) and highest resolution (binning small). Exposure was used for 30 seconds per minute until the peak intensity of the bioluminescent signal began to decrease. Each image was saved for subsequent analysis. Imaging was performed in a blinded manner: the researcher performing the imaging was unaware of the identity of the experimental conditions of the transplanted cells.

[0173] Bioluminescence image analysis :Analysis of each image was performed using Living Image software, version 4.0 (Caliper Life Sciences). A manually generated circle was placed over the region of interest and sized to completely enclose the limb or designated area of the recipient mouse. Similarly, the background region of interest was placed in the area of the mouse outside the transplanted leg.

[0174] Tissue collection :The TA muscle was carefully dissected away from the bone, weighed, and placed in a 0.5% PFA solution overnight for fixation. Next, the muscle was transferred to a 20% sucrose solution for 3 hours or until the muscle reached its saturation point and began to sink. The tissue was then embedded in optimal cutting temperature (OCT) medium, frozen, and stored at -80 °C until sectioning. Sectioning was performed on a Leica CM3050S cryostat set to generate 10-μm sections. The sections were mounted on Fisherbrand Colorfrost slides. These slides were stored at -20 °C until immunohistochemical examination could be performed.

[0175] Histology : TA muscle was fixed with 0.5% electron microscopy grade paraformaldehyde for 5 hours and then transferred to 20% sucrose overnight. Next, the muscle was frozen in OCT and cryosectioned at a thickness of 10 μm and stained. Samples were processed according to the manufacturer's recommended protocol for colorimetric staining with hematoxylin-eosin (Sigma) or Gomorri trichrome (Richard-Allan Scientific).

[0176] MuSC immunostaining : A 1-hour blocking step with 20% donkey serum / 0.3% Triton in PBS was used to prevent unwanted primary antibody binding for all samples. The primary antibody was applied and incubated overnight at 4 °C in 20% donkey serum / 0.3% Triton in PBS. After four washes with 0.3% PBST, the fluorescent-conjugated secondary antibody was added and incubated at room temperature for 1 hour in 0.3% PBST. After three additional rinses, each slide was mounted using Fluoview mounting medium.

[0177] Antibody : The following antibodies were used in this study. The source of each antibody is indicated. Mouse: GFP (Invitrogen, #A11122, 1:250); luciferase (Sigma-Aldrich, #L0159, 1:200); collagen I (Cedarlane Labs, #CL50151AP, 1:200); HSP47 (Abcam, #ab77609, 1:200).

[0178] Imaging : Samples were imaged using either a standard fluorescence microscope and a 10× or 20× air objective lens. Volocity imaging software was used to adjust the excitation and emission filters, which came with pre-programmed AlexaFluor filter settings and were used whenever possible. The total exposure time was optimized in the first round of imaging and then maintained constant throughout subsequent imaging.

[0179] Image analysis : Using ImageJ, by using the color threshold plugin, the percentage of the area composed of collagen was calculated to create a mask of only the area where collagen was positive. Next, the area was divided by the total area of the sample found using the freehand drawing tool. All other analyses were performed using Volocity software, the fibers were manually counted using the freehand drawing tool, and the number of nuclei, eMHC+ fibers, neuromuscular junctions, and blood vessels were manually counted.

[0180] Lentiviral transduction : The luciferase and GFP protein reporters were subcloned into a third-generation HIV-1 lentiviral vector (CD51X DPS, SystemBio). For transduction of freshly isolated MuSCs, cells were seeded at a density of 30,000 - 40,000 cells per well on poly-D-lysine (Millipore Sigma, A-003-E) and ECM-coated 8-well chamber slides (Millipore Sigma, PEZGS0896), and incubated with 5 μl of concentrated virus per well and 8 μg / mL polybrene (Santa Cruz Biotechnology, sc-134220). The plates were spun at 3200 g for 5 minutes and then at 2500 g for 1 hour at 25 °C. Next, the cells were washed twice with fresh medium, scraped from the plates, and resuspended in the final volume according to the experimental conditions.

[0181] MitoTracker staining and flow cytometry analysis : MuSCs undergoing reprogramming and controls were washed twice with pure HamsF10 (without serum or pen / strep). Then, the MuSCs were stained with 0.5 μM MitoTracker Green FM (ThermoFisher, M7514) and DAPI for 30 minutes at 37 °C, washed three times with pure HamsF10, and analyzed using a BD FACSAria III flow cytometer.

[0182] Statistical analysisUnless otherwise stated, all statistical analyses were performed using MATLAB R2017a (MathWorks software) or GraphPad Prism 5 (GraphPad software). For statistical analysis, the t-test was used. All error bars represent s.e.m.; *p < 0.05; **p < 0.001; ***p < 0.0001.

[0183] Although the preferred embodiments of the present disclosure have been described and recited, it is recognized that various changes can be made therein without departing from the spirit and scope of the present disclosure.

[0184] [Table 1] TIFF0007696406000002.tif229164TIFF0007696406000003.tif168155

[0185] P embodiment

[0186] Embodiment P1. A method for rejuvenating cells, comprising: a) Transfecting one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors into the cells, wherein the transfecting step is performed once a day for at least 2 days and no more than 4 days; and b) Translating the one or more non-integrating messenger RNAs to produce one or more cell reprogramming factors in the cells, resulting in transient reprogramming of the cells, wherein the cells rejuvenate without dedifferentiating into stem cells.

[0187] Embodiment P2. The method according to Embodiment P1, wherein the one or more cell reprogramming factors are selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0188] Embodiment P3. The method according to Embodiment P2, wherein the cell reprogramming factors include OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0189] Embodiment P4. The method according to Embodiment P1, wherein the cell is a mammalian cell.

[0190] Embodiment P5. The method according to Embodiment P4, wherein the cell is a human cell.

[0191] Embodiment P6. The method according to Embodiment P1, wherein the cell is derived from an elderly subject.

[0192] Embodiment P7. The method according to Embodiment P1, wherein the cell is a fibroblast, an endothelial cell, a chondrocyte, or a skeletal muscle stem cell.

[0193] Embodiment P8. The method according to Embodiment P1, wherein the transient reprogramming results in increased expression of HP1 gamma, H3K9me3, lamin support protein LAP2 alpha, and SIRT1 protein, decreased nuclear folding, decreased vesicle formation, increased cellular autophagosome formation, increased chymotrypsin-like proteasome activity, increased mitochondrial membrane potential, or decreased reactive oxygen species (ROS).

[0194] Embodiment P9. The method according to Embodiment P1, wherein the cell is within a tissue or an organ.

[0195] Embodiment P10. The method according to Embodiment P9, wherein the transient reprogramming reduces the number of senescent cells within the tissue or organ.

[0196] Embodiment P11. The method according to Embodiment P9, wherein the transient reprogramming reduces the expression of GMSCF, IL18, and TNFα.

[0197] Embodiment P12. The method according to Embodiment P9, wherein the treatment restores the function of the cells within the tissue or organ, increases the differentiation ability, enhances the survival rate, or increases the replication ability or lifespan.

[0198] Embodiment P13. The method according to Embodiment P1, which is performed in vitro, ex vivo or in vivo.

[0199] Embodiment P14. The method according to Embodiment P1, wherein the step of transfection is performed once a day for 3 days or 4 days.

[0200] Embodiment P15. A method for treating an age-related disease or condition in a subject, comprising: a) transfecting one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors into cells in need of rejuvenation, either in vivo or ex vivo, wherein the transfection step is performed once a day for at least 2 days and no more than 4 days; and b) expressing one or more cell reprogramming factors in the cells to effect transient reprogramming of the cells, wherein the cells rejuvenate without dedifferentiating into stem cells.

[0201] Embodiment P16. The method according to Embodiment P15, wherein the one or more cell reprogramming factors are selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

[0202] Embodiment P17. The method according to Embodiment P16, wherein the cell reprogramming factors include OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

[0203] Embodiment P18. The method according to Embodiment P15, further comprising transplanting the rejuvenated cells into the subject.

[0204] Embodiment P19. The method according to Embodiment P15, wherein the age-related disease or condition is a degenerative disease.

[0205] Embodiment P20. The method according to Embodiment P15, wherein the age-related disease or condition is a neurodegenerative disease or a musculoskeletal disorder.

[0206] A method for treating a disease or disorder involving cartilage degeneration, comprising: a) transfecting one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors into chondrocytes in need of rejuvenation, either in vivo or ex vivo, wherein the transfecting step is performed once a day for at least 2 days and no more than 4 days; and b) expressing one or more cell reprogramming factors in the chondrocytes to effect transient reprogramming of the chondrocytes, such that the chondrocytes rejuvenate without dedifferentiating into stem cells.

[0207] Embodiment P22. The method according to embodiment P21, wherein the one or more cell reprogramming factors are selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0208] Embodiment P23. The method according to embodiment P22, wherein the cell reprogramming factors include OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0209] Embodiment P24. The method according to embodiment P21, wherein the disease or disorder involving cartilage degeneration is arthritis.

[0210] Embodiment P25. The method according to embodiment P24, wherein the arthritis is osteoarthritis or rheumatoid arthritis.

[0211] Embodiment P26. The method according to embodiment P21, wherein the treatment reduces inflammation in the subject.

[0212] Embodiment P27. The method according to embodiment P21, wherein the transfecting step is performed ex vivo, and the rejuvenated chondrocytes are transplanted into the joints of the subject's arthritis.

[0213] Embodiment P28. The method according to embodiment P27, wherein the chondrocytes are isolated from a cartilage sample obtained from the subject.

[0214] Embodiment P29. The method according to embodiment P21, wherein the treatment reduces the expression of RANKL, iNOS, IL6, IL8, BDNF, IFNα, IFNγ and LIF by chondrocytes and increases the expression of SOX9 and COL2A1.

[0215] Embodiment P30. The method according to embodiment P21, wherein the subject is an elderly subject.

[0216] Embodiment P31. The method according to embodiment P21, wherein the subject is a mammalian subject.

[0217] Embodiment P32. The method according to embodiment P31, wherein the mammalian subject is a human subject.

[0218] Embodiment P33. A method for treating a disease or disorder involving muscle degeneration in a subject, comprising: a) transfecting one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors into skeletal muscle stem cells in vivo or ex vivo, wherein the transfecting step is performed once a day for at least 2 days and 4 days or less; and b) expressing one or more cell reprogramming factors in the skeletal muscle stem cells, resulting in transient reprogramming of the skeletal muscle stem cells, wherein the skeletal muscle stem cells rejuvenate without losing their ability to differentiate into muscle cells.

[0219] Embodiment P34. The method according to embodiment P33, wherein the one or more cell reprogramming factors are selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

[0220] Embodiment P35. The method according to embodiment P34, wherein the cell reprogramming factors comprise OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

[0221] The method according to Embodiment P33, wherein the step of transfecting is performed ex vivo, and the rejuvenated skeletal muscle stem cells are transplanted into the muscle in the subject that requires repair or regeneration.

[0222] The method according to Embodiment P33, wherein the skeletal muscle stem cells are isolated from a muscle sample obtained from the subject.

[0223] The method according to Embodiment P33, wherein the treatment results in the regeneration of muscle fibers.

[0224] The method according to Embodiment P33, wherein the treatment restores the differentiation ability of the skeletal muscle stem cells.

[0225] The method according to Embodiment P33, wherein the subject is an elderly subject.

[0226] The method according to Embodiment P33, wherein the subject is a mammalian subject.

[0227] The method according to Embodiment P41, wherein the mammalian subject is a human subject.

[0228] Embodiment

[0229] A method for rejuvenating cells, comprising the step of transfecting one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors into the cells for no more than 5 consecutive days, thereby producing rejuvenated cells.

[0230] The method according to Embodiment 1, wherein the transcriptome profile of the rejuvenated cells becomes similar to the transcriptome profile of young cells.

[0231] Method according to embodiment 2, wherein the transcriptome profile of the rejuvenated cells comprises an increase in gene expression of one or more genes selected from RPL37, RHOA, SRSF3, EPHB4, ARHGAP18, RPL31, FKBP2, MAP1LC3B2, Elf1, Phf8, Pol2s2, Taf1 and Sin3a.

[0232] Method according to any one of the preceding embodiments, wherein the rejuvenated cells exhibit increased gene expression of one or more nuclear and / or epigenetic markers as compared to a reference value.

[0233] Method according to embodiment 4, wherein the marker is selected from HP1 gamma, H3K9me3, laminin support protein LAP2 alpha and SIRT1 protein.

[0234] Method according to any one of the preceding embodiments, wherein the rejuvenated cells exhibit increased proteolytic activity as compared to a reference value.

[0235] Method according to embodiment 6, wherein the increased proteolytic activity is measured as increased cellular autophagosome formation, increased chymotrypsin-like proteasome activity, or a combination thereof.

[0236] Method according to any one of the preceding embodiments, wherein the rejuvenated cells exhibit improved mitochondrial health and function as compared to a reference value.

[0237] Method according to embodiment 8, wherein the improved mitochondrial health and function is measured as increased mitochondrial membrane potential, decreased reactive oxygen species (ROS), or a combination thereof.

[0238] Method according to any one of the preceding embodiments, wherein the rejuvenated cells exhibit decreased expression of one or more SASP cytokines as compared to a reference value.

[0239] Embodiment 11. The method according to Embodiment 10, wherein the SASP cytokine comprises one or more of IL18, IL1A, GROA, IL22, and IL9.

[0240] Embodiment 12. The method according to any one of the preceding embodiments, wherein the rejuvenated cells exhibit reversal of the methylation landscape.

[0241] Embodiment 13. The method according to Embodiment 12, wherein the reversal of the methylation landscape is measured by estimation using the Horvath clock.

[0242] Embodiment 14. The method according to any one of Embodiments 4 to 13, wherein the reference value is obtained from aged cells.

[0243] Embodiment 15. The method according to any one of the preceding embodiments, wherein the step of transfecting the cells with messenger RNA comprises a method selected from lipofectamine and LT-1-mediated transfection, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, electroporation, encapsulation of mRNA in liposomes, and direct microinjection.

[0244] Embodiment 16. The method according to any one of the preceding embodiments, wherein one or more cell reprogramming factors are selected from OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0245] Embodiment 17. The method according to any one of the preceding embodiments, wherein one or more cell reprogramming factors comprise OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0246] Embodiment 18. The method according to any one of the preceding embodiments, wherein the cells are mammalian cells.

[0247] Method according to any one of the preceding embodiments, wherein the cell is a human cell.

[0248] Method according to any one of the preceding embodiments, wherein the cell is derived from an elderly subject.

[0249] Method according to any one of the preceding embodiments, wherein the cell is selected from fibroblasts, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells and corneal epithelial cells.

[0250] Method according to embodiment 21, wherein the cell is a mesenchymal stem cell.

[0251] Method according to embodiment 22, wherein the rejuvenated mesenchymal stem cells exhibit a decrease in aging parameters (p16, p21 and positive SAβGal staining), increased cell proliferation, and / or a decrease in ROS levels.

[0252] Method according to any one of the preceding embodiments, which is performed in vitro, ex vivo or in vivo.

[0253] Method according to embodiment 24, which is performed in vivo.

[0254] Method according to embodiment 25, wherein the cell is in a tissue or organ.

[0255] Method according to any one of embodiments 25 to 27, which reduces the number of aged cells in a tissue or organ.

[0256] Method according to any one of embodiments 25 to 27, which reduces the expression of one or more of IL18, IL1A, GROA, IL22 and IL9.

[0257] Embodiment 29. The method according to any one of the preceding embodiments, which restores the function of cells, increases the differentiation ability, enhances the survival rate, increases the replication ability or lifespan, or a combination thereof.

[0258] Embodiment 30. The method according to any one of Embodiments 1 to 24, wherein the step of transfection is performed once a day for 5 days.

[0259] Embodiment 31. The method according to any one of Embodiments 1 to 24, wherein the step of transfection is performed once a day for 4 days.

[0260] Embodiment 32. The method according to any one of Embodiments 1 to 24, wherein the step of transfection is performed once a day for 3 days.

[0261] Embodiment 33. The method according to any one of Embodiments 1 to 24, wherein the step of transfection is performed once a day for 2 days.

[0262] Embodiment 34. A method for treating an age-related disease or condition, a cartilage degeneration disorder, a neurodegenerative disorder and / or a musculoskeletal dysfunction of a subject, comprising the step of administering a therapeutically effective amount of cells, wherein the cells comprise one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors.

[0263] Embodiment 35. The method according to Embodiment 34, wherein the one or more cell reprogramming factors are selected from OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

[0264] Embodiment 36. The method according to any one of Embodiments 34 to 35, wherein the one or more cell reprogramming factors comprise OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

[0265] Embodiment 37. The method according to any one of Embodiments 34 to 36, wherein the subject has an age-related disease or condition.

[0266] Embodiment 38. The method according to Embodiment 34, wherein the age-related disease or condition is selected from eye, skin, or musculoskeletal dysfunction.

[0267] Embodiment 39. The method according to any one of Embodiments 34 to 36, wherein the subject has a cartilage degeneration disorder.

[0268] Embodiment 40. The method according to Embodiment 39, wherein the disorder is selected from arthritis, chondrophasia, spondylosis, ankylosing spondylitis, lupus erythematosus, relapsing polychondritis, and Sjögren's syndrome.

[0269] Embodiment 41. The method according to any one of Embodiments 39 or 40, wherein the treatment reduces the expression of inflammatory factors and / or increases ATP and collagen metabolism.

[0270] Embodiment 42. The method according to Embodiment 41, wherein the inflammatory factor is selected from RANKL, iNOS2, IL6, IFNα, MCP3, and MIP1A.

[0271] Embodiment 43. The method according to Embodiment 42, wherein ATP and collagen metabolism are measured by one or more of increased ATP levels, decreased ROS, increased SOD2, increased COL2A1, and overall proliferation by chondrocytes.

[0272] Embodiment 44. The method according to any one of Embodiments 34 to 36, wherein the subject has musculoskeletal dysfunction.

[0273] Embodiment 45. The method according to any one of Embodiments 34 to 44, wherein the step of administering a therapeutically effective amount of cells includes injection or surgical implantation.

[0274] Embodiment 46. The method according to any one of Embodiments 34 to 45, wherein the therapeutically effective amount of rejuvenated cells is selected from fibroblasts, endothelial cells, chondrocytes, skeletal muscle stem cells, keratinocytes, mesenchymal stem cells, and corneal epithelial cells.

[0275] Embodiment 47. The method according to Embodiment 46, wherein the therapeutically effective amount of rejuvenated cells is corneal epithelial cells.

[0276] Embodiment 48. The method according to Embodiment 47, wherein the rejuvenated corneal epithelium exhibits a decrease in aging parameters.

[0277] Embodiment 49. The method according to Embodiment 48, wherein the aging parameter includes one or more of the expression of p21 and p16, mitochondrial neogenesis PGC1α, and the expression of the inflammatory factor IL8.

[0278] Embodiment 50. A method for treating an age-related disease or condition of a subject, a cartilage degeneration disorder, and / or treating a subject having skeletal muscle dysfunction, the method comprising administering a therapeutically effective amount of one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors.

[0279] Embodiment 51. The method according to Embodiment 50, wherein the one or more cell reprogramming factors are selected from OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0280] Embodiment 52. The method according to any one of Embodiments 50 to 51 to 48, wherein the one or more cell reprogramming factors include OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0281] Embodiment 53. The method according to any one of Embodiments 50 to 52, wherein the subject has an age-related disease or condition.

[0282] Embodiment 54. The method according to Embodiment 53, wherein the age-related disease or condition is selected from eye, skin, or skeletal muscle dysfunction.

[0283] Embodiment 55. The method according to any one of Embodiments 50 to 52, wherein the subject has a cartilage degeneration disorder.

[0284] Embodiment 56. The method according to Embodiment 55, wherein the disorder is selected from arthritis, chondrophasia, spondylarthritis, ankylosing spondylitis, lupus erythematosus, relapsing polychondritis, and Sjögren's syndrome.

[0285] Embodiment 57. The method according to any one of Embodiments 50 to 52, wherein the subject has a musculoskeletal dysfunction.

[0286] Embodiment 58. The method according to any one of Embodiments 50 to 57, wherein the step of administering a therapeutically effective amount of one or more non-integrating messenger RNAs comprises direct injection into the target cells.

[0287] Embodiment 59. The method according to Embodiment 58, wherein the target cells are selected from epithelial cells, endothelial cells, connective tissue cells, muscle cells, and nervous system cells.

[0288] Embodiment 60. A method for rejuvenating an engineered tissue ex vivo, comprising transfecting the tissue with one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors for a continuous period of 5 days or less, thereby producing a rejuvenated engineered tissue.

[0289] Embodiment 61. The method according to Embodiment 60, wherein the engineered tissue exhibits aging parameters, a decrease in inflammation-promoting factors, an improvement in histological scores, or a combination thereof.

[0290] Embodiment 62. The method according to any one of Embodiments 60 or 61, wherein the engineered tissue is engineered skin tissue.

[0291] Embodiment 63. The method according to any one of Embodiments 60 to 62, wherein the aging parameter is selected from p16, positive SAβGal staining, and pro-inflammatory factors IL8 and MMP1.

[0292] Embodiment 64. The method according to any one of Embodiments 60 to 63, wherein the histological score includes morphology, organization, and / or quality.

[0293] Embodiment 65. A pharmaceutical composition comprising rejuvenated cells, wherein the rejuvenated cells are obtained by a step of transfecting a cell with one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors for a continuous period of 5 days or less.

[0294] Embodiment 66. The method according to any one of the preceding embodiments, wherein the one or more cell reprogramming factors are selected from OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

[0295] Embodiment 67. The composition according to Embodiment 65 or 66, wherein the cell exhibits one or more of increased expression of HP1 gamma, H3K9me3, LAP2 alpha, SIRT1, increased mitochondrial membrane potential, decreased reactive oxygen species, and decreased expression of SASP cytokines.

[0296] Embodiment 68. The composition according to Embodiment 67, wherein the SASP cytokine includes one or more of IL18, IL1A, GROA, IL22, and IL9.

[0297] Embodiment 69. The composition according to any one of Embodiments 65 to 68, further comprising one or more additional components selected from nutrients, cytokines, growth factors, extracellular matrix (ECM) components, antibiotics, antioxidants, and immunosuppressants.

[0298] Embodiment 70. The composition according to any one of Embodiments 65 to 69, further comprising a pharmaceutically acceptable carrier.

[0299] Composition according to any one of embodiments 65 to 70, wherein the cells are autologous or allogeneic.

Claims

1. A composition comprising cells of a therapeutically effective amount, wherein the cells are transfected with one or more non-integrating messenger RNAs encoding one or more cell reprogramming factors for a period sufficient to rejuvenate the cells but not so long as to dedifferentiate into pluripotent stem cells, the composition is for treating an age-related disease or condition, a cartilage degeneration disorder, a neurodegenerative disorder, and / or a musculoskeletal dysfunction in a subject, the one or more cell reprogramming factors are selected from the group consisting of Oct4, Sox2, Klf4, cMyc, Lin28, and NANOG, the composition.

2. The composition according to claim 1, wherein the age-related disease or condition is a skin disease or condition, an eye disease or condition, a respiratory disease or condition, or a musculoskeletal disease or condition.

3. The composition according to claim 2, wherein the skin disease or condition is skin atrophy, skin elastolysis, skin wrinkles, sebaceous gland hyperplasia, sebaceous gland hypoplasia, senile lentigines, abnormal pigmentation, white hair, alopecia, hair thinning, or chronic skin ulcers.

4. The composition according to claim 2, wherein the eye disease or condition is age-related macular degeneration, glaucoma, cataract, dry eye, diabetic retinopathy, or vision loss.

5. The composition according to claim 2, wherein the respiratory disease or condition is pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, chronic bronchitis, pulmonary embolism, lung cancer, or pulmonary infection.

6. The composition according to claim 2, wherein the musculoskeletal disease or condition is arthritis, osteoporosis, multiple myeloma, gout, Paget's disease, fracture, myelodysplastic syndrome, ankylosis, diffuse idiopathic skeletal hyperostosis, hematogenous osteomyelitis, muscle atrophy, peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis, Duchenne muscular dystrophy, primary lateral sclerosis, or myasthenia gravis.

7. The composition according to claim 1, wherein the cartilage degeneration disorder is arthritis. Claim 8 The composition according to claim 7, wherein the arthritis is osteoarthritis or rheumatoid arthritis.

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